Efficacy of Dietary Supplementation with Phytase and Citric Acid on Whole-Body Composition and Hematological Parameters of Cyprinus carpio and Cirrhinus mrigala Fingerlings Fed on Canola Meal-Based Diet

Muhammad Zubair ul Hassan Arsalan1, Syed Makhdoom Hussain2*,

Abdullah Ijaz Hussain3, Majid Hussain4, Muhammad Asrar2, Zeeshan Yousaf2 and Aumme Adeeba Bano2

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

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

3Department of Chemistry, Government College University Faisalabad, Pakistan

4Department of Fisheries and Aquaculture, University of Okara, Okara, Pakistan

ABSTRACT

A 90-day feeding trial was designated to evaluate how canola meal (CM)-based diet supplemented with phytase (PHY) and citric acid (CA) affects whole-body composition and hematological parameters in Cirrhinus mrigala and Cyprinus carpio fingerlings. Sixteen experimental diets (T1─T16) with varied CA (0 and 2.5%) and PHY (0 and 750 FTU/kg) levels were prepared. The fingerlings were fed at the rate of 5.0% of live fish body mass. Fifteen fish were randomly stocked into triplicate tanks for each of sixteen test diets. Significant (p<0.05) increase in crude protein (CP) and crude fat (CF) was noticed in fish fed T12 (2.5% CA and 750 FTU/kg PHY) diets. Comparison of treatments showed maximum values of RBCs (3.57×106 mm-3 and 3.18×106 mm-3), PLT (83.32 and 80.35), Hb (8.92g/100ml and 8.62g/100ml) and PCV (29.07% and 30.07%) in C. mrigala and C. carpio fingerlings, respectively, when fed with T12 diets. Conclusively, a 50% CM-based diet with 2.5% CA and 750 FTU/kg PHY supplementation, performed better in terms of whole-body composition and hematological parameters in C. mrigala and C. carpio fingerlings.

Article Information

Received 01 January 2023

Revised 20 March 2023

Accepted 03 April 2023

Available online 26 July 2023

(early access)

Published 19 July 2024

Authors’ Contribution

MZH conducted the study and wrote the manuscript. SMH acquired funds, administered and supervised the project. AIH and MH analyzed the data. MA, ZY and AAB edited and reviewed the manuscript.

Key words

Citric acid, Phytase, Canola meal, Whole-body composition, Hematology

DOI: https://dx.doi.org/10.17582/journal.pjz/20230101160121

* Corresponding author: [email protected]

0030-9923/2024/0005-2163 $ 9.00/0

Copyright 2024 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

Aquaculture has become the primary means of acquiring aquatic food due to a decline in wild fisheries sources (Gong et al., 2021). According to FAO’s most recent global aquaculture statistics, the yield of farmed aquatic animals has reached about 82.1 million metric tons, with an average annual growth rate of 5.3% from 2011 to 2018 (FAO, 2020). This change is intimately related to the expansion of intensive aquaculture practice and the accessibility of high quality aqua-feeds. The significant protein source in aqua-feeds is fishmeal (FM), but due to the limited supply of natural resources, there are growing efforts to identify other substitutes, like phyto-proteins, to replace FM. Aquaculture producers and nutritionists now place a great deal of attention on plant proteins due to their accessibility, environment friendliness, sustainability and relative affordability compared to FM protein (Abdel-Latif et al., 2022).

Canola meal (CM) is a highly nutritious plant protein (33-45%) obtained from plant Brassica napus (Glencross, 2016), with well-balanced amino acids (methionine, lysine, and cysteine), minerals and vitamins (thiamine, choline, niacin, biotin, riboflavin) (Mohammadi et al., 2016). All over the world, it is cheaper than FM with easy availability and serves as the primary source of protein in feed formulations (Mushtaq et al., 2006), but one significant drawback is the existence of anti-nutritional factors like tannins, glucosinolates, and phytate that restrict its usage in fish feed (Von Danwitz and Schulz, 2020). Phytate, the primary phosphorus storage compound, has a significant chelating capability to make bonds with starch, protein, and minerals in intestine, reducing the bioavailability of these nutrients (Kies et al., 2001). Therefore, for the efficient use of plant-based proteins in aqua-feed, it is crucial to identify and destroy anti-nutritional factors that prevent nutrient consumption.

Exogenous enzymes, i.e., phytase (PHY), may be employed in fish diets to boost the nutritional value of plant-based protein sources and inactivate anti-nutritional factors (Dalsgaard et al., 2012). PHY hydrolyzes phytate’s phosphate group, lowers its affinity, and forces it to release nutrients in order to improve nutrient assimilation and decrease the nitrogen and phosphorus discharge (Kumar et al., 2010). Organic acids supplementation is another way to enhance plant protein quality (Reda et al., 2016; Hussain et al., 2017). Citric acid (CA) is an organic acid with strong buffering ability and particular taste that has widely been applied in aqua-diets (Hossain et al., 2007). By preserving a healthy gut pH, it increases the effectiveness of both exogenous and endogenous phytases. It also improves feed utilization and serves as an antimicrobial agent (Shah et al., 2015). CA inclusion has shown improved growth performance, feed intake and specific growth rate in many species such as, rainbow trout, beluga sturgeon, and tilapia (Pandey and Satoh, 2008; Li et al., 2009; Khajepour and Hosseini, 2012).

Pakistan is one of the countries having freshwater and marine water resources that support a wide range of highly nutritious and economically valuable freshwater fishes (Hussain et al., 2011). Cirrhinus mrigala, also referred to as mori, is a freshwater species widely cultivated in Pakistan. It has high market value and is grown semi-intensively in polyculture systems for the better success of carp culture system (Abid and Ahmed, 2009). Cyprinus carpio, commonly known as gulfam, is cultured in Asia, Eastern Europe and other parts of the world (Khan et al., 2016). This freshwater fish tolerates a wide range of temperature, salinity and pH. It is one of the most commonly farmed fish of world with characteristics of fast growth rate and good protein source for human food (Sugiura et al., 2001). Therefore, this study was designed with the aim of evaluating the importance of PHY and CA supplementation in plant-based diets as well as their effects on body composition and hematology of C. mrigala and C. carpio fingerlings.

To observe the physiologic variations in fish in response to particular diets, the hematological parameters, mainly, hemoglobin (Hb), white blood cells (WBCs), red blood cells (RBCs), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), mean cell volume (MCV) and lymphocyte count are observed (Shah and Altindağ, 2004). Studies regarding the effects of exogenous enzyme supplementation on fish hematological status are not widely available. Thus, this study was designed to evaluate the interaction between exogenous enzyme supplementation and hematology of fish.

MATERIALS AND METHODS

Test diets formulation

Sixteen isoenergetic and isonitrogenous experimental diets were prepared. CM was used to substitute 45.77% of FM protein and CA and PHY were added at levels of 0 and 2.5% as well as 0 and 750 FTU/kg, respectively (Table I). All ingredients of feed were ground finely, mixed properly along with CA, pelleted via 3 mm die and sprayed with PHY dissolved in 50 ml distilled water as stated by Robinson et al. (2002). Pellets were dried before being placed in sealed plastic jars and kept at below 20℃ for the period of the feeding trial.

Experimental fishes and rearing conditions

A 90-day feeding trial was organized. Fingerlings of C. mrigala and C. carpio were bought from the Government Fish Seed Hatchery, Faisalabad, Pakistan and were acclimated to laboratory environment. These were fed on basal diet round the clock. Following a two-week acclimation period, 15 fish were stocked randomly in experimental tanks, each tank with 70 L water capacity, having triplicates per test diet. These were fed twice a day, at 0900 and 1700 h. to apparent satiety. After 2 h. of feeding period, the tanks were washed and refilled with water. Quality of water was checked daily by observing parameters like dissolved oxygen, pH, and water temperature.

Sample collection and chemical analysis

After 90-day experimental period, fingerlings were fasted for 24 h. Five fingerlings were picked randomly from each experimental tank for whole-body proximate analysis by standard protocols of AOAC (2005). Feed and fish samples were completely oven dried at 105oC and homogenized using a mortar and pestle before being inspected. Moisture content estimation was done after drying. Micro Kjeldahl apparatus was used to assess crude protein (CP) (N 6.25) and petroleum ether extraction system was utilized to determine crude fat (CF) (Soxtec HT2 1045 system). Ash was assessed by sample ignition at 550 ℃ for about 12 h in an electric furnace (Eyela-TMF 3100).

 

Table I. Ingredient’s composition (%) of test diets.

Test diets	Replacement levels of fishmeal protein (%)	Fish meal	Can-ola meal	Phytase (FTU /kg)	Citric acid (%)	Wheat flour	Rice polish	Fish oil	*Vitamins Premix	**Minerals Premix	Chro-mic oxide
T1	0	48	18	0	0	22	3	6	1	1	1
T2					2.5	19.5
T3				750	0	22
T4					2.5	19.5
T5	25	36	35	0	0	17
T6					2.5	14.5
T7				750	0	17
T8					2.5	14.5
T9	50	24	53	0	0	11
T10					2.5	8.5
T11				750	0	11
T12					2.5	8.5
T13	75	12	71	0	0	5
T14					2.5	2.5
T15				750	0	5
T16					2.5	2.5

 

*Each 100Kg of Vitamins premix contains: Vit A 2000,000IU, Vit B6 600 mg, Vit D 400,000 IU, Vit B12 2,000 mg, Vit E 4000 mg, Vit C 2,000 mg, Vit K3 900 mg, Folic acid 200 mg, Vit B1 200 mg, Calcium pantothenate 2,000 mg, Vit B2 3000 mg, Nicotinic acid 10,000 mg. **Each Kg mineral premix contains: Na 45g, Ca 155g, Zn 3000 mg, Fe 1000 mg,Cu, 600mg, Mg, 55g, P, 135g, Mn, 2000mg, I, 40mg, Co, 40mg, Se, 3mg.

Blood sample collection and hematological assay

Fingerlings were tranquilized with 150 mg tricane methane sulfonate solution to take blood samples from a fish’s caudal vein (Wagner et al., 1997) after the 90-day experimental period. The samples of blood were transferred to the Molcare Lab, Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan. Utilizing an automatic cell counter, the hematological indices, including Hb, WBCs, RBCs, MCH, MCV, and MCHC, were calculated. PLT and PCV were measured by following methods of Brahimi et al. (2009) and using a micro-hematocrit reader, PCV was calculated while being spun at 10,500 rpm. By following formulae, the MCH, MCV, and MCHC were estimated as well.

MCHC = Hb / PCV × 100

MCH = Hb / RBC × 10

MCV = PCV / RBC × 10

Statistical analysis

The one-way analysis of variance was used to analyze the data on body composition and hematological indices (Steel et al., 1996). The Tukey’s Honesty Significant Difference Test was utilized for comparison of differences between the samples, and considered significant at (p<0.05) (Snedecor and Cochran, 1989). The CoStat Computer Package (Version 6.303, CA, Monterey, PMB 320, 93940 USA) was utilized for statistical analysis.

RESULTS

Whole-body composition

Tables II and III showed the whole-body composition of C. mrigala and C. carpio fingerlings fed CM-based diets with varying supplemented levels of CA and PHY. There were significant (p<0.05) differences between body composition of fingerlings fed different test diets in case of CF, CP, ash, and moisture contents. The whole-body composition findings evaluated that the C. mrigala and C. carpio fingerlings fed T12 diet exhibited significantly (p<0.05) higher levels of CP (20.19% and 18.05%), ash (2.55% and 2.49%) and moisture (74.52% and 76.50%) while lower lipid levels (2.74% and 2.97%), respectively. It was noted that 50% CM level in diet T12 was adequate to improve the carcass protein compared to T1 diet without CA and PHY supplementation.

Hematological parameters

The hematological indices of C. mrigala and C. carpio fingerlings fed diets with distinct replacement levels of CM and provided with varying CA and PHY levels are shown in Tables IV and V. The fingerlings of C. mrigala and C. carpio fed on test diet T12 gave the highest

 

Table II. Analyzed whole-body composition of C. mrigala fingerlings fed canola meal-based diet supplemented with citric acid (CA) and phytase (PHY).

Fishmeal replacement levels (%)	PHY	CA	Test diets	Protein		Fat		Ash		Moisture
0	0	0	T1	13.86p		9.85a		2.22e		74.08d
		2.5	T2	14.49n		9.09c		2.29c		74.14c
	750	0	T3	15.01m		8.51d		2.31c		74.17c
		2.5	T4	17.78g		5.49j		2.44b		74.30b
25	0	0	T5	14.29o		9.35b		2.25d		74.11c
		2.5	T6	16.84i		6.52h		2.40b		74.24c
	750	0	T7	17.37h		5.93i		2.42b		74.28b
		2.5	T8	19.18d		3.97m		2.49b		74.36b
50	0	0	T9	15.41l		8.06e		2.33c		74.20c
		2.5	T10	16.45j		6.96g		2.38b		74.22c
	750	0	T11	19.91b		3.16o		2.54a		74.39b
		2.5	T12	20.19a		2.74p		2.55a		74.52a
75	0	0	T13	15.88k		7.57f		2.35c		74.21c
		2.5	T14	18.25f		4.97k		2.46b		74.32b
	750	0	T15	18.87e		4.32l		2.47b		74.33b
		2.5	T16	19.49c		3.61m		2.53a		74.37b
Pool Standard Error (PSE)					0.04		0.03		0.02		0.03

 

Means within columns having different superscripts are significantly different at p<0.05. Data are means of three replicates.

 

Table III. Analyzed whole-body composition of C. carpio fingerlings fed canola meal-based diet supplemented with citric acid (CA) and phytase (PHY).

Fishmeal replacement levels (%)	PHY	CA	Test diets	Protein	Fat	Ash	Moisture
0	0	0	T1	14.75m	12.81a	1.00p	71.44p
		2.5	T2	15.38l	10.82c	1.05n	72.74n
	750	0	T3	15.78k	10.10d	1.08m	73.04m
		2.5	T4	16.87g	6.13j	2.03g	74.97g
25	0	0	T5	14.75m	6.13j	1.03o	72.04o
		2.5	T6	16.27i	12.18b	1.68j	74.07j
	750	0	T7	16.57h	7.98g	1.98h	74.77h
		2.5	T8	17.38d	6.68i	2.39c	75.67d
50	0	0	T9	16.27i	4.56l	1.78i	74.37i
		2.5	T10	16.03j	7.58h	1.48k	73.74k
	750	0	T11	17.84b	8.75f	2.47b	76.67a
		2.5	T12	18.05a	3.03n	2.49a	76.50b
75	0	0	T13	16.08j	2.97o	1.28l	73.34l
		2.5	T14	17.03f	9.30e	2.13f	75.07f
	750	0	T15	17.18e	5.22k	2.33e	75.27e
		2.5	T16	17.58c	4.02m	2.43d	75.97c
Pool Standard Error (PSE)				0.14	0.14	0.01	0.01

 

Means within columns having different superscripts are significantly different at p<0.05. Data are means of three replicates.

 

 

 

value of RBCs (3.57×106 mm-3 and 3.18×106 mm-3), respectively. In case of C. mrigala fingerlings, the highest value of WBCs (8.85×103 mm-3) was found when fed with diet T15 (75% FM replacement with 0% CA and 750 FTU/kg PHY) while in C. carpio fingerlings, highest value of WBCs (8.19×103 mm-3) was at T12 level. The fingerlings of C. mrigala and C. carpio fed with T12 diet showed the highest value of PLT (83.32 and 80.35), Hb (8.92 g/100ml and 8.68g/100ml) and PCV (29.07% and 30.07%), respectively. These all values were significantly (p<0.05) different from fishes given other test diets. The highest amount of MCH (74.59 pg and 72.41 pg) and MCV (209.69 fl and 209.79 fl) were found when fingerlings fed on test diet T10 (50% FM replacement level with 2.5% CA and 0 FTU/kg PHY), respectively whereas, the lowest amount of MCH (24.47 pg) and MCV (81.26 fl) were noted in C. carpio fingerlings fed T1 (0 % CA and 0 PHY) and T2 (2.5 % CA and 0 FTU/kg PHY).

DISCUSSION

The whole-body composition has a direct influence on the quality of aquatic food items. In order to use fish more effectively, it is helpful to have detailed understanding of its body composition. Body composition is undoubtedly regarded as a valuable method to estimate the physiological state and health of the fish, alongside hematological analysis (Fazio et al., 2019). Age, sex, nutritional status, digestive function, and other factors, all have an impact on the biochemical makeup of the fish’s entire body (Abdel-Tawwab et al., 2006). Phytate contents are usually present in plant-based diets and make these diets non digestible. Basically, it chelates with important nutrients i.e., fat, protein, and minerals. Consequently, fish become unable to consume them, ensuing deficient fish body composition (Cao et al., 2007).

In this study, the body composition of both fishes was improved when fed with varying levels of CM-based diets supplied with PHY and CA. The results showed that CA and PHY addition is important for dissociation of phytate complexes ensuing higher retention of nutrients in body of fish as compared to those fed control diet (without CA and PHY supplementation). Best values of CP, CF and carcass ash were found in C. mrigala (20.19%), (2.74%) (2.55%), respectively and in case of C. carpio (18.05%), (2.97%), (2.49%), respectively when fed on T12 diet whereas the lowest values of CP (13.86%), CF (9.85%) and ash (2.22%) of C. mrigala and (14.75%), (12.81%) and (1%) of C. carpio, respectively was recorded when both fishes were fed on control diet (T1). The results of body composition of this study were also corroborated by Ahmad et al. (2023) which suggested that when Labeo rohita fed on 50% replacement level of cottonseed meal (CSM)-based diets provided with 2.5% CA and 750 FTU/kg PHY, they showed highest amount of CP, CF, and ash contents in comparison to those fed on other test diets. Similarly, Khajepour et al. (2012) also found improved muscle proximate composition of C. carpio when fed on 3% CA and 500 FTU/kg PHY supplemented soybean meal (SBM)-based diet. On contrary, Afzal et al. (2019) noticed a non-significant interaction between both supplements i.e., CA and PHY, to improve fat and protein contents in fish body. They found that the SBM-based diets supplemented with CA and PHY separately, gave better results than control diet (with no supplement).

In current study, CA supplementation alone significantly enhanced (p<0.05) the muscle CP, ash and dry matter, while reduced the CF level. The positive impact of CA on reduction of body fat might be due to its impact on phosphorous retention. This decreased fat content in fish body due to utilization of CA supplemented plant-based diet depicts that more phosphorous is available to improve fat oxidation, hence providing more energy for better growth performance of fish (Shirmohammad et al., 2003). Our results were also in line with Khajepour and Hosseini (2012) who detected increase in muscle CP and ash levels in Huso huso when fed on 2 and 3% CA supplemented test diets. CA may decrease the gut pH helping in the dissociation of chelated phytate nutrient complexes that leads to improved retention of these nutrients in fish body (Afzal et al., 2019).

Like CA, PHY supplementation also individually improves (p<0.05) the proximate composition of muscle of fish. In accordance with this study, Sardar et al. (2007) also found increase in CP and decrease in CF levels in body of C. carpio due to PHY supplementation in feed. It is due to PHY enzyme’s ability to hydrolyze phytate and release the chelated nutrients making them more accessible to fish (Afzal et al., 2019). In another study, Shirmohammad et al. (2003) also found that SBM-based diet provided with 3% CA and 1000 FTU/kg level of PHY synergistically improved ash contents but could not increase protein and lipid contents in C. carpio. The combination of CA and PHY resulted in a non-significant reduction in body fat content. This could be due to change in pH of digestive tract, which improved PHY performance. A combination of PHY and CA is more numerically effective than either one alone.

In present study, the moisture level in body composition of C. mrigala and C. carpio was 74.52% and 76.50%, respectively. PHY supplemented diets can improve fish body mineralization by increasing nutrient retention. It is especially during the developing stage (Cao et al., 2007). Nuez-Ortin (2011) and Reda et al. (2016) also reported that Nile tilapia fed on acidified diet significantly improved the chemical composition of fish body. When Nile tilapia were fed acidified diets, they retained significantly higher protein contents. Because crude proteins are more digestible at greater CA levels, they retained longer in the fish body. The rate of pepsinogen conversion to pepsin was raised when CA supplementation in diet was increased, resulting in higher pepsin activity (Kirchgessner and Roth, 1985). The outcomes of multiple research experiments are variable due to changes in diet ingredients, feed formulation processes, ecological variations and species differences, thus demanding more exploration. This study granted the valuable effects of dietary supplementation of both CA and PHY in C. mrigala and C. carpio diet and their role in improving the body composition and fish health status. Hence, 50% CM-based diet with CA and PHY supplementation could be added to aqua-feeds to improve body composition of cultured C. mrigala and C. carpio through dietary management.

Variations in hematological indices are key signs, utilized for evaluation of stress, nutritional and health status (Yilgmaz et al., 2016; Khalafalla et al., 2020) as well as physiological changes in fishes (Ranjan et al., 2020). Therefore, monitoring hematological indices may reveal the fish’s immune status in response to changes especially related to nutrition (Fazio et al., 2019). In fish, hematological studies are necessary to check the health status of fish and to ensure that the formulated diets are of good quality (Shahzad et al., 2016).

According to the current study, highest values of blood parameters were found when both fishes fed on test diet T12 having 50% replacement level of CM with 2.5% CA and 750 FTU/kg PHY showing that PHY and CA supplementation in plant-based diets synergistically improved the hematological status of the fishes. In a parallel study, Baruah et al. (2007) also found that 3% CA and 500 FTU/kg PHY supplementation in SBM-based diet synergistically improved the RBCs, WBCs, Hb hematocrit volume of L. rohita juveniles.

The chelated phytate complex in plant by-products has an impact on overall fish performance, including hematology (Ehsani and Torki, 2010). Because phytate chelates with iron, found in RBCs, it may reduce overall oxygen carrying capacity of RBCs (Spinelli et al., 1983). Well-known features of PHY are that, it showed better nutrient digestibility and hematology of Indian major carps when fed plant-based diets (Hussain et al., 2011, 2016; Shahzad et al., 2017). PHY supplementation has been suggested as an effective immune system stimulant in monogastric animals that results in a higher number of monocytes and a higher production of blood cells (Ehsani and Torki, 2010). Shahzad et al. (2020) found that C. catla fingerlings showed improved values of RBCs, WBCs and Hb when fed on moringa-based diet having 900 FTU/kg PHY whereas the poorest values were noted when fed on control diet. Hb level in olive flounder (Paralichthys olivaceus) fed on PHY supplemented SBM diet was greater than the fish fed on control diet (Yoo and Bai, 2014).

CA is already approved for use in animal diets, and previous studies have shown that it is safe for the target fish species (EFSA, 2015). Various hematological parameters in fish blood were improved as a result of diet acidification. Dietary acidification had a beneficial effect on fish blood parameters (Hb, RBCs, WBCs, PLT, MCH and MCV) as noted by Reda et al. (2016). In present study, WBCs in C. mrigala showed increasing trend as CM level increased but in the case of C. carpio, WBC count decreased when CM increased in the experimental diet. High WBC count is generally related to microbial invasion or due to foreign bodies or antigens found in blood circulation. Similar findings were also noted in other research studies (Bello et al., 2013; Hussain et al., 2018). In the current research, significant increases in WBCs number were observed in fishes fed with PHY supplemented diets as also noted by Ranjan et al. (2020) indicating the improved levels of immunity in fish. Significant increase in number of RBCs and WBCs along with Hb value might be the result of increased haemo-concentration (Ranjan et al., 2020). The possible reason for this might be due to water movement from blood plasma to the muscle cell (Wilson and Taylor, 1993). In current findings, the increased number of RBCs was found in fingerlings fed with enzyme supplemented CM-based diets. These improved RBCs could deduce a good immune response (Jiang et al., 2007). As there is not much insight into developed relation between enzyme supplementation in fish feed and fish hematological state, so more research is necessary to demonstrate the mode of action (Ranjan et al., 2020).

In the current study, PHY alone improved numbers of WBCs and MCHC of C. mrigala fish when fed test diets T15, having 75% replacement levels of CM with 0% CA and 750 FTU/kg of PHY enzyme. In contrast, Baruah et al. (2007) noticed non-significant impacts (p>0.05) on WBCs in L. rohita fed SBM-based diet with 500 FTU/kg PHY. Similar to our results, WBCs were maximum in C. carpio fed on soy-protein based diet with 500 FTU/kg PHY supplementation (Sardar et al., 2007). Maximum WBCs count was found in Nile tilapia fed on SBM-based diet with 500 FTU/kg PHY in comparison with fish fed on similar level of PHY supplementation with jatropha meal-based diet (Kumar et al., 2011).

The findings of present research demonstrated that CA individually enhanced the MCH and MCV values of both fishes C. mrigala and C. carpio when fed on test diet T10, having 50% CM-based diet with 2.5% CA and without PHY. Hussain et al. (2021) also observed that 3% CA significantly improved the hematological parameters of C. mrigala fingerlings fed Moringa oleifera-based diets. In contrary to our results, acidification of diet with CA had no significant (p>0.05) impact on RBCs, WBCs, MCH, MCV, and MCHC. Thus, at 3% CA level, concentration of Hb and Hct were significantly (p<0.05) greater than the control level (Khajepour et al., 2011). The information about MCHC and MCH has a key role in anemia analysis in many species and are recognized to be symptomatic of RBCs count and blood oxygen carrying capacity in fish body (Houston, 1997). These indices exhibited no distinction between different groups of this trial, showing that blood oxygen carrying capacity for different metabolic processes was not influenced.

The fish hematology is influenced by a number of aspects, including fish species, age, size, environmental conditions, physiological state and dietary practice i.e., quantity and quality of feed, protein sources, dietary ingredients, vitamins and probiotics etc. (Barnhart, 1969; Osuigwe et al., 2005). This important part of hematological study is the minimum explored area by researchers and it needs a rigorous research work to find out the probable interaction of PHY enzyme with hematological parameters of fish (Shahzad et al., 2016; Ranjan et al., 2020).

CONCLUSION

It is concluded that a 50% CM-based diet with 2.5% CA and 750 FTU/kg PHY supplementation, improved the whole-body composition and hematological parameters in C. mrigala and C. carpio fingerlings. Thus, dietary supplementation of CA and PHY in plant-based diets is effective to increase their use in aqua-feeds.

Acknowledgement

The authors are thankful to HEC Pakistan for funding Department of Zoology, Government College University, Faisalabad for provision of facilities for this research.

Funding

The authors are grateful to Higher Education Commission (HEC) Islamabad, Pakistan for its support by providing Project# 5649/Punjab/NRPU/R&D/HEC/2016 and Project# 20-4892/NRPU/R&D/HEC/14/1145 to conduct this research work.

IRB approval

The procedures and methods used in the study followed the ethical guidelines of Government College University Faisalabad.

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

Abdel-Latif, H.M., Abdel-Daim, M.M., Shukry, M., Nowosad, J. and Kucharczyk, D., 2022. Benefits and applications of Moringa oleifera as a plant protein source in aquafeed: A review. Aquaculture, 547: 737369. https://doi.org/10.1016/j.aquaculture.2021.737369

Abdel-Tawwab, M., Khattab, Y.A., Ahmad, M.H. and Shalaby, A.M., 2006. Compensatory growth, feed utilization, whole-body composition, and hematological changes in starved juvenile Nile tilapia, Oreochromis niloticus (L.). J. appl. Aquacult., 18: 17-36. https://doi.org/10.1300/J028v18n03_02

Abid, M. and Ahmed, M.S., 2009. Growth response of Labeo rohita fingerlings fed with different feeding regimes under intensive rearing. J. Anim. Pl. Sci., 19: 45-49.

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. Pakistan J. Zool., 51: 1741. https://doi.org/10.17582/journal.pjz/2019.51.5.1741.1750

Ahmad, B., Hussain, S.M., Ali, S., Arsalan, M.Z.H. and Sharif, A., 2023. Efficacy of acidified phytase supplemented cottonseed meal based diets on growth performance and proximate composition of Labeo rohita fingerlings. Braz. J. Biol., 83: e247791. https://doi.org/10.1590/1519-6984.247791

AOAC (Association of Official Analytical Chemists), 2005. Official methods of analysis. 15th Ed. Association of Official Analytical chemists, Washington, D.C. USA., pp. 1094.

Barnhart, R.A., 1969. Effects of certain variables on hematological characteristics of rainbow trout. Trans. Am. Fish. Soc., 98: 411-418. https://doi.org/10.1577/1548-8659(1969)98[411:EOCVOH]2.0.CO;2

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

Bello, M.A.A., Souza, D.G.F., Faria, V.P., Prado, E.J.R., Moraes, F.R. and Onaka, E.M., 2013. Haematological response of Curimbas prochilodus lineatus, naturally infected with Neoechinorhynchus curemai. J. Fish Biol., 82: 1403-1410. https://doi.org/10.1111/jfb.12060

Brahimi, M., Osmani, S., Arabi, A., Enta-Soltan, B., Taghezout, Z., Elkahili, B.S. and Bekadja, M.A., 2009. The estimation of platelet counts from a blood smear on the basis of the red cell: Platelet ratio. Turk. J. Hematol., 26: 21-24.

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., Verlhac, V., Hjermitslev, N.H., Ekmann, K.S., Fischer, M., Klausen, M. and Pedersen, P.B., 2012. Effects of exogenous enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed diets with high inclusion of plant-based protein. Anim. Feed Sci. Technol., 171: 181–191. https://doi.org/10.1016/j.anifeedsci.2011.10.005

EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2015. Scientific Opinion on the safety and efficacy of citric acid when used as a technological additive (acidity regulator) for all animal species. ESFA J., 13: 4010. https://doi.org/10.2903/j.efsa.2015.4010

Ehsani, M. and Torki, M., 2010. Effects of dietary inclusion of guar meal supplemented by β-mannanase on performance of laying hens, egg quality characteristics and diacritical count of white blood cells. Am. J. Anim. Vet. Sci., 5: 237–243. https://doi.org/10.3844/ajavsp.2010.237.243

FAO, 2020. The state of world fisheries and aquaculture. Sustainability in action; FAO: Rome, Italy.

Fazio, F., Iaria, C., Saoca, C., Costa, A., Piccione, G. and Spanò, N., 2019. Effect of dietary vitamin C supplementation on the blood parameters of striped bass Morone saxatilis (Walbaum, 1752). Turk. J. Fish. aquat. Sci., 20: 491-497. https://doi.org/10.4194/1303-2712-v20_6_07

Glencross, B., 2016. Understanding the nutritional and biological constraints of ingredients to optimize their application in aquaculture feeds. In: Aquafeed formulation (ed. S.F. Nates). Academic Press, New York. pp. 32-73.

Gong, W., Gao, S., Zhu, Y., Wang, G., Zhang, K., Li, Z., Yu, E., Tian, J., Xie, Y., Xie, J. and Ni, J., 2021. Effect of the aerobic denitrifying bacterium Pseudomonas furukawaii ZS1 on microbiota compositions in grass carp culture water. Water, 13: 1329. https://doi.org/10.3390/w13101329

Hossain, M.A., Pandey, A. and Satoh, S., 2007. Effects of organic acids on growth and phosphorus utilization in red sea bream Pagrus major. Fish. Sci., 73: 1309-1317.

Houston, A.H., 1997. Are the classical hematological variables acceptable indicators of fish health? Trans. Am. Fish. Soc., 126: 879-894. https://doi.org/10.1577/1548-8659(1997)126<0879:RATCHV>2.3.CO;2

Hussain, S., Afzal, M., Rana, S.A., Javid, A. and Iqbal, M., 2011. Effect of phytase supplementation on growth performance and nutrient digestibility of Labeo rohita fingerlings fed on corn gluten meal-based diets. Int. J. Agric. Biol., 13: 916-922.

Hussain, S.M., Ahmad, S., Shahzad, M.M., Arsalan, M.Z.H., Riaz, D., Ahmad, N. and Ahmed, A.W., 2016. Mineral digestibility of Labeo rohita fingerlings fed on cottonseed meal based diets supplemented with citric acid and phytase enzyme. Int. J. Biosci., 8: 25-35. https://doi.org/10.12692/ijb/8.2.25-35

Hussain, M., Hussain, S.M., Iqbal, R., Javid, A., Shahzad, M.M., Arsalan, M.Z. and Riaz, D., 2017. Effect of citric acid acidified Moringa oleifera seed meal based diet on nutrients digestibility and growth performance of Cirrhinus mrigala fingerlings. Int. J. Agric. Biol., 19: 719-725. https://doi.org/10.17957/IJAB/15.0346

Hussain, S.M., Ahmad, N., Javid, A., Shahzad, M.M., Hussain, M. and Arsalan, M.Z.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, M., Hussain, S.M., Iqbal, R., Shahzad, M.M., Shah, S.Z.H., Akram, A.M. and Arsalan, M.Z.H., 2021. Effect of citric acid acidified Moringa oleifera seed meal based diet on minerals absorption, carcass composition and hematological indices of Cirrhinus mrigala fingerlings. Pakistan J. Zool., 54: 1737-1745. https://doi.org/10.17582/journal.pjz/20190531050527

Jiang, N., Tan, N.S., Ho, B. and Ding, J.L., 2007. Respiratory protein generated reactive oxygen species as an antimicrobial strategy. Nat. Immunol., 8: 1114-1122. https://doi.org/10.1038/ni1501

Khajepour, F. and Hosseini, S.A., 2012. Citric acid improves growth performance and phosphorus digestibility in Beluga (Huso huso) fed diets where soybean meal partly replaced fishmeal. Anim. Feed sci. Technol., 171: 68-73. https://doi.org/10.1016/j.anifeedsci.2011.10.001

Khajepour, F., Hosseini, S.A., and Hoseini, S.M., 2011. Study on some hematological and biochemical parameters of juvenile beluga (Huso huso) fed citric acid supplemented diet. Glob. Vet., 7: 361-364.

Khajepour, F., Hosseini, S.A. and Imanpour, M.R., 2012. Dietary crude protein, citric acid and microbial phytase and their interacts to influence growth performance, muscle proximate composition and hematocrite of common carp, Cyprinus carpio L juvenile. World J. Zool., 7: 118−122.

Khalafalla, M.M., Ibrahim, S.A., Zayed, M.M., Awad, M.N. and Mohamed, R.A., 2020. Effect of a dietary mixture of beneficial bacteria on growth performance, health condition, chemical composition, and water quality of Nile Tilapia, Oreochromis niloticus fingerlings. J. Aquat. Fd. Prod. Technol., 29: 823-835. https://doi.org/10.1080/10498850.2020.1764685

Khan, M.N., Shahzad, K., Chatta, A., Sohail, M., Piria, M. and Treer, T., 2016. A review of the introduction of common carp Cyprinus carpio Pakistan: Origin, purpose, impact, and management. Croatian J. Fish., 74: 71–80. https://doi.org/10.1515/cjf-2016-0016

Kies, A.K., Van Hemert, K.H.F. and Sauer, W.C., 2001. Effect of phytase on protein and amino acid digestibility and energy utilization. Worlds Poult. Sci. J., 57: 109-126. https://doi.org/10.1079/WPS20010009

Kirchgessner, M., and Roth, H.P., 1985. Influence of zinc depletion and zinc status on serum growth hormone levels in rats. Biol. Trace elem. Res., 7: 263-268. https://doi.org/10.1007/BF02989251

Kumar, V., Makkar, H.P., Devappa, R.K. and Becker, K., 2011. 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.). Fd. chem. Toxicol., 49: 2144-2156. https://doi.org/10.1016/j.fct.2011.05.029

Kumar, V., Sinha, A.K., Makkar, H.P. and Becker, K., 2010. Dietary roles of phytate and phytase in human nutrition: A review. Fd. Chem., 120: 945-959. https://doi.org/10.1016/j.foodchem.2009.11.052

Li, J.S., Li, J.L., and Wu, T.T., 2009. Effects of non-starch polysaccharides enzyme, phytase and citric acid on activities of endogenous digestive enzymes of tilapia (Oreochromis niloticus × Oreochromis aureus). Aquacult. Nutr., 15: 415-420. https://doi.org/10.1111/j.1365-2095.2008.00606.x

Mohammadi, M., Sarsangi, H., Mashaei, N., Rajabipour, F., Bitarat, A. and Hafeziyeh, M., 2016. Canola substitution in Nile tilapia Oreochromis niloticus diets. J. Cell Sci., 7: 256–260. https://doi.org/10.4172/2157-7013.1000256

Mushtaq, T., Sarwar, M., Ahmad, G., Nisa, M.U. and Jamil, A., 2006. The influence of exogenous multienzyme preparation and graded levels of digestible lysine in sunflower meal-based diets on the performance of young broiler chicks two weeks posthatching. Poult. Sci., 85: 2180-2185. https://doi.org/10.1093/ps/85.12.2180

Nuez-Ortin, W.G., 2011. Gustor-Aqua: An effective solution to optimize health status and nutrient utilization. Int. Aquafeed, May–June, 18-20.

Osuigwe, D.I., Obiekezie, A.I. and Onuoha, G.C., 2005. Some hematological changes in hybrid catfish (Heterobranchus longifilis x Clarias gariepinus) fed different dietary levels of raw and boiled jackbean (Canavalia ensiformis) seed meal. Afr. J. Biotechnol., 4: 1017-1021.

Pandey, A., and Satoh, S., 2008. Effects of organic acids on growth and phosphorus utilization in rainbow trout Oncorhynchus mykiss. Fish. Sci., 74: 867-874. https://doi.org/10.1111/j.1444-2906.2008.01601.x

Ranjan, A., Kumar, S., Sahu, N.P., Jain, K.K. and Deo, A.D., 2020. Exogenous phytase and xylanase supplementation of formulated diets for rohu (Labeo rohita): Impact on haematology, histology and IGF I gene expression. Fish Physiol. Biochem., 47: 49-58. https://doi.org/10.1007/s10695-020-00893-7

Reda, R.M., Mahmoud, R., Selim, K.M. and El-Araby, I.E., 2016. Effects of dietary acidifiers on growth, hematology, immune response and disease resistance of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol., 50: 255-262. https://doi.org/10.1016/j.fsi.2016.01.040

Robinson, E.H., Li, M.H. and Manning, B.B., 2002. Comparison of microbial phytase and dicalcium phosphate for growth and bone mineralization of pond-raised channel catfish, Ictalurus punctatus. J. appl. Aquacult., 12: 81-88. https://doi.org/10.1300/J028v12n03_08

Sardar, P., Randhawa, H.S., Abid, M. and Prabhakar, S.K., 2007. Effect of dietary microbial phytase supplementation on growth performance, nutrient utilization, body compositions and haemato-biochemical profiles of Cyprinus carpio (L.) fingerlings fed soyprotein-based diet. Aquacult. Nutr., 13: 444–456. https://doi.org/10.1111/j.1365-2095.2007.00497.x

Shah, S.L. and Altindağ, A., 2004. Hematological parameters of tench (Tinca tinca L.) after acute and chronic exposure to lethal and sublethal mercury treatments. Bull. environ. Contam. Toxicol., 73: 911–918. https://doi.org/10.1007/s00128-004-0513-y

Shah, S.Z.H., Afzal, M., Fatima, M., Hussain, S.M., Bhatti, M.A., and Hayat, H.F., 2015. Supplementation of phytase and citric acid to soybean meal based diet enhance muscle mineralization of rohu, Labeo rohita, juveniles. J. Sci., 5: 796-800.

Shahzad, M.M., Hussain, S.M., Hussain, M., Tariq, M., Ahmed, N., Furqan, M. and Rafique, T., 2020. Improvement in overall performance of Catla catla fingerlings fed phytase included low cost plant by products-based diet. Saudi J. biol. Sci., 27: 2089-2096. https://doi.org/10.1016/j.sjbs.2020.06.012

Shahzad, M.M., Hussain, S.M., Jabeen, F., Hussain, A.I., Ahmad, S., Ashraf, A. and Arsalan, M.Z.U.H., 2017. Effect of phytase supplementation on mineral digestibility to Catla catla fingerlings fed Moringa oleifera leaf meal based test diets. Punjab Univ. J. Zool., 32: 65-73.

Shahzad, M.M., Hussain, S.M., Jabeen, F., Hussain, A.I., Arsalan, M.Z.H., Ahmad, N., Rehan, M.M.H. and Riaz, D., 2016. Carcass composition and hematological study of Catla catla fingerlings fed on phytase supplemented Moringa oleifera leaf meal (MOLM) based diet. J. Biodivers. environ. Sci., 9: 57- 68.

Shirmohammad, F., Porreza, J. and Mahbobi, N., 2003. Effect of supplemental citric and phytase on growth performance and body composition of common carp (Cyprinus carpio). Iran agric. Res., 22: 138-152.

Snedecor, G.W. and Cochran, W.G., 1989. Statistical methods, 8th Edn. Iowa State Univ. Press, Ames, Iowa, 54: 71-82. https://doi.org/10.1016/0044-8486(83)90153-9

Spinelli, J., Houle, C.R. and Wekell, J.C., 1983. The effect of phytates on the growth of rainbow trout (Salmo gairdneri) fed purified diets containing varying quantities of calcium and magnesium. Aquaculture, 30: 71-83. https://doi.org/10.1016/0044-8486(83)90153-9

Steel, R.G.D., Torrie, J.H. and Dickey, D.A., 1996. Principles and procedures of statistics (3rd ed.). McGraw Hill international Book Co. Inc., New York. USA, pp. 336-352.

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. Aquac. Res., 32: 583–592. https://doi.org/10.1046/j.1365-2109.2001.00581.x

Von Danwitz, A. and Schulz, C., 2020. Effects of dietary rapeseed glucosinolates, sinapic acid and phytic acid on feed intake, growth performance and fish health in turbot (Psetta maxima L.). Aquaculture, 516: 734624. https://doi.org/10.1016/j.aquaculture.2019.734624

Wagner, E.J., Jensen, T., Arndt, R., Routledge, M.D. and Brodwich, Q., 1997. Effects of rearing density upon cutthroat hematology, hatchery performance, fin erosion and general health and condition. Prog. Fish-Culturist, 59: 73-187. https://doi.org/10.1577/1548-8640(1997)059<0173:EORDUC>2.3.CO;2

Wilson, R.W. and Taylor, E.W., 1993. The physiological responses of freshwater rainbow trout, Oncorhynchus mykiss, during acutely lethal copper exposure. J. comp. Physiol. B, 163: 38-47. https://doi.org/10.1007/BF00309663

Yilgmaz, S., Ergün, S. and Celik, E.S., 2016. Effect of dietary spice supplementations on welfare status of sea bass, Dicentrarchus labrax L. Proc. natl. Acad. Sci. India B Biol. Sci., 86: 229–237. https://doi.org/10.1007/s40011-014-0444-2

Yoo, G. and Bai, S.C., 2014. Effect of dietary microbial phytase supplementation on the bioavailability of phosphorus in juvenile olive flounder (Paralichthys olivaleus) fed soybean based diets. J. Fish. aquat. Sci., 17: 319–324. https://doi.org/10.5657/FAS.2014.0319