A Comparison between dietary effects of Cuminum cyminum essential oil and Cuminum cyminum essential oil, loaded with iron nanoparticles, on growth performance, immunity and antioxidant indicators of white leg shrimp (Litopenaeus vannamei)
Abstract: This study evaluates the effect of dietary cumin essential oil's (CEO) and cumin essential oil's loaded iron nanoparticles (CEO‐loaded NPs) on growth performance, haematological parameters and antioxidant status of white leg shrimp. Shrimps with the initial weight of 3.38 ± 0.014 g were allocated into 15 fibreglass tanks (300 L, 60 shrimps per tank) and were fed with five experimental diets containing 0 g/kg CEO (the control diet), 5 g/kg CEO, 10 g/kg CEO, 5 g/kg CEO‐NPs and 10 g/kg CEO‐NPs for 56 days. After 56 days of culture, the shrimps fed with CEO and CEO‐loaded NPs supplemented diets showed a more significant increase in growth performance (p < .05) than in the control diet. Total haemocyte count (THC) value, large‐granule cells (LGC), hyaline cells (HC)%, superoxide dismutase (SOD) and Alkaline phosphatase (AKP) activity were significantly higher (p < .05) in the shrimps fed with CEO and CEO‐loaded NPs supplemented diets than the shrimps fed with the control diet. Glutathione peroxidase (GPX) enzyme activities along with the glucose and cholesterol concentrations significantly decreased in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets more than those fed with the control diet. The results suggest that the dietary CEO‐loaded NPs can improve the growth performance, the health status and the antioxidant capacity of L. vannamei.
antioxidant status; cholesterol; cumin (Cuminum cyminum); glucose; Litopenaeus vannamei; nanoparticles
INTRODUCTION
The White leg shrimp Litopenaeus vannamei, an important rearing species, is native to the west coast of Latin America in the Pacific Ocean. The species is the main candidate in crustacean's culture in most developing countries, mainly due to its highly fed efficiency, growth performance and resistance to unsuitable environmental conditions. Over the past two decades, Iran's shrimp farming industry has been affected by the occurrence of various diseases such as white spot virus disease and various bacterial diseases that can result in inevitable losses (Lem, [25] ).
Today, aquaculture has gained a special place among human food sources. One of the basic practices used to supply human's need to protein is rearing fish. Therefore, accompanied by the increase in human population in recent decades, most researchers have attempted to present more innovative ways for increasing production in the shortest possible time with the least side effects in fish and this topic which requires too much attention has focused on the use of additives biological diets, such as enzymes, probiotics, proteins, and medicinal plants.
Medicinal herbs have been originally used as a dietary supplement in aquaculture (Ji et al., [19] ) and in many studies, the effects of different plant species (flowers, leaves, seeds and roots) have been reported in the forms of improvement in growth, antioxidant status, none specific immune response and increase in survival rate in commercial crustacean species such as Litopenaeus vannamei (Wang, Liang, Duan, Niu, Wang, Huang, and Lin, [44] ; Yudiati, Isnansetyo, Murwantoko, Triyanto, & Handayani, [48] ), Fenneropenaeus indicus (Vaseeharan, Sai Prasad, Ramasamy, & Brennan, [42] ), Macrobrachium rosenbergii (Liu et al., [27] ) and Penaeus indicus (Immanuel, Vincybai, Sivaram, Palavesam, & Marian, [18] ). Crustacean defence has an innate and acquired structure made up of cellular and humeral immune mechanisms that cellular immune responses perform in different types of cells in the haemolymph. But the humeral immune response (a kind of cellular response) is characterized by secretion and in turn, inhibits the growth of bacteria associated with antibacterial properties. Finally, both defence mechanisms result in elimination of involving pathogens in the synchronized activities.
Cuminum cyminum (Cumin) is a medicinal plant that is widely used in traditional medicine and is available as a dietary supplement used in medical science. Traditionally, diversity of active compounds was used in treatment of various diseases. Likewise, alkaloid, coumarin, anthraquinone, flavonoid, glycoside, protein, resin, saponin, tannin and steroid were the major components of the essential oil extracted from cumin (Al‐Snafi, [6] ). In addition, further studies reported its various pharmacological actions including antifungal, antimicrobial, insecticidal, anti‐inflammatory, analgesic, antioxidant, anticancer, antidiabetic, antiplatelet aggregation, hypotensive, bronchodilatory, immunological, contraceptive, antiamyloidogenic, antiosteoporotic, aldose reductase, alpha‐glucosidase and tyrosinase inhibitory effects and protective and central nervous effects (Al‐Snafi, [6] ; Kedia, Prakash, Mishra, & Dubey, [21] ).
Today, iron oxide nanoparticles (Fe2O3 NPs) are used extensively in biological and non‐biological processes in industry, medicine and agriculture because of the low toxicity, physicochemical and superparamagnetic properties such as surface area and volume ratio (Ali et al., [5] ).
Previous studies have shown that the antioxidant defence system developed in animals includes antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and some functional molecules (e.g., albumin, ceruloplasmin and ascorbic acid) to stabilize the minimum levels of ROS in the cell (Garcia‐Triana, Zenteno‐Savin, Peregrino‐Uriarte, & Yepiz‐Plascencia, [15] ; Sheikhzadeh, Tayefi‐Nasrabadi, Khani Oushani, & Najafi Enferadi, [38] ; Zhang et al., [49] ).
Analysis of serological and haematological parameters is one of the most informative methods of monitoring the physiological status, because these parameters are specific among different species and sexes and provide a diagnostic tool to assess possible dysfunction and detect the metabolic disturbances as well as the infectious and non‐infectious aetiologies (Akrami et al., [4] ).
However, to the best of our knowledge, little is known on the effects of Cumin essential oil and the applications of CEO‐loaded nanoparticles on the physiological status of shrimp. Therefore, in this study, it was the first attempt to investigate the effects of dietary supplementation of Cumin essential oil and CEO‐loaded nanoparticles on growth parameters, survival and antioxidant enzymes activities and some haematological parameters of cultured Litopenaeus vannamei.
MATERIALS AND METHODS
Diet formulation, preparation and body composition
The experimental diets' formulations are given in Table . In this study, Cumin essential oil (CEO) was brought from a local company (Beiza Co., Iran). Cumin essential oil‐loaded iron nanoparticles (CEO‐NPs) were synthesized and prepared based on the previously described method (Khajeh & Sanchooli, [22] ).
Formulation and proximate composition of experimental diets (g/kg) for L. vannamei, used for the different CEO and CEO‐loaded NPs diets for 56‐day feeding trial
| Ingredient | Control g/kg | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| 5 | 10 | 5 | 10 |
|---|
| Fish meal | 350 | 350 | 350 | 350 | 350 |
| Shrimp meal | 150 | 150 | 150 | 150 | 150 |
| Wheat flour | 150 | 150 | 150 | 150 | 150 |
| Soybean flour | 210 | 210 | 210 | 210 | 210 |
| Fish oil | 40 | 40 | 40 | 40 | 40 |
| Lecithin soybean | 5 | 5 | 5 | 5 | 5 |
| Gluten wheat | 20 | 20 | 20 | 20 | 20 |
| Mineral premix | 20 | 20 | 20 | 20 | 20 |
| Vitamin premix | 20 | 20 | 20 | 20 | 20 |
| Binder | 20 | 15 | 10 | 15 | 10 |
| Cellulose | 15 | 15 | 15 | 15 | 15 |
| CEO | 0 | 5 | 10 | 0 | 0 |
| CEO‐loaded NPs | 0 | 0 | 0 | 5 | 10 |
| Proximate composition (g/kg DM) |
| Protein | 382 | 388 | 389 | 383 | 388 |
| Fat | 91 | 90 | 90 | 91 | 90 |
| Moisture | 71 | 70 | 72 | 70 | 70 |
| Ash | 90 | 88 | 87 | 90 | 89 |
1 DM, dry matter.
- 2 Provided per kilogram of feed: vitamin D, 1.28 mg; vitamin E, 136 mg; thiamine, 8 mg; riboflavin, 16 mg; 3 pyrodoxine‐Cl,16 mg; vitamin C, 80 mg; calcium pantothenate, 17.4 mg; biotin, 0.16 mg; folic acid, 4.8 mg; niacin, 40 mg; vitamin B, 0.016 mg; menadione bisulphite, 20 mg.
- 3 Provided per kilogram of feed: magnesium, 625 mg; potassium, 509 mg; calcium, 465 mg; zinc, 100 mg; iron, 62 mg; manganese, 13 mg; copper, 6 mg.
For preparation of CEO‐NPs, magnetic nanoparticles were first prepared by coprecipitation of Fe+2 and Fe+3 ions with the ammonia solution and then, they were treated under hydrothermal conditions. The ferrous and ferric chlorides (mole ratio 1:2) were mixed in 25 ml water at a concentration of 0.3 mol−1 iron ions, and the pH was maintained constant around 10.5. By adding 5 ml NH4OH solution, the chemical precipitation was achieved at ambient temperature under vigorous stirring. The precipitates were warmed at 80°C in 30 min to eliminate the residual solvent, and then, they were washed several times with high purity deionized water and ethanol. For binding of CEO, a suitable amount of dried nano‐sized magnetic particles was poured in a Teflon beaker and then, the CEO solution was added. The mixture was gently agitated by a Teflon rod, so the solvent evaporated slowly. The modified magnetic nanoparticles were then warmed at 90°C for 1 hr to eliminate the residual solvent completely. Next, CEO and CEO‐NPs were supplemented in the basal diet at the levels of 5 g/kg and 10 g/kg (control), CEO (5 g/kg), CEO (10 g/kg), CEO‐NPs (5 g/kg and CEO‐NPs (10 g/kg). The cumin supplementation doses in diets were further selected based on the result of the previous study (Yılmaz, Ergun, & Soytas, [46] ). The diet preparation was performed according to Wang et al. ([43] ).
Shrimp and experimental design
White leg shrimp with the mean weight of 3.38 ± 0.014 g (mean ± SD) was obtained from the shrimp farm of Tiabe, Iran. The shrimps were fed with the basal diet for one week to acclimate to the experimental conditions prior to the beginning of the feeding trial. The shrimps were cultured in 15 fibreglass tanks (300 L, with the water depth of 1 m, i.e., 60 shrimps per tank) and were then divided into five groups with three replicates for each group. During 56 days, shrimp was fed four times daily at 08:00, 13:00, 17:00 and 21:00 with various trial diets calculated based on 4% of the body weight. During the trial period, water temperature, pH, salinity, dissolved oxygen and ammonia–nitrogen were 30 ± 1°C, 7.8 ± 0.2, 34%±2%, 5.2 ± 0.4 mg/L and 0.05 ± 0.02 mg/L, respectively. At the end of the trial, that is, 24 hr before harvesting, the shrimps were fasted and then the total number and the mean weight of the shrimps in all treatment groups were measured. Survival rate (SR), final body weight (FBW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) were also calculated based on the equations described in previous studies
(Lin, Lin, Yang, Li, & Luo, [26] ; Zhang, Liu, Tian, Yang, Liang, Yue, and Xu, [49] ).
Here, Wt and W0 are the final and the initial body weights (g), respectively, t is the duration of the experimental days, N0 is the initial number of the shrimps and Nt is the final number of the shrimps.
Twenty‐four hours after the end of the experimental period, the analysis of the shrimps' carcass (moisture, crud protein, crude lipid and ash) was carried out with five shrimps per tank and according to the previously described methods (Zhang, Liu, Tian, Yang, Liang, Yue, and Xu, [49] ).
Assessments of biochemical constituents and immune parameters
In the end of the feeding trial, to measure the total haemocyte count (THC), haemolymph was extracted from the pericardial cavity using a 1 ml syringe containing an anticoagulant with a 26‐gauge needle (four shrimps from each treatment) (Estrada, Velazquez, Rodriguez‐Jaramillo, & Ascencio, [14] ). Measurement of THC was carried out by placing an anticoagulant–haemolymph drop on a Buker haemocytometer under the optical microscope and the haemocytes were counted in 0.1 mm3. A different type of haemocytes including small‐granule cells (SGC), large‐granule cells (LGC) and hyaline cells (HC) was determined according to the previously described method (Estrada et al., [14] ). The percentage of each type of cell was calculated based on the total number of the cells.
Total protein plasma (TPP) was measured in the samples of the mixture anticoagulant and haemolymph collection (three shrimps from each treatment) according to the explained method (Chen, Chen, Lin, Yeh, & Huang, [11] ).
At the end of the experimental period, nine shrimps were randomly sampled from each tank haemolymph and the samples were obtained from the pericardial cavity using a 1 ml syringe with a 26‐gauge needle. Then, they were pooled in a 1.5 ml microtube and were centrifuged at 10,000 rpm for 10 min. Next, the serum samples were isolated and stored at 4°C (Yu, Li, Lin, Wen, & Ma, [47] ). Finally, glucose, total cholesterol and albumin were determined by the use of commercial kits (Pars Azmoon Co., Tehran, Iran) according to the manufacturer's instructions.
Alkaline phosphatase (AKP) activity enzyme was determined as a method previously described by Lin et al. ([26] ). In this method, 2 ml p‐nitrophenyl phosphate was incubated in glycine‐NaOH buffer‐pH 9 with 100 μl of crude enzyme solutions for 30 min at 37°C. Then, 2.9 ml of 0.1 N NaOH was added to the previous solution and the absorbance was determined spectrophotometrically at 405 nm and the activity was reported as mg/ml released p‐nitrophenyl.
The activities of SOD and GPX were measured according to the manufacturer's instruction of antioxidant Enzyme ZellBio (GmbH, Germany) Test Kits. For analysis of SOD and GPX, the hepatopancreas samples were homogenized briefly in the phosphate buffer (0.025 m KH2PO4, 0.025 m Na2PO4.12H2O, pH 6.4).
Total SOD activity in supernatant samples was measured following the method described by Jing and Zhao ([20] ). The absorbance was measured at 420 nm. One unit of SOD activity was defined as the amount of SOD required to inhibit the rate of xanthine reduction by 50% in a 1 ml reaction system. Special SOD activity was expressed as a unit per mg of protein.
Glutathione peroxidase activity was determined by quantifying the rate of H2O2‐induced oxidation of GSH to the oxidized glutathione (GSSG). A yellow product with absorbance at 412 nm was formed as GSH reacts with dithiobisnitrobenzoic acid (Xia & Zhu, [45] ). One unit (U) of GPX was defined as the amount that reduces the level of GSH by 1 mm in 5 min per mg hepatopancreas protein. GPX activity in hepatopancreas was expressed as U/mg protein.
Statistical analysis
Data were tested for normality and homogeneity of variance before the application of the parametric tests. All data were subjected to one‐way analysis of variance (ANOVA) at the level of significance (.05). Differences between the means were tested by Tukey's multiple range tests with SPSS (version 17.0) software for the windows.
RESULTS
Growth performance and body composition
The effects of different dietary CEO and CEO‐loaded NPs doses on growth performance and survival rate were presented in Table . After a 64‐day feeding trial, FBW, WG and SGR of the shrimp‐fed CEO and CEO‐loaded NPs supplemented diets were significantly higher (p < .05) than those of the shrimp‐fed control diet. FCR of the shrimp‐fed CEO and CEO‐loaded NPs supplemented diets improved more significantly (p < .05) in comparison with the control. The highest growth indices and the best improvement FCR were observed in the shrimp‐fed 10 g/kg CEO‐loaded NPs supplemented diet (Table ). The shrimps' survival rate among all treatment groups had no significant difference (p > .05) and varied between 93.77% and 95.33% (Table ).
Growth parameters and survival rate of L. vannamei fed with different CEO and CEO‐loaded NPs diets for 56‐day feeding trial (data expressed as M ± SE)
| Dietary group | Control | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| Parameters | 5 | 10 | 5 | 10 |
|---|
| IBW g | 0.03 ± 3.43 | 0.03 ± 3.39 | 0/03 ± 3.42 | 0.02 ± 3.31 | 0.03 ± 3.33 |
| FBW g | 8.63 ± 0.05a | 9.17 ± 0.03b | 9.12 ± 0.07b | 9.33 ± 0.05c | 9.46 ± 0.05c |
| WG % | 151.91 ± 1.37 | 171.12 ± 2.73b | 166.12 ± 1.82b | 181.46 ± 1.15c | 183.97 ± 1.77c |
| SGR % day−1 | 1.64 ± 0.01a | 1.77 ± 0.02b | 1.74 ± 0.01b | 1.84 ± 0.01c | 1.86 ± 0.01c |
| FCR | 1.87 ± 0.01c | 1.65 ± 0.02b | 1.69 ± 0.01b | 1.58 ± 0.01a | 1.55 ± 0.01a |
| Survival rate % | 93.77 ± 3.07 | 94.66 ± 4.01 | 93.88 ± 2.94 | 95.33 ± 2.55 | 95.33 ± 4.19 |
4 Means marked by different letters are significantly different (p < .05).
The body composition of L. vannamei among all treatment groups is shown in Table . The crude protein, crude lipid and ash and moisture content had no significant difference (p > .05) in all treatment groups.
Whole body composition of L. vannamei fed with different levels of CEO and CEO‐loaded NPs for 56 days (data expressed as M ± SE)
| Dietary group | Control | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| Parameters (% dry weight basis) | 5 g/kg | 10 g/kg | 5 g/kg | 10 g/kg |
|---|
| Crude protein | 849.5 ± 11.5 | 85.7 ± 15 | 854.5 ± 15.4 | 877 ± 12 | 855.1 ± 13.4 |
| Crude lipid | 53.4 ± 1.1 | 53.7 ± 1.0 | 55 ± 1.9 | 54.5 ± 2.4 | 54.8 ± 2.2 |
| Ash | 61 ± 3.3 | 64.2 ± 4.3 | 61.5 ± 4.4 | 61.6 ± 2.8 | 64.4 ± 2.5 |
| Moisture (% wet weight basis) | 71.65 ± 1.75 | 70.65 ± 1.70 | 69.50 ± 1.21 | 70.20 ± 1.26 | 69.70 ± 1.21 |
Haematological indices and immune parameters
Fifty‐six days after the feeding trial, the effects of different levels of dietary CEO and CEO‐loaded NPs on the THC and different types of haemocytes parameters (SGC, LGC and HC %) were presented in Table . THC value, HC and LGC% were significantly higher (p < .05) in shrimp‐fed CEO and CEO‐loaded NPs supplemented diets than those shrimps fed with the control diet, and the highest value of THC, HC and LGC% was recorded in the shrimps fed with the 10 g/kg CEO supplemented diet (26.55 ± 1.16, 18.33 ± 0.33, and 28.67 ± 0.66, respectively).
Haemocytes parameters (total and differential counts) in L. vannamei fed with different CEO and CEO‐loaded NPs diets levels for 56‐day feeding trial (data expressed as M ± SE)
| Dietary group | Control | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| Parameters | 5 | 10 | 5 | 10 |
|---|
| THC (×105cell/ml) | 19.45 ± 0.25a | 23.55 ± 0.39b | 23.20 ± 0.31b | 24.22 ± 0.20b | 26.55 ± 1.16c |
| HC% | 11.50 ± 0.52a | 12.00 ± 0.47a | 13.33 ± 0.33b | 14.00 ± 0.57b | 18.33 ± 0.33c |
| SGC% | 59.00 ± 0.57 | 59.67 ± 0.33 | 58.67 ± 0.33 | 59.00 ± 0.57 | 60.33 ± 1.20 |
| LGC% | 22.67 ± 0.33a | 27.33 ± 0.33b | 27.67 ± 0.66b | 28.33 ± 0.33b | 28.67 ± 0.66b |
5 Means marked by different letters are significantly different (p < .05).
Activities of SOD, GPX and AKP are shown in Table . During the 64 days of the feeding trial, more significant differences (p < .05) were found in SOD in the shrimps fed with the 10 g/kg CEO, 5 g/kg and 10 g/kg CEO‐loaded NPs supplemented diets in compared with the control. The maximum value of SOD activity was observed in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet (39.77 ± 1.39 U/mg). The GPX activity significantly increased (p < .05) in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets compared with the control. The highest value of GPX activity was recorded in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet (335.89 ± 1.70 U/mg). Compared with the control, supplementation of CEO and CEO‐loaded NPs to diets significantly increased the AKP activity in 64 days (p < .05). The highest of AKP activity was also obtained in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet (4.4 ± 0.41 U/ml).
Antioxidant capacity parameters and metabolic enzyme in L. vannamei fed with different CEO and CEO‐loaded NPs diets levels for 56‐day feeding trial (data expressed as M ± SE)
| Dietary group | Control | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| Parameters | 5 | 10 | 5 | 10 |
|---|
| SOD (U/mg) | 32.74 ± 2.10a | 32.29 ± 1.54a | 36.43 ± 1.92b | 35.95 ± 1.71b | 39.77 ± 1.39c |
| GPX (U/mg) | 313.79 ± 2.80a | 305.00 ± 2.04a | 303.55 ± 1.28b | 306.97 ± 3.34b | 300.89 ± 1.70c |
| AKP (U/ml) | 2.10 ± 0.20a | 2.5 ± 0.33b | 3.1 ± 0.27b | 3.6 ± 0.31bc | 4.4 ± 0.41c |
6 Means marked by different letters are significantly different (p < .05).
Glucose, cholesterol, albumin and total protein concentrations in the shrimps treated during 64 days of the feeding trial were presented in Table . Glucose and cholesterol concentrations significantly decreased (p < .05) in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets compared with the control. Then, the maximum concentrations of glucose and cholesterol were observed in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet (19.20 ± 1.53 and 10.20 ± 2.82 mg/dl, respectively).
Biochemical parameters of haemolymph in L. vannamei fed with different CEO and CEO‐loaded NPs diets levels for 56‐day feeding trial (data expressed as M ± SE)
| Dietary group | Control | CEO g/kg | CEO‐loaded NPs g/kg |
|---|
| Parameters | 5 | 10 | 5 | 10 |
|---|
| Glucose (mg/dl) | 34.87 ± 2.13a | 29.13 ± 0.67b | 20.20 ± 2.18c | 25.27 ± 2.49b | 19.20 ± 1.53c |
| Cholesterol (mg/dl) | 25.07 ± 4.37a | 18.73 ± 2.10b | 14.13 ± 2.10c | 14.9 ± 1.77c | 10.20 ± 2.82d |
| Albumin (g/dl) | 2.40 ± 0.20 | 2.13 ± 0.07 | 2.40 ± 0.12 | 2.00 ± 0.12 | 2.13 ± 0.13 |
| Total protein (g/dl) | 11.21 ± 0.63 | 9.73 ± 0.59 | 10.67 ± 0.87 | 9.68 ± 0.79 | 10.30 ± 0.37 |
7 Means marked by different letters are significantly different (p < .05).
DISCUSSION
Cumin, as a spice, has a long tradition of consumption and is cultivated in India and the Mediterranean countries (Yılmaz, Ergun, and Soytas, [46] ), yet it is currently processed in different forms (e.g., seed, powder and essential oil) and is consumed as the most popular feed additive for humans as well as animals (Azeez, [8] ; Yılmaz, Ergun, and Soytas, [46] ). The results of the present study indicated that the dietary CEO and CEO‐loaded NPs levels of 5 g/kg and 10 g/kg could significantly improve the growth performance of L. vannamei. The previous studies demonstrated that dietary cumin, caraway and funnel (belonging to the family Apiacea) could significantly enhance the growth performance of fishes (Abd El Hakim, Ahmad, Azab, Lashien, & Baghdady, [1] ; Ahmad & Tawwad, [3] ; Yılmaz, Ergun, and Soytas, [46] ). However, the results of the present study indicated that iron NPs and cumin complex have a synergism effect on the growth performance and the physiological status of L. vannamei. Moreover, the maximum improvement in growth indices was observed in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet compared with the control. On the other hand, Srinivasan, Saravana Bhavan, Rajkumar, Satgurunathan, and Muralisankar ([41] ) evaluated the use of Fe2O3 NPs in the diet of giant freshwater prawn (Macrobrachium rosenbergii) postlarvae for 90 days, and found that Fe2O3 NPs' supplementation (up to 20 mg/kg) was suitable for maintenance and could improve the growth performance in M. rosenbergii. The growth‐promoting effects of cumin may be explained by the various active compounds (for example: thymol, menthol, HUFA and PUFA) which lead to their positive effects on the digestion, absorption and assimilation of the nutrients (Azeez, [8] ; Srinivasan, [40] ; Yılmaz et al., [46] ). Furthermore, it demonstrated that cumin can act as an attractant spice (Harada, [17] )in which its odour can cause the presence of cumin aldehyde‐induce olfactory organ. In turn, it can also explain the enhanced growth. Bhosale, Bhilave, and Nadaf ([9] ) reported that cumin is able to induce the secretion of pancreatic enzymes and the nutrient digestion and assimilation. However, FCR improvement in the shrimps treated with CEO and CEO‐loaded NPs supplemented diets suggested that the increasing digestibility and in turn the energetic benefits could increase the growth performance, possibly due to its appetite characteristics. It also demonstrated an improvement in the digestive activity and the absorptive capacity of the intestine related to the anti‐inflammatory and antiproliferative effects of the medicinal plants (Pérez‐Sánchez, Borrás‐Linares, Barrajón‐Catalán, Arráez‐Román, González‐Álvarez, Ibáñez,Segura‐Carretero, Bermejo, Micol, [35] ).
Haematological indices also play a key role as an indicator of health as well as the physiological status of an organism (Chu, Chen, & Huang, [12] ). Haemocytes density is affected by the type of sex, moulting, development, reproductive status and nutrition (Song, Yu, Lien, Huang, & Lin, [39] ). In the present study, THC, HC, SGC and LGC increased more significantly in the shrimps fed with the CEO and CEO‐loaded NPs doses than the control suggesting the positive effect of cumin and cumin+NPs on non‐specific immune responses. In line with this conclusion, Srinivasan et al. ([41] ) reported that the contents of THC, HC, SGC and LGC increased in the M. rosenbergii fed 20 mg/kg Fe2O3 NPs supplemented diet. Therefore, it appears that the essential oil of cumin and iron nanoparticles have synergistic effects and can lead to a maximum increase in haemocytes in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet. In continue, many studies have focused on commercial fish, yet current knowledge is very limited for economically marine invertebrates, especially for shrimps. It is demonstrated that optimum levels of dietary additive as nano‐sized forms have produced better results on haemocytes available in M. rosenbergii (Muralisankar, Bhavan, Radhakrishnan, Seenivasan, & Srinivasan, [33] ; Muralisankar et al., [32] ).
Glutathione peroxidase and SOD are two major physiological antioxidant enzymes acting against lipid proxidation and free radicals that provide protection of mechanisms within the tissue damaged by a radical process and phagocytosis (Abhijith, Ramesh, & Poopal, [2] ). As shown in Table , maximum GPX and SOD enzyme activities are found in the shrimps fed with the 10 g/kg CEO‐loaded NPs supplemented diet. Contrary to some previous reports, our finding did not exhibit any significant difference or significant decrease in GPX and SOD activities after the shrimps were fed with the dietary supplemented with Rhadiola rosea and astaxanthin (Wang et al., [44] ; Zhang et al., [49] ). Similar results were also found in Peneaus monodon treated with Psidium guajara leaf extract and Litopeneaus vannamei treated with polysaccharides from mycelia of Cordyceps sinensis (Deng et al., [13] ; Zhou, Wang, Chu, & Luo, [50] ). The fact that GPX and SOD activities increased together can enhance the activity of NADPH oxidase, which is responsible for scavenging superoxide anion (Zhang, Liu, Tian, Yang, Liang, Yue, and Xu, [49] ). In addition, ripe cumin seeds are a richer source of iron and zinc than other medical herbal seeds which can be very important in the antioxidant defence system of organisms (Powell, [36] ).
Therefore, the results suggested that cumin components are probably able to increase the antioxidant status in the treated shrimps of this research, and NPs have a synergic effect on inducing the antioxidant activities of the system.
Alkaline phosphatase induces transfer activity and mainly leakages from the lining of the bile canaliculi and the sinusoidal surface of hepatocytes that normally secretes through the bile from the liver (Gharaei, Ghaffari, Keyvanshokooh, & Akrami, [16] ). The liver produces more AKP than other organs and bones (Shahsavani, Mohri, & Gholipour Kanani, [37] ) and acts as a regulator in phosphorylation and dephosphorylation processes in the immune system of white leg shrimps (Zhou et al., [50] ). Increasing AKP in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets suggests that cumin accompanied with NPs could be used as a non‐specific immunological stimulant for shrimps. Similar results were reported by Deng, Wang, Tao, Li, Wang, Wang, and Wu ([13] ) and Liu, Jiang, Mou, Wang, and Guan ([28] ) who conducted surveys on the effect of Cordyceps sinensis polysaccharides, seaweed polysaccharides and C. militaris polysaccharides on shrimps which could enhance the level of AKP. The results may due to the fact that after absorption, tannin (or other components) is readily decomposed in biological systems by non‐specific esterases and the entrance of their hydrolysis products into blood and, especially in the liver and kidney (Makkar, Blümmel, Borowy, & Becker, [29] ).
Glucose is one of the most important sugary compounds in shrimp haemolymph. Glucose levels in the shrimp haemolymph are considered a good indicator of short‐ and long‐term stresses (Mercier et al., [31] ) and also act as an indicator of the nutritional condition (Pascual, Gaxiola, & Rosas, [34] ). In fact, glucose has an important metabolic role in all organisms and is one of the most appropriate physiological indices used to investigate stress incidents and increase their incidence when they occur. As it is shown in Table , glucose and cholesterol levels decrease more significantly in the shrimps fed with the CEO and CEO‐loaded NPs supplemented diets than in the control. It is demonstrated that cumin can decrease blood glucose by increasing insulin secretion (Lee, [24] ), so it can be said that essential oil cumin may contribute to the reduction in blood glucose by affecting the metabolism of insulin and carbohydrate metabolism (Meral, Donmez, Baydas, Belgo, & Kanter, [30] ). Cholesterol in crustaceans is used as a constituent of cellular membranes, subcellular structures and as a precursor of steroid hormones and moulting hormones such as ecdysone. In the present study, the decrease reported in cholesterol concentrations in blood, prior to ecdysis, was due to construction of cell membranes and to cellular growth, whereas its higher and lower concentrations were recorded before ecdysis and in intermolt stage, respectively (Bonilla‐Gómez et al., [10] ). Decreasing cholesterol level in the shrimps treated with the CEO and CEO‐loaded NPs may be due to the increased metabolism of cholesterol and lipids in the liver under the effect of the active compounds of cumin. In parallel with this result, some researcher reported that supplementation of cumin essential oil in diet resulted in a decreased cholesterol level in blood of common carps, chickens and mice (Ardakani, Akbarian, & Nazarian, [7] ; Kochhar, [23] ). However, albumin and total protein content in haemolymph were not affected by CEO and CEO‐loaded NPs supplementation; therefore, it cannot be considered as a good indicator of a better immunological and nutritional status under the trial conditions of the present study.
ACKNOWLEDGEMENTS
We thank Mr. Khandan Barani and Mr. Rahdari for their time and energy, Mrs. Miri for her technical assistance in experimental analysis and all staff of Hamoon International Wetland Research Institute for corporation. The research project was funded by University of Zabol (Grant cod: UOZ‐GR‐9517‐16).
[
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]
By M. Sheikh Asadi; A. Gharaei; J. Mirdar Harijani and A. Arshadi
