Dietary Supplementation of Citric Acid and Phytase in Plant-Based Diets Improves Mineral Bioavailability in Common Carp, Cyprinus carpio

Syed Makhdoom Hussain1*, Muhammad Zubair-ul-Hassan Arsalan2,

Nisar Ahmad3, Bilal Ahmad4, Sadia Tabassum1 and Basharat Ali5

1Fish Nutrition Lab, Department of Zoology, Government College University, Faisalabad

2Department of Life Sciences, Khawaja Fareed University of Engineering and Information Technology, Rahim Yar Khan

3Department of Zoology, University of Jhang, Jhang

4Department of Zoology, The Islamia University Bahawalpur, Bahawalnagar Campus,

5Department of Agriculture, Khawaja Fareed University of Engineering and Information Technology, Rahim Yar Khan

ABSTRACT

The common carp (Cyprinus carpio) is an agastric fish, having digestive tract pH above 6 and cannot digest phytate properly found in plant-based diets. Phytate acts as an anti-nutrient, as it chelates with minerals to form insoluble mineral-phytate complexes and reduce their bioavailability. Therefore, in this study citric acid (CA) and exogenous phytase (PHY) were incorporated with canola meal-based (CM) diets to check their effects on mineral availability and digestibility in C. carpio fingerlings. Four CM-based diets were prepared by substituting CM for fishmeal at 0, 25, 50, and 75% levels, in which PHY (0 and 750 FTU kg-1) and CA (0 and 2.5%) were added at two levels, creating a total of sixteen test diets labelled from T1 to T16. For 8-week, common carp were fed on test diets about 2.5% of their live body weight. Fecal samples were collected from each tank twice daily to assess mineral digestibility. The results showed that the highest apparent digestibility coefficients for minerals were observed in fish fed on T12 diet (50% CM, 2.5% CA and 750 FTUkg-1 PHY), This combination synergistically improved the digestibility of minerals, such as calcium (63%), phosphorus (70%), sodium (60%), potassium (70%), magnesium (68%), iron (73%), copper (73%), manganese (67%), and zinc (63%). These findings suggested that supplementing CM based diets with CA and PHY effectively released chelated minerals from phytate complexes, thereby enhancing their availability for C. carpio. This approach can contribute to improve fish performance and overall nutritional efficiency. Incorporating CA and PHY in CM based diets holds promise for optimizing mineral digestibility in fish.

Article Information

Received 03 August 2023

Revised 21 October 2023

Accepted 09 November 2023

Available online 11 January 2024

(early access)

Published 29 April 2025

Authors’ Contribution

SMH: Conceptualization,

supervision. MZHA: Formal analysis,

investigation, writing - original draft.NA: Methodology, software. BA, ST, BA: Writing review and editing.

Key words

Cyprinus carpio, Canola meal, Phytase, Citric acid, Mineral digestibility, Supplements

DOI: https://dx.doi.org/10.17582/journal.pjz/20230803073318

* Corresponding author: [email protected]

0030-9923/2025/0003-1141 $ 9.00/00

Copyright 2025 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

The common carp (Cyprinus carpio) is a significant economic fish species that is well known for its superior nutrition, fastest growth rate, and long standing breeding history. C. carpio is an omnivorous bottom feeder carp that feeds on plants, small invertebrates as well as detritus (Adámek et al., 2019; Selamoglu et al., 2015). It is a major contributor to freshwater aquaculture (Nakajima et al., 2019). In 2018, the production of common carp made up 7.7% (4.2 Mt) of all fish produced globally (Eljasik et al., 2022). Carp aquaculture was predicted to consume 27% (13.5 Mt) of aqua-feed globally, in 2015. Around 37.5% (5.1 Mt) of this aqua-feed intended for carp farming worldwide was consumed by common carp alone (FAO FishStat, 2017). Fishmeal is considered as an ideal source of protein in aqua-feed owing to its well-balanced amino acids, fatty acids and mineral content. It has less carbohydrates and anti-nutrients with higher palatability as well as digestibility (Gatlin et al., 2007). Over the years, expensive fishmeal inclusion rates have significantly reduced in feed based aquaculture industry (Olsen and Hasan, 2012), that resulted in extensive research and usage of different plant protein sources.

Canola meal (CM), a plant protein source, has significant potential to replace FM. It is a by-product of the oil-processing industry, having comparatively higher protein level (~ 38%), a balanced amino acid composition and lower production cost (Enami, 2011). However, there are number of anti-nutritional factors in CM i.e., fiber, glucosinolates, phenolic compounds and phytic acid that limit its use, particularly in animal nutrition. Phytate or phytic acid, a reservoir of phosphorus, is found in CM about 4-5% and lower its nutritive value by making bonds with multivalent cations i.e., Zn, Fe and Ca and hence, lowering their availability to animals (Al-Asheh and Duvnjak, 1994; Sarfraz et al., 2020).

Microbial phytase (PHY) separates minerals and phosphorus, lowering inositol phosphate from phytate. Monogastric and agastric animals show natural deficiency of PHY due to absence of PHY producing microbes. Therefore, the dietary incorporation of exogenous PHY to plant-based fish diet improves the minerals bioavailability and digestibility, which results in less nutrient loss through excretion in the feces, reducing water body pollution (Priya et al., 2023). PHY needs an optimum range pH (2.5-5.5) to function effectively. Fish without stomachs, such as the common carp, have digestive systems with neutral to alkaline pH. This huge pH difference between the optimum PHY activity and intestine of agastric fish is a main reason of poor efficiency of PHY (Kumar et al., 2012; Sarfraz et al., 2020).

Organic acids i.e., citric acid (CA) can be used as feed additive to increase the efficiency and utilization of PHY because these organic acids reduce the acidity of feed, lower the digestive tract pH and boost the efficiency of supplemented PHY (Baruah et al., 2005; Kumar et al., 2012). Decrease in pH by organic acids may directly increase the phytate solubility, and dissociate mineral complexes, improving mineral availability and absorption in gut (Canibe et al., 2005). PHY and CA has been proposed as promising feed additive to improve mineral availability and digestibility, improving general fish health. Therefore, the present research work aimed to examine the individual and synergistic impacts of dietary PHY and CA on the digestibility and absorption of minerals present in CM based diets of C. carpio, which is a significant aquaculture fish species worldwide.

MATERIALS AND METHODS

Fish and experimental conditions

The C. carpio fingerlings were obtained from the Government Fish Hatchery in Faisalabad. Prior to start of the trial, a disinfection process was conducted on the fingerlings. This involved immersing them in a sodium chloride (NaCl) solution with a concentration of 5g/L for a specific duration. After the disinfection process, fingerlings were shifted to cement tanks for two weeks for acclimatization, fed on basal diet once daily. Throughout the experiment, water quality indicators i.e., temperature, pH, and dissolved oxygen were carefully monitored regularly.

Feed ingredients and test diets

All ingredients were sourced from the local commercial feed market. Prior to formulation, the chemical composition of these feed ingredients was analyzed using standard methods provided by AOAC (2005). CM, the basic test ingredient, was used to replace fishmeal at levels of 0, 25, 50, and 75%, resulting four CM based test diets, in which PHY (0 and 750 FTU kg-1) and CA (0% and 2.5%) at two levels were incorporated, resulting in a total of sixteen test diets labeled from T1 to T16 (Table I). After undergoing the grinding process, the feed ingredients were sieved through a mesh with a size of 0.5mm to ensure a consistent particle size. Subsequently, these ingredients were mixed in a food mixer for approximately 5 min. During the mixing process, fish oil was added gradually to mixture. After that to form a suitable dough, about 10-15% water was added. Pellets were formed via pelleting machine, then dried and placed at 4℃ to be used in trial.

Plan of feeding and sample collection

Each experimental diet was tested in triplicates (16 experimental diets × 3 tanks). Each tank was initially stocked with 15 fingerlings. The feeding schedule was two times a day (at 8:00 am and 2:00 pm). The fingerlings were given their referred diets, ~ 5% of their live body weight. Following 2h. of feeding, fecal matter was collected via specially designed V-shaped water tanks. Great care was taken during fecal collection to avoid the breakdown of fecal strings and nutrient leaching. The fecal material collected from the tanks was carefully dried in an oven set at 60°C to remove moisture content. Subsequently, the dried fecal material was finely ground into a powder to ensure homogeneity and facilitate chemical analysis. The powdered samples were then stored in the laboratory under appropriate conditions to maintain their integrity for future analysis.

Mineral analysis of feed and feces

Before analysis, test diets and feces samples were individually homogenized using a mortar and pestle to ensure consistency. The analysis was done by following standard procedures outlined by AOAC (2005). To estimate

 

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

Test diets	Fishmeal replace-ment levels (%)	Fish-meal	Canola meal	Phytase (FTU kg-1)	Citric acid (%)	Rice polish	Wheat flour	Fish oil	Vitamins premix*	Minerals premix**	Chromic oxide
T1	0%	48	18	0	0	3	22	6	1	1	1
T2		48	18		2.5	3	19.5	6	1	1	1
T3		48	18	750	0	3	22	6	1	1	1
T4		48	18		2.5	3	19.5	6	1	1	1
T5	25%	36	35	0	0	3	17	6	1	1	1
T6		36	35		2.5	3	14.5	6	1	1	1
T7		36	35	750	0	3	17	6	1	1	1
T8		36	35		2.5	3	14.5	6	1	1	1
T9	50%	24	53	0	0	3	11	6	1	1	1
T10		24	53		2.5	3	8.5	6	1	1	1
T11		24	53	750	0	3	11	6	1	1	1
T12		24	53		2.5	3	8.5	6	1	1	1
T13	75%	12	71	0	0	3	5	6	1	1	1
T14		12	71		2.5	3	2.5	6	1	1	1
T15		12	71	750	0	3	5	6	1	1	1
T16		12	71		2.5	3	2.5	6	1	1	1

 

Wheat flour was substituted for phytase and citric acid. *Vitamin (Vit.) premix kg-1: Vit. A: 15,000,000 IU, Vit. B12: 40 mg, Vit. C: 15,000 mg, Vit. D3: 3,000,000 IU, B2: 7000 mg, Vit. E:30000 IU, Vit. B6: 4000 mg, Vit. Ca pantothenate: 12,000 mg, Vit. K3: 8000 mg, Folic acid: 1500 mg, Nicotinic acid: 60,000 mg. **Mineral premix kg-1: Fe: 1000 mg, Ca: 155 g, P: 135 g, Se: 3 mg, Na: 45 g, Co: 40 mg, Zn:3000mg, Cu: 600 mg, Mn: 2000 mg, I: 40 mg, Mg: 55 g.

 

mineral content, the samples were digested individually in a mixture of perchloric acid and boiling nitric acid (1:2 ratios). Following dilution using distilled water, mineral content was evaluated using an Atomic Absorption Spectrophotometer (Hitachi Polarized Atomic Absorption Spectrometer, Z-8200). Calibrated standards formed with commercially accessible standards from AppliChem® Gmbh Ottoweg4, DE-64291 Darmstadt, Germany, were used for estimating mineral contents. For phosphorus content, a UV/VIS spectrophotometer at 720nm absorbance was used for calorimetric assessment via ammonium molybdate reagent. Sodium and potassium content was measured utilizing a flame photometer (Jenway PFP-7, UK). The chromic oxide, an inert marker, in test diets and feces was estimated by Spectrophotometer at 370nm absorbance after oxidation via perchloric acid (Divakaran et al., 2002).

Calculation of apparent mineral digestibility coefficients

The apparent digestibility coefficients (ADC%) of the minerals in diets were estimated by the following standard formula (NRC, 1993):

ADC (%) = [100- (100 × (Percentage marker in diet × Percentage nutrient in feces)/ (Percentage marker in feces × Percentage nutrient in diet))]

Statistical analysis

One way analysis of variance (ANOVA) was utilized for analysis of data on mineral digestibility and absorption. To determine significant variations, Tukey’s honestly significant difference test was applied, with a significance level set at P<0.05 (Snedecor and Cochran, 1989). For the statistical assay, the Co-Stat Computer Package of a version 6.303, PMB 320, Monterey, CA, 93940 USA) was utilized.

RESULTS

Significant differences in mineral compositions between the test diets and feces samples were discovered by their analysis. Tables II, III, and IV outline the primary effects of PHY, CA, and their interaction on the minerals composition of test diets, feces and minerals digestibility in C. carpio fingerlings. The mineral digestibility of C. carpio fingerlings significantly improved (P<0.05) with the incorporation of CA and PHY in CM based diets. In comparison, the T12 diet showed the maximum values of mineral digestibility with notable percentages recorded for Ca (63.12%), P (70.78%), Na% (60.56%), K (70.76%), Mg (68.07%), Fe (73.14%), Cu (73.18%), Mn (67.19%), and Zn (63.29%), which were significantly (P<0.05) higher

 

Table II. Effect of dietary supplementation of citric acid and phytase in plant based diet on mineral composition (%) of Cyprinus carpio.

Test diets	Fish meal replace-ment levels (%)	Phy-tase (FTU kg-1)	Citric acid (%)	Ca	Na	K	Fe	Cu	Zn	Mn	P	Mg	Al	Cr
T1	0%	0	0	3.20	0.24	0.260	0.019	0.0113	0.029	0.017	0.515	0.0055	0.00018	0.032
T2			2.5	3.20	0.21	0.263	0.013	0.0115	0.029	0.012	0.515	0.0054	0.00018	0.031
T3		750	0	3.21	0.23	0.267	0.012	0.0121	0.029	0.012	0.515	0.0051	0.00018	0.031
T4			2.5	3.16	0.24	0.267	0.012	0.0124	0.029	0.012	0.516	0.0055	0.00018	0.033
T5	25%	0	0	3.15	0.24	0.250	0.012	0.0122	0.029	0.012	0.513	0.0221	0.00018	0.028
T6			2.5	3.11	0.27	0.570	0.012	0.0122	0.029	0.012	0.514	0.0055	0.00042	0.029
T7		750	0	3.12	0.26	0.253	0.012	0.0125	0.028	0.012	0.514	0.0051	0.00018	0.025
T8			2.5	3.16	0.23	0.250	0.012	0.0117	0.029	0.012	0.515	0.0058	0.00018	0.024
T9	50%	0	0	3.15	0.24	0.237	0.012	0.0118	0.029	0.012	0.514	0.0055	0.00018	0.029
T10			2.5	3.17	0.27	0.250	0.012	0.0113	0.028	0.012	0.543	0.0053	0.00018	0.029
T11		750	0	3.14	0.25	0.253	0.012	0.0125	0.028	0.012	0.545	0.0056	0.00018	0.037
T12			2.5	3.18	0.22	0.247	0.012	0.0146	0.029	0.012	0.541	0.0056	0.00018	0.032
T13	75%	0	0	3.14	0.23	0.257	0.012	0.0116	0.029	0.012	0.552	0.0055	0.00018	0.032
T14			2.5	3.16	0.28	0.243	0.012	0.0118	0.029	0.012	0.543	0.0056	0.00018	0.029
T15		750	0	3.14	0.24	0.247	0.012	0.0114	0.029	0.012	0.512	0.0057	0.00018	0.015
T16			2.5	3.13	0.25	0.253	0.012	0.0163	0.029	0.012	0.531	0.0057	0.00018	0.036
Pool Standard Error (PSE)				0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01

 

At P<0.05, means within columns with distinct superscripts differ significantly. Data are the means of three replicas.

 

Table III. Effect of canola meal-based diets supplemented with phytase and citric acid on the minerals composition (%) in feces of C. carpio fingerlings.

Test diets

Fish meal replace-ment levels (%)

Phy-tase (FTU kg-1)

Citric acid (%)

Ca

Na

K

Fe

Cu

Zn

Mn

P

Mg

Al

Cr

T1

0%

0

0

1.45c

0.10a

0.12b

0.004c

0.008a

0.021f

0.007a

0.36g

0.011b

0.0005a

0.021d

T2

2.5

1.22d

0.13a

0.08c

0.008b

0.004a

0.044d

0.006a

0.38f

0.003h

0.0001a

0.020d

T3

750

0

1.45c

0.11a

0.20a

0.009b

0.005a

0.010j

0.006a

0.32j

0.004g

0.0002a

0.013d

T4

2.5

1.52c

0.13a

0.12b

0.006b

0.006a

0.053a

0.007a

0.44b

0.006e

0.0002a

0.024d

T5

25%

0

0

1.66c

0.13a

0.15b

0.040a

0.007a

0.028e

0.008a

0.43c

0.005f

0.0003a

0.036c

T6

2.5

1.45c

0.12a

0.12b

0.004c

0.005a

0.051b

0.006a

0.35h

0.004g

0.0006a

0.037c

T7

750

0

1.32c

0.10a

0.08c

0.038a

0.004a

0.020f

0.006a

0.38f

0.003h

0.0001a

0.016d

T8

2.5

1.41c

0.11a

0.12b

0.006b

0.005a

0.010j

0.006a

0.33i

0.003h

0.0002a

0.018d

T9

50%

0

0

1.60c

0.12a

0.12b

0.006b

0.005a

0.047c

0.007a

0.42d

0.007d

0.0002a

0.151b

T10

2.5

1.88a

0.14a

0.15b

0.007b

0.005a

0.019f

0.008a

0.44b

0.007d

0.0002a

0.032c

T11

750

0

1.45c

0.11a

0.12b

0.004c

0.005a

0.015h

0.007a

0.31k

0.019a

0.0006a

0.021d

T12

2.5

1.21d

0.10a

0.08c

0.008b

0.007a

0.010j

0.007a

0.28l

0.004g

0.0001a

0.020d

T13

75%

0

0

1.38c

0.10a

0.13b

0.039a

0.005a

0.009k

0.006a

0.39e

0.003h

0.0002a

0.018d

T14

2.5

1.59c

0.11a

0.12b

0.006b

0.004a

0.013i

0.006a

0.45a

0.009c

0.0002a

0.176a

T15

750

0

1.91a

0.12a

0.15b

0.006b

0.006a

0.017g

0.007a

0.45a

0.006e

0.0003a

0.035c

T16

2.5

1.44c

0.13a

0.12b

0.003c

0.005a

0.012i

0.008a

0.35h

0.007d

0.0003a

0.021d

Pool standard error (PSE)

0.07

0.01

0.01

0.01

0.01

0.02

0.01

0.01

0.01

0.01

0.01

 

At P<0.05, means within columns with distinct superscripts differ significantly. Data are the means of three replicas.

 

Table IV. Effect of canola meal-based diets supplemented with phytase and citric acid on the Apparent digestibility coefficients of minerals by C. carpio fingerlings.

Test diets

Fish meal replace-ment levels (%)

Phy-tase (FTU kg-1)

Citric acid (%)

Ca

Na

K

Fe

Cu

Zn

Mn

P

Mg

Al

Cr

T1

0%

0

0

51.50d

33.17f

42.95g

53.86b

43.15e

51.46d

32.97j

34.65g

44.65e

36.41d

32.14f

T2

2.5

58.79b

50.56b

49.43d

52.14b

49.85c

60.29b

41.87h

43.08e

46.41d

46.50b

37.55e

T3

750

0

57.94c

44.00d

45.79f

49.52c

45.38d

62.62a

44.92g

44.36e

41.68f

35.04d

42.71c

T4

2.5

49.88d

42.88d

43.45g

45.37d

43.23e

52.08d

36.80i

40.74f

38.96g

33.93d

35.88f

T5

25%

0

0

40.56f

42.69d

39.80h

35.75i

38.79e

41.00f

31.40j

37.13f

37.66g

32.97d

33.07f

T6

2.5

49.83d

43.17d

42.62g

49.19c

41.14e

51.46d

41.97h

40.65f

44.62e

36.08d

34.81f

T7

750

0

57.45c

50.56b

51.76c

43.48e

47.18d

58.29b

47.86f

45.68e

45.61d

42.50c

45.55b

T8

2.5

58.94b

49.33b

51.46c

46.85d

39.05e

60.96b

50.25e

44.99e

41.21f

38.38d

38.71e

T9

50%

0

0

49.54d

46.88c

45.12f

42.70e

37.24e

49.74e

45.80g

38.81f

41.60f

33.26d

43.88c

T10

2.5

45.56e

41.69d

41.46g

39.42g

33.79f

42.67f

50.40e

39.73f

37.36g

36.64d

40.07d

T11

750

0

50.16d

38.84e

48.95d

45.86d

41.13e

55.46c

56.97b

59.60b

54.58b

48.41b

38.81e

T12

2.5

63.12a

60.56a

70.76a

73.14a

73.18a

63.29a

67.19a

70.78a

68.07a

59.84a

52.69a

T13

75%

0

0

58.61b

44.33d

55.13b

40.52f

46.05d

59.29b

48.91f

48.45d

41.67f

33.04d

38.38e

T14

2.5

45.88c

41.21d

54.45b

45.37d

40.57e

52.74d

50.47e

38.91f

39.26g

28.93e

35.67f

T15

750

0

40.22f

50.35b

48.46d

38.75h

36.48e

51.67d

54.06c

40.13f

38.18g

26.64e

38.85e

T16

2.5

49.50d

59.17a

47.28e

49.86c

60.39b

48.46e

52.63d

54.32c

50.23c

39.74d

34.88f

Pool standard error (PSE)

1.84

1.43

1.57

2.13

2.10

1.64

1.52

1.40

1.82

2.28

1.82

 

At P<0.05, means within columns with distinct superscripts differ significantly. Data are the means of three replicas.

 

when compared to control (without any supplementation) and other experimental diets. Thus, supplementation with 2.5% CA and 750 FTU kg-1 PHY in 50% CM based diet was optimum to improve the fish performance in terms of minerals digestibility. The T11 diet (750 FTU kg-1 PHY in 50% CM based diet) also showed the second-lowest mineral excretion in the feces, representing high digestibility of minerals.

DISCUSSION

Fishmeal is a common protein source from animals in the aquaculture feeding because of its good amino acid content, higher palatability, and enhanced digestibility. However, the usage of fishmeal in aquatic diets increases costs of production as stocks of wild fish are depleting. As a result, there is a pressing requirement to look for novel and less expensive alternate sources of protein (Mugwanya et al., 2023). Currently, plant proteins are increasingly being used as fishmeal alternatives in aquatic diets because of their higher nutritional value, widespread availability, and lower cost. CM, recognized for its superior mineral content, has emerged as a plant protein source of choice to substitute fishmeal in fish diets. Its utilization provides a promising solution for maintaining adequate mineral resources in fish feed formulations. However, an anti-nutritional factor phytate is present in plants that hamper the availability and absorption of minerals in fish, by forming complexes with minerals, limiting their bioavailability (Selamoglu et al., 2015; Rizwanuddin et al., 2023). In the current study, CM based diets having identical mineral compositions were fed to C. carpio. However, CA and PHY were supplemented in these diets to improve the mineral digestibility in C. carpio fingerlings.

According to current findings, the 50% substitution of CM for fishmeal with 2.5% CA and 750 FTU kg-1 PHY supplements emerged as the most optimal combination that reacted synergistically to reduce the excretion of essential minerals into the aquatic environment through feces and improve the minerals bioavailability. Consistent with the findings of the present study, Shah et al. (2021) also observed a reduction in mineral excretion (P, K, Mg, Na, Mn, Fe, and Cu) when Labeo rohita fingerlings were fed a soybean meal-based diet combined with 3% CA and 1000 FTU/kg PHY. This indicates that the combined supplementation of CA and PHY synergistically decreases mineral excretion in fish feces and enhances mineral digestibility. Similarly, Afzal et al. (2020) discovered significant improvements in the digestibility of minerals, i.e., Ca, Cu, Fe, P, Na, and K in L. rohita given a sunflower meal-based diets with 2% CA and 1000 FTU/kg PHY. In another study by Afzal et al. (2019), the digestibility of Ca, Cu, Fe, K, Mg, Mn, P, and Zn in L. rohita juveniles was significantly enhanced (P<0.05) fed with soybean meal based diet combined with 2% CA and 1000 FTU/kg PHY compared to the control diet. Similarly, Hussain et al. (2018) reported the highest apparent digestibility coefficients (ADC%) of minerals (Na: 64%, Fe: 64%, Cu: 68%, Zn: 74%, Ca: 68%, K: 62%, Mg: 53%, P: 77%, and Mn: 67%) in Cirrhinus mrigala fingerlings fed a corn gluten meal-based diet with CA 5% and PHY 500 FTU/kg. They suggested that the use of CA and PHY increased the availability of minerals for fish by releasing chelated minerals, resulting in reduced excretion of essential minerals into the aquatic environment and, consequently, mitigating aquatic pollution. Several studies have consistently demonstrated that the synergistic supplementation of PHY and CA in diets based on plant proteins enhances the availability of minerals for fish. Examples include diets based on canola meal with 3% CA and 1000 FTU/kg PHY for L. rohita fingerlings (Habib et al., 2018), sunflower meal with 2% CA and 1000 FTU/kg PHY for L. rohita juveniles (Rabia et al., 2017), cottonseed meal with 4% CA and 400 FTU/kg PHY for L. rohita (Hussain et al., 2016), and soybean meal with 3% CA and 1000 FTU/kg PHY for L. rohita (Shah et al., 2016; Baruah et al., 2007).

In present study, it was observed that supplementation of CM with CA and PHY resulted in the maximum digestibility of various minerals, including Ca, K, Fe, Cu, Mn, P, Mg, and Al, in C. carpio fingerlings. This improved mineral digestibility proved the effective phytic acid hydrolysis by PHY. The addition of CA enhanced digestion and lowered the pH of the gastrointestinal tract, providing optimal conditions for PHY activity. As a result, the minerals became more bioavailable to C. carpio fingerlings, leading to improved growth performance. Furthermore, the synergistic incorporation of CA and PHY resulted in the minimal excretion of essential minerals into the aquatic ecosystem compared to the control diet without any supplementation. Similar findings have been reported by several researchers in L. rohita (Baruah et al., 2007; Shah et al., 2016, 2021; Rabia et al., 2017; Habib et al., 2018; Afzal et al., 2019, 2020) and C. mrigala (Hussain et al., 2018). In conclusion, the CA and PHY addition facilitated the dissociation of phytate chelated structure, leading to the release and assimilation of essential minerals in fish. This resulted in improved mineral digestibility and reduced excretion of essential minerals through feces. The findings of this study aligned with previous research demonstrating that the supplementation of plant-based diets with CA enhances mineral absorption in fish (Sugiura et al., 2001).

The current findings revealed a positive correlation between CA and PHY, highlighting their synergistic impact on mineral absorption. It is plausible that CA played a crucial role in optimizing the pH levels within the fish’s gastrointestinal tract, facilitating the efficient functioning of PHY and thereby maximizing the mineral digestion process. Furthermore, demonstrated that diet acidification led to a reduction in the rate of stomach emptying, allowing for an extended time frame for PHY to exert its effects. Notably, prior research conducted by Baruah et al. (2007) and Sugiura et al. (2001) has also documented enhanced mineral absorption in response to the incorporation of CA and PHY in plant-based diets, further validating the beneficial impacts of these supplements.

The current study revealed that the digestibility of minerals was significantly lower in the group fed the control diet (without CA or PHY) compared to the groups fed diets having CA or PHY or both. Similarly, Zhang et al. (2016) found improvement in minerals bioavailability in Large yellow croaker (Larimichthys crocea) fed citric acid treated soybean meal-based diets. Notably, the improved digestibility of phosphorus (P) was found to have a positive impact on the digestibility of other minerals, including Ca, K, Fe, Cu, Mn, Mg, and Al. This finding aligns with previous research highlighting the beneficial role of PHY supplementation in releasing chelated phosphorus and enhancing mineral availability in various fishes (Baruah et al., 2007; Cao et al., 2007; Dalsgaard et al., 2009; Pontes et al., 2021; Hussain et al., 2022). In a study by Nwanna and Schwarz (2007), it was discovered that fish groups supplemented with PHY exhibited higher P digestibility and reduced P discharge compared to the control group. The incorporation of PHY in plant-based diets has shown promising results in producing less expensive and environmentally friendly aqua-feeds by enhancing nutrients digestibility and minimizing nutrient discharge into the aquatic environment (Priya et al., 2023). Therefore, the supplementation of PHY in plant meal-based diets is expected to contribute to the reduction of aquatic pollution.

CONCLUSION

In conclusion, the findings of this study showed that the T12 diet, which involved a 50% replacement level of CM supplemented with CA and PHY, demonstrated a significant enhancement in mineral digestibility for C. carpio fingerlings. This finding underscores the potential of incorporating plant by-products like CM as a viable and sustainable alternative to fishmeal in the development of environment friendly and cost-effective aqua feed for C. carpio fingerlings. Additionally, the PHY and CA supplements in plant-based diets maximize their effectiveness by reducing the anti-nutritional components and boosting mineral bioavailability.

Funding

The funds for this project were provided by HEC Islamabad, Pakistan for its consistent assistance by providing Project# 5649/Punjab/NRPU/R & D/HEC/2016 and Project# 20-4892/NRPU/R & D/HEC/14/1145 to be able to manage this research work.

Ethical statement

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

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Adámek, Z., Mössmer, M. and Hauber, M., 2019. Current principles and issues affecting organic carp (Cyprinus carpio) pond farming. Aquaculture, 512: 734261. https://doi.org/10.1016/j.aquaculture.2019.734261

Afzal, M., Fatima, M., Qamar, A., Farhan, M. and Shah, S.Z.H., 2020. Growth, muscle proximate composition and whole-body nutrient status of Labeo rohita fed acidified and phytase pre-treated sunflower meal-based diets. Pakistan J. Zool., 52: 937. https://doi.org/10.17582/journal.pjz/20190408080431

Afzal, M., Sultana, N., Hassan, A., Shah, S.Z.H., Fatima, M., Hussain, S.M. and Hussain, M., 2019. Effectiveness of acidification and phytase pretreatment on growth performance, muscle proximate composition and nutrient digestibility of rohu (Labeo rohita, Hamilton 1822) juveniles fed soybean meal-based diet. Pakisatn J. Zool., 51: 1741. https://doi.org/10.17582/journal.pjz/2019.51.5.1741.1750

Al-Asheh, S. and Duvnjak, Z., 1994. The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal by Aspergillus carbonarius during a solid state fermentation process. Biotechnol. Lett., 16: 183-188. https://doi.org/10.1007/BF01021668

AOAC, 2005. Official methods of analysis. 18th edn. Association of Official Analytical Chemists; Arlington, VA, USA.

Baruah, K., Pal, A.K., Sahu, N.P., Jain, K.K., Mukherjee, S.C. and Debnath, D., 2005. Dietary protein level, microbial phytase, citric acid and their interactions on bone mineralization of Labeo rohita (Hamilton) juveniles. Aquacult. Res., 36: 803-812. https://doi.org/10.1111/j.1365-2109.2005.01290.x

Baruah, K., Sahu, N.P., Pal, A.K., Debnath, D. and Yengkokpam, S.A., 2007. Interactions of dietary microbial phytase, citric acid and crude protein level on mineral utilization by rohu, Labeo rohita (Hamilton) juveniles. J. World Aquacult. Soc., 38: 238-249. https://doi.org/10.1111/j.1749-7345.2007.00092.x

Canibe, N., Højberg, O., Højsgaard, S. and Jensen, B.B., 2005. Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs. J. Anim. Sci., 83: 1287-1302. https://doi.org/10.2527/2005.8361287x

Cao, L., Wang, W., Yang, C., Yang, Y., Diana, J., Yakupitiyage, A., Luo, Z. and Li, D., 2007. Application of microbial phytase in fish feed. Enzyme Microb. Technol., 40: 497–507. https://doi.org/10.1016/j.enzmictec.2007.01.007

Dalsgaard, J., Ekmann, K.S., Pedersen, P.B. and Verlhac, V., 2009. Effect of supplemented fungal phytase on performance and phosphorus availability by phosphorus-depleted juvenile rainbow trout (Oncorhynchus mykiss), and on the magnitude and composition of phosphorus waste output. Aquaculture, 286: 105-112. https://doi.org/10.1016/j.aquaculture.2008.09.007

Divakaran, S., Leonard, G.O., and Lan, P.F., 2002. Note on the methods for determination of chromic oxide in shrimp feeds. J. Agric. Fd. Chem., 50: 464-467. https://doi.org/10.1021/jf011112s

Eljasik, P., Panicz, R., Sobczak, M. and Sadowski, J., 2022. Key performance indicators of common carp (Cyprinus carpio L.) wintering in a pond and RAS under different feeding schemes. Sustainability, 14: 3724. https://doi.org/10.3390/su14073724

Enami, H.R., 2011. A review of using canola/rapeseed meal in aquaculture feeding. J. Fish aquat. Sci., 6: 22-36. https://doi.org/10.3923/jfas.2011.22.36

FAO FishStat, 2017. Fisheries and aquaculture software. FishStat plus universal software for fishery statistical time series. FAO Fisheries and Aquaculture Department [online], Rome. Available from http://www.fao.org/fishery/.

Gatlin III, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W. and Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: A review. Aquacult. Res., 38: 551-579. https://doi.org/10.1111/j.1365-2109.2007.01704.x

Habib, R.Z., Afzal, M., Shah, S.Z.H., Fatima, M., Bilal, M. and Hussain, S.M., 2018. Potential of phytase and citric acid treated canola meal-based diet to enhance the minerals digestibility in Labeo rohita fingerlings. Pakistan J. Zool., 50: 2045-2050. https://doi.org/10.17582/journal.pjz/2018.50.6.2045.2050

Hussain, S.M., Afzal, M., Nasir, S., Javid, A., Azmat, H., Mamoona, M.S., Shah, S.H.Z., Hussain, M., Mustafa, I. and Iqbal, M., 2016. Role of phytase supplementation in improving nutrient digestibility and growth performance for Labeo rohita fingerlings fed on canola meal-based diet. J. appl. Anim. Res., 45: 15-21. https://doi.org/10.1080/09712119.2015.1091331

Hussain, S.M., Ahmad, N., Javid, A., Shahzad, M.M., Hussain, M. and Arsalan, M.Z.U.H., 2018. Effects of phytase and citric acid supplemented corn gluten (30%) meal-based diets on the mineral digestibility of Cirrhinus mrigala fingerlings. Turk. J. Fish. Aquat. Sci., 18: 501-507.

Hussain, S.M., Hanif, S., Sharif, A., Bashir, F. and Iqbal, H.M., 2022. Unrevealing the sources and catalytic functions of phytase with multipurpose characteristics. Catal. Lett., 152: 1-14. https://doi.org/10.1007/s10562-021-03752-z

Kumar, V., Sinha, A.K., Makkar, H.P.S., De Boeck, G. and Becker, K., 2012. Phytate and phytase in fish nutrition. J. Anim. Physiol. Anim. Nutr., 96: 335-364. https://doi.org/10.1111/j.1439-0396.2011.01169.x

Mugwanya, M., Dawood, M.A., Kimera, F. and Sewilam, H., 2023. Replacement of fish meal with fermented plant proteins in the aquafeed industry: A systematic review and meta‐analysis. Rev. Aquac., 15: 62-88. https://doi.org/10.1111/raq.12701

Nakajima, T., Hudson, M.J., Uchiyama, J., Makibayashi, K. and Zhang, J., 2019. Common carp aquaculture in Neolithic China dates back 8,000 years. Nat. Ecol. Evol., 3: 1415-1418. https://doi.org/10.1038/s41559-019-0974-3

National Research Council, 1993. Nutrient requirements of fish. National Academies Press.

Nwanna, L.C. and Schwarz, F.J., 2007. Effect of supplemental phytase on growth, phosphorus digestibility and bone mineralization of common carp (Cyprinus carpio L.). Aquacult. Res., 38: 1037-1044. https://doi.org/10.1111/j.1365-2109.2007.01752.x

Olsen, R.L. and Hasan, M.R., 2012. A limited supply of fishmeal: Impact on future increases in global aquaculture production. Trends Fd. Sci., 27: 120-128. https://doi.org/10.1016/j.tifs.2012.06.003

Pontes, T.C., Brito, J.M.D., Wernick, B., Urbich, A.V., Panaczevicz, P.A.P., Miranda, J.A.G. and Furuya, W.M., 2021. Top‐sprayed phytase enhances the digestibility of energy, protein, amino acids and minerals, and reduces phosphorus output in Nile tilapia fed all‐vegetable diets. Aquacult. Res., 52: 6562-6570. https://doi.org/10.1111/are.15527

Priya, V.I., Pragya, G.R.K., Singh, B., Sharma, J.G. and Giri, B., 2023. Role of microbial phytases in improving fish health. Rev. Aquacult., pp. 1-12.

Rabia, S., Afzal, M. and Shah, S.Z.H., 2017. Nutrient digestibility performance by rohu (Labeo rohita) juveniles fed acidified and phytase pre-treated sunflower meal-based diet. J. appl. Anim. Res., 45: 331-335. https://doi.org/10.1080/09712119.2016.1190731

Rizwanuddin, S., Kumar, V., Naik, B., Singh, P., Mishra, S., Rustagi, S. and Kumar, V., 2023. Microbial phytase: Their sources, production, and role in the enhancement of nutritional aspects of food and feed additives. J. Agric. Fd. Res., 12: 100559. https://doi.org/10.1016/j.jafr.2023.100559

Sarfraz, Q., Hussain, S.M., Sharif, A., Selamoglu, Z., Bashir, F. and Ahsan, S., 2020. Potential of phytase supplemented Moringa oleifera leaf meal-based diet on mineral digestibility of Oreochromis niloticus fingerlings. J. Surv. Fish. Sci., 6: 65-77. https://doi.org/10.18331/SFS2020.6.2.8

Selamoglu, Z., Duran, A., Gulhan, M.F. and Erdemli, M.E., 2015. Effects of propolis on biochemical and microbiological parameters in carp (Cyprinus carpio) fillets exposed to arsenic. Iran. J. Fish. Sci., 14: 896-907.

Shah, S.Z.H., Afzal, M., Akmal, A., Fatima, M. and Hussain, S.M., 2016. Effect of citric acid and phytase on growth performance and mineralization of Labeo rohita juveniles fed soybean meal-based diet. Int. J. Agric. Biol., 18: 111-116. https://doi.org/10.17957/IJAB/15.0071

Shah, S.Z.H., Afzal, M., Fatima, M., Hussain, S.M. and Ahmed, T., 2021. Nutrient digestibility in Labeo rohita fingerlings fed citric acid acidified phytase sprayed diet. Pakistan J. Zool., 53: 459. https://doi.org/10.17582/journal.pjz/20150923011725

Snedecor, G.W. and Cochran, W.G., 1989. Statistical methods, 8th ed. Iowa State Univ. Press, Ames, 11 Online First Article Iowa, 54: 71-82.

Sugiura, S.H., Gabaudan, J., Dong, F.M. and Hardy, R.W., 2001. Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout Oncorhynchus mykiss (Walbaum) fed soybean meal-based diets. Aquacult. Res., 32: 583–592. https://doi.org/10.1046/j.1365-2109.2001.00581.x

Zhang, H., Yi, L., Sun, R., Zhou, H., Xu, W., Zhang, W. and Mai, K., 2016. Effects of dietary citric acid on growth performance, mineral status and intestinal digestive enzyme activities of large yellow croaker Larimichthys crocea (Richardson, 1846) fed high plant protein diets. Aquaculture, 453: 147-153. https://doi.org/10.1016/j.aquaculture.2015.11.032