Effects of free and nanoencapsulated garlic essential oil on growth performance and immune functions in broiler chickens
Abstract
The aim of this research was to determine the effect of free and nanoencapsulated garlic essential oil (GEO) on performance, serum biochemistry, and immune functions. Broiler chickens (900 males 1-day-old, Ross 308) were randomly assigned to six treatment diets (0, 75, or 150 mg/kg free GEO and 0 [containing chitosan], 75, or 150 mg/kg nanoencapsulated GEO) in a 2 × 3 factorial arrangement of treatments. The inclusion of nanoencapsulated GEO with a concentration of 75 mg/kg significantly increased the growth performance (p < 0.05) of the birds fed the diet containing free GEO (75 mg/kg). Also, GEO nanocapsules significantly reduced serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) compared to the control diet (p < 0.05) (42 days), sheep red blood cell (SRBC) antibody titer (35 and 42 days), heterophilus (H): lymphocyte (L), and H ratio, 2,4-dinitrochlorobenzene (DNCB) (42 days) (p < 0.05). In conclusion, the findings show that the amount of 75 (mg/kg) of the nanoencapsulated GEO, compared to the free GEO, causes more growth performance and strengthens the immune system of broiler chickens.
1 INTRODUCTION
The harmful effects caused by frequent use of antibiotics, for example, the possibility of bacterial resistance and long-term shelf life in animal food products, have led to restrictions on antibiotics used to promote most regions. Removing antibiotics can have adverse effects on poultry health and increase production costs. Banning the use of antibiotics as a useful food additive has led to the expansion of the use of compounds such as probiotics, prebiotics, enzymes, organic acids, medicinal plants, and their derivatives (Brenes & Roura, 2010).
Herbal essences are concentrated hydrophobic liquids that contain aromatic volatile compounds. These substances and their compounds are secondary metabolites of plants whose antimicrobial properties have long been recognized. However, they are easily oxidized by light and heat because they have unsaturated carbon chains. In addition, their poor solubility in water limits their use in biological fluids because it prevents them from being absorbed, thereby reducing their biological access. Therefore, these compounds may not reach the ends of the intestine if a proper method is not used (Zhang et al., 2014). Meanwhile, numerous studies have been conducted using different herbal essences or mixtures of them in in vitro conditions that have had contradictory results.
One of the antibacterial, anti-inflammatory, antiseptic, anti-parasitic, antioxidant volatile oils from garlic (Allium sativum) contains bioactive components (strong sulfur) such as allicin, diallyl monosulfide, diallyl disulfide, diallyl trisulfide, and S-allyl cysteine have properties, such as delaying lipid oxidation (Shang et al., 2019).
Some studies on animals demonstrated that garlic has strengthening effects on the immune system (Stanaćev & Plavša, 2011). Onibi et al. (2009) reported that the sulfur compounds in garlic can modulate the immune system and garlic essential oil (GEO) increases the proliferation of lymphocytes and macrophages. They stated that antioxidant and antimicrobial compounds of garlic can be effective in controlling diseases and increasing the resistance level of the bird's body.
Nanoencapsulation is a new technology in the poultry industry that protects herbal essential oils against environmental factors, such as high temperature, high humidity, dryness, and volatility of essential oils. In this method, small particles of material are placed in the core, and capsule-shaped walls surround them. In some research, chitosan has been used as one of the polymer nanocarriers for the controlled release of aromatic substances. Recently, chitosan has received a lot of attention to encapsulating bioactive compounds, because it is generally known to be safe and has superior biological properties, such as degradability, biocompatibility, and nontoxicity (Estevinho et al., 2013).
The essence of the garlic medicinal plant encapsulated with biopolymers chitosan and alginate as the natural polymers have properties, such as being non-toxic to body cells, biodegradability, compatibility with the body, and adhering to the gastrointestinal wall cells. In addition, as the chitosan used in the encapsulation is a positively charged molecule, it can bind to the negatively charged bacteria on its surface and prevent them from growing. Chitosan also increases the amount of terpenoids and phenolic compounds. Previously, chitosan nanoparticle encapsulation of thyme oil resulted in the improvement of broiler performance and health via effects on the gut microflora (Hosseini & Meimandipour, 2018).
Since essential oils need to be released under controlled conditions due to their volatile compounds and also being insoluble in water, the use of nanoencapsulation of GEO by chitosan in effective transfer and increasing its effect period can be important from the viewpoint of innovation. Therefore, the current research was conducted with the aim of determining the effects of free and nanoencapsulated GEO supplement diets on the growth performance of broiler chickens, in terms of serum biochemistry, hematologic parameters, and immune functions.
2 MATERIALS AND METHODS
Distillation of volatile oil was conducted using plant materials and with the help of a Clonger distillation machine (Herbal Elixir Company, Mashhad, Iran). Chitosan was used for nanoencapsulation, prepared using crab shell and sodium pentabasic triphosphate (TPP, Sigma Chemical Company [St. Louis, MO, USA]). Acetic acid and ethanol were provided by Merck (Merck Animal Health, Madison, NJ, USA).
2.1 Nanoencapsulation of GEO
GEO nanoencapsulation was performed using the ionic gel method based on the method described by Hosseini and Meimandipour (2018). Chitosan with an average molecular weight of 190 to 310 KDa was used to make essential oil nanocapsules. First, 0.1 g of chitosan was mixed in 100 mL of 1% (v/v) acetic acid at room temperature (RT) for 12 h on a shaker until chitosan was completely dissolved. Then, 10 mL pentabasic sodium triphosphate solution (1% w/v) was added to 25 mL chitosan solution slowly, drop by drop, and stirred for 1 h until the crosslinks between the particles were formed, and finally, microparticles of chitosan-tri-pentabasic phosphate (C-TPP) were produced. Finally, the desired concentrations of the essential oil were prepared by adding C-TPP microparticles to the essential oil and gently stirring at RT. The nanoencapsulated essential oil was stored in the refrigerator until use.
2.2 GC–MS analysis of GEO
Gas chromatography/mass spectrometry (GC/MS) analysis was performed using an Agilent gas chromatograph (7890B) connected to a mass detector (model 5977A; Agilent Technologies, USA). The injector temperature was 270°C, and the oven temperature was programmed from 60°C (0 min) to 200°C at 5°C/min; m × 0.25 mm ID 0.25 μm (Agilent Technologies) were equipped. Helium was chosen as the carrier (flow rate = 1 mL/min, injection volume = 1 μL). The mass spectrometer was set to 70 eV in electron impact (EI) mode. Also, the interface temperature was set to 280°C, and the mass range was 35 to 500 m/z. The constituents were identified by comparing the mass spectra with the database of the GC–MS system and the quat retention index (Adams & Sparkman, 2007).
2.3 Experimental birds
Ross 308 male broiler chickens (Ross 308, 900 chickens, 1 day old), from a hatchery with an average body weight of 44 ± 2g, were transferred to Tehran-Iran Animal Science Research Institute. Chickens were randomly placed in 40 pens (100 × 300 × 300 cm). Birds were allowed access to food and water during the feeding period. At the beginning of the experiment, the temperature was maintained at 33°C, and on the 21st day, it gradually decreased to 25°C and then remained stable. All animal-handling procedures have been reviewed by the Institutional Animal Care and Use of Sichuan Agricultural University (Review Number: IR.UK.VE.REC.13398.023).
2.4 Chemical composition of GEO
Different GEO components were identified by GC/MS (Table 1). Principal components of the garlic oil were diallyl trisulfide (32.82%), diallyl disulfide (20.18%), methylallyl disulfide (7.18%), and allyl n-propyl sulfide (3.53%). Other compounds were negligible or unknown.
| Number | Retention time | Compounds | % total |
|---|---|---|---|
| 1 | 3.2 | Propylene | 0.01 |
| 3 | 3.4 | Allyl n-propyl sulfide | 3.53 |
| 4 | 4.4 | 3-Methylthiophene | 0.10 |
| 5 | 5.1 | Methyl allyl disulfide | 7.18 |
| 6 | 7.8 | Diallyl disulfide | 20.18 |
| 7 | 11.3 | Diallyl trisulfide | 32.82 |
2.5 Diets and treatments
This research was conducted as a completely randomized design with five replications of 30 birds, three levels of GEO (0, 75, or 150 mg/kg) and with a factorial arrangement of 2 × 3 with two forms of GEO (free and nanoencapsulated). The treatment diets were (1) base without any additives (negative control) (Ctrl 1), (2) base supplemented with 75 mg/kg free GEO, (3) base supplemented with 150 mg/kg GEO free, (4) base without GEO nanocapsules (containing 150 mg/kg chitosan) (Ctrl 2), (5) base supplemented with 75 mg/kg nanoencapsulated GEO, (6) base supplemented with 150 mg/kg GEO nanoencapsulated. Near-infrared reflectance spectroscopy (NIRS) was used to estimate the amino acid (AA) content of soybean and corn samples. Amino NIRS is a service for the rapid analysis of AA in raw feed materials provided by Evonic (Table 2). Experimental diets were prepared from 1 to 14, 15 to 28, and 29 to 42 days based on corn and soybean meal (Table 3). The diets were formulated according to the requirement of Ras 308 strain using UFFDA software.
| Content (% as is) | ||
|---|---|---|
| Parameter | Soybean meal | Corn |
| Methionine | 0.548 | 0.119 |
| Crude protein (% as is) | 42.25 | 7.08 |
| Methionine + Cystine | 1.04 | 0.278 |
| Threonine | 1.40 | 0.228 |
| Lysine | 2.45 | 0.201 |
| Tryptophan | 0.501 | 0.045 |
| Isoleucine | 1.69 | 0.238 |
| Leucine | 2.87 | 0.756 |
| Arginine | 2.92 | 0.304 |
| Phenylalanine | 1.88 | 0.320 |
| Histidine | 1.06 | 0.229 |
- a NIRS: near-infrared reflectance spectroscopy.
| Ingredients (% diet) | Starter (Day 1–14) | Grower (Day 15–28) | Finisher (Day 29–42) |
|---|---|---|---|
| Corn | 52.37 | 55.60 | 60.80 |
| Soybean meal | 39.00 | 38.30 | 33.60 |
| Corn gluten | 2.50 | 0.000 | 0.000 |
| Dicalcium phosphate | 1.60 | 1.30 | 1.10 |
| Calcium carbonate | 1.25 | 1.20 | 1.10 |
| Common salt | 0.255 | 0.255 | 0.255 |
| DL-methionine | 0.320 | 0.300 | 0.300 |
| Sodium bicarbonate | 0.200 | 0.200 | 0.200 |
| Soybean oil | 1.65 | 2.10 | 1.90 |
| HCl-lysine | 0.250 | 0.140 | 0.140 |
| Vitamin and mineral premixa | 0.500 | 0.500 | 0.500 |
| L-threonine | 0.100 | 0.100 | 0.100 |
| Phytase 10,000 (hostazym-p) | 0.005 | 0.005 | 0.005 |
| Calculated chemical composition (%) | |||
| Crude protein (%) | 22.09 | 20.33 | 18.72 |
| Metabolizable energy (MJ/kg) | 12.20 | 12.34 | 12.57 |
| Dry mater (%) | 87.37 | 86.09 | 86.23 |
| Threonine (%) | 0.838 | 0.788 | 0.734 |
| Methionine (%) | 0.628 | 0.575 | 0.555 |
| Methionine + cysteine (%) | 0.925 | 0.849 | 0.815 |
| Arginine (%) | 1.36 | 1.30 | 1.18 |
| Tryptophan (%) | 0.228 | 0.218 | 0.197 |
| Lysine (%) | 1.26 | 1.14 | 1.04 |
| Calcium (%) | 1.01 | 0.928 | 0.835 |
| Linoleic acid (%) | 1.94 | 1.93 | 1.78 |
| Available phosphorous (%) | 0.503 | 0.449 | 0.406 |
| Na (%) | 0.170 | 0.170 | 0.170 |
- a Provided per kg of diet: folic acid: 1.6 mg, cyanocobalamin: 0.017 mg, biotin: 0.18 mg, niacin: 35 mg, thiamin: 2.5 mg, K (menadione): 3 mg, E (dL-alpha-tocopheryl acetate): 43.55 mg, A (trans-retinyl acetate): 3 mg, D3 (cholecalciferol): 0.112 mg, riboflavin: 5.2 mg, pantothenic acid: 18 mg, pyridoxine: 3.2 mg, copper: 16 mg, iron: 20 mg, manganese: 120 mg, choline choloride: 800 mg and antioxidant: 2.5 mg. Provided per kg of diet: iodine: 1.25 mg, selenium: 0.3 mg and zinc: 110 mg.
2.6 Performance parameters
All chickens of each treatment were weighed at the end of each period. The feeding of the birds was stopped 3 h before weighing. The body weight gain (BWG) of each period was calculated from the difference between the end and beginning weights of chickens in each breeding period. The feed intake (FI) of each period was determined by subtracting the amount of feed remaining at the end of each rearing period from the total feed given during the period. Finally, the feed conversion ratio (FCR) was calculated for each period by dividing the average FI by the average BWG of chickens.
2.7 Serum biochemical parameters
Randomly in each replication, 2 mL of blood was collected from the wing vein (42 days) from 10 selected chickens to determine serum characteristics. Plasma was separated by centrifugation for 15 min at 3000°C and stored at −20°C. The concentrations of total cholesterol (CHOL), serum glucose, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in serum samples were measured using Pars Azmoon laboratory kits and spectrophotometer model CLima 617 Spain (Mohammadi & Ansari Pirsaraei, 2014).
2.8 Immunology
In each cage, at the age of 28 and 35 days old, 1 mL of sheep red blood cells (7% suspension, sheep red blood cell (SRBC)) was injected into the wing vein of two chickens. Blood samples were collected 35 and 42 days after injection. Serum was obtained by centrifugation at 25°C at 1500×g for 15 min and stored at −30°C until assay. The hemagglutination assay was used to measure the total antibody response (IgM + IgG) produced against SRBC. To differentiate between the levels of IgG and IgM, the IgG component was isolated as the 2-mercaptoethanol-resistant antibody. The IgM level was then determined by subtracting the IgG value from the total antibody response, as IgM is sensitive to 2-mercaptoethanol (Cheema et al., 2003).
The number of heterophilus (H) and lymphocyte (L) was determined according to the method of Gross and Siegel (1983) in 42 days. With the injection of phytohemagglutinin (PHA), the effect of treatment on cutaneous basophil hypersensitivity (CBH) in chickens was evaluated using the thickness of the web of the right toe before the injection and at 24 and 48 h after the injection.
At the age of 31 and 42 days, 100 μg of PHA (suspended in 0.1 mL of sterile saline) was injected into the right foot of the bird, between the third and fourth toes. At 31 and 42 days, 0.25 mL of 2, 4-dinitrochlorobenzene (DNCB) solution (10 mg/mL) was spread in a medium consisting of a mixture of acetone and olive oil (4:1) and kept on a 10 cm area of the skin without feathers on the right side, two birds on each side. Skin swelling was calculated by measuring the skin thickness before challenge and 24 and 48 h after challenge using a digital caliper.
2.9 Statistical analysis
The data obtained from the present experiment were analyzed using SAS statistical software (SAS Institute, 2004) and the general linear model (GLM) method, and the mean of the treatments were compared using Duncan's test at a 5% probability level. The statistical model of the design was Yijk = μ + Ai + Bj + AiBj + eijk. In this model, Yijk is the value of each observation, μ is the average of observations, Ai is the essential oil forms (free and nanoencapsulated), Bj is the essential oil levels, AiBj is the interactive effect of essential oil form and the levels, and eijk is the experimental error effect.
3 RESULTS
3.1 Chicken performance
The main effects of GEO shape and level and their interaction on BWG, FI, and FCR in broilers are shown in Table 4. The interaction effect of GEO shape and level on FI in the period of 15 to 28 days was significant (p < 0.05). EO level was 75 mg/kg in 29–42 days and 150 mg/kg in 15–28 days and 1–42 days and showed the lowest FI (p < 0.05). Birds fed with GEO nanocapsules had the lowest FCR values on Days 1–14, 15–28, and 1–42, indicating more efficient growth, the interaction between GEO levels × shape was significant for FCR. At the same time, the lowest FCR values in all periods were for birds fed with 75 mg/kg GEO (p < 0.05).
| Treatment | Main effects | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| GEO form | GEO levels (mg/kg) | p-Value | |||||||||
| Free | Nanoencapsulation | SEM | 0 | 75 | 150 | SEM | GEO form | GEO levels | Form × levels | ||
BWG (g/b/d) |
1–14 days | 23.19b | 24.24a | 0.302 | 22.96b | 24.76a | 23.41b | 0.370 | 0.022 | 0.006 | 0.896 |
| 15–28 days | 53.09b | 56.32a | 0.583 | 54.43 | 55.76 | 53.92 | 0.715 | 0.001 | 0.192 | 0.564 | |
| 29–42 days | 74.94 | 74.37 | 0.885 | 72.32b | 76.09a | 75.57b | 1.08 | 0.653 | 0.045 | 0.0001 | |
| 1–42 days | 50.41b | 51.60a | 0.477 | 49.90b | 52.23a | 50.98ab | 0.585 | 0.034 | 0.003 | 0.037 | |
FI (g/b/d) |
1–14 days | 28.50 | 24.48 | 0.318 | 28.31 | 28.41 | 28.75 | 0.390 | 0.958 | 0.712 | 0.277 |
| 15–28 days | 95.57a | 88.50b | 0.853 | 100.49a | 89.85b | 85.77c | 1.04 | 0.0001 | 0.0001 | 0.001 | |
| 29–42 days | 152.15 | 151.38 | 0.823 | 154.39a | 148.04b | 152.87a | 1.00 | 0.515 | 0.0001 | 0.130 | |
| 1–42 days | 92.10 | 89.13 | 0.719 | 94.40a | 88.68ab | 89.15b | 0.881 | 0.298 | 0.025 | 0.862 | |
FCR |
1–14 days | 1.23a | 1.17b | 0.014 | 1.23a | 1.14b | 1.23a | 0.017 | 0.014 | 0.002 | 0.659 |
| 15–28 days | 1.80a | 1.57b | 0.016 | 1.85a | 1.61b | 1.59b | 0.019 | 0.0001 | 0.0001 | 0.0001 | |
| 29–42 days | 2.03 | 2.04 | 0.022 | 2.13a | 1.95b | 2.03b | 0.027 | 0.685 | 0.0001 | 0.001 | |
| 1–42 days | 1.81a | 1.61b | 0.013 | 1.79a | 1.63b | 1.63b | 0.016 | 0.001 | 0.0001 | 0.023 | |
- Note: a,b,cIn each row, means without superscript or with at least one same superscript do not differ significantly at p > 0.05.
- Abbreviations: BWG, body weight gain; FCR, feed conversion ratio; FI, feed intake; SEM, standard error of the mean.
3.2 Serum biochemical parameters
The effects of different forms of GEO supplementation on Day 42 on glucose, TG, CHOL, HDL-C, and LDL-C levels are shown in Table 5. Dietary supplements with the control group significantly (p < 0.05) increased CHOL and LDL-C in the serum as well as the free GEO group compared to the nanoencapsulated GEO group compared to other treatments. A significant interaction between the shape and level of GEO on serum TG, serum CHOL, and LDL-C has been observed (p < 0.05).
| Main effects | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment | GEO form | GEO levels (mg/kg) | p-Value | |||||||
| Free | Nanoencapsulation | SEM | 0 | 75 | 150 | SEM | GEO form | GEO levels | Form × levels | |
| Glucose (mg/dL) | 171.73 | 182.18 | 5.90 | 188.77 | 167.50 | 174.60 | 7.23 | 0.223 | 0.128 | 0.369 |
| TG (mg/dL) | 94.11b | 120.93a | 4.84 | 114.67 | 99.10 | 108.80 | 5.93 | 0.001 | 0.195 | 0.001 |
| CHOL (mg/dL) | 140.00 | 140.13 | 2.58 | 144.90a | 133.00b | 141.30ab | 3.16 | 0.971 | 0.034 | 0.0001 |
| HDL-C (mg/dL) | 51.25 | 51.03 | 0.214 | 51.39 | 50.96 | 51.06 | 0.262 | 0.480 | 0.485 | 0.168 |
| LDL-C (mg/dL) | 157.17b | 158.83a | 0.359 | 159.98a | 157.52b | 159.50b | 0.440 | 0.003 | 0.0001 | 0.001 |
- Note: a,bIn each row, means without superscript or with at least one same superscript do not differ significantly at p > .05.
- Abbreviations: CHOL, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SEM, standard error of the mean; TG, triglyceride.
3.3 Immunological tests
Seven days after the first and second SRBC injection, anti-SRBC and IgM increased with the group fed GEO with nanocapsules. The lowest IgG and the highest IgM were observed in control-fed birds 7 days after the second SRBC. Table 6 shows the effects of GEO shape and level and their interaction on antibody response to SRBC at 35 and 42 days. Seven days after the second SRBC injection, the interaction between shape × GEO level was dominant, and there was no significant difference between treatments. However, a significant interaction was observed between EO shape and level on anti-SRBC titers in the first SRBC injection (p < 0.05).
| Treatment | Main effects | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| GEO form | SEM | GEO levels (mg/kg) | SEM | p-Value | ||||||
| Free | Nanoencapsulation | 0 | 75 | 150 | GEO form | GEO levels | Form × levels | |||
| 7 days after first SRBC injection | ||||||||||
| ImT | 3.46b | 4.13a | 0.194 | 3.50 | 4.30 | 3.60 | 0.238 | 0.023 | 0.052 | 0.012 |
| IgG | 3.00 | 3.06 | 0.166 | 2.70 | 3.20 | 3.20 | 0.204 | 0.780 | 0.157 | 0.239 |
| IgM | 0.466b | 1.06a | 0.170 | 0.800 | 1.10 | 0.400 | 0.208 | 0.020 | 0.078 | 0.147 |
| 7 days after second SRBC injection | ||||||||||
| ImT | 4.60b | 5.66a | 0.149 | 5.10 | 5.30 | 5.00 | 0.182 | 0.001 | 0.506 | 0.506 |
| IgG | 4.06 | 4.26 | 0.133 | 3.50b | 4.50a | 4.50a | 0.163 | 0.299 | 0.001 | 0.243 |
| IgM | 0.533b | 1.40a | 0.202 | 1.60a | 0.800ab | 0.500b | 0.248 | 0.006 | 0.013 | 0.947 |
- Note: a,bIn each row, means without superscript or with at least one same superscript do not differ significantly at p > .05.
- Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; IgT, immunoglobulin total; SEM, standard error of the mean.
The H count and H:L ratio on Day 42 of the growth period were significantly decreased in the nanoencapsulated GEO group (Table 7). Tables 8 and 9 show the skin's response to swelling of DNCB and toe web by PHA shape and GEO level and their interaction in 31- and 42-day-old broiler chickens. PHA and DNCB levels were higher for chickens fed with 100 mg/kg nanoencapsulated GEO (p < 0.05) on Day 31 (24 h post-challenge). Also, there was a significant interaction between GEO form and surface-on-skin response to DNCB at 48 h post-challenge (Day 42).
| Treatment | Main effects | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| GEO form | SEM | GEO levels (mg/kg) | SEM | p-Value | ||||||
| Free | Nanoencapsulation | 0 | 75 | 150 | GEO form | GEO levels | Form × levels | |||
| H, % | 15.33a | 13.80b | 0.391 | 15.20 | 14.40 | 14.10 | 0.479 | 0.011 | 0.265 | 0.134 |
| L, % | 84.73 | 85.76 | 0.476 | 84.75 | 85.20 | 85.80 | 0.583 | 0.138 | 0.455 | 0.285 |
| H:L | 0.181a | 0.161b | 0.005 | 0.179 | 0.169 | 0.164 | 0.006 | 0.011 | 0.261 | 0.117 |
- Note: a,bIn each row, means without superscript or with at least one same superscript do not differ significantly at p > .05.
- Abbreviations: H, heterophilus; H:L, heterophilus:lymphocyte ratio; L, lymphocyte; SEM, standard error of the mean.
| Treatment | Main effects | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| GEO form | SEM | GEO levels (mg/kg) | SEM | p-Value | ||||||
| Free | Nanoencapsulation | 0 | 75 | 150 | GEO form | GEO levels | Form × levels | |||
| 24 h post-challenge | ||||||||||
| PHA | 1.70 | 1.75 | 0.069 | 1.53b | 1.88a | 1.76ab | 0.085 | 0.590 | 0.022 | 0.953 |
| DNCB | 1.44 | 1.52 | 0.031 | 1.42b | 1.56a | 1.46ab | 0.038 | 0.095 | 0.040 | 0.937 |
| 48 h post-challenge | ||||||||||
| PHA | 0.791 | 0.826 | 0.043 | 0.809 | 0.854 | 0.762 | 0.052 | 0.578 | 0.481 | 0.460 |
| DNCB | 0.702 | 0.753 | 0.023 | 0.723 | 0.758 | 0.702 | 0.029 | 0.144 | 0.400 | 0.333 |
- Note: a,bIn each row, means without superscript or with at least one same superscript do not differ significantly at p > .05.
- Abbreviations: DNCB, 2, 4-dinitrochlorobenzene; PHA, phytohemagglutinin; SEM, standard error of the mean.
| Treatment | Main effects | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Essential oil form | Essential oil levels (mg/kg) | p-Value | ||||||||
| Free | Nanoencapsulation | SEM | 0 | 75 | 150 | SEM | EO form | EO levels | Form × levels | |
| 24 h post-challenge | ||||||||||
| PHA | 1.52 | 1.60 | 0.040 | 1.55 | 1.62 | 1.52 | 0.049 | 0.192 | 0.357 | 0.546 |
| DNCB | 1.50b | 1.62a | 0.039 | 1.52 | 1.55 | 1.61 | 0.048 | 0.043 | 0.421 | 0.842 |
| 48 h post-challenge | ||||||||||
| PHA | 0.904 | 1.01 | 0.044 | 0.985 | 1.02 | 0.870 | 0.054 | 0.093 | 0.141 | 0.842 |
| DNCB | 0.720 | 0.825 | 0.038 | 0.719 | 0.844 | 0.753 | 0.047 | 0.066 | 0.178 | 0.049 |
- Note: a,bIn each row, means without superscript or with at least one same superscript do not differ significantly at p > 0.05.
- Abbreviations: DNCB, 2, 4-dinitrochlorobenzene; PHA, phytohemagglutinin; SEM, standard error of the mean.
4 DISCUSSION
Diallyl trisulfide, diallyl disulfide, methyl allyl disulfide, and allyl n-propyl sulfide were determined to be the major components of GEO, whereas other compounds were unknown. Many of these components have been reported in the past as major GEO escape components (Abu-Lafi et al., 2004).
No information was found about the effect of nanoencapsulated GEO on chicken performance and healthy intestinal function. Beneficial effects of free-form GEO dietary supplements have been reported. Karangiya et al. (2016) reported that feeding a diet supplemented with 1% sun-dried and ground garlic resulted in higher performance compared to the control diet, which was due to the presence of plant secondary metabolites including isoprene derivatives and flavonoids, and their effects were attributed to intestinal microflora and nutrient metabolism, which can increase the secretion of endogenous enzymes, thus improving performance. The use of garlic powder in the diet also improved digestibility in broilers (Isa, 2011).
Williams (2001) reported that herbal essential oils might stimulate nutrient digestibility by enhancing digestive enzymes production and promoting liver function. Improved performance of birds fed high levels of garlic in diet can also be attributed to the antibacterial properties of allicin and 1, 8-cineole compounds in garlic extract. These compounds improve intestinal microflora and consequently increase weight gain by reducing competition for nutrients between host and pathogens. Effective components of medicinal herbs and spices not only stimulate digestion but also contribute to the well-being of animals and improve their performance by affecting other physiological functions (Lewis et al., 2003).
Nanoencapsulation is a method that protects free essential oils from harmful factors, including excessive temperature, moisture, and drying, which can lead to controlled release under particular conditions (Hosseini & Meimandipour, 2018). In the present study, the inclusion of nanoencapsulated GEO at a concentration of 150 mg/kg in diets from 1 to 42 days significantly decreased BWG and increased FCR compared to its lower level of the nanoencapsulated counterpart (75 mg/kg), while it had no effect. FI seems to be the main reason for these observations because of the high amount of nanoencapsulated GEO (150 mg/kg) in the diet. Therefore, nanoencapsulation can improve growth through improved essential oil delivery and antimicrobial properties of chitosan (Hosseini & Meimandipour, 2018; Meimandipour et al., 2017). This finding suggests that lower level of essential oil is needed by protecting essential oil using nanoencapsulation technology.
The current study found that GEO in free form decreased blood TG and LDL-C levels in broilers (Table 5). The effects of garlic on blood lipids have been attributed to the inhibition of the liver enzymes (Squalene Monooxygenase and HMG-CoA reductase) responsible for CHOL synthesis (Rahman & Lowe, 2006). The effect of consuming garlic on lowering CHOL level is dependent on the presence of water-soluble sulfur compounds in garlic, especially s-allyl-L cysteine Yeh and Liu (2001). However, CHOL-lowering mechanisms of probiotics have not been fully determined. Multiple mechanisms, such as imbibition of CHOL by binding and incorporation into the cell membrane, hydrolase, and the prohibition of hepatic CHOL synthesis by short-chain fatty acids, have been proposed (Cho & Kim, 2015). The results of this study reverberate those of Cho and Kim (2015) who also found improved efficacy of probiotics on total CHOL and LDL-C relative to HDL-C rates.
In the present study, various treatments such as GEO in the form of nanocapsules exerted positive effects on the immune system of vaccinated chickens by increasing the anti-SRBC IgM response (Table 6). These results mirror those of Rahimi et al. (2011) who also found that 0.1% garlic extract supplementation improved the antibody response to SRBC. Ao et al. (2011) found that fermented garlic powder improved the immune system function by increasing IgG and lymphocyte levels. Effective compounds found in medicinal plant essential oil can affect the immune system. Garlic is an excellent source of selenium, which is an important cofactor of glutathione peroxidase, which is essential for fighting and preventing cell damage (Surai, 2006). In addition, Lau et al. (1991) reported that sulfur compounds in garlic had immune-modulating properties. Immune system stimulants such as garlic can directly produce antimicrobial molecules and activate nonspecific defense mechanisms by a direct effect on receptors and intracellular gene activator release (Bricknell & Dalmo, 2005).
The current study found that dietary treatment affects H and H:L ratio. Mohebbifar and Torki (2011) did not find any effect of garlic supplementation (200 mg/kg diet) on heterophils count in Ross broiler chickens. In the present study, broilers given diet supplemented with nanoencapsulated GEO in both levels showed numerically lower percentages of H and consequently H:L ratio. This seems to be associated with efficient delivery of GEO via nanoencapsulation that decreases the level of stress compared to other treatments. Birds under stress have been reported to show that an increase in the H:L ratio is a more reliable predictor of mild and moderate stress than plasma cortisone concentration (Maxwell, 1993).
In the present study, nanoencapsulated GEO improved cellular responses in birds at 31 and 42 days. Although very few papers were found on the effect of garlic on cellular immune response, previous studies showed that feeding broiler chickens with other phenolic compounds, such as thymol and carvacrol (Hashemipour et al., 2013) increased the CBH response to PHA-P injection. Basophils and mast cells play a major role in allergy. In fact, by increasing inflammatory cells, these cells increase the secretion of fluids at the site of inflammation and facilitate the recruitment and entry of other immune cells such as macrophages and lymphocytes to the site of infection (Holgate, 2000). The main cells involved in the CBH response are basophils and mast cells. Further research is needed to investigate these GEO-encapsulated features.
The most obvious finding from this study is that the inclusion of nanoencapsulated GEO at 75 mg/kg in the diet improved both the performance and immunity of broilers compared to the control-fed group. Therefore, the above treatments can be recommended as an alternative to antibiotics to increase broiler performance. Furthermore, compared to the use of free-form GEO, nanoencapsulation halved the surface area of GEO for dietary supplements possibly through increased GEO shelf life, effective delivery, and synergistic effects in combination with chitosan.
ACKNOWLEDGMENTS
This research was done with the personal funding of the authors and the authors thank all the teams who worked on the experiments and provided results during this study.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
