Aquaculture Reports 20 (2021) 100738

Available online 29 May 20212352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Dietary nucleotides enhanced growth performance, carcass composition, blood biochemical, and histology features of European sea bass, Dicentrarchus labrax L

Fawzy I. Magouz a, Mohamed M. Abdel-Rahim b, Ayman M. Lotfy b, Amira Mosbah a, Mohamed Alkafafy c, Hani Sewilam d,e, Mahmoud A.O. Dawood a,* a Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt b Fish Rearing Laboratory, Aquaculture Division, National Institute of Oceanography and Fisheries (NIOF), Anfoushy, Alexandria, Egypt c Department of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif, 21944, Saudi Arabia d The Center for Applied Research on the Environment and Sustainability, The American University in Cairo, 11835, Cairo, Egypt e Department of Engineering Hydrology, RWTH Aachen University, Aachen, Germany

A R T I C L E I N F O

Keywords: Dicentrarchus labrax Feed additives Growth performance Liver enzymatic profile Histology

A B S T R A C T

Nucleotides (NT), as a modern feed additive, are effective in many vital functions of cultured aquatic organisms. An experiment was performed to evaluate the effects of dietary nucleotides on the growth performance, carcass composition, liver enzymatic profile, liver, intestine, and spleen histology in European sea bass, Dicentrarchus labrax (initial weight 29.56 ± 0.083 g/fish). Four isonitrogenous (46 % crude protein) and isolipidic (16 % crude lipid) experimental diets were supplemented with different levels of nucleotides, representing N0, N500, N1000, and N1500 treatments, respectively. Each diet was fed to three net enclosures (experimental net cage: 1 × 1 × 1 m) with 0.5 m3 water volume, and each net enclosure contained eight fish. The fish were fed to satiation for 56 days. The results showed significant (P < 0.05) differences in growth performance and feed utilization indices in favor of N500 among all the treatments (P < 0.05). Seabass fed N0 and N1500 had the significantly lowest results. No significant (P < 0.05) differences in survival were detected. Fish fed diets sup-plemented with nucleotides exhibited significantly lower values of alkaline phosphatase activity (ALP), aspartate aminotransferase activity (AST), alanine aminotransferase activity (ALT), and bilirubin (BIL) compared with the control. The intestine, liver, and spleen histology showed the best development in N500 fed group fish compared with the other treatment, mainly N0 and N1500. In conclusion, the present experiment showed that 500 mg/kg of dietary nucleotides is the optimum level for the best growth performance, health condition, and digestive organs development of D. labrax.

1. Introduction

With the increased development of the fish farming industry in most countries globally and growing production strategies to rationalize water consumption, many problems and infectious diseases began to appear in the epidemic spread, especially in intensive fish farming (Dawood et al., 2021; Gewaily et al., 2021). The use of antibiotics to control fish diseases has regularly been managed for decades (Ahma-difar et al., 2020; El Asely et al., 2020). However, the negative impacts of antibiotics application were documented, such as the appearance of antibiotic-resistant bacterial strains, the residual accumulation in edible seafood, human health, the depression of immune systems and the

environment (Zaineldin et al., 2021). For the previous negative impacts, the application of antibiotics in aquaculture has been banned in different countries globally, such as Europe, and strict regulations were forced in the United States and other countries (Abdo et al., 2021; Carbone and Faggio, 2016).

Consequently, there is an encouraging need for emerging alternate safe feedstuffs to antibiotics to control aquatic diseases without causing any negative impact on the environment (Dawood et al., 2020; Gatlin, 2002; Ringo et al., 2012). Nucleotides are vital metabolites that are involved in nearly all cellular processes (Liu, 2016). They play essential roles in structural, metabolic, functions, and energetic regulatory pro-cesses (Ridwanudin, Haga et al., 2019). Nucleotides have several

* Corresponding author. E-mail address: Mahmoud.dawood@agr.kfs.edu.eg (M.A.O. Dawood).

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https://doi.org/10.1016/j.aqrep.2021.100738 Received 19 February 2021; Received in revised form 20 May 2021; Accepted 25 May 2021

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physiological functions, including cell signaling, cellular agonists, and co-enzyme components (Carver and Walker, 1995; Liu, 2016). It has been theorized that tissues, immune cells, and gastrointestinal cells require nucleotides during the early stages of development with a high metabolism or fast growth (Ringo et al., 2012; Liu, 2016). The results of the research confirm the multiple benefits of the dietary addition of a nucleotide in many aquatic organisms, such as meagre, Argyrosomus regius (Saenz de Rodriganez et al., 2013), gilthead seabream, Sparus aurata (El-Nokrashy et al., 2020, 2021), turbot, Scophthalmus maximus L. (Peng et al., 2013), red drum, Sciaenops ocellatus (Cheng et al., 2011), Coho salmon, Salmo salar L. (Burrells et al., 2001a), Atlantic salmon (Burrells et al., 2001b), rainbow trout, Oncorhynchus mykiss (Mohebbi et al., 2013; Tahmasebi-Kohyani and Keyvanshokooh, 2011), Labeo rohita (Baidya et al., 2015), common carp, Cyprinus carpio L. (Sakai et al., 2001), Ancherythroculter nigrocauda (Yin et al., 2015), grouper, Epi-nephelus malabaricus (Lin et al., 2009), hybrid tilapia, Oreochromis nilo-ticus ♀ × O. aureus ♂ (Xu et al., 2015), hybrid striped bass, Morone chrysops × M. saxatilis (Li et al., 2004), and zebrafish (Guo et al., 2017).

In light of the potential role of nucleotides in improving the growth performance, immune responses, disease resistance, intestinal micro-biota, and gastrointestinal morphology and physiology in many aquatic organisms (Tahmasebi-Kohyani and Keyvanshokooh, 2011; Guo et al., 2019; El-Nokrashy et al., 2020, 2021). The objective of the experiment was to investigate growth and nutrient utilization responses, carcass composition, liver enzymatic profile, and the histology of liver, intes-tine, and spleen in juvenile European sea bass, Dicentrarchus labrax fed different levels of nucleotides.

2. Material and methods

2.1. Experimental facilities

This experiment was conducted at El-Max Research Station, National Institute of Oceanography and Fisheries (NIOF), Alexandria, Egypt. Underground saltwater with a salinity of 32 ppt was used. Water tem-perature, dissolved oxygen, pH, and ammonia were monitored during the experiment. Water temperature ranged from 26 ± 0.61 ◦C, salinity 32 ± 0.5 ppt, pH 7.9 ± 0.1, dissolved oxygen > 5.8 ± 0.22 ppm. The daily water exchange rate was 20 % /pond /day, and fish were kept under the natural light regime.

2.2. Experimental fish

Ninty six juvenile European sea bass, Dicentrarchus labrax L. with an average initial body weight of 29.56 ± 0.083 g/fish obtained from a commercial marine fish farm, Egypt used. The juveniles were stocked into four cement ponds (each with 24 m3). Each cement pond was installed with three net enclosures (experimental net cage: 1 × 1 × 1 m) with 0.5 m3 water volume, and each net enclosure was stocked with eight fish.

2.3. Experimental treatments and feeding regime

Four levels of nucleotides (NucleoforceFish™; Bioiberica® Spain; https://www.bioiberica.com/en/products) (0, 500, 1000, and 1500 mg/kg) were added to the basic commercial diet (Skretting feed) purchased from Skretting Egypt (https://www.skretting.com/en-EG/) with 46 % crude protein (CP), 16 % crude lipids, 3.03 % fiber, 492.64 kcal/100-gram gross energy, and 93.23 N: C ratio (mg CP: Kcal). The doses of nucleotides were proposed based on the previous studies conducted by El-Nokrashy et al. (2020) and El-Nokrashy et al. (2021). NucleoforceFish™ contains 34 % of free nucleotides from inactivated yeast 92 extract, 80 % pyrimidine, and 20 % purine (El-Nokrashy et al., 2020). Diets were abbreviated as N0, N500, N1000, and N1500. The basic diet was finely ground, mixed with four levels of nucleotides. After the addition of NucleoforceFish™ and hot water, the diets were pressed

using an electric kitchen meat grinder (Moulinex 1600 W, France). Feed strands were then dried at 45 ◦C for 12 h. The dried pellets were sieved to appropriate sizes using different feed sieves. The dried pellets were packaged and stored at − 20 ◦C until use. Fish were fed to satiation for 56 days, six days a week. The experiment was carried during the period from 1st July – 27th August 2019. Fish in each enclosure were weighed biweekly to follow up the growth performance and visually check up the health status.

2.4. Fish and feed analytical methods

At the beginning and end of the experiment, samples of the whole fish body and feed were collected to analyze the diets and fish’s proxi-mate compositions, including the moisture, protein, lipid, fiber, and ash contents according to AOAC (2006) methodology. In brief, the moisture content evaluated using oven drying at 105 ◦C till reaching the constant dry weight. Ash content was detected using a muffle furnace at 550 ◦C for 6 h. Crude protein was analyzed using the Micro-Kjeldahl method using Kjeltech autoanalyzer (Model 1030, Tecator, Hoganas, Sweden). Total lipids content was determined by petroleum ether extraction in the Soxhlet apparatus for 16 h.

2.5. Growth performance and feed utilization indices

At the end of the experiment, fish were collected, counted, and weighed. The growth performance and feed utilization parameters were determined as follows:

Average daily gain (gm∕fish∕day), ADG = Wt − W0∕days

Specific growth rate (%∕day), SGR = 100 × lnWt − lnW0 ∕days

where: W0: initial fish weight (g); Wt: final fish weight (g); ln: natural logarithm.

Survival rate (%) = 100 × (Final number of fish ∕ initial number of fish).

Feed conversion ratio, FCR = feed intake (g) as dry weight ∕ weight gain (g).

Protein efficiency ratio, PER = weight gain (g) ∕ protein intake (g).

Protein productive value (%), PPV = 100 × (protein gain (g) ∕ protein intake (g)).

Energy gain, Kcal = Et − E0

where: Et: Energy content in the fish carcass (Kcal) at the end E0: Energy content in the fish carcass (Kcal) at the start Energy utilization (%), EU = 100 × (energy gain (kcal) ∕ energy

intake (kcal)). Carcass energy (kcal∕100 g DM) = (protein% × 5.64) + (ether

extract% × 9.44) (Bonaldo et al., 2010).

2.6. Biometric indices

Four fish from each net enclosure were sacrificed to obtain their final biological records, including liver and viscera weights to determine hepatosomatic (HSI), viscerosomatic (VSI) indices, as follows: Hep-atosomatic index, HSI = 100 × [liver weight (g)/ total body weight (g)]; Viscerosomatic index, VSI = 100 × [Total weigh of all viscera (g) / total body weight of the fish before removal of the viscera (g)].

2.7. Serum analyses

Blood samples for enzymatic assays and transaminase activities were collected. Fish were anesthetized using clove oil at the concentration of 0.3 mL/L before blood sampling. The blood samples of four different fish groups were collected from the caudal vein using a 21 Gauge hypoder-mic needle and a 5 mL disposable syringe and transferred into a non-

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heparinized bottle, allowed to clot at room temperature. The serum was then separated by centrifugation at 3000 rpm for 10 min (Hettich Zen-trifuges, ROTINA 380) and stored at – 20 ◦C. Alkaline phosphatase ac-tivity (ALP), aspartate aminotransferase activity (AST), alanine aminotransferase activity (ALT), bilirubin (BIL), and creatinine (mg/dL) were quantitatively estimated according to the method of Henry (1964). ALP (U/I) was determined by the colorimetric method, according to Bergmeyer and Bernt (1974). AST (U/I) and ALT (U/I) were determined by the colorimetric method, according to Reitman and Frankel (1957). Creatinine was determined, according to Pincus (1996).

2.8. Histopathological examination of intestine, liver, and spleen

Four randomly selected fish from each treatment were anesthetized using clove oil (0.3 mL/L). Tissue samples from the intestine, liver, and spleen were collected and fixed in neutral buffer formaldehyde solution. After complete fixation, the tissue samples were dehydrated in ascending concentration of ethanol followed by clearing in xylene and embedding in paraffin wax. Paraffin blocks were sectioned at 5 μm thickness using a rotary microtome (Leica 2025) and stained with He-matoxylin and Eosin (H&E), and examined under a light microscope (Bancroft and Gamble, 2007).

2.9. Statistical analysis

Results are given as the mean ± SE. Data were statistically analyzed with a one-way analysis of variance (ANOVA) using the software SPSS (Standard Version 22.0 SPSS Inc. Chicago, Illinois). Duncan’s multiple range test was used to compare differences between means when sig-nificant F values were observed at P < 0.05 level.

3. Results

3.1. Growth performance

The growth performance and survival of European sea bass were significantly affected by the inoculation level of nucleotides in the experimental diets (Table 1). The values of final weight, gain, average daily gain (ADG), and specific growth rate (SGR) were significantly (P <0.05) increased in nucleotides-supplemented diets compared with the control diet. However, the best values were recorded in N500. The survival rate was also increased in nucleotides-supplemented diets compared with the control diet but without significant differences (P <0.05). The increased survival rates were 16.67, 16.67, and 8.34 % in N500, N1000, and N1500 compared with the control. Viscerosomatic index (VSI) decreased significantly in N500 group fish, while the hep-atosomatic index (HSI) did not affect by the dietary addition of nucleotides.

Feed utilization indices of European sea bass were significantly

improved in nucleotides-supplemented diets compared with the control diet (Table 1). Data of feed conversion ratio (FCR), the protein efficiency ratio (PER), protein productive value (PPV), energy gain, and energy utilization showed better (P < 0.05) values in the N500 group compared with the other treatments. The percentage of improvement in N500 compared with the control group was 20, 25.3, 25.9, 20.7, and 25 % regarding FCR, PER, PPV, energy gain, and energy utilization, respec-tively. The increment in nucleotide addition did not significantly (P <0.05) gave better results than the N500 group.

3.2. Carcass composition

Data of initial and final carcass composition of European sea bass were shown in Table 2. In general, there were significant (P < 0.05) differences among treatments. Treatment N1500 exhibited the lowest significant (P < 0.05) values regarding protein, lipids, carcass energy content, and highest ash and fiber values. Further, the levels of nucle-otides (1000 and 1500 mg/kg) exhibited lower ether extract values than fish of the control and N500 groups (P < 0.05).

3.3. Blood biochemistry

Data of serum analyses of European seabass are presented in Table 3. Values of alkaline phosphatase activity (ALP), aspartate aminotrans-ferase activity (AST), alanine aminotransferase activity (ALT), bilirubin (BIL), and creatinine were significantly (P < 0.05) different among treatments. The levels of liver enzyme function (ALT, AST, and ALP) showed a marked reduction (P < 0.05) in sea bass fed dietary nucleo-tides compared to the control diet. Values recorded in N500 were the lowest significant values compared with the other treatments. The reduction ratio in ALT, AST, ALP, and BIL enzymes recorded in N500 compared to N0 were 51.2, 47.9, 29.9, and 35.4 %, respectively.

3.4. Histopathology

Fish fed the basal, N500, N1000, and N1500 diets had normal in-testinal villar structures. Further, the intestinal villi length was markedly increased by N500, N1000, and N1500 diets (Fig. 1).

All groups displayed normal hepatic structure with mild hepatocytic cloudy swelling and congestion of main blood vessels in the control group. Besides, N500, N1000, and N1500 groups had diffuse hepatocytic vacuolations that may be attributed to glycogen deposition. Fish fed N1000 showing mild congestion both in main blood vessels and sinu-soids (Fig. 2).

The control, N500, N1000, and N1500 groups show normal splenic structure with occasional focci on red pulp depletion. Spleens of fish fed the N1000 diet showing mild congestion, and those fed the N1500 diet had moderate congestion of main blood vessels (Fig. 3).

Table 1 Effect of different levels of nucleotides on the growth performance, survival, viscerosomatic index, and hepatosomatic index of European sea bass.

Treatments N0 N500 N1000 N1500

Initial weight (g/fish) 29.29 ± 0.28 29.57 ± 0.08 29.67 ± 0.11 29.70 ± 0.07 Final weight (g/fish) 62.05 ± 0.22c 69.42 ± 0.69a 64.80 ± 0.60b 63.94 ± 0.38b

Gain (g/fish) 32.75 ± 0.07c 39.85 ± 0.64a 35.13 ± 0.54b 34.24 ± 0.32b

ADG (g/fish/day) 0.58 ± 0.00c 0.71 ± 0.01a 0.63 ± 0.01b 0.61 ± 0.01b

SGR (%/day) 1.34 ± 0.01c 1.52 ± 0.02a 1.39 ± 0.01b 1.37 ± 0.01bc

Survival (%) 83.33 ± 11.02 100.0 ± 0.00 100.0 ± 0.00 91.67 ± 4.17 Viscerosomatic index 12.59 ± 0.496a 10.20 ± 0.567b 10.92 ± 0.858ab 11.47 ± 0.334ab

Hepatosomatic index 2.31 ± 0.24 2.34 ± 0.15 2.68 ± 0.24 3.10 ± 0.34 Feed conversion ratio 1.45 ± 0.01a 1.16 ± 0.02d 1.30 ± 0.01c 1.38 ± 0.02b

Protein efficiency ratio 1.50 ± 0.012d 1.88 ± 0.029a 1.67 ± 0.015b 1.57 ± 0.019c

Protein productive value 8.79 ± 0.21c 11.07 ± 0.14a 9.58 ± 0.11b 9.24 ± 0.14bc

Energy gain, Kcal 18.14 ± 0.32b 21.89 ± 0.31a 18.45 ± 0.29b 18.54 ± 0.51b

Energy Utilization, % 7.73 ± 0.19b 9.66 ± 0.14a 8.20 ± 0.07b 7.95 ± 0.19b

Values represent the mean ± SE. Different superscript letters in each row indicate significant differences (P < 0.05).

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4. Discussion

The dietary addition of nucleotides in aquafeed was found to in-crease the efficacy of utilizing the low fish meal (FM)-alternative diets, increase the growth and health status of aquatic animals and reduce FM- protein sources (Hossain et al., 2020). There has been evidence of beneficial effects on fish performance in several fish species (Krüger and van der Werf, 2018). In the present study, a significant improvement in growth indices, feed utilization, and sea bass survival was recorded when fish were fed NT-supplemented diets, especially in the N500 group

compared with the control diet. Like the present study, gilthead sea bream fed the dietary addition of nucleotides at 500 mg/kg exhibited the highest growth performance, survival, and feed efficiency compared with NT non-supplemented diets and those fed 250 mg/kg (El-Nokrashy et al., 2020, 2021). The same direction of the increase in growth per-formance and feed utilization was found in red drum (Cheng et al., 2011), turbot (Peng et al., 2013), rainbow trout (Hunt et al., 2014; Liu, 2016), amberjack (Hossain et al., 2017c), red sea bream (Hossain et al., 2016, 2017a, 2017b), zebrafish (Guo et al., 2017), Nile tilapia (Barros et al., 2015; Kader et al., 2018), and hybrid tilapia (Xu et al., 2015). To

Table 2 Effect of different levels of nucleotides on the carcass composition of European sea bass.

Treatment Dry matter, % Protein, % Ether extract, % Ash, % Fiber, % Carcass energy, Kcal/100gm

Initial 32.73 ± 0.18 19.97 ± 0.12 7.13 ± 0.07 4.08 ± 0.02 0.114 179.94 ± 0.21

Final

N0 31.66 ± 0.23b 19.55 ± 0.02a 7.41 ± 0.02a 4.05 ± 0.03c 0.173b 180.17 ± 0.29a

N500 31.52 ± 0.10b 19.54 ± 0.02a 7.36 ± 0.05a 4.55 ± 0.07b 0.143c 179.66 ± 0.38a

N1000 31.12 ± 0.02b 19.60 ± 0.01a 7.17 ± 0.04b 4.00 ± 0.01c 0.123d 178.18 ± 0.29b

N1500 32.33 ± 0.29a 19.13 ± 0.07b 7.04 ± 0.08b 5.98 ± 0.07a 0.223a 174.32 ± 0.36c

Values represent the mean ± SE. Different superscript letters in each column indicate significant differences (P < 0.05).

Table 3 Effect of different levels of nucleotides on serum analyses of European sea bass.

Treatments ALT (mg/dL) AST (mg/dL) ALP (mg/dL) BIL (mg/dL) Creatinine (mg/dL)

N0 582.00 ± 2.89a 480.00 ± 2.31a 87.00 ± 0.58a 1.13 ± 0.03a 0.47 ± 0.03bc

N500 280.67 ± 1.20d 250.33 ± 0.88d 61.33 ± 0.88c 0.73 ± 0.03c 0.83 ± 0.03a

N1000 377.00 ± 1.53c 370.33 ± 0.88c 63.33 ± 0.33c 0.87 ± 0.03b 0.57 ± 0.03b

N1500 429.33 ± 2.60b 394.67 ± 3.84b 70.33 ± 0.88b 1.03 ± 0.03a 0.37 ± 0.03c

Values represent the mean ± SE. Different superscript letters in each column indicate significant differences (P < 0.05). Alkaline phosphatase activity (ALP), aspartate aminotransferase activity (AST), alanine aminotransferase activity (ALT), and bilirubin (BIL).

Fig. 1. Intestine of European sea bass, Dicentrarchus labrax belonging to the fish groups fed nucleotides at 0, 500, 1000, and 1500 mg/kg feed. H&E staining, magnification × 200.

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Fig. 2. Liver of European sea bass, Dicentrarchus labrax belonging to the fish groups fed nucleotides at 0, 500, 1000, and 1500 mg/kg feed. H&E staining, magnification × 200.

Fig. 3. Spleen of European sea bass, Dicentrarchus labrax belonging to the fish groups fed nucleotides at 0, 500, 1000, and 1500 mg/kg feed. H&E staining, magnification × 200.

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explain the mode of action of nucleotides as a growth promoter, Asa-duzzaman et al. (2017) described that supplementation of nucleotides in juvenile Nile tilapia improved the muscle growth through encouraging hyperplasia, hypertrophy, and upregulating the primary growth-related gene expression (GH, GHR-1, IGFs, MRFs, Pax7 and myostatin).

In contradiction to the findings of this study, Tulli et al. (2005) found that nucleic acid supplemented diets did not significantly affect final weight, specific growth rate, feed intake, feed efficiency, and protein efficiency ratio in sea bass treated with RNA extract. Also, Ridwanudin et al. (2019) found that after 15 weeks of feeding rainbow trout on nucleotides supplemented diets, there was no significant difference among dietary treatments on growth performance and feed utilization. In the same direction, Bowyer et al. (2019) found no significant differ-ences in sea bass fish performance after feeding with nucleotides mixture (Laltide®) at 0.15 and 0.30 %. In the present study, increasing the nucleotides level from 500 mg/kg to 1000 and 1500 mg/kg feed did not improve growth or feed utilization. On the contrary, it led to a decrease in the previous values. This is consistent with Clifford and Story (1976), who stated that excessive dietary nucleotides in monogastric animals might have toxic impacts leading to disorder in protein, lipid, and carbohydrate metabolism because of the deficient level of uricase activity, the enzyme involved in nucleotide metabolism. Results of vis-cerosomatic index (VSI) and hepatosomatic index (HSI) obtained in the current study agreed with the findings of El-Nokrashy et al. (2020) in gilthead sea bream. The addition of dietary nucleotides at 500 mg/kg reduced the viscerosomatic index. In agreement with our study, Bae et al. (2020) found no significant differences in HSI in olive flounder, Paralichthys olivaceus fed yeast extract nucleotides at 4 g/kg. However, they also found no significant differences in VSI fed yeast extract nu-cleotides at 4 g/kg.

In the current study, the addition of NT did not improve the carcass composition of sea bass. The same results were confirmed in red drum, Sciaenops ocellatus (Liu et al., 2003; Li et al., 2007), in gilthead seabream (Oliva-Teles et al., 2006), red sea bream, Pagrus major (Hossain et al., 2016), amberjack (Hossain et al., 2017c), and sea bass (Bowyer et al., 2019). However, fish-fed nucleotides supplemented diet exhibited higher protein and lipids in their muscles, as stated in Ancherythroculter nigrocauda by Yin et al. (2015) and in gilthead sea bream by El-Nokrashy et al. (2020). The previous authors found that Sparus aurata fed diets contained 25 % fish meal exhibited higher protein content and lowered lipids content than the diets containing 0% FM irrespective of nucleo-tides levels. There are numerous gaps in the efficacy of nucleotide on the performance of farmed aquatic species, and their body composition was significantly recorded. The variation depends on fish species, source of nucleotide, feed quality, the fishmeal content, and the addition level (Li and Gatlin, 2006; Bowyer et al., 2019). For example, Do Huu et al. (2012) stated that fish diets with non-fishmeal ingredients often contain relatively low amounts of nucleotides, negatively affecting fish perfor-mance. The same conclusion was observed by better diets that included higher fishmeal levels (El-Nokrashy et al., 2020).

Fish blood analysis is one of the most important criteria for judging the health status of fish. The results showed that the addition of nucle-otide at a concentration of 500 mg/kg feed led to a significant decrease in the serum content of alkaline phosphatase activity (ALP), aspartate aminotransferase activity (AST), alanine aminotransferase activity (ALT), and bilirubin (BIL). Simultaneously, the level of creatinine in fish blood was increased significantly compared to the control diet. The current study results agreed with Tahmasebi-Kohyani et al. (2012), who found that the serum enzymes content of ALT, AST, ALP in rainbow trout fed the nucleotide-supplemented diets were significantly lower than fish fed the control diet. The previous authors stated that the decline in serum enzymes could explain the potential beneficial role of the dietary nucleotides in improving liver function. Rising the enzymes mentioned above in response to nutritional and/or environmental agents may be considered a sign of liver dysfunction (Talas and Gulhan, 2009).

The positive results of the current study concerning the growth

performance, survival, and feed utilization attributed primary to the positive development in the histology of the intestine, liver, and spleen. In this regard, Guo et al. (2019) stated that the intestine, with its structural integrity and mucosal immune components, serves as the first protection line against aquatic pathogens, besides its vital role in feed utilization (Doan et al., 2018). The assessment of histological develop-ment of digestive organs in Cyprinus carpio fed extruded feed containing fishmeal offered valued data regarding digestive capacity and possible health impacts (Markovic et al., 2012).

5. Conclusion

The present study showed that the addition of 500 mg/kg of dietary NucleoforceFish® is the optimum level for the highest growth perfor-mance, feed utilization, fish health, and lower values of ALT, AST, ALP, and BIL enzymes. The digestive organs (intestine, liver, and spleen) showed the best development in N500 fed group fish compared with the other treatment, mainly N0 and N1500. The authors recommend dietary addition of 500 mg/kg NucleoforceFish® in sea bass feeds to obtain the maximum benefits.

Author statement

Conceptualization, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Amira Mosbah, Mahmoud A.O. Dawood; Formal analysis, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Amira Mosbah; Funding acquisition, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Amira Mosbah, Mohamed Alkafafy, Hani Sewilam, Mahmoud A.O. Dawood; Investigation, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Amira Mosbah, Mahmoud A.O. Dawood; Methodology, Fawzy I. Magouz, Mohamed M. Abdel- Rahim, Ayman M. Lotfy, Amira Mosbah, Mahmoud A.O. Dawood; Su-pervision, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Mahmoud A.O. Dawood; Writing—original draft, Fawzy I. Magouz, Mohamed M. Abdel-Rahim, Ayman M. Lotfy, Amira Mosbah, Mohamed Alkafafy, Hani Sewilam, Mahmoud A.O. Dawood.

Funding

The current work was funded by Taif University Researchers Sup-porting Project number (TURSP - 2020/57), Taif university, Taif, Saudi Arabia.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgments

Taif University Researchers Supporting Project number (TURSP- 2020/57), Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.

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