Fish and Shellfish Immunology 97 (2020) 367–374
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Dietary supplementation of guanidinoacetic acid improves growth, biochemical parameters, antioxidant capacity and cytokine responses in Nile tilapia (Oreochromis niloticus)
Abeer Azizaa,1, Rania Mahmouda, Eman Zahranb,∗,1, Hossam Gadallac a
Department of Nutrition and Nutritional Deﬁciency Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt Department of Internal Medicine, Infectious and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt c Clinical Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amino acid Fish health Oxidative stress Pro-inﬂammatory gene expression
A total of 180 unsexed Nile Tilapia ﬁsh (initial weight, 21 g) fed isonitrogenous (32%), isocaloric (3000 kcal/kg) diets containing diﬀerent levels of guanidinoacetic acid (GAA) at levels of (GAA1, 0.06%, GAA2, 0.12%, GAA3, 0.18%); for 60 days. Results showed higher ﬁnal body weight (FBW) and body weight gain (BWG) in groups supplemented with diﬀerent levels of GAA. Speciﬁc growth rate (SGR) was the highest in groups supplemented with 0.12% and 0.18% GAA. Lipid % of whole-body composition was higher in all groups excluding GAA3 group. Serum creatine kinase (CK) activity, cholesterol, and creatinine levels showed a marked signiﬁcant (P < 0.05) increase in all GAA supplemented groups compared to the control one. Triglycerides level demonstrated a higher elevation (P < 0.05) in both GAA2 and GAA3 supplemented groups. No signiﬁcant observed in total protein, albumin, globulin, and A/G ratio. Lipid peroxidation marker (malondialdehyde/MDA) is markedly decreased along with a signiﬁcant increase of superoxide dismutase (SOD), reduced glutathione (GSH), and nitric oxide (NO) levels in both GAA2 and GAA3 compared to other groups. Similarly, interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) gene expression levels were downregulated along with upregulation of transforming growth factor β1 (TGF-β1) at higher GAA levels, particularly at 0.18%. Our ﬁndings give important insights for the growth promoting, antioxidant and immunomodulatory eﬀects of GAA supplemented diet particularly at level of 0.18%.
1. Introduction Aquaculture industry is a rapidly growing sector playing a part in global food safety and security . To meet the increased population demand, ﬁsh farming experienced signiﬁcant expansion and production is rapidly increased . There are several biotic and abiotic factors aﬀecting the production of healthy ﬁsh. One of these essential factors is the temperature; it inﬂuences the pathogenic microbial growth, infections prognosis, metabolism and oxygen demands [3,4]. Supplementation of nutrients or special additives is known to inﬂuence ﬁsh growth and immune response [5–10]. The regulation and use of creatine are known to rely on body temperature, which in ﬁsh is dependent on the surrounding temperature . In this context, creatine has shown to assist in osmoregulation; besides, possessing anti-inﬂammatory properties and modulating cytokines production [11–13]. Nevertheless, creatine as a feed additive has several disadvantages as instability and
high cost, as compared to GAA, which is more stable and less expensive . Therefore, potential dietary supplementation of GAA has been suggested to spare the animal's need for creatine . GAA is converted into creatine via guanidinoacetic methyltransferase (GAMT) and then phosphorylated to phosphocreatine . Arginine and glycine are the substrate for the production of GAA and, consequently, creatine and typically found in higher concentrations in feedstuﬀs such as animal by-products meal than in corn and soybean meal [14,17,18]. The marked growth promoting eﬀect of creatine may be explained merely on an arginine-sparing basis; because there is no need for GAA formation when creatine is supplemented to a diet, the precursors of GAA production and creatine formation are able to be spared for use elsewhere in the body. In regard to GAA supplementation, both arginine and glycine can be used for other functions in the body, such as protein accretion, nitric oxide production, or de novo amino acid synthesis [19,20] which conﬁrmed via studies by Tong and
Corresponding author. E-mail addresses: [email protected]
, [email protected]
(E. Zahran). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fsi.2019.12.052 Received 30 August 2019; Received in revised form 14 December 2019; Accepted 17 December 2019 Available online 19 December 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.
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Barbul  where dietary GAA might additionally aﬀect growth and NO-mediated functions by making extra arginine. GAA dietary supplementation has shown to have diﬀerent alternative physiological roles such as stimulation of hormonal release and neuromodulation, changing metabolic usage of arginine, and balance of oxidant-antioxidant status . Recently, GAA is used as a feed additive for chickens and pigs to improve growth, breast meat yield, feed conversion ratio, as well as used to improve reproductive parameters and postnatal progeny performance in quails [22–24]. The chemical composition of GAA may act as a direct pro-oxidant since it generates superoxide anion, which is a potent free radical by donating an electron from its conjugate base [16,25]. However, it may exert an indirect antioxidant eﬀect through its related metabolites including creatine and arginine which are able to quench free radicals . GAA dietary supplementation in Cherry Valley duck improved the antioxidant capacity especially when supplemented at a level of 0.05% . Additionally, signiﬁcant enhancement of plasma T-AOC and CAT activity associated with decreased MDA concentration were noted in growing-ﬁnishing pigs fed GAA-containing diets . Pro-inﬂammatory cytokines; Tumor necrosis factor-α (TNF-a) and interleukin-1 (IL1) are released as part of the innate immune response in ﬁsh and mammals . Events of uncontrolled and excessive proinﬂammatory cytokines production can lead to tissue injury and dysfunction in rodents . Anti-inﬂammatory cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) can control the production of pro-inﬂammatory cytokines to limit extensive inﬂammatory responses in ﬁsh . However, many studies concerned the eﬀect of dietary supplementation of GAA, the current literature about the eﬀects of GAA dietary supplementation in ﬁsh are still limited. To the best of our knowledge, the present study is the ﬁrst to elucidate the eﬀects of GAA on selected arrays of signiﬁcant biomarkers in ﬁsh. This study was undertaken to better understand the potential role of GAA dietary supplementation at diﬀerent levels on growth performance, biochemical parameters, oxidative stress, and cytokines gene expression analysis in Nile tilapia.
Table 1 Ingredients (%) and nutrients composition of experimental diets. Ingredients %
Yellow corn (8.5%) Soybean meal (44%) Fish meal Wheat bran Corn glutein Gelatin Oil Minerals and vitamins premixa Salt Vitamin C Antioxidant Dicalcium phosphate Methionine
11.97 20.3 20 40 1 2 3 1 0.3 0.1 0.02 0.1 0.21
Chemical composition (analyzed) DE (Kcal/kg) (calculated) CP % Ash % EE %
3009 32.05 7.27 6.03
a Trace minerals & vitamins premixes were prepared to cover the levels of the microminerals & vitamins for tilapia ﬁsh as recommended by (NRC, 1993). Vitamins premix (IU or mg/kg diet); vit. A 5000, Vit. D3 1000, vit. E 20, vit. k3 2, vit. B1 2, vit. B2 5, vit. B6 1.5, vit. B12 0.02, Pantothenic acid 10, Folic acid 1, Biotin 0.15, Niacid 30. Mineral mixture (mg/kg diet); Fe 40, Mn 80, Cu 4, Zn 50, I 0.5, Co 0.2 & Se 0.2.
range (pH – 7.4, dissolved oxygen – 6.5 mg/L, and temperature – 25 ± 1 °C). Daily cleaning for each aquarium was carried out at 50% water changes to avoid metabolite accumulations (static-renewal system).
2.3. Growth parameter measurements The following equations were used to evaluate ﬁsh growth performance:
2. Materials and methods
Weight gain (g) = Mean ﬁnal weight (g) - Mean initial weight (g)
2.1. Diet preparation
Feed conversation ration (FCR) = Total dry weight of feed (g)/body weight gain (g)
Four isonitrogenous (32% CP) isocaloric (3000 Kcal DE/Kg) diets were formulated (Table 1) to satisfy the nutritional requirements of Nile tilapia (O. niloticus) according to NRC . Four diets were formulated; control (with no GAA supplementation/GAA0) and other three diets supplemented with diﬀerent levels of GAA (GAA1/0.06%, GAA2/ 0.12%, GAA3/0.18%); all diets ingredients were identical except GAA levels. Diets were prepared in the form of a water-stable sinking pellet and stored in plastic bags in the refrigerator during the time of use. The proximate analysis of diets was performed according to the standard methods of AOAC ; moisture after drying in a hot air oven at 105 °C until constant weight, crude protein (N x 6.25) by Kjeldahl method after acid digestion, ash content by incineration in a muﬄe furnace at 500 °C for 18 h.
Speciﬁc growth rate (SGR %/day) = Loge ﬁnal weight (g) − Loge initial weight (g) × 100)/t (experimental period in days) Protein eﬃciency ratio (PER) = Wet weight gain (g) /crude protein fed (g)
2.4. Sample collection At the end of the trial (60 days), ﬁsh were euthanized with tricaine methanesulphonate (MS-222, Finquel®, Argent) buﬀered with sodium bicarbonate. Blood samples were withdrawn and collected by venipuncture (3 ﬁsh/tank, 9 ﬁsh/group); and collected into plain centrifuge tube, left to clot at room temperature for 10 min, and then left at 4 °C for 4 h before centrifuging at 1198×g for 10 min, then serum was collected and stored at −20 °C until analysis. Randomly, ﬁve ﬁsh per aquarium from all experimental groups were minced for whole body approximate chemical analysis. Liver tissue samples were collected; divided into two portions, one portion was kept in RNA Later® (Qiagen) at −80 °C till analysis. The other portion was processed for measuring oxidant and antioxidant biomarkers.
2.2. Experimental design One hundred and eighty unsexed Nile tilapia ﬁsh weighing 21.20 ± 1.78 gm were received from a private ﬁsh farm in Kafr ElSheikh Governorate, Egypt, and stocked in glass aquarium tanks (80 × 35 × 40 cm), 15 ﬁsh in each, in triplicate. Fish fed at 3% body weight twice daily (9 h and 17 h) for 60 days as a suﬃcient time period for determining the eﬀect of the diets on the growth performances of ﬁsh. Fish were kept under 12-h daylight and dark photoperiod. Water quality was measured twice weekly via water quality test kits (Aquarium Pharmaceuticals, Inc.), and maintained within the normal 368
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conditions used. The expression of each gene was ﬁrst normalized to that of EF-1α, and presented as a fold change by calculating the average expression level of the GAA supplemented samples divided by that of the controls as described previously .
2.5. Serum biochemical parameters Serum samples were analyzed spectrophotometrically (BM Co. Germany, 5010) for estimation of Creatine Kinase (CK) level by using the commercial kit provided by ELITech company according to Aldrich et al. . The serum total protein (TP) and albumin (Alb) were evaluated using (Stanbio laboratory) USA kits according to Doumas et al. . Globulin concentration in serum was calculated by subtracting albumin from total proteins after that, albumin to globulin ratio (A/G ratio) was then calculated according to Kaneko et al. . Creatinine (Cr) was assayed by using ready-made kits manufactured by (Human Company Germany) as described elsewhere [35,36]. Cholesterol, triglycerides and High-density lipoprotein (HDL) cholesterol were measured according to Young  using kits presented by (Spinreact) Spain.
2.9. Statistical analysis Data were subjected to a one-way ANOVA to test the eﬀect of different levels of GAA on growth performance, serum metabolites, oxidant/antioxidant enzymes and cytokines gene expression. Data were analyzed using statistical SPSS v20 (SPSS Inc, Chicago, Illinois). Diﬀerences between means were compared using Duncan's multiple range test at the signiﬁcance of diﬀerences (P < 0.05) among dietary treatments. 3. Results
2.6. Oxidant and antioxidant biomarkers 3.1. Growth performance and whole-body composition analysis One gram of liver was washed and homogenized in ice-cold phosphate-buﬀered saline (PBS) (pH 7.4). The liver homogenate was centrifuged at 4 °C for 15 min at 1198×g (Centrikon H-401 centrifuge), and fresh supernatants were aliquoted and stored at −80 °C. MDA and antioxidant markers; GSH, SOD, and NO were spectrophotometrically determined (Photometer 5010, Photometer, BM Co. Germany) by enzymatic colorimetric method using commercially available kits (Biodiagnostic, Egypt).
The growth performance parameters of Nile tilapia fed the experimental diets supplemented with diﬀerent levels of GAA are presented in Table (2). Our results revealed that the FBW and SGR of Nile tilapia in GAA2 and GAA3 groups were signiﬁcantly (P > 0.05) higher than the control and GAA1 groups. While WG was signiﬁcantly increased in all GAA supplemented groups compared to the control. PER decreased signiﬁcantly in GAA3 compared to other groups; however, FCR showed no signiﬁcant diﬀerences among all groups. Lipid content was signiﬁcantly increased in all groups compared to GAA3, while Ash % signiﬁcantly increased in GAA2 and GAA3 groups compared to other groups. Whereas, moisture and CP % didn't show any statistical differences among all groups (Table 3).
2.7. Extraction of total RNA and cDNA synthesis Total RNA was extracted from 50 mg of liver samples using a Qiagen RNeasy® kit according to the manufacturer's instructions and was puriﬁed and quantiﬁed by a NanoDrop Reader (Thermo Fisher Scientiﬁc, USA), a 260/280 nm absorbance ratio of 1.8–2.0 indicates a pure RNA sample and RNA samples stored at −80 °C until use. 5 μg of total RNA was mixed with Oligo-dT28VN (Sigma–Aldrich) primers and reverse transcribed into cDNA using RevertAid < SUP > TM < / SUP > Reverse Transcriptase (Fermentas), following the manufacturer's instructions, and stored at −20 °C
3.2. Serum biochemical analysis Serum biochemical parameters are presented in Figure (1). CK activity was increased signiﬁcantly in GAA2 and GAA3, compared to other groups with no statistical diﬀerence between them. Cholesterol and creatinine levels were in the same pattern where a signiﬁcant increase in all GAA supplemented groups compared to control, being the higher signiﬁcantly levels in GAA2 and GAA3 groups compared to other groups. Triglycerides level revealed a higher elevation in both GAA2 and GAA3 groups compared to the GAA1 group, and a signiﬁcant increase in GAA3 compared to the control group as well. No statistically signiﬁcant changes were found in total protein, albumin, globulin and A/G ratio levels among all groups Fig. 2.
2.8. Real-time quantitative PCR (RT-qPCR) RT-qPCR was performed using Immolase (Bioline) and SYBR Green ﬂuorescent tag (Invitrogen) with a LightCycler® 480 Real-Time PCR System (Roche), as described previously [38,39]. The ampliﬁcation program proceeded at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s; annealing at 60 °C for 30 s and extension at 72 °C for 30 s. After the cycling protocol, the melting curves were obtained to assess the speciﬁcity. The expression of pro-inﬂammatory cytokines IL-1β, TNF-α, TFG-β1 and the housekeeping gene elongation factor-1α (EF-1α), was examined. The primers for real-time PCR are detailed in Table 2, with at least one primer of a pair designed to cross an intron so that genomic DNA could not be ampliﬁed under the PCR
3.3. Oxidative and antioxidant biomarkers The eﬀect of GAA dietary supplementation on oxidant/antioxidant status is presented in Fig. 3. MDA was markedly decreased in GAA3 compared to GAA2 and in both groups compared to GAA1 and control groups with no statistical changes between the later. However, NO level
Table 2 Growth performance of Nile tilapia fed with the control diet or diets supplemented with diﬀerent levels of guanidinoacetic acid (GAA). Data were presented as the mean of nine ﬁsh ± SEM. Values with a diﬀerent letter superscript in same row indicate signiﬁcant diﬀerence between groups (P < 0.05). Experimental diets parameters
Initial weight (g) Final weight (g) Body wt gain (g) FCR SGR PER
21.20 ± 1.78 51.55 ± 1.48b 30.38 ± 1.62b 2.13 ± 0.055 1.47 ± 0.098b 1.45 ± 0.037a
20.90 ± 0.99 56.43 ± 1.62ab 35.53 ± 1.14a 2.00 ± 0.051 1.53 ± 0.077b 1.49 ± 0.025a
21.10 ± 1.04 57.30 ± 1.44a 36.20 ± 0.61a 2.23 ± 0.037 1.60 ± 0.074a 1.39 ± 0.01b
21.11 ± 0.91 56.94 ± 1.27a 35.83 ± 0.72a 2.26 ± 0.038 1.58 ± 0.070b 1.39 ± 0.02b
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Table 3 Body composition of Nile tilapia (on dry matter basis) fed with the control diet or diets supplemented with diﬀerent levels of guanidinoacetic acid (GAA). Experimental diets parameters
Moisture % CP % Lipid % Ash %
76.56 60.52 10.98 25.50
± ± ± ±
0.19 0.62 0.07a 0.10c
75.91 61.24 10.62 25.14
75.46 60.35 10.01 26.64
77.17 ± 0.81 60.51 ± 0.69 8.82 ± 0.31b 27.67 ± 0.30a
± ± ± ±
0.51 0.92 0.03a 0.38c
± ± ± ±
0.48 1.18 0.89a 0.15b
Data were presented as the mean of nine ﬁsh ± SEM. Values with a diﬀerent letter superscript in same row indicate signiﬁcant diﬀerence between groups (P < 0.05).
in GAA2 and GAA3 compared to the control group. While the level of GAA1 showed, no statistical changes compared to other groups. On the other side, the TGF-β1 mRNA level was upregulated (P < 0.05) in GAA3 compared to GAA1 and control groups with no statistical changes between the later. While TGF-β1 mRNA level in GAA2 showed no statistical changes compared to other groups.
exhibited a higher elevation in GAA3 compared to GAA2 and in both groups compared to GAA1 and control groups with no statistical changes between the later. Similarly, GSH increased signiﬁcantly in GAA3 compared to GAA2 and in both groups compared to GAA1 and control groups with a signiﬁcant statistical change between the later. SOD level was peaked signiﬁcantly in GAA2 and GAA3 compared to GAA1 and control groups with no statistical changes between the later.
4. Discussion 3.4. Real-time PCR analysis for cytokines gene expression GAA supplementation was able to induce favorable changes in Nile tilapia ﬁsh as observed in our study. In the current study, GAA supplementation at a level of 0.12 and 0.18% was able to improve growth performance in the form of FBW, WG, and SGR. PER showed a slight reduction in GAA2 and GAA3 compared to other groups, suggesting that ﬁsh were not able to utilize dietary protein once after reaching the optimum protein level. Thus, the excess dietary protein could be
Liver cytokine gene expression levels in GAA supplemented groups are presented in Fig. 4. IL-1β mRNA level signiﬁcantly (P < 0.05) downregulated in GAA3 compared to GAA1 and control groups with no statistical changes between the latter. While the level of GAA2 showed, no statistical changes compared to other groups. TNF-α mRNA level had a similar pattern, where its level was signiﬁcantly downregulated
Fig. 1. Serum creatine kinase (CK), creatinine (Cr), cholesterol, and triglycerides levels in Nile tilapia fed with the control diet or diets supplemented with guanidinoacetic acid at levels of 0.06% (GAA1), 0.12% (GAA2) or 0.18% (GAA3) for 60 days. Data is expressed as the mean ± SEM of nine ﬁsh. Diﬀerent superscript letters are statistically signiﬁcant (P < 0.05) between diﬀerent groups, as determined by one-way ANOVA. 370
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Fig. 2. Serum total protein, albumin, globulin levels, and A/G ratio in Nile tilapia fed with the control diet or diets supplemented with guanidinoacetic acid at levels of 0.06% (GAA1), 0.12% (GAA2) or 0.18% (GAA3) for 60 days. Data is expressed as the mean ± SEM of nine ﬁsh. Diﬀerent superscript letters are statistically signiﬁcant (P < 0.05) between diﬀerent groups, as determined by one-way ANOVA.
that have anabolic functions in synthesis of DNA, RNA, and proteins , along with its hydration eﬀect on the muscle cells, bringing water into the cells, which promotes protein synthesis, reduces proteolysis, and enhances glycogen synthesis . In addition, several studies reported that GAA supplementation has a role in increasing insulin-like growth factor 1 (IGF-1) in plasma, which led to the enhancement of protein synthesis and growth improvement [45,46]. Further, GAA supplementation could improve energy utilization with subsequent enhanced body weight [47,48]. Our ﬁndings are also in line with several studies conducted in broilers and other animals that revealed the growth promoting eﬀect of GAA; for example, GAA supplementation at 0.4 g/kg to soybean mealbased diets improved the growth of bullfrog . Moreover, Murakami et al.  reported improvement of weight gain and feed conversion in oﬀspring of breeder quail at 35 d of age supplemented with GAA at
deaminated and catabolized to provide energy; thereby, reducing its eﬃciency. There are several factors that could contribute to the growth improvement observed in our study, like the sparing of the endogenous synthesis of GAA from arginine and glycine, thereby providing more arginine and glycine available for body protein or endogenous amino acids synthesis, leading to growth enhancement. Besides, GAA is an immediate precursor of creatine and its phosphorylated derivative, phosphocreatine-a, a rapidly mobilizable reserve of high energy phosphates [41,42]. Since creatine acts as an intracellular signal-coupling enhanced muscle activity and enhanced muscle growth, it could subsequently lead to additional energy for cellular bioenergetics. Thus, the GAA growth promoting eﬀect could be attributed to the enhanced creatine synthesis [16,43]. Furthermore, GAA favored the production of growth promoting polyamines (putrescine, spermidine and spermine),
Fig. 3. Malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH) and superoxide dismutase (SOD) levels in Nile tilapia fed with the control diet or diets supplemented with guanidinoacetic acid at levels of 0.06% (GAA1), 0.12% (GAA2) or 0.18% (GAA3) for 60 days. Data is expressed as the mean ± SEM of nine ﬁsh. Diﬀerent superscript letters are statistically signiﬁcant (P < 0.05) between diﬀerent groups, as determined by one-way ANOVA. 371
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Fig. 4. Gene expression of cytokines (IL-1β, TNF-α, and TGF- β1) determined by real-time PCR in liver of Nile tilapia fed with the control diet or diets supplemented with guanidinoacetic acid at levels of 0.06% (GAA1), 0.12% (GAA2) or 0.18% (GAA3) for 60 days. Data is expressed as the mean ± SEM of nine ﬁsh. Diﬀerent superscript letters are statistically signiﬁcant (P < 0.05) between diﬀerent groups, as determined by one-way ANOVA.
ﬁnding was in parallel with Tossenberger et al. and Nasiroleslami et al. [54,55], who found that CK level was increased after GAA supplementation in the broiler diet. Similar to our ﬁnding, GAA dietary supplementation at 1200 mg/kg in broiler resulted in a signiﬁcant increase in CK . Several studies reported the increased level of CK after creatine supplementation or its precursor (GAA) as a consequence of the increased total creatine pool in the muscle and plasma [16,56–58]. Additionally, CK has a contributing role in the PCr shuttle of phosphoryl groups and, consequently, the maintenance of high rates of ATP production and overall energy metabolism . Therefore, in our study, the role of GAA supplementation in energy utilization might be associated with a rise in CK level. As observed, the creatinine level was increased in both GAA2 and GAA3 groups compared to others, which correlate to the increased CK level as well. This is consistent with the study conducted by Zeng et al.  who observed the similar increased level of creatinine in bullfrog Rana (Lithobates catesbeiana) fed GAA supplemented soybean meal diet at 0.6 g/kg for 8 weeks. Similarly, Michiels et al.  found that GAA supplementation increased the level of creatine deposition in muscles and subsequently increased the creatinine level. The levels of total protein, albumin, globulin, and A/G ratio in serum showed no signiﬁcant diﬀerence among the diﬀerent GAA treatments, which could be necessary for evaluating renal function, which coincided with other previous studies [47,54,60,61]. In the current study, cholesterol level was elevated signiﬁcantly in all GAA supplemented groups compared to control, being the higher signiﬁcantly levels in GAA2 and GAA3 groups. However, triglycerides levels revealed a higher elevation in both GAA2 and GAA3 groups compared to the GAA1 group; and a signiﬁcant increase in GAA3 compared to the control group as well. Our results could be associated with the increased lipid content of GAA1 and GAA2 in this study. Besides, it could be attributed to the increased creatine level due to GAA supplementation, which has a potential role in activating proteins of lipid metabolism such as [Apolipoprotein A (Apo-A) and 14 kDa Apo-
level 0.14%. Supplementation of GAA at level 0.06 and 0.12% improved gain: feed by an average of 2.3% compared with the negative control in broiler chickens . Similarly, Mousavi et al.  reported that the feed conversion ratio was improved with 0.06% GAA supplementation from d 0 to 40 to a nutritionally-complete diet in ﬁnishing pigs. GAA supplementation at 0.1% increased signiﬁcantly average daily feed intake and average daily gain in ﬁnishing pigs . GAA supplementation provided better performance in broilers fed with diets based on corn and soybean meal with adequate amounts of arginine (1.39% initial phase to 1.068% ﬁnal phase) . This was inconsistent with Dilger et al.  who reported that GAA supplementation to broiler Arg-deﬁcient diets at 0.12% resulted in improvement of their growth performance compared to 0.15% creatine and 0.25% arginine supplementation. As observed in our study, ash% were increased in higher levels of GAA supplemented groups compared to others and lipid was also increased in other groups compared to GAA3, with no signiﬁcant changes in moisture and CP values. These ﬁndings are inconsistent with Zeng et al.  who reported non-signiﬁcant diﬀerences in the whole-body composition of bullfrog Rana (Lithobates) fed plant-based diets supplemented with diﬀerent levels of GAA (0.2, 0.4, 0.6, 0.8 g/kg diet). Similarly, no signiﬁcant changes were noticed in the muscle composition of Jian carp fed GAA-containing diets at levels 250, 500, 1000 mg/kg diet . In respect to lipid %, we observed a signiﬁcant decrease in lipid % of the whole body in GAA3 groups compared to others. This was supported by Esser et al. , who noted a signiﬁcant decrease in the abdominal fat deposition in broilers fed diets supplemented with GAA. GAA is a natural precursor to creatine in the body. Creatine considered as an essential molecule in energy homeostasis; via the creatine and phosphocreatine (PCr) system . Creatinine Kinase enzyme functions to buﬀer changes in ATP during the altered energy. Thus, changes in CK activity usually reﬂect muscle energy storage and conversion status . In our study, CK enzyme activity exhibited a signiﬁcant increase in GAA2 and GAA3 groups compared to others. This 372
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A which could be involved in the cholesterol transport and thus lipid metabolism [62–64]. Similar to our results, Cobb broiler chicks fed creatine at 0.1% demonstrated a higher level of triglyceride after 21 and 42 days . The state of oxidative damage results in over-production of ROS that promote lipid peroxidation responses, which are reﬂected by the induction of MDA content in plasma and tissues  besides, antioxidant enzymes are increased as a protective response against the highly produced ROS . In the current study, GAA 2 and GAA3 exhibited a decreased in the oxidative stress represented by the MDA level being the highest eﬀect in GAA3. the antioxidant enzymes, SOD, NO and GSH levels were in a similar pattern, where they signiﬁcantly increased in the higher supplemented doses of GAA compared to the lower and GAA1 groups. This could be due to the pure form of GAA could be as a pro-oxidant agent and hinder the antioxidant ability due to the presence of guanidinium ion of its conjugate base . On the other side, GAA could serve indirectly as an antioxidant agent owing to its metabolites of creatine and arginine having the ability to scavenge free radicals [22,68,69]. Our results in accordance with previous studies [22,26,70] reported that GAA dietary supplementation in Cherry Valley ducks and growing-ﬁnishing pigs was able to improve the antioxidant enzymes and manipulate the oxidative stress through lowering MDA level. These results suggest the favorable action of GAA dietary supplementation on the oxidant-antioxidant mechanism; since it was reported that exogenous GAA may act as pro-oxidant or antioxidant agent . Cytokines are considered as immune system regulators in ﬁsh. Our results demonstrated a signiﬁcant downregulation of IL-1β and TNFαmRNA level and upregulation of the TGF-β1 mRNA level in GAA3 compared to control groups. However, IL-1β and TGF-β1 mRNA levels showed no statistical changes in GAA2; and the same for TNF-α mRNA level in GAA1, compared to other groups. It is postulated that creatine and arginine (the metabolites of GAA) have a potential role in improving ﬁsh immune response, particularly the inﬂammatory response and cytokine modulation [13,71–73]. Therefore, increase the biosynthesis of both metabolites upon GAA supplementation further support our ﬁndings regarding the anti-inﬂammatory modulation of GAA. Similarly, Zhao et al.  showed that Jian carp fed diets containing isoleucine at 7.0–11.9 g/kg was able to lower the intestinal TNF-α mRNA levels than those fed other dietary isoleucine levels. Consistent with our results, Hu et al.  reported dietary glutamine supplementation has the capacity to signiﬁcantly downregulate levels of TNFα and IL-10 mRNA, whereas the level of TGF-β2 mRNA was upregulated in the head kidney of juvenile Jian carp. 5. Conclusion This study concludes that dietary supplementation of GAA, particularly at level 0.18% in Nile tilapia, markedly improved the weight gain, serum biochemical parameters, and showed the potential roles of GAA as an antioxidant and anti-inﬂammatory agent. Our study opens new potential outcomes in further advancing the usage of GAA as a feed additive in ﬁsh. However, further investigations are needed to elucidate the precise mechanism involved in energy utilization and muscle quality and tissue histology. Acknowledgments This research received no speciﬁc grant from any funding agency in the public, commercial, or not-for-proﬁt sectors. References  D.C. Little, R. Newton, M. Beveridge, Aquaculture: a rapidly growing and signiﬁcant source of sustainable food? Status, transitions and potential, J Proc. Nutrition Society (2016) 274–286.
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