The effects of microencapsulated garlic (Allium sativum) extract on growth performance, body composition, immune response and antioxidant status of rainbow trout (Oncorhynchus mykiss) juveniles

ABSTRACT

This study investigates the effects of microencapsulated garlic (Allium sativum) extract (G) on growth, immune response, and antioxidant status of rainbow trout juveniles. A total of 300 fish (13.05 ± 0.36 g) were divided into five groups (three replicates per group) and fed with the experimental diets containing G (0, 0.25, 0.5, 1, and 2%) for 40 days. The results indicated that dietary supplementation with garlic could significantly improve growth performance, Feed conversion ratio (FCR) fish fed with G3 diet compared to control. Proximate composition of fish showed significantly higher protein content and lower lipid content in the fish fed G3 diet compared to control. Significantly higher total serum protein and lysozyme activity were found in the fish fed diet supplemented with 0.5% garlic extract, whereas the activities of liver enzymes (ALP, AST, ALT) were significantly lower in the fish fed diet containing garlic extract than in fish fed the control diet. The highest superoxide dismutase (SOD) and catalase (CAT) activities were in fish fed diet supplemented with garlic extract. Therefore, the current research suggests that microencapsulated garlic extract could be considered as a good food additive to improve growth, non-specific immune response, and antioxidant capacity of rainbow trout.

KEYWORDS:

• Rainbow trout
• Allium sativum
• growth
• immune response
• antioxidant capacity

1. Introduction

Aquaculture is one of the most important animal culture systems and food production sectors in the world (Food and Agriculture Organization of the United Nations 2018). Therefore, the global interest is focused on the development of new strategies that allow to improve its sustainability and profitability while reducing its environmental impact. One way to improve growth, antioxidant capacity, immune response, and resistance of aquatic animals to stress or infectious diseases is the inclusion of herbal supplements to the diet (Sivaram et al. 2004; Inoue et al. 2016; Öz et al. 2018; Foysal et al. 2019; Yousefi et al. 2020), as a possible alternative to synthetic substances in order to increase the safety of derived food products and meet the consumer expectations (Hoseini and Yousefi 2019).

Garlic (Allium sativum family Liliaceae) is known as a prophylactic as well as a therapeutic medicinal plant in different communities. Allicin (allyl 2-propenethiosulfinate or diallyl thiosulfinate) is the principal bioactive compound which is present in the aqueous extract of garlic or raw garlic homogenate. When garlic is chopped or crushed, allinase enzyme is activated and produce allicin from alliin (present in intact garlic). Other available compounds in garlic homogenate are included 1-propenyl allyl thiosulfonate, allyl methyl thiosulfonate, (E, Z)-4,5,9-trithiadodeca-l,6,11-triene 9-oxide (ajoene), and y-L-glutamyl-S-alkyl-L-cysteine (Bayan et al. 2014). Moreover, garlic contains vitamins including A, C, and B as well as linoleic acid, and valuable ingredients such as iodine salts and silicates which have positive effects on circulatory and skeletal systems (Labrador et al. 2016). The antioxidant properties of garlic have been proven to be due to two types of substances including flavonoids and sulfur containing compounds (4,5 dithia 1,7 octadiene and s allyl cysteine) (Kim et al. 1997; Sharma et al. 2010). Garlic has shown antimicrobial, antihypertensive, hepatoprotective and antioxidant properties (Kumar and Berwal 1998; Suetsuna 1998; Wang et al. 1998). Also, it possesses enhancing immune activities that include the promotion of lymphocyte proliferation, cytokine release, phagocytosis, and natural killer cell activity (Kyo et al. 1998). Several previous studies have demonstrated the garlic ability to improve immune system capability (Nya et al. 2010; Nwabueze 2012), antioxidant capacity (Drobiova et al. 2011), growth performance and feed utilization (Lee et al. 2012), body content and enzyme activity (Esmaeili et al. 2017), egg fertilization and larval hatching (Marengoni et al. 2017) in different aquatic animals. However, these properties have not yet been reported for microencapsulated garlic extract.

In the recent years, the importance of encapsulation technologies has been increased in the food industry, particularly in the development of ingredients to design functional and/or healthy foods (Encina et al. 2016). Microencapsulation is a smart way to ameliorate the properties of bio-actives in terms of structuration/functionalization (e.g., converting oil and extract into powder form). This technology is applicable to a broad spectrum of compounds, from small active molecules (e.g. vitamins, minerals, flavors, oils) to bigger ones (e.g. biopolymers, enzymes, proteins) and even microorganisms.

Rainbow trout (Oncorhynchus mykiss) is the main cultured freshwater fish species worldwide. Also, it has been recognized as the main aquaculture species farmed in Iran with high economic value. The aim of this study was to investigate the effects of microencapsulated garlic (A. sativum) extract on growth performance, body content, and immune and antioxidant status of rainbow trout juveniles.

2. Materials and methods

2.1. Animal and Experimental condition

Rainbow trout juveniles with an average weight of 13.05 ± 0.36 g (mean ± S. D.) were obtained from a local commercial hatchery (Zaringol, Iran) and transferred to the Aquatic laboratory of Gonbad Kavous University, Iran. Before the experiment, fish were acclimated in two concrete tanks (Water volume of 400 liters) and fed with a basal diet for two weeks, four times per day. The experiment was carried out in a fresh-water flow system equipped with 15 plastic tanks with 50 liters capacity (three replicates/dietary treatment).

During the experiment, water temperature (17.10 ± 0.74 °C) and dissolved oxygen (8.12 ± 1.02 mg L^-1) were measured with Multiparameter Hack (Model 2000). Electrical conductivity (874.57 ± 17.38 µS cm^-1) was tested by Martini instrument (Model EC60, Italy) and pH (8.09 ± 0.15) was recorded using a Palin test Device (Model 7500, England). The Photoperiod of tanks was 10 h darkness and 14 h light with the light period since 06:00to 20:00. All these parameters were controlled and maintained constant during the experiment.

2.2. Diet preparation and feeding trial design

Fresh garlic (A. sativum) was purchased from a fruit store in Golestan Province, Iran. The garlic was peeled and then was powdered in Oven (ON-11E). Garlic powder was mixed with ethanol (purity of 70%) on a shaker at room temperature for 48 hours. The solution was passed through a Whatman filter paper (42 microns) and placed in Rotary (HS-200S, Korea) at 75 °C for one hour to remove the alcohol. Then, the extract was placed in oven at 38 °C for 30 minutes. In order to encapsulate garlic extract, 30 g maltodextrin and 10 g Arabic gum was mixed with 60 g distilled water at 70 to80 °C and then homogenized (IKA T 25 digital ULTRA, Germany) for one hour by homogenizer at a round of 7000 g. The material was stored at 60 °C inside the Ben-Marie (Memert. WNB 14, Germany) for 24 hours. Coating materials and garlic extract (3:1 ratio) were mixed for 30 minutes (Mahdavee Khazaei et al. 2014). Microencapsulated garlic extract was preserved in freeze dryer (Alpha-2 LD plus, Germany) for 24 hours.

A commercial diet with 2.2 mm size (Bezae Company, Iran) was employed as the experimental diet. The analyzed content was as follows: crude protein 44- 45%, crude fat 14- 14.5%, moisture 10%, crude fiber 2- 2.2%, absorbable phosphor 0.8%, and digestible energy 4300 kcal/kg. The feed was crushed and the microencapsulated garlic extract was added to it at the concentrations of 0 (G0), 0.25 (G1), 0.5 (G2), 1 (G3) and 2% (G4). After mixing, the material was broken up, sieved to convenient pellet size and stored at 4 °C (Gallardo et al. 2002). The 15 experimental tanks were randomly assigned to the 5 dietary treatments (three tanks/treatment) and fish (20 fish/tank) were fed at the level of 3% body weight/day, four times daily at 06:00 h, 11:00 h, 16:00 h and 20:00 h for 40 days.

2.3. Sample collection

At the end of the feeding experiment, animals were fasted for 24 h and were profoundly anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222, 200 mg L⁻¹ water). Nine anesthetized fish per treatment (three fish per replicate) were randomly captured and blood was collected from the caudal vein by heparinized syringe containing heparin sodium salt at the concentration of 5000 IU in 1 ml. Blood samples were centrifuged at 4000 ×g for 10 min, plasma samples collected and stored at −80 °C for subsequent analyses of biochemical, immunological and antioxidant parameters. Then, total length and final body weight were measured for all fish in each tank.

2.4. Growth and feed efficiency

Growth and feed utilization indices were calculated as follows (Öz et al. 2018): weight gain (WG g) = Wt – W0, weight specific growth rate (SGRW % day^-1)   =100 (ln Wt – ln W0) t^-1, length specific growth rate (SGRL % day^-1)   =100 (ln Lt – ln L0) t^-1, feed conversion ratio (FCR) = (dry feed intake (g)) (wet weight gain (g))^-1, protein efficiency ratio (PER) = (weight gain (g)) (protein fed (g))^-1, condition factor (CF) =Wt L^-3, viscero-somatic index (VSI) = 100 [(visceral weight (g)) (body weight (g))^-1], hepato-somatic index (HSI) = 100 [(liver weight (g)) (body weight (g))^-1], where Wt is fish body weight at day t and W0 at day 0, t (days) is the duration of experiment, L is fish total length. Average carcass weight (ACW) was also measured.

2.5. Biochemical, immune, and antioxidant parameters

Protein concentration in plasma was quantified by using the Bradford (1976) method, with bovine serum albumin as a standard. Albumin content was determined following the method of Doumas et al. (1997). Globulin content (subtracting albumin from the total proteins) was calculated as described by Kumar et al. (2005). Serum lysozyme activity was measured using the turbidimetric method described by Ellis et al (1990) . Plasma glucose was measured by the glucose oxidase method reported by Sacks (1999).

Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were measured according to the methods described by Thomas (1998) and Moss et al (1999).

Total superoxide dismutase (SOD) (EC1.15.1.1) activity was determined using the method of Marklund and Marklund (1974). Catalase (CAT) (EC1.11.1.6) activity was measured following the reduction of hydrogen peroxide at 240 nm and using the extinction coefficient 0.04 mM⁻¹ cm⁻¹ (Beers and Sizer 1952). Malondialdehyde (MDA) concentration was measured colorimetrically according to the the Buege and Aust (1978).

2.6. Whole body composition

Three fish from each tank (9 fish/dietary treatment) were randomly selected for evaluating of whole-body proximate composition. Protein, lipid, moisture, and ash were quantified according to the standard methods (AOAC 2000): crude protein by analysis of nitrogen (N, 6.25) content and the Kjeldahl method (Kjeltec 1030 Auto Analyzer, Tector, Sweden) crude lipid by petroleum ether extraction using the Soxhlet method (model 1043 Extraction Unit; Tecator, Sweden) (Folch et al. 1957); moisture by drying an oven at 105 °C to a constant weight, and ash by ignition at 550 °C for 24 h (Heraeus, Germany). The fiber was analyzed using a Fibertec System Tector 1010 (Sweden).

2.7. Statistical analyses

The data obtained in this experiment were tested for their normality using the Kolmogorov–Smirnov test. One-way ANOVA was used for comparison of variances and Duncan test was used for comparison of means between experimental groups (p<0.05). The statistical analyses were performed using SPSS software version 23.0.

3. Results

3.1. Growth performance

The growth and feeding performance data of rainbow trout fed with the diets containing the different levels of microencapsulated garlic extract are shown in Table 1. Growth performance enhanced with increasing garlic extract supplementation levels (P<0.05). The final weight was significantly higher in G3 and G4 compared to the other treatments (P<0.05). Best specific growth ratio (SGR) was obtained in fish submitted to G3 and G4 treatments (P<0.05). G3 diet induced the lowest feed conversion ratio, resulting significantly better than G0 and G1 treatments (P<0.05), but not significantly different than G2 and G4 treatments (P>0.05). The inclusion of microencapsulated garlic extract in the diet had no significant effect on CF, HSI and VSI of rainbow trout (P>0.05). Survival percentage was 100% in all experimental groups (Table 1).

Table 1. Growth performance and feed efficiency of rainbow trout fed diets supplemented with microencapsulated garlic extract different levels.

Parameters	G0	G1	G2	G3	G4
FW (g)	46.53 ± 3.56^b	47.25 ± 2.19^b	51.76 ± 4.29^ab	55.87 ± 3.91^a	54.37 ± 3.70^a
FL (cm)	15.90 ± 0.69^b	15.98 ± 0.44^b	16.51 ± 0.66^ab	17.34 ± 0.51^a	17.10 ± 0.73^a
ADG (%)	83.70 ± 8.91^b	85.51 ± 5.49^b	96.76 ± 10.73^ab	107.06 ± 9.79^a	103.31 ± 9.27^a
SGR (% day^−1)	3.17 ± 0.19^b	3.21 ± 0.11^b	3.43 ± 0.20^ab	3.63 ± 0.17^a	3.56 ± 0.17^a
CF (%)	1.15 ± 0.08^a	1.15 ± 0.06^a	1.14 ± 0.06^a	1.07 ± 0.06^a	1.08 ± 0.07^a
FCR	1.31 ± 0.18^a	1.19 ± 0.09^ab	1.00 ± 0.11^b	0.99 ± 0.10^b	1.00 ± 0.07^b
FCE	77.25 ± 10.45^c	84.24 ± 6.77^bc	100.3 ± 11.41^ab	101.78 ± 10.76^a	99.66 ± 7.49^ab
PER	1.10 ± 0.08^b	1.12 ± 0.05^b	1.23 ± 0.10^ab	1.33 ± 0.09^a	1.29 ± 0.08^a
LER	3.10 ± 0.23^b	3.15 ± 0.14^b	3.45 ± 0.28^ab	3.72 ± 0.26^a	3.62 ± 0.24^a
HSI	2.05 ± 0.56^a	2.00 ± 0.46^a	2.12 ± 0.25^a	1.81 ± 0.18^a	1.99 ± 0.37^a
VSI	13.16 ± 4.82^a	12.80 ± 4.93^a	11.58 ± 1.81^a	11.62 ± 1.73^a	11.55 ± 2.22^a
ACW (g)	39.26 ± 4.21^b	40.23 ± 2.95^b	44.49 ± 3.74^ab	47.78 ± 3.94^a	46.97 ± 3.62^a
Survival (%)	100	100	100	100	100

Data are expressed as mean ± SD of three replicate tanks. Mean values with different superscripts are significantly different. The significance level is defined as P < 0.05 (Duncan’ test).

3.2. Whole body composition

Whole body compositions of rainbow trout fed with the experimental diets containing graded levels of microencapsulated garlic extract are presented in Table 2. The crude protein and fiber content were significantly higher in fish fed G3 diet compared to the control group (P<0.05). The lowest amount of crude lipid was observed in fish fed G3 diet, resulting significantly lower than that detected in control group and in fish fed the other diets (P<0.05). The test diets induced significant differences in dry matter and ash among the experimental groups (P<0.05).

Table 2. Whole body composition (%) of the rainbow trout fed diets supplemented with microencapsulated garlic extract at different levels.

Parameters	G0	G1	G2	G3	G4
Dry Matter	23.56 ± 0.22^cd	25.66 ± 0.39^a	23.14 ± 0.14^d	24.33 ± 0.21^bc	24.89 ± 0.75^ab
Crude Protein (%)	64.92 ± 0.38^abc	64.05 ± 0.58^c	65.23 ± 0.40^ab	65.89 ± 0.15^a	64.17 ± 0.35^bc
Crude Lipid (%)	23.52 ± 0.47^a	23.39 ± 0.47^a	23.13 ± 0.26^a	21.94 ± 0.16^b	23.40 ± 0.32^a
Fiber (%)	0.76 ± 0.03^b	0.59 ± 0.02^c	0.63 ± 0.04^bc	1.03 ± 0.09^a	0.71 ± 0.02^bc
Ash	10.69 ± 0.32^bc	11.33 ± 0.18^a	10.25 ± 0.29^c	10.46 ± 0.19^c	11.13 ± 0.14^ab

Data are expressed as mean ± SD of three replicate tanks. Mean values with different superscripts are significantly different. The significance level is defined as P<0.05 (Duncan’ test; n=9).

3.3. Biochemical and immune parameters

Plasma biochemical and immune parameters of rainbow trout fed diets with different levels of microencapsulated garlic extract are shown in Table 3. A significant difference in glucose level was detected among the experimental groups (P<0.05). Fish fed G2 and G3 diets showed lower glucose levels compared to the control group (P<0.05). Total protein and albumin contents in fish fed G2 and G3 diets were significantly higher than those measured in the other groups (P<0.05). The highest lysozyme activity was observed in G1 and G2 treatments, whereas the lowest lysozyme activity was recorded in fish fed the control diet. The activity of liver enzymes (ALP, AST and ALT) resulted significantly decreased in rainbow trout fed diets supplemented with microencapsulated garlic extract compared to rainbow trout fed the control diet (P<0.05).

Table 3. Plasma biochemical and immune parameters of rainbow trout fed diets supplemented with microencapsulated garlic extract at different levels.

Parameters	G0	G1	G2	G3	G4
Glucose (mg/dl)	129.00 ± 4.38^b	124.61 ± 3.15^bc	117.82 ± 2.97^c	109.21 ± 6.01^d	139.02 ± 2.63^a
Total Protein (g/dl)	2.93 ± 0.06^b	3.07 ± 0.14^b	3.42 ± 0.07^a	3.34 ± 0.04^a	3.03 ± 0.08^b
Albumin (g/dl)	1.22 ± 0.04^b	1.24 ± 0.01^b	1.30 ± 0.02^a	1.31 ± 0.01^a	1.27 ± 0.03^ab
Lysozyme (u/ml)	25.49 ± 1.79^c	35.7 ± 1.53^a	36.89 ± 0.81^a	32.6 ± 2.37^b	31.13 ± 1.04^b
ALP (u/l)	934.50 ± 16.62^a	524.92 ± 38.56^c	766.41 ± 32.42^b	542.22 ± 38.38^c	809.55 ± 17.77^b
AST (u/l)	689.70 ± 18.16^a	421.26 ± 11.70^d	561.84 ± 13.98^c	369.49 ± 15.30^e	605.36 ± 17.62^b
ALT (u/l)	22.60 ± 1.29^a	14.08 ± 1.89^c	16.88 ± 0.45^b	10.14 ± 0.33^d	16.02 ± 1.02^bc

Data are expressed as mean ± SD of three replicate tanks. Mean values with different superscripts are significantly different. The significance level is defined as P < 0.05 (Duncan’ test; n=9).

3.4. Antioxidant status

The activity of plasma antioxidant enzymes is displayed in Table 4. The SOD activity exhibited a significant increase in rainbow trout fed diets supplemented with microencapsulated garlic extract compared to rainbow trout fed the control diet, in an extract concentration-dependent manner from G0 to G3 (P<0.05). No significant difference was observed in SOD activity among G2, G3 and G4 treatments (p>0.05). Catalase activity was significantly different among rainbow trout fed diet with microencapsulated garlic extract and control diet (P<0.05). The MDA level was significantly lower in fish fed with garlic supplemented diets than in fish fed the control diet (P<0.05) and the lowest MDA concentration was found in fish fed G2 diet.

Table 4. Plasma antioxidant enzyme activity of rainbow trout fed with experimental diets supplemented with microencapsulated garlic extract at different levels.

Parameters	G0	G1	G2	G3	G4
SOD (µmol/l)	48.26 ± 2.92^c	53.28 ± 2.69^b	58.64 ± 3.49^a	62.10 ± 1.54^a	59.63 ± 2.08^a
Catalase (u/ml)	110.87 ± 2.94^c	137.16 ± 2.49^ab	141.98 ± 2.06^a	133.22 ± 3.98^b	134.37 ± 3.62^b
MDA (µmol/l)	101.89 ± 4.77^a	80.25 ± 3.76^b	66.50 ± 4.24^c	79.24 ± 3.31^b	84.80 ± 3.21^b

Data are expressed as mean ± SD of three replicate tanks. Mean values with different superscripts are significantly different. The significance level is defined as P < 0.05 (Duncan’ test; n=9).

4. Discussion

The application of medicinal plants is one of the most interesting approaches to promote growth performance and strengthen of immune responses in aquatic animals (Fazelan et al. 2020). Garlic, Allium sativum, has been widely reported to enhance growth, activity of non-specific defenses of immune system and antioxidant enzymes in mammals (Drobiova et al. 2011; Samolińska et al. 2020) and proved to reduce free fatty acids (Öz 2018). It is also reported that dietary garlic improved growth, survival, immune response (Talpur and Ikhwanuddin 2012), antioxidant status (Metwally 2009; Mohebbi et al. 2012), and digestive enzyme activities which enhanced digestion and absorption of nutrients essential for fish growth (Chitsaz et al. 2018). Therefore, the use of garlic in aquaculture is powerfully recommended.

The use of encapsulation in order to incorporate additives in commercial diet of fish is an important strategy for aquaculture industry (Vanegas-Espinoza et al. 2019). Cyprinid larvae fed diet supplemented with hydrosoluble vitamins and minerals had poor growth and survival, due to leaching of the additives (Petitjean and Csengeri 1995). These must be protected and microencapsulation seems a good method for protection. Petitjean and Csengeri (1995) obtained microcapsules of polyvitamin solution of 10- 20 μm diameters with a good yield for cyprinid larvae to avoid leaching. Aragão et al. (2014) showed that taurine should be previously encapsulated in diets for Senegalese sole. Microencapsulation of taurine in diets improves its metabolic availability (Aragão et al. 2014). Also, microencapsulation can be used as anthocyanins (roselle calyx extracts, Hibiscus sabdariffa) carrier during fish diet manufacture being a practical alternative to provide natural pigments that improve different growth parameters and skin pigmentation of fantail goldfish (Carassius auratus) (Vanegas-Espinoza et al. 2019). The present study investigated for the first time the impact of microencapsulated garlic extract on growth, whole body composition, non-specific immune response, and antioxidant status of rainbow trout.

In this research, zootechnical indices (fish length and fish body weight) and feed efficiency showed a significant increase in rainbow trout fed diet supplemented with 1% microencapsulated garlic extract (G3) compared to fish fed the control diet (G0). Similarly, other studies demonstrated that diets with garlic content caused a significant improvement of growth performance, feed conversion ratio, and protein efficiency in rainbow trout (Nya and Austin 2009; Farahi et al. 2010; Esmaeili et al. 2017; Büyükdeveci et al. 2018). Moreover, the beneficial effects of garlic on growth parameters and nutritional conditions have been previously reported in different fish species such as Carassius auratus (Sasmal et al. 2005), Oreochromis niloticus (Metwally 2009), Salmo caspius (Zaefarian et al. 2017), and Lates calcarifer (Talpur and Ikhwanuddin 2012). The effects of garlic on animal growth may be related to allicin (Erguig et al. 2015). Moreover, dietary garlic was beneficial for rainbow trout in terms of promotion growth and inducing changes in the intestinal microbiota (Etyemez Büyükdeveci et al. 2018). It is also reported that dietary garlic extract could significantly improve immune responses, villus height, digestive enzyme activities and growth performance in Nile tilapia, Oreochromis niloticus (Supa-aksorn et al. 2017). Inoue et al (2016) reported that the inclusion of 0, 15, 30 and 45 g of fresh garlic per kg in the diet did not affect growth, food conversion, and body condition of Colossoma macropomum (tambaqui). In addition, hybrid tilapia juveniles (O. niloticus x O. aureus) fed with garlic supplemented diets showed a decreased trend in weight gain compared to fish fed the control diet (Ndong and Fall 2011). The contradiction in these reports may be due to differences in fish species, consumed amounts and diet formulation.

The dietary supplementation of 1% garlic extract has led to a significant increase in fish crude protein content and a significant decrease in fish crude lipid content. Similarly, a diet containing 30g/kg of garlic powder increased protein content and reduced fat content of brown trout (Salmo trutta caspius) (Zaefarian et al. 2017). Also, Shalaby et al. (2006) showed that the crude protein content in whole fish increased significantly in the group fed on 30 g A. sativum/kg diet, while total lipids decreased significantly in the same group. Meanwhile, Ajiboye et al (2016) reported that increasing quantities of dried garlic (0, 1, 2 and 3%) in the diet of Monosex Tilapia zillii caused an evident increase of protein level and decrease of fat level. This situation may be due to increased digestibility and absorption of garlic nutritional content.

Garlic is considered as an important vegetable that should be used in humans and animals diets. At end of the feeding trial, higher plasma proteins, albumin, and lysozyme activity were observed in fish fed diets with 0.5 and 1% microencapsulated garlic extract in comparison with fish fed the control diet, suggesting the effectiveness of garlic extract in the health promotion of Salmonids. Also, fish fed G2 and G3 diets showed lower glucose levels compared to the control group. Similarly, serum total protein level was significantly higher and serum glucose level was lower in fish fed on diets containing garlic as compared to the control group (Metwally 2009; Talpur and Ikhwanuddin 2012). Lysozyme (N-acetylmuramide glucanohydrolase or muramidase), as one constituent of the innate immunity system by their anti-inflammatory and bactericidal properties contributes to non-specific immunity of organisms including fishes (Thanikachalam et al. 2010; Ángeles Esteban 2012; Yu et al. 2013; Hoseinifar et al. 2020). Motlagh et al. (2020) showed that administration of 0.15 mL of garlic extract per kg feed significantly increased the skin mucus lysozyme activity and values of ACH50 in Guppy, Poecilia reticulata. According to Talpur and Ikhwanuddin (2012), erythrocytes, leucocytes, haematocrit, haemoglobin, phagocytic activity, respiratory burst, lysozyme, anti-protease and bactericidal activities were enhanced in garlic-fed groups compared to the control. The active ingredients present in the medicinal herbs have a key role to enhance the fish immunity (Rajabiesterabadi et al. 2020). Garlic contains an active compound, allicin, which has an effect on different enzymes that can affect metabolism of virulent bacteria (Ankri and Mirelman 1999). Garlic also hampers the growth of those bacteria that are resistance to certain antibiotics (Arora and Kaur 1999). Therefore, the non-specific immunity of fish and prevention of bacterial infections in aquaculture could be obtained through the addition of garlic in the diet (Talpur and Ikhwanuddin 2012).

Garlic and its extracts have been successfully used as environmentally friendly immunostimulants to control of pathogens (Vaseeharan and Thaya 2014). The development of immune protection to Aeromonas hydrophila infection after the dietary application of garlic as an immunostimulant was studied in the work involving rainbow trout reported by Nya and Austin (2011). Fourteen days after the stopping of feeding, mortality rates of 12% and 16% were recorded in fish which received 0.5 g and 1.0 g of garlic 100 g⁻¹ of feed, respectively, compared to 84% mortality in the control. According to Sahu et al. (2007), superoxide anion production, lysozyme, serum bactericidal, serum protein and albumin were improved in Labeo rohita fed dietary garlic compared with the control. It is also reported that Echinacea and garlic supplemented diet improved growth, survival and resistance to Aeromonas hydrophila and cold stress in Nile tilapia (Aly and Mohamed 2010). These results indicate that dietary garlic stimulates the immunity and makes fish more resistant to stress and infection.

Antioxidant enzymes such as superoxide dismutase and catalase constitute the first line of enzymatic defense mechanism against free radicals to maintain the complex immune system of fish (Deng et al. 2015; Ghelichpour et al. 2019). According to the results obtained by Benkeblia (2005), Allium species contain a variety of components act as the powerful antioxidants. Garlic extract exerts antioxidant action (SOD, CAT, and GPX) by scavenging reactive oxygen species (ROS) and enhancing the cellular antioxidant enzymes (Metwally 2009). The results of the present study showed a significant increase of plasma SOD and catalase activities and a significant decrease of plasma malondialdehyde level in rainbow trout fed diets supplemented with microencapsulated garlic extract. Similarly, previous investigations reported that dietary garlic can improve the antioxidant status of O. mykiss, Cyprinus carpio and O. niloticus as evidenced by decreased thiobarbituric acid in serum and tissues (Mohebbi et al. 2012; Naeiji et al. 2013; Mahmoud et al. 2019). Kumar et al. (2009) reported that garlic extract reduces oxidative stress caused by cadmium in freshwater catfish (Clarias batrachus). Metwally (2009) also indicated that GPx, SOD, and catalase activities were significantly increased and plasma MDA levels decreased in fish fed on garlic than the control group.

According to our results, there have been reports of the use of some medicinal herbs in the diet to improve the antioxidant and immune system in different fish species.Sarhadi et al. (2020) reported that dietary artemisia (Artemisia annua) leaves extract supplementation significantly improved serum lysozyme, immunoglobulin, protease and catalase of common carp after an 8-week feeding period. Yousefi et al. (2020) found improvement in the plasma globulin, total Ig, lysozyme, and ACH50 of common carp fed with lavender (Lavandula angustifolia) extract supplemented diets for 70 days. Every component of the immune system has its own inherent protective value, and the final combination of these components is likely to be related to a satisfactory immune response (Whyte 2007).

In conclusion, the results showed that microencapsulated garlic extract can improve the growth performance, immune system and antioxidant enzymes status of rainbow trout. Therefore, it can be concluded that microencapsulated garlic extract has considerable potential as a good food additive in fish culture.

Disclosure statement

No potential conflict of interest was reported by the authors.