Impact of polyculture in aquaponics on the hemato-serological and health status of Nile tilapia (Oreochromis niloticus) and carp (Cyprinus carpio)

ABSTRACT Aquaponics is an innovative, eco-friendly, and sustainable food production system that combines hydroponic crop production with recirculating aquaculture. This study aimed to assess the haematological, biochemical, and health status of Nile tilapia and common carp in monoculture and polyculture systems. A total of 800 (Oreochromis niloticus) and (Cyprinus carpio) with an average body weight of 1.1± 0.2 g were used. The fish were divided into three groups: (I) contained Nile tilapia only (monoculture), (II) contained common carp only (monoculture), and (III) contained polycultured tilapia and carp. The results of the study revealed a decrease in red blood cell counts, haemoglobin levels, mean corpuscular haemoglobin concentration, and hematocrit in monoculture tilapia blood, indicating significant differences when compared to polyculture tilapia blood. The biochemical parameters except total protein, glucose, and urea were significantly higher in the blood serum of polyculture carp compared to tilapia. Immunoglobulin variables and lysozyme activity were the same in the blood of fishes, confirming that Nile tilapia and common carp were still fit with a good immunological status under monoculture and polyculture in an aquaponic system. Conclusion: Nile tilapia and common carp cultivated in aquaponic systems (monoculture and polyculture) still fall within the normal range for haematology and biochemistry.


Introduction
The Food and Agriculture Organization of the United Nations (FAO) reports that aquaculture has expanded more quickly than other significant food production sectors, with the goal of fulfilling rising global demand for fish while protecting natural fish populations [1].Currently, improving culture systems is necessary to increase fish production in both quantity and quality while reducing aquaculture's environmental impact [2].Hundreds of millions of people throughout the world rely on fisheries and aquaculture for food, nutrition, money, and livelihood, with global aquaculture production reaching 80.37 million metric tonnes in 2016 [1].In Egypt, aquaculture is regarded as the only viable option for increasing fish output [3], contributing significantly to our national economy, nutrition, job creation, income, and foreign exchange earnings.It has a lot of promise in terms of meeting rising seafood and animal protein needs.The country's increased fish output through aquaculture might be regarded as a recent success story in feeding Egypt's people [4].
Aquaculture in Egypt could meet the rising need for protein, according to the current fish output, and has been experiencing unprecedented growth during the last decade [5,6].Aquaculture necessitates a large amount of water, which raises issues in the context of the worldwide water problem.A typical moderately intensive aquaculture sector uses roughly 10.3 m 3 of water per kilogram of fish produced [7].Egypt's water scarcity is steadily worsening, posing a severe danger to the country's food security.Geopolitical factors, population expansion, and climate change are all factors that encourage people in Egypt to seek innovative solutions to the problem of feeding future generations.Aquaponics is a good alternative answer that presented itself powerfully as part of the hunt for unorthodox solutions to Egypt's food dilemma.Aquaponics is the combination of aquaculture and hydroponics.It is the symbiotic cultivation of aquatic animals and plants, benefiting the economic yield [8].
One of the 21st century's most effective and environmentally friendly farming techniques is aquaponics [9,10].Moreover, it is the solution to many of today's global problems, having a huge potential to become the future farming method that provides year-round production of high-quality fish and vegetables in a sustainable way [10,11].Polyculture is the practice of cultivating more than one species of aquatic organisms in the same pond or tank with different food habits [12].Polyculture fits into the principles of sustainable aquaculture, since it aims at reducing the environmental impact of the activity by improving feeding efficiency and increasing producer income by rearing together two or more species that do not compete for the same feed resources [13].According to Landau, Ponce and Mehrim [14][15][16] polyculture is one strategy for increasing fish production yields by maximizing the use of available resources.Furthermore, polyculture has the potential to improve long-term ecological stability and function [16,17].Little study has been done on polyculture in recirculating systems, including aquaponics.Polyculture would enhance aquaponics by producing a variety of fish products for local consumption.Polyculture in aquaponics, particularly in coupled single recirculation systems, poses significant challenges but also offers promising opportunities to improve aquaponics as a sustainable food production system [18].There needs to be much more experimentation to come up with some ideal species for polyculture in aquaponics.
Blood characteristics are effective and sensitive indices for monitoring physiological changes in fish.Analysis of blood indices has proven to be a valuable approach for examining the health status of farmed fish, providing reliable information on metabolic disorders [19], becoming a basic part of fish health monitoring programmes [20].Hence, as a result, the current study was conducted to promote the sustainability of Egyptian agro-ecosystems by promoting innovative solutions for competitive food production systems for the first-time using polyculture in aquaponics, with the goal of determining the effect on hematology, biochemical parameters, and immune status in Nile tilapia and common carp in polyculture systems.

Materials and methods
The current experiment was carried out in Al-Azhar Aquaponics (ASTAF-PRO aquaponics) at the Department of Zoology, Faculty of Science, Al-Azhar University (Assiut branch), Assiut, Egypt.

Experimental design
A total of 800 apparently healthy Nile tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) were obtained from a private hatchery in Kafr El-Sheikh city, Kafr El-Sheikh Governorate, Egypt with an average body weight of 1.1 ± 0.2 g.Prior to the experiment, fish were acclimated in the ASTAF-PRO aquaponic system for 21 days.The design of the ASTAF-PRO aquaponic unit was based on Kloas [21] considering the modifications of Osman [10].In brief, it consisted of three rearing tanks (each = 1 m 3 ), a sedimentation tank (1 m 3 ), bio-filter (1 m 3 ), three hydroponic trenches, two separate pump units, and one one-way valve (Figure 1) in a wooden greenhouse.
The experiment consisted of three groups in two replicates.The first group (I) contained Nile tilapia that was pure (monoculture, (200 fish for both, replicates).The second group (II) contained common carp that were pure (monoculture, 200 fish for both, replicates).The third group (III) contained polycultured Nile tilapia and common carp 400 fish for both, replicates).All fish were fed 7% of their body weight twice daily [22] at 9:00 AM and 4:00 PM, six days a week.The diet containing 30% protein used in the experiment was formulated to cover all nutrients required for the tilapia and common carp as recommended by [22].They were reared for three months, between September 1 and 30 November 2020.

Biochemical analysis
Blood samples were collected through cardiac puncture as described by [25].No anaesthetic was given to the fish, as anesthesia can affect blood parameters.The blood samples were then allowed to coagulate for 15-20 min at 4°C prior to centrifugation for 20 min at 3000 rpm to separate serum.The serum was stored at −20°C until use for biochemical and immunological analyses.Serum total protein, glucose, cholesterol, triglyceride, creatinine, urea, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were calculated according to the methods of [26].

Statistical analysis
A database was created using Excel 2013 software to perform the first analysis using descriptive statistics.Data were presented as mean ± SD (standard deviation).The results were subjected to a one-way analysis of variance (ANOVA) to test the effect of treatment inclusion on fish performance.Data were analyzed using SPSS (1997) program, Version 16.Differences between means were compared using Duncan's (1955) multiple range tests at p < 0.05 level.

Hematological parameters
Table 1 shows the average values for red blood cells count (RBCs), (Hb), (MCV), (MCH), (MCHC), (Hct), (WBCs), lymphocytes, neutrophils, eosinophils, and monocytes in monoculture and polyculture Tilapia increased in an aquaponic system.After three months of exposure, statistical analysis revealed a decrease in all treated RBCs, Hb, MCHC (excluding the third month), Hct, lymphocytes (except the 1 st month), and eosinophils (except the 3 rd month) in monoculture tilapia blood, indicating significant differences when compared to polyculture tilapia blood.However, the values for all treated MCV, MCH, WBCs, neutrophils, and monocytes (except the 1 st month) in polyculture tilapia blood were significantly lower than those for monoculture tilapia (Table 1).The blood parameters of monoculture and polyculture common carp reared in an aquaponic system are shown in Table 2. Statistically, all parameters in Table 2 exhibited significant differences in the blood of monoculture and polyculture carp after three months of experiment, with the exception of RBCs in polyculture common carp, eosinophils (%) in monoculture common carp, and Hb in monoculture and polyculture.

Blood biochemistry
In the blood serum of tilapia and common carp reared in the aquaponic system (monoculture and polyculture), the average concentrations of total protein, glucose, cholesterol, triglycerides, creatinine, urea, ALT, and AST are shown in (Tables 3  and 4).Tables 3 and 4 show non-significant (P < 0.05) changes in the average values of identified biochemical parameters between tilapia and common carp raised in monoculture after three months of exposure (excluding cholesterol and triglycerides).All biochemical parameters (except total protein, glucose, and urea (excluding the third month)) were significantly (P 0.01) higher in the blood serum of polyculture common carp compared to tilapia (Tables 3 and 4).

Immunological responses
Figures 2 and 3 compare the differences in selected immunological parameters in the   blood of monosex Nile tilapia and common carp are reared in aquaponic systems (monoculture and polyculture).In both monoculture and polyculture, the means of IgM levels were nonsignificant for two fish species.Furthermore, lysozyme activity in monoculture for tilapia was non-significant, whereas lysozyme activity was significant in monoculture for carp and polyculture for tilapia and carp (Figures 2 and 3).

Haematological parameters
Blood parameter studies have proven to be a useful tool for identifying the association between blood characteristics with habitat and species adaptability to the environment, as well as monitoring fish health [28,29].Hematology is becoming increasingly important in aquaculture due to its ease of use and dependability in monitoring the health of cultivated species [30].
Numerous studies have shown that the peripheral RBCs, Hb, Hct and MCHC of fish displayed decrease incidence under stressed field and lab conditions [31].The present study showed that RBC, Hb, MCHC, Hct, lymphocytes (%), and eosinophils (%) were higher in monoculture tilapia than in polyculture.The rest of parameters, namely MCV, MCH, WBCs, neutrophils, and monocytes were at higher levels in polycultured tilapia.The all resulted hematological parameters were within normal ranges for healthy Nile tilapia reared in monoculture and polyculture, and they were in line with values given by other authors for the same fish in different locations [32].This implies that the fish had perfect health during the research.In addition, neither species felt severely threatened by the other's existence.This is in contrast to the findings of Hrubec [32], who found the adapted social interactions between fishes can negatively affect fish growth by causing competition for food and/or space.
In the current study, there were notable variations in the blood indices of monoculture and polyculture carp, except for RBCs in polyculture common carp, eosinophils (%) in monoculture common carp, and Hb in both monoculture and polyculture.These results appear to demonstrate that rearing common carp in an aquaponic environment (mono or polyculture) has no negative  effects on hematological parameters or the health status.In addition, the common carp elevated in polyculture have considerably higher white blood cell counts (WBCs).A rise in the white blood cell count could be a sign that the common carp immune system is working well [33].

Blood biochemistry
Blood biochemical indices are useful for determining the health status of fish following different feeding trials [34].There are differences in biochemical characteristics between species, and numerous biotic and abiotic factors, such as water quality parameters, can have an impact on them [35].Overall, the blood biochemical values found in the current study were within the acceptable limits for Nile tilapia and common carp.Since total serum protein is one of the most stable blood components and is only mildly changed by numerous factors, it is commonly used to evaluate a fish's physiological state [36].In the present study, non-significant differences were observed in the total protein of the monosex Nile tilapia and common carp reared in monoculture and polyculture.These findings suggest that fish health status was nearly the same for monocultured and polycultured is reared in aquaponic systems [10].
Plasma glucose levels in fish increase during stress, probably due to the action of catecholamine on stored glycogen in the liver and other tissues [35].It was noted that fish raised in polyculture had blood glucose levels that were noticeably higher (Nile tilapia and common carp) than in the blood of fish reared in monoculture, but, still within the normal range for both, mono and polycultured fish.In the current study, monocultured Nile tilapia, compared to polycultured Nile tilapia, recorded higher cholesterol levels in their blood.While the opposite occurred in common carp.Anorexia, diabetes, liver malfunction, and malabsorption of fat, which are signs of abnormal glucose and cholesterol levels in the blood, were ruled out because glucose and cholesterol levels were within the usual range [35,37].The triglycerides recorded the same trend as being higher in the blood of monocultured Nile tilapia compared to polycultured Nile tilapia, and vice versa in common carp.Since the muscle is a vital organ closely linked to the turnover of amino acids, the increase in protein levels in fish blood is probably a result of the increase in triglyceride levels [35].
Uncertainty surrounds the physiological functions of urea, uric acid, and creatinine.They are, however, useful indicators of fish gill and kidney health [38], feed consumption [39], and amino acid (arginine) requirements [40].More than 50% of the nitrogenous waste discharged by the fish kidney is made up of creatine [38].In the present study, it was shown that the average values of creatinine and urea decreased between monoculture and polyculture in Nile tilapia and common carp, implying good health status during this experiment.These results agree with those of Sánchez-Muros [41].AST and ALT levels in the blood serum are indicators of continuing chemical processes within animal body [42].Of the enzyme activities under the current investigation, an unequivocal difference was observed in ALT and AST activities, with mean values higher in Nile tilapia reared in monoculture compared to polyculture.On contrast, the mean values of ALT and AST were lower in common carp reared in polyculture.These results are in accordance with Osman [10], who found significant changes in the levels of ALT and AST in the blood of fish reared in aquaponic systems.Our finding may be explained by the fact that both enzyme levels were within the normal range in aquaponic systems with acceptable water quality, which may have an immune-potentiating effect.

Immunological responses
The nonspecific immune system of fish is considered to be the first line of defense against invading pathogens [6].In the present study, IgM levels exhibited the same level in the blood of fish reared in monoculture and polyculture, confirming that the monosex Nile tilapia and common carp were still fit and had a good immunological status under aquaponic conditions.
Innate immunity depends heavily on the enzyme lysozyme [43].Mammals and other higher vertebrates rely on both innate and adaptive immunity to defend against pathogens, but fish mainly rely on innate immunity [44].Fish must have strong inherent defenses since they dwell in water that is full of several diseases [44].Lysozyme is found in fish in sites where there is a high risk of bacterial infection, such as the gills, skin, gastro-intestinal system, and eggs.According to Saurabh and Sahoo [44], lysozyme is mostly present in the gills, serum, and liver of tilapia (Oreochromis mossambicus); however, O. niloticus has the highest concentration of lysozyme in its serum [43].Stress lowers immunity and tolerance to illnesses (including bacterial infections) [45].Studies in O. niloticus show that persistent stress decreases lysozyme activity [43,46].In contrast to the current study, non-stressed monocultures and polycultures of tilapia and carp have higher levels of lysozyme activity.

Conclusion
Fish polyculture is critical for the nutritional and economic aspects of fish production.Based on the findings of this study, it is possible to conclude that the hematological, blood biochemical, and health status data suggest an improvement in fish health status and an improvement in the innate immune system in monosex O. niloticus and common carp reared in aquaponics (monoculture and polyculture).

Figure 2 .
Figure 2. Immunological parameters in the blood of Nile Tilapia (O.niloticus) cultured in monoculture and polyculture systems for three months.

Figure 3 .
Figure 3. Immunological parameters in the blood of common carp (C.carpio) cultured in monoculture and polyculture systems for three months.

Table 1 .
Haematological parameters (mean ±SD) of Nile Tilapia (O.niloticus) cultured in monoculture and polyculture systems for three months.

Table 2 .
Haematological parameters (mean ±SD) of common carp (C.carpio) cultured in monoculture and polyculture systems for three months.

Table 3 .
Biochemical parameters (mean ±SD) of Nile Tilapia (O.niloticus) cultured in monoculture and polyculture systems for three months.

Table 4 .
Biochemical parameters (mean ±SD) of common carp (C.carpio) cultured in monoculture and polyculture systems for three months.
bcMeans in the same row with different superscript letters are significantly different (P<0.05).