A blend of microencapsulated essential oils improves performance, economic index, meat fatty acid profile, carcass characteristics, and immune index of broilers

Abstract

This experiment investigated the impact of the blend of microencapsulated essential oils (BMEOs) on performance, economic index, meat fatty acid profile, carcass characteristics, and immune index in broilers. In a completely randomized design, a total of 640 (mixed-sex) one-day-old Ross 308 broilers were divided into 4 treatments with 10 replicates (16 chicks/replicate) for 42 days. Treatments were basal diet supplemented with BMEOs at levels of 0 (control), 300 (BMEOs-300), 600 (BMEOs-600), and 900 (BMEOs-900) mg/kg of diet. BMEOs contain equal ratios of EOs of eucalyptus (compounds: α-pinene, γ-terpinene, 1,8-cineol, limonene, and α-terpineol), oregano (compounds: carvacrol, thymol, ρ-cymene, linalool, ethyl caprate, ledol, and β-bisabolene), rosemary (compounds: 1,8-cineol, α-pinene, borneol, camphor, verbenone, bornyl acetate, limonene, camphene, and β-myrcene), and thyme (main compounds: thymol, carvacrol, ρ-cymene, γ-terpinene, and β-caryophyllene). The result showed that during the whole experimental period, the BMEOs-900 treatment increased (P < 0.05) average daily body weight gain (ADBWG; 69.3 vs. 62.0 g/chick), European production efficiency factor (EPEF) (437.9 vs. 359.9), European broiler index (EBI) (269.8 vs. 221.3), and decreased (P = 0.001) feed conversion ratio (FCR) (1.60 vs. 1.75 g/g) compared to the control and other treatments. The diet containing BMEOs-900 increased the relative weight of the bursa of Fabricius (57.3%), spleen (51.6%), and thymus (123.2%) as immune organs, proventriculus (34.2%), and pancreas (49%) (P < 0.05), as well as lowered the relative weight of the abdominal fat (75.1%, P = 0.001) compared with broilers in the control and other treatments. Broilers in the BMEOs-900 group showed higher (P = 0.001) levels of the total polyunsaturated fatty acids (ΣPUFA; 1.33-fold in breast, 1.26-fold in thigh), Σ(omega-6) n-6 PUFA (1.20-fold in breast, 1.35-fold in thigh), Σ(omega-3) n-3 PUFA (1.45-fold in breast, 1.76-fold in thigh), and PUFA/saturated fatty acid (SFA) ratio (1.74-fold in breast, 1.45-fold in thigh), as well as lower (P = 0.001) levels of ΣSFA (1.30-fold in breast, 1.16-fold in thigh) and n-6 PUFA/n-3 PUFA (1.21-fold in breast, 1.33-fold in thigh) ratio in breast and thigh meat compared to the control and other treatments. Therefore, the active and phenolic components of EOs may have a positive effect on the broiler’s body fatty acid profile by increasing its body antioxidant function and the fatty acid β-oxidation process. In conclusion, based on the observations in this trial, BMEOs-900 could serve as a feed additive to enhance growth performance, economic index, meat and carcass quality, and boost immune status in poultry diets.

The blend of microencapsulated essential oils improves performance and economic index, meat fatty acid profile, as well as carcass characteristics and the immune index in broilers.

Introduction

The alarming increase in antibiotic resistance among pathogens in animals and poultry has raised concerns about the potential transfer of this resistance through the food chain to human pathogens. At least about 61% of all human pathogens originate from animals, making this a significant public health issue (Ambrosio et al. 2017). However, removing antibiotic growth promoters (AGPs) may result in higher production costs. The push for alternatives to AGPs has prompted nutritionists and feed manufacturers to explore phytogenic compounds as one possible option (Safari et al. 2016a). Additionally, poultry products should ensure low lipid levels and a higher content of polyunsaturated fatty acids (PUFA), such as omega-3 (n-3) PUFA, a balanced omega-6 (n-6) PUFA/n-3 PUFA ratio, and an appropriate PUFA/saturated fatty acids (SFA) ratio (PUFA/SFA) (Shen et al. 2019). It has been suggested that they can lower the risk of some metabolic diseases, including diabetes, hypertension, coronary artery disease, autoimmune, and inflammatory diseases (Shen et al. 2019). Modifying the fatty acid composition of meat through feeding provided acceptable results in poultry (Shen et al. 2019). As an alternative to antibiotics, the essential oils (EOs) are volatile materials with odorous impacts, hydrophobic esters, short-chain lipophilic compounds, and low molecular weight aliphatic hydrocarbons, such as phenols and aldehydes (Ebani et al. 2016; Cui et al. 2019). EOs are aromatic, oily liquids extracted from various parts of the plant (Ebani et al. 2016; Cui et al. 2019). Bioactive compounds of essential oils have various beneficial effects on poultry. For example, it has been reported that the addition of thymol and carvacrol to broiler diets may improve intestinal morphology and function, as well as absorption and transport capacity, and improve barrier function and inflammation (Li et al. 2023). Therefore, it can enhance growth performance. In addition, 1,8-Cineol has a protective effect against oxidative stress in broilers (Di et al. 2022), and α-Pinene decreases quorum sensing and the colonization of broilers by Campylobacter jejuni (Šimunović et al. 2020). Phytogenic feed additives, derived from plants such as EOs, whether as a single compound or in multiple combinations, are natural, less toxic, residue-free, and could serve as acceptable feed additives for animals and poultry instead of inorganic antibiotics or organic chemicals. Additionally, EOs demonstrate biological activities that stimulate nutrient digestibility, exhibit antimicrobial effects, reduce inflammation, promote growth, regulate immune function, provide antioxidant, antiprotozoal, and antifungal benefits (Ebani et al. 2016; Cui et al. 2019). Moreover, supplementing plant EOs in a non-coated form at high doses, due to the active molecules and strong smell, repellent smell, and bitter taste, decreases the palatability and feed intake, which may impact poultry performance (Farouk et al. 2020; Temiz and Özturk 2021; Paul et al. 2023). Therefore, microencapsulation of EO is a technique used for various purposes, including increasing the stability and bioavailability of bioactive components, solidifying liquids, preventing the loss of volatile compounds, and masking undesirable tastes and odors. Also, extending the effect period, preserving their structure, reducing loss during feed processing, lowering the effective dose, decreasing oxidation, mitigating the negative effects of environmental factors, and enabling controlled release to target tissues are the targets of microencapsulation of EO (Temiz and Özturk 2021; Paul et al. 2023). Moreover, non-coated EOs are primarily metabolized in the proximal part of the gastrointestinal tract and rarely reach the distal part of the gastrointestinal tract. In contrast, the microencapsulated EOs may reach the lower digestive tract without being impacted by the acidic and other special environmental conditions of the upper digestive tract. This could lead to better absorption of nutrients, resulting in products with high bioavailability, as well as enhanced intestinal health and performance, and reduced survival rates of enteric pathogens (Temiz and Özturk 2021; Paul et al. 2023). Among the potential candidates are the EOs of the oregano (Origanum vulgare L.), rosemary (Rosmarinus officinalis L.), thyme (­Thymus vulgaris L.), and eucalyptus (Eucalyptus globulus). Their main biologically active compounds are as follows: thymol, carvacrol, 1,8‐cineol, α-pinene, ρ-cymene, γ-terpinene, β-caryophyllene, limonene, α-terpineol, linalool, ethyl caprate, ledol, β-bisabolene, borneol, camphor, verbenone, bornyl acetate, camphene, and β-myrcene (shown in Table 1). Previous studies have identified the positive effects of supplementing the volatile EOs, microencapsulated and/or encapsulated EOs in broilers’ diets, such as improving the growth performance parameters, carcass characteristics, immune organs index and fatty acid profile of meat (Cross et al. 2007; Hashemipour et al. 2013; Hafeez et al. 2016; Kumar et al. 2018; El-Ashram and Abdelhafez 2020; Galli et al. 2020; Mohebodini et al. 2021; Amer et al. 2022; Islam et al. 2022; Johnson et al. 2022; Nameghi et al. 2022; Seidavi et al. 2022; Meligy et al. 2023; Vlaicu et al. 2023). As far as the writers are aware, there is no information about the formation of the blend of microencapsulated essential oils (BMEOs) from thyme, rosemary, oregano, and eucalyptus, and its effect on the performance, economic index, meat fatty acid profile, carcass characteristics, and immune index in broilers. Therefore, this study aimed to produce BMEOs and investigate these parameters.

Table 1.

Analysis of the chemical compositions (%) of the various essential oils (EOs).

Compound	Oregano EO	Thyme EO	Eucalyptus EO	Rosemary EO
Carvacrol	62.11	51.31	ND	ND
Thymol	5.41	21.12	ND	ND
ρ-Cymene	7.234	9.422	3.641	2.761
Linalool	3.521	2.536	0.102	2.127
Ethyl caprate	3.567	ND	ND	ND
α-Pinene	1.785	0.972	13.16	16.01
β-Myrcene	2.125	1.012	0.843	2.015
γ-Terpinene	3.743	5.402	6.572	2.072
Ledol	1.451	ND	ND	ND
β-Bisabolene	1.632	ND	ND	ND
(E)-β-Farnesene	0.152	ND	ND	ND
α-Terpinene	0.994	0.721	0.947	1.866
β-Pinene	0.314	1.071	0.928	0.887
Camphene	0.271	0.196	0.124	3.147
1,8‐Cineol	0.134	0.637	63.31	40.67
β-Caryophyllene	1.311	2.031	1.016	2.136
1-Octen-3-ol	0.446	0.384	ND	ND
Borneol	0.362	1.012	1.245	5.635
cis-Sabinenehydrat	0.251	ND	ND	ND
α-Phellandrene	0.182	0.124	0.571	0.443
1-Terpinen-4-ol	0.421	ND	0.567	ND
α-Thujene	0.743	0.357	0.401	0.072
Limonene	ND	0.152	1.532	4.158
α-Terpineol	ND	0.233	3.017	ND
Terpinolene	ND	ND	0.764	0.405
Viridiflorene	ND	ND	0.211	ND
δ- 3- Carene	ND	ND	ND	1.637
Camphor	ND	ND	ND	5.105
Verbenone	ND	ND	ND	4.751
Bornyl acetate	ND	ND	ND	2.442
Total of identified compositions	98.159	98.692	98.951	98.339

ND: Not detected.

Materials and methods

All experimental protocols and sample collection methods used in the current study were approved by the Animal Ethics Committee of the University of Guilan (code H98718/S16), Rasht, Iran.

Preparation, chemical compositions, and microencapsulation of EOs

Four EOs, including thyme, rosemary, oregano, and eucalyptus, were bought directly from the Barij Essence Company (Kashan, Iran). Gas chromatography-mass spectrometry (GC-MS) (GC, Hewlett-Packard, HP-6890, Palo Alto, CA, USA; MS, Hewlett-Packard 7953, CA, USA) was used to determine the chemical compositions of EOs using the previously described method (Puvača et al. 2021), and the data can be found in Table 1. Briefly, the Agilent HP-6890 GC equipped with a 7953 EI/CI mass spectrometer detector (MSD) and flame ionization detector (FID) connected by a capillary flow technology 2-way splitter with make-up was used for the GC-MS analysis. The capillary column HP-5MS (30 m in length, internal diameter 0.25 mm, film thickness 0.25 μm) was used. The GC oven’s temperature was set vary from 60 to 300°C at a rate of 3°C per minute and maintained for a duration of 15 minutes. Helium was utilized as the carrier gas at a flow rate of 1.0 mL/min at 16.255 psi (constant pressure mode). Also, 1 μL of the EO sample was injected into the GC-MS using a split-splitless injector. The split ratio was 1:50. The splitless mode was used to analyze the sample. Both the injector and the detector were operating at 300°C. MS data were collected in EI mode with a scan range of 30–550 m/z, an ionization source temperature of 230°C, and a quadruple temperature of 150°C; the solvent delay was 3 minutes. The EO compounds were identified in the analyses by comparing their mass spectra and linear retention indices (with respect to C8–C36 n-alkanes on the HP-5MSI column) with those of authentic standards from the NIST 21, NIST 11, NIST 107, and NIST 14 databases.

The EOs were microencapsulated using an ionic gelation process, following the method previously described (Sawtarie et al. 2017). Briefly, deionized water was used for all steps of the EO microencapsulation. The chitosan (CS) with medium molecular weight as a coating polymer and sodium triphosphate pentabasic (TPP) were bought from Sigma-Aldrich. CS was dissolved in 1% (w/v) acetic acid solution at a concentration of 1 mg/mL and then sonicated. The solution was stirred overnight at room temperature for complete dispersion of CS. Then, before being sterilized at 121°C for 15 min to remove impurities, the produced solution was filtered (filter paper; Calbiochem-Novabiochem Crop, USA). The dropwise increase of 10 ml TPP-solution (1 mg/mL) to a 25 mL CS solution at pH 5, under constant stirring conditions at room temperature. This resulted in the formation of CS-TPP particles by the ionic gelation mechanism. For the production of CS-TPP particles loaded with EO, 20% (w/v) EO was added to the CS-solution before adding the TPP-solution. Microencapsulation efficiency was above 96%, and the size of microencapsulated EOs was 4 ± 0.3 μm.

Broilers, diets, and management

The study was carried out at the poultry research farm and the laboratory for feeding of the University of Guilan in Rasht, Iran. Based on a completely randomized design, a total of 640 1-day-old mixed-sex broiler chickens (Ross-308) were obtained from a commercial hatchery. Broiler chickens with outlier initial body weight (BW) (above or below the flock mean BW ±1 standard deviation) were removed to achieve initial BW uniformity. Birds with similar average initial body weight (42.3 ± 1.0 g) were randomly allocated to 4 treatments of 10 replicate pens each containing 16 birds (8 male and 8 female). Each pen (1 m wide, 1.85 m long, and 1 m high) was equipped with nipple drinkers, a hanging feed dish, and a fresh wood shavings litter, and the birds were allowed to consume feed (mash form diet) and water ad libitum for a 42-day experiment. The guidelines of the Ross 308 Breeding Guidance Manual on temperature, relative humidity, photoperiod, and household environmental conditions were followed (Aviagen 2019). The broilers received vaccinations against Newcastle (Lasota; days 1, 8, and 18), infectious bronchitis (H120; day 1), influenza (H5; day 1), and Gumboro (IBD 071 IR; day 16). The vaccines have all been acquired from the Razi Institute of Serums and Vaccines (Karaj, Iran). As shown in Table 2, the basal diets were formulated according to the Ross 308 broiler chicken nutrient requirements guidelines (Aviagen 2019). The broiler chicks received the experimental diets in three rearing phases, including starter (1–10 days), grower (11–24 days), and finisher (25–42 days). The following is how the experimental dietary treatments were created. A basal diet consisting of soybean meal and corn containing a blend of microencapsulated essential oils (BMEOs) at levels of 0 (control), 300 (BMEOs-300), 600 (BMEOs-600), and 900 (BMEOs-900) mg/kg of diet. BMEOs contain an equal ratio of eucalyptus, oregano, rosemary, and thyme EOs.

Table 2.

Ingredients and nutrient composition of the basal diets.

Ingredients (% as-fed basis)	Starter (day 1–10)	Grower (day 11–24)	Finisher (day 25–42)
Corn	52.96	56.51	60.23
Soybean meal (CP, 44%)	40.12	35.25	31.68
Corn gluten meal	0	1	0
Soybean oil	2.44	3.18	4.34
Dicalcium phosphate	1.9	1.7	1.52
Calcium carbonate	0.95	0.88	0.82
Sodium bicarbonate	0.21	0.19	0.30
Sodium chloride	0.23	0.24	0.13
Mineral and Vitamin Premixa	0.5	0.5	0.5
DL-Met	0.34	0.28	0.26
L-Lys HCL	0.24	0.20	0.17
L-Thr	0.11	0.07	0.05
Total	100	100	100
Nutrient composition
Metabolizable energy, kcal/kg	2900	3000	3100
Crude protein, %	22.23	20.81	18.89
Lys, %	1.39	1.25	1.12
Met, %	0.69	0.62	0.57
Met + Cys, %	1.04	0.96	0.88
Thr, %	0.94	0.85	0.76
Calcium, %	0.93	0.84	0.77
Available phosphorus, %	0.46	0.42	0.38
Sodium, %	0.16	0.16	0.16
Chloride, %	0.23	0.23	0.23

^aSupplied per kg diet: vitamin E, 36.75 U; vitamin A, 11000 U; vitamin B2, 5.25 mg; vitamin K3, 3.4 mg; vitamin B1,1.98 mg; vitamin D3, 5000 U; vitamin B6, 2.87 mg; vitamin B12, 0.024 mg; choline, 800 mg; pantothenic acid, 10.5 mg; biotin, 0.105 mg; folic acid, 1.2 mg; niacin, 31.5 mg; selenium, 0.3 mg; iron, 50 mg; zinc, 100 mg; I, 1.3 mg; copper, 12 mg; manganese, 120 mg; antioxidant, 100 mg.

Performance and economic index

The BW, feed intake (FI), average daily BW gain (ADBWG), average daily FI (ADFI), and feed conversion ratio (FCR) of birds of each replicate within each treatment were recorded weekly, and periodically for the starter, grower, and finisher phases, as well as the entire experimental period. Every day, the mortality rate in each replicate was noted. At the end of the experiment (d 42), the economic index, including the European Production Efficiency Factor (EPEF) and the European Broiler Index (EBI), was calculated using the following formulas (Safari et al. 2024a).

$$\begin{array}{l}EPEF=\left[(Viability\%)\times (BW(kg)/(Age(d))\right.\\ \left.\times (FCR(kgfeed/kggain))\right]\times 100.\end{array}$$

$$\mathit{EBI} = \left[\left(\mathit{Viability}\%\right)\times (\mathit{average} \mathit{daily} \mathit{gain}\left(g/\mathit{chick}/\mathit{day}\right)\right)/(\mathit{FCR} \left(kg \mathit{feed}/kg \mathit{gain}\right))\times 10]$$

Carcass characteristics and immune organs

On the 42nd day of the experiment, four birds per replicate were randomly selected based on their weight, which was closest to the pen average BW, and weighed individually before slaughter and defeathering. The weight of the eviscerated carcass, pancreas, proventriculus, heart, abdominal fat, gizzard, liver, and immune organs (bursa of Fabricius, spleen, and thymus) was weighed and calculated as a relative percentage of live BW ([(g of organ weight/g of live BW) × 100]). The weight of the breast and thigh was expressed as a percentage of the eviscerated carcass weight ([(g of breast or thigh weight/g of eviscerated carcass weight) × 100]) (Safari et al. 2024b).

Determination of fatty acid profiles of breast and thigh meat

On day 42, four birds (closest to the average BW of each replicate) from each replicate were slaughtered. The left-hand breast and thigh meat were sampled and vacuum packaged in plastic bags, then stored at -20°C. The fatty acid profile of the breast and thigh muscles was determined by gas ­chromatography (Hewlett-Packard, HP-6890, Palo Alto, CA, USA) method, which was described previously (Wu et al. 2022).

Statistical analysis

Shapiro-Wilk’s test verified the distribution normality, and Levene’s test verified the variance homogeneity of the data. The one-way ANOVA model was used for analyzing the data of meat fatty acid profile, carcass characteristics, economic index, and immune index in a completely randomized design by the General Linear Model (GLM) procedure in SAS (Statistical Analysis System) software (SAS 9.2; Institute Inc., Cary, NC, USA). Data related to the performance were analyzed using the repeated measures model with the mixed procedure (PROC MIXED) of SAS software (SAS 9.2; Institute Inc., Cary, NC, USA). This accounts for variation over time, dietary treatment as a fixed effect, and pen as a random effect. Each pen in each treatment served as the experimental unit for performance and economic index, while the individual sampled broiler was set as the experimental unit for meat fatty acid profile, carcass characteristics, and immune index statistical analysis. Significant differences among experimental treatment means were identified by post-hoc Tukey’s multiple-range tests and declared at P <0.05.

Results

Performance and economic index

As shown in Table 3, during the starter and finisher phases, the BMEOs-900 treatment increased (P < 0.05) ADBWG but reduced (P < 0.05) FCR compared to the control and other treatments. In the current study, ADFI was not influenced by various dietary treatments (P > 0.05). During the grower phase, no treatment impact was observed on FCR (P > 0.05); however, at the same time, the BMEOs-900 treatment increased (P < 0.05) ADBWG compared with the BMEOs-300 and control groups. During the whole experimental period (day 1–42), the BMEOs-900 treatment increased (P < 0.05) ADBWG and decreased (P < 0.05) FCR compared to the control and all other treatments. During the entire grow-out period (day 1–42), BMEOs-600 treatment had a higher (P < 0.05) economic index, including EPEF and EBI, compared with the control and BMEOs-300 treatments; while BMEOs-900 had the highest (P < 0.05) EPEF and EBI in comparison to the control and all other treatments.

Table 3.

The effect of different concentrations of a blend of microencapsulated essential oils (BMEOs) supplementation on the performance and economic index of broilers (n = 160 broilers per treatment).

Parameters1	Treatments2
	Control	BMEOs-300	BMEOs-600	BMEOs-900	SEM	P-value
Initial BW, g/chick	42.00	41.91	41.86	41.89	0.070	0.502
Starter phase (day 1–10)
ADFI, g/chick	30.10	30.72	30.71	31.15	0.650	0.461
ADBWG, g/chick	22.34b	22.93b	23.21b	26.16a	0.468	0.001
FCR, g/g	1.34a	1.34a	1.32a	1.19b	0.037	0.003
Grower phase (day 11–24)
ADFI, g/chick	89.50	90.05	91.14	91.25	2.602	0.892
ADBWG, g/chick	60.21b	60.86b	62.11ab	65.74a	1.785	0.004
FCR, g/g	1.49	1.48	1.47	1.39	0.063	0.372
Finisher phase (day 25–42)
ADFI, g/chick	167.60	168.86	171.29	171.72	3.037	0.402
ADBWG, g/chick	85.58b	86.42b	89.07b	96.11a	1.846	0.001
FCR, g/g	1.95a	1.95a	1.92a	1.78b	0.026	0.001
Overall phase (day 1–42)
ADFI, g/chick	108.83	109.70	111.10	111.43	1.614	0.326
ADBWG, g/chick	62.07c	62.78c	64.40b	69.33a	0.532	0.001
FCR, g/g	1.75a	1.74a	1.72a	1.60b	0.018	0.001
Economic index (day 1–42)
EPEF	359.90c	365.25c	379.34b	437.93a	5.249	0.001
EBI	221.37c	224.71c	233.47b	269.83a	3.257	0.001

^{a, b, c}Significant differences exist among means with different superscripts within a row (P <0.05); SEM, Standard error mean.

¹BW, Body weight; ADFI, Average daily feed intake; ADBWG, Average daily body weight gain; FCR, Feed conversion ratio; EPEF, European production efficiency factor; EBI, European broiler index.

²Control, Basal diet (without BMEOs); BMEOs-300, Basal diet contains 300 mg/kg BMEOs; BMEOs-600, Basal diet contains 600 mg/kg BMEOs; BMEOs-900, Basal diet contains 900 mg/kg BMEOs.

Carcass characteristics and immune organs

As illustrated in Table 4, dietary supplementation with various levels of BMEOs had no significant effect (P > 0.05) on the relative weights of the carcass, breast, thigh, heart, gizzard, and liver as compared with the control group. Broilers fed the diet containing BMEOs-900 had a significantly higher relative weight of the immune organs (bursa of Fabricius, spleen, and thymus), pancreas, and proventriculus compared to the control and all other treatments (P < 0.05). The lowest relative weight of the abdominal fat was found in birds fed on diets containing BMEOs-900 compared to the control and all other treatments (P < 0.05).

Table 4.

The effect of different concentrations of a blend of microencapsulated essential oils (BMEOs) on the relative weight of the carcass, internal, and immune organs of broilers (n = 4 broilers per replicate) at 42 d of age.

Parameters1	Treatments2
	Control	BMEOs-300	BMEOs-600	BMEOs-900	SEM	P-value
Carcass and internal organs
Carcass	74.84	75.10	77.24	77.25	0.402	0.074
Breast	23.66	24.15	24.08	25.31	0.335	0.244
Thigh	22.41	22.92	23.12	24.03	0.319	0.367
Heart	0.527	0.524	0.547	0.552	0.006	0.361
Gizzard	1.51	1.52	1.54	1.55	0.024	0.911
Liver	1.68	1.68	1.70	1.70	0.032	0.986
Abdominal fat	2.33a	2.21a	1.63b	1.33c	0.041	0.001
Proventriculus	0.368b	0.372b	0.380b	0.494a	0.005	0.001
Pancreas	0.200c	0.203c	0.248b	0.298a	0.003	0.001
Immune organs
Bursa of Fabricius	0.190c	0.193c	0.238b	0.299a	0.003	0.001
Thymus	0.301c	0.310c	0.515b	0.672a	0.012	0.001
Spleen	0.120c	0.121bc	0.130b	0.182a	0.002	0.001

^{a, b, c}Significant differences exist among means with different superscripts within a row (P <0.05); SEM, Standard error mean.

¹Breast and thigh were calculated as a relative percentage of eviscerated carcass weight, while other parameters were calculated as a relative percentage of live body weight.

²Control, Basal diet (without BMEOs); BMEOs-300, Basal diet contains 300 mg/kg BMEOs; BMEOs-600, Basal diet contains 600 mg/kg BMEOs; BMEOs-900, Basal diet contains 900 mg/kg BMEOs.

Fatty acid profile of breast and thigh meat

As noted in Table 5, the concentration of total monounsaturated fatty acids (ΣMUFA) was not influenced by dietary treatments in breast and thigh meat (P > 0.05). Broilers in the BMEOs-900 group showed higher (P < 0.05) levels of the total PUFA (ΣPUFA), Σn-6 PUFA, and Σn-3 PUFA, as well as lower (P < 0.05) levels of total SFA (ΣSFA) and n-6PUFA/n-3PUFA ratio in breast and thigh meat compared to the control and other treatments. In comparison to the control and the other groups, the highest PUFA/SFA ratio in the breast and thigh meat was found in the BMEOs-900 treatment (P < 0.05).

Table 5.

The effect of different concentrations of a blend of microencapsulated essential oils (BMEOs) on fatty acid profile (% of total fatty acid) in the breast and thigh meat of broilers (n = 4 broilers per replicate) at 42 d of age.

Parameters1	Treatments2
	Control	BMEOs-300	BMEOs-600	BMEOs-900	SEM	P-value
Breast meet
ΣSFA	39.46a	39.20a	38.88a	30.30b	0.400	0.001
ΣPUFA	20.19b	20.29b	21.01b	26.94a	0.286	0.001
ΣMUFA	32.72	33.11	34.20	34.31	0.366	0.192
Σn-6 PUFA	19.74b	19.86b	20.04b	23.86a	0.211	0.001
Σn-3 PUFA	2.62b	2.67b	2.71b	3.82a	0.068	0.001
PUFA/SFA ratio	0.511b	0.517b	0.540b	0.891a	0.013	0.001
n-6PUFA/n-3PUFA ratio	7.85a	7.78a	7.66a	6.48b	0.116	0.001
Thigh meet
ΣSFA	37.33a	37.11a	36.86a	32.15b	0.335	0.001
ΣPUFA	22.95b	23.45b	28.10a	28.92a	0.320	0.001
ΣMUFA	34.22	34.46	35.58	36.26	0.415	0.215
Σn-6 PUFA	17.86b	18.34b	19.12b	24.21a	0.296	0.001
Σn-3 PUFA	2.15b	2.25b	2.38b	3.80a	0.071	0.001
PUFA/SFA ratio	0.620c	0.631c	0.763b	0.902a	0.010	0.001
n-6PUFA/n-3PUFA ratio	8.72a	8.64a	8.21a	6.51b	0.149	0.001

^{a, b, c}Significant differences exist among means with different superscripts within a row (P <0.05); SEM, Standard error mean.

¹SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acids; PUFA, Polyunsaturated fatty acids.

²Control, Basal diet (without BMEOs); BMEOs-300, Basal diet contains 300 mg/kg BMEOs; BMEOs-600, Basal diet contains 600 mg/kg BMEOs; BMEOs-900, Basal diet contains 900 mg/kg BMEOs.

Discussion

Performance and economic index

The results from our study revealed that the diet supplemented with BMEOs-900 significantly increased ADBWG, EPEF, and EBI compared to the control and other treatments, while also decreasing FCR over the entire period. The results of this study are consistent with those of previous studies, where in diet of broilers included amounts of 300 and 1,000 mg/kg of oregano, eucalyptus, and thyme EOs (Cross et al. 2007; El-Ashram and Abdelhafez 2020; Nameghi et al. 2022; Seidavi et al. 2022), 100 and 150 mg/kg microencapsulated and encapsulated thyme, oregano, and rosemary EOs (Hafeez et al. 2016; Islam et al. 2022), and 400 mg/kg of encapsulated EOs containing oregano EO, compared to 200 mg/kg (Meligy et al. 2023) increased BW, BWG, and EPEF, as well as decreased FCR. In the present study, no difference was found for ADFI among treatments. Similarly, 0.05 and 1%, as well as 100–300 mg/kg thyme powder and/or EO (Placha et al. 2019; El-Ashram and Abdelhafez 2020; Fawaz et al. 2021) in the diet of broilers did not influence the FI. Our observations do not agree with the previous studies, which showed that the 200 mg/kg mixture of thymol and carvacrol (Hashemipour et al. 2013) and 1,000 mg/kg thyme EO (Cross et al. 2007) in the diet of broilers decreased FI. Although in contrast with the current study, an earlier study demonstrated that dietary supplementation of eucalyptus EO at 250, 750, and 1000 mg/kg did not affect the growth performance parameters of broilers in the starter phase; consistent with our findings, improved growth performance in the grower phase, the finisher phase, and the whole period (Mohebodini et al. 2021).

Two indices, EPEF and EBI, have been used to assess the economic efficiency of broiler performance in recent years (Safari et al. 2024a). Uniform broiler BWG, healthy flock, good rearing technical, better production, and economic efficiency are all indicated by higher EPEF and EBI values. The key factor in these two economic indicators is the FCR, since feed represents about 70% of the cost in current large broiler-rearing systems (Safari et al. 2024a). However, the following factors may be responsible for improvements in the performance and economic index by BMEOs-900. The improved ADBWG in the current study was probably caused by increased nutrient utilization because BMEOs-900 supplementation decreased FCR without affecting the broilers’ ADFI. In this case, it has been reported that the EOs, by decreasing the crypt depth and increasing villus height, improve the gastrointestinal tract morphology and absorptive surface area, and the birds’ general health is maintained. This increases nutrient digestibility (Amer et al. 2023). Additionally, the enhanced growth performance may result from higher secretion of growth-related hormones by feeding EOs (Amer et al. 2023; He et al. 2023). Furthermore, EOs increasing digestive enzyme secretion from the pancreas and intestinal mucosa, such as trypsin, lipase, and amylase, results in increased secretion of bile acid and saliva, and increases the secretion and flow of gastric juices, all of which improve the nutrient digestibility of dry and organic matter (Hafeez et al. 2016; Amer et al. 2023; Rafeeq et al. 2023). In this connection, in the current research, the growth-promoting effect during the starter phase demonstrated that BMEOs-900 improved young animals’ nutrient utilization, which is an important outcome because endogenous enzyme secretion, such as lipase, is insufficient during this crucial period. Moreover, EOs by reducing the intestinal pH, improve beneficial gut microbiota, and also, by their lipophilic property, modify the permeability of pathogenic bacterial cell membranes, which leads to the leakage of essential cations throughout the membrane and bacterial death (Seidavi et al. 2022). In addition, EOs can stimulate glycolysis and increase energy utilization for production and, ultimately, growth (Mpofu et al. 2016). Furthermore, the EOs’ active and phenolic ingredients, including carvacrol, thymol, and trans-anethole, enhance feed digestibility and metabolic processes, which raises the liver’s levels of gluconeogenic enzymes (eg glucose-6-phosphatase and fructose 1,6-bisphosphatase) (El-Ashram and Abdelhafez 2020). Additionally, phenolic compounds of EOs improve the GIT’s ability to better ferment digesta, which increases nutrient absorption, and have either direct or indirect anabolic effects on host tissues (Mahfuz et al. 2021). Moreover, EOs enhance immune response and increase antioxidant status (Rafeeq et al. 2023). Different outcomes of the impacts of various EO types on broiler growth performance in various studies may be due to dose variations, form of used EOs, active components of the EOs mixture, animal age, differences in bird breed, the period of supplementation, the stress factors, overall management system, the environmental factors, and diet composition.

Carcass characteristics and immune organs

In the present research, compared to the control, BMEOs-300, and BMEOs-600 groups, the highest relative weight of immune organs (spleen, bursa of Fabricius, and thymus), proventriculus, and pancreas, as well as the lowest abdominal fat relative weight, were found in broilers fed the diet supplementing BMEOs-900. In agreement with our results, it has been reported that 0.750% of the blend of thyme and rosemary powders (Nameghi et al. 2022; Seidavi et al. 2022), 150 mg/kg microencapsulated EOs containing oregano and rosemary (Islam et al. 2022), and 150 and 200 ppm of the blend of thyme and eucalyptus EOs (Hesabi Nameghi et al. 2019) in the broiler’s diet reduced the relative weight of abdominal fat and increased the relative weight of the immune organs. Similarly, it has been reported that 0.275 g/kg of dietary oregano EO increased the relative weights of the proventriculus of laying hens (Johnson et al. 2022). In the present experiment, the relative weights of the liver, gizzard, heart, thigh, breast, and carcass were unaffected by dietary treatments. Contrary to our present findings, it has been shown that 0.275 g/kg of dietary oregano EO increased the entire GIT and gizzard relative weights of laying hens (Johnson et al. 2022); also in broilers, the relative weight of the immune organs (Mohebodini et al. 2021) was not affected by feeding amounts of either 150 mg/kg of dietary microencapsulated EOs containing oregano and rosemary or 0, 250, 750, and 1000 mg/kg of dietary eucalyptus EO.

Broiler processors and consumers do not favor excess body fat, particularly abdominal fat, in poultry. As a result, successful poultry meat production largely hinges on enhancing muscle mass in the carcass while minimizing abdominal fat. The following factors may contribute to the generally proposed mechanisms of action of BMEOs-900 in reducing abdominal fat. BMEOs-900 improve hypercholesterolemia by increasing the conversion of endogenous cholesterol into bile acid and lowering the absorption of exogenous cholesterol, which decreases the accumulation of abdominal fat (Nameghi et al. 2022). In addition, increasing feed digestibility due to the increased secretion of bile acids and enhanced endogenous digestive enzyme production, including trypsin, lipase, and amylase, by phytogenic components (Abdel-Wareth et al. 2019). Furthermore, BMEOs’ antioxidant properties and the short-chain fatty acids produced by the gut microbiota prevent fat from building up on the carcass (Abdel-Wareth et al. 2019). Moreover, in poultry, more of the lipids that build up in the adipose tissues are fatty acids that are derived from blood triglycerides. The reduction in abdominal fat and circulating blood triglycerides can be attributed to the down-regulatory effect of phenols, flavonoids, and chemical compounds of EOs such as thymol and carvacrol (main compounds of thyme and oregano EOs) on 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductases, the rate-limiting enzyme of fat and cholesterol synthesis, or enhanced lipoprotein catabolism (Nameghi et al. 2022). In line with the reasons mentioned above, the mechanism of action of thymol and carvacrol in reducing abdominal fat is reported as follows. The possible mechanisms for the body fat reduction effects of thymol are attributed to its antitachycardial, anti-apoptotic, antihypertrophic, and anti-lecithin cholesterol acyl transferase inhibitory activities in the liver, as well as its antihyperlipidemic and free radical scavenging properties (Meeran et al. 2015). Moreover, it has been shown that carvacrol may prevent adipocyte differentiation and fat accumulation between cells in mice. Additionally, in visceral adipose tissue of mice, carvacrol reduced the expression of adipogenesis-related genes, including the fibroblast growth factor receptor (Gholami-Ahangaran et al. 2022).

No significant differences in the relative weights of the liver and heart suggest that BMEOs up to 900 mg/kg of diet may not harm broilers. The rise in the proventriculus and pancreas relative weights could be attributed to enhanced specific enzymatic activity resulting from the supplementation of BMEOs-900 (Johnson et al. 2022), which is justified by improved performance in broilers fed BMEOs-900. The poultry body’s cellular and humoral immune function may improve with growth and development of the immune organs, and an increase in their relative weights. Since the increase in their relative weight, follicle size, and count is associated with the rise in production of T and B lymphocytes and the level of immunoglobulin synthesis (El-Kholy et al. 2021). The bursa of Fabricius is considered a main lymphoid organ and serves as a site for the development and differentiation of B-lymphocytes, which primarily contribute to humoral immunity (El-Kholy et al. 2021). The spleen, as the largest peripheral immune organ in poultry, is typically called a secondary lymphoid organ and plays a key role in leukocyte maturation, lymphocyte proliferation, cytokine synthesis, and activation of the inflammatory process (El-Kholy et al. 2021). T-cell differentiation occurs in the thymus. Once in the thymus, lymphatic stem cells mature and differentiate into mature T-cells (Hesabi Nameghi et al. 2019).

Several mechanisms have been suggested for BMEOs-900 for promoting the growth of broiler immune organs, which improve the body’s immune system, including: The bioactive components of BMEOs, such as thymol and carvacrol, have antioxidant, anti-inflammatory, and antimicrobial properties that may have a beneficial impact on the development and activation of the immune organs (Hesabi Nameghi et al. 2019). In addition, EO supplements aid in the body’s defense against bacterial and viral infections by stimulating B- and T-lymphocytes and generating IgG and IgM antibodies (El-Kholy et al. 2021). Furthermore, another possible explanation is that a higher level of EO may increase nutrient digestion. As a result, more nutrients reach lymphocytes through the bloodstream, stimulating lymphocyte proliferation and the growth of immune organs (Zhang et al. 2022). Moreover, the EOs assessed in this experiment may incite nonspecific immune cells’ phagocytic activity, boosting neutrophil and monocyte activity as well as the gut’s production of mucin and IgA. Also, it has been reported that 1,8-Cineol (the main component of eucalyptus and rosemary EOs), by enhancing the expression of antioxidant enzymes and decreasing the oxidative stress as well as PI3K/AKT pathway, alleviates apoptosis and necroptosis in the bursa of Fabricius in chickens (Liu et al. 2021). These could be beneficial for the health, growth, and development of the bursa of Fabricius, and finally for the immune system response. Additionally, EOs and their constituents, such as α-pinene, have a positive effect on intestinal microflora population function (Šimunović et al. 2020). Therefore, the capability of intestinal beneficial microorganisms in modulating the immune response of broilers could be a reason for the increased relative weight of immune organs. However, histological experiments of immune organs are required to further validate the results reported in the current study.

Fatty acid profile of breast and thigh meat

In the current research, broilers fed the diet containing BMEOs-900 showed significantly higher concentrations of the ΣPUFA, Σn-6 PUFA, Σn-3 PUFA, and PUFA/SFA ratio, as well as lower levels of the ΣSFA and n-6 PUFA/n-3 PUFA ratio in breast and thigh meat in comparison to the control and other treatments. The findings of our research are also similar to the results of previous researchers, which illustrated that supplementation of broilers’ diet with 0.05% thyme EO (Vlaicu et al. 2023), 100 mg/kg of microencapsulated phytogenic containing thymol and carvacrol (Galli et al. 2020), 200, 400, or 600 mg/kg lavender EO (Amer et al. 2022), and 1000 mg/kg of eucalyptus EO (Mohebodini et al. 2021) increased levels of ΣPUFA, n-6 PUFA, n-3 PUFA, and PUFA/SFA ratio, as well as decreased concentrations of ΣSFA and the n-6/n-3 ratio in the breast and thigh meat. Similarly, a phytogenic product (thymol and carvacrol) at 200 mg/kg of broiler diet increased ΣPUFA while decreasing ΣSFA levels in serum and thigh muscle; however, in contrast to the findings of the current study, it did not affect breast meat (Hashemipour et al. 2013). In contrast with this study, it has been shown that supplementation of broilers’ diet with EOs did not affect ΣPUFA and ΣSFA concentration in breast or thigh muscles (Kumar et al. 2018; Placha et al. 2019). In the present experiment, our findings showed that there was no significant difference in the ΣMUFA level of breast and thigh among the various dietary treatments. The results of the present research are consistent with those of previous studies, where the use of EOs in the diet of broilers did not affect the ΣMUFA levels in breast or thigh muscles (Hashemipour et al. 2013; Kumar et al. 2018; Mohebodini et al. 2021).

ΣPUFA and ΣMUFA must be provided in foods because they cannot be synthesized in the human body, yet are necessary for health. ΣPUFA are part of phospholipids, which are present in cell membranes and act as building blocks for eicosanoids, a class of inflammatory mediators that resemble hormones (Demirci et al. 2019; Safari et al. 2025). Also, ΣPUFA, including n-3 PUFA and n-6 PUFA, can effectively lower body fat, control autogenic immunity, enhance growth and development, and prevent some chronic diseases, which have positive effects on the brain and are beneficial to human health (Safari et al. 2016b; Wu et al. 2022). ΣMUFA could boost memory, decrease blood sugar, and enhance high-density lipoprotein (HDL) with beneficial effects on health (Safari et al. 2016a; Wu et al. 2022). In contrast, the hypercholesterolemic effects of the ΣSFA, which are associated with coronary artery disease, make it extremely important (Hashemipour et al. 2013). The PUFA/SFA and/or n-6/n-3 ratios serve as crucial markers for assessing the nutritional quality of meat. The WHO advises a range of 5–10:1 for the n-6/n-3 ratios, and the relevant PUFA/SFA ratios should be higher than 0.4 (Wu et al. 2022).

In the current study, the breast meat ratios of n-6/n-3 and PUFA/SFAs were 6.48–7.85 and 0.511–0.891, respectively. The thigh meat ratios of n-6/n-3 and PUFA/SFAs were 6.51–8.72 and 0.620–0.902, respectively. In this study, the PUFA/SFA and n-6/n-3 ratios are all within the appropriate range that the WHO recommends. This showed that dietary addition of various levels of BMEOs not only did not have harmful impacts on the meat fatty acids profile of broilers, but also that BMEOs-900 improved the broilers’ meat fatty acid content.

There are several possible mechanisms of how the BMEOs-900 increased the meat PUFA content, which are listed below. The high PUFA levels due to EOs may result from the antioxidant properties of EOs such as flavonoids and terpenoids, which enhance broilers’ antioxidant systems, including SOD, and prevent lipid peroxidation of tissue lipids, particularly PUFA. In this case, one of the factors in EO components that gives it its antioxidant properties is the phenolic OH group. It acts as a donor of hydrogen and counteracts the peroxyl radicals generated in the first stage of lipid peroxidation (Hashemipour et al. 2013). In addition, the meat enrichment with PUFA in the EOs-fed broilers can be attributed to the composition of EOs’ fatty acids (Amer et al. 2022). Moreover, the higher PUFA in meat might be due to the active components of EOs, which might lead to the fatty acid β-oxidation process, thereby reducing the fat deposition (Omidi et al. 2020). Furthermore, the fatty acid synthase enzyme and lipid homeostasis may have improved the fatty acid level of broiler meat, which may account for the anti-hyperlipidaemic action of medicinal plants’ phenolic acids (Omidi et al. 2020). Also, the phenolic compounds (such as carvacrol, thymol, flavonoids, and terpenoids) in EOs are anti-inflammatory and antibacterial, which can modify the gut microbial flora. Therefore, the impact is to suppress the activity of the hydrogenated microorganisms, which are responsible for producing certain fatty acids in meat (Wu et al. 2022). Additionally, the high level of PUFA in meat is due to the activity of specific enzymes, including delta-5 (Δ5) and delta-6 (Δ6) desaturases, which enhance elongation and desaturation (Amer et al. 2022). As a mechanism of this action, it could explain that although animals cannot synthesize essential fatty acids (EFA), linoleic acid (n-6 PUFA; 18:2), and α-linolenic acid (n-3 PUFA; 18:3) from acetyl-CoA, they can convert EFA supplied by the diet to more unsaturated FA with a longer carbon chain. This process is catalyzed by the enzymes that elongate and desaturate. The Δ5 and Δ6 desaturases, which introduce double bonds into EFA to produce long-chain PUFA. For example, synthesis of eicosapentaenoic acid (n-3 PUFA; 20:5) and docosahexaenoic acid (n-3 PUFA; 22:6) from α-linolenic acid is achieved by a sequence of desaturation and chain elongation steps, controlled by the enzymes Δ5 and Δ6 desaturases and elongases (Boschetti et al. 2016). It has been reported that saturated and trans fats inhibit the Δ5 and Δ6-pathways, limiting long-chain FA levels, but the inclusion of antioxidant components (such as EOs) in the diet modulates the Δ5 and Δ6-desaturase pathway favorably and enhances PUFAs levels in poultry products (Vlaicu et al. 2021). Moreover, an increase in ΣPUFA content by EOs causes a decrease in ΣSFA level in meat lipids (Hashemipour et al. 2013). Furthermore, as observed in the current study, the higher level of n-3 PUFA could increase the PUFA/SFA ratio and reduce the n-6/n-3 level ratio, which is beneficial to human health (Wu et al. 2022). Humans are not able to synthesize Σn-3PUFAs in their bodies; therefore, broiler meat could be a good source of these compounds as a widely consumed food in the human diet. A lower ratio of n-6PUFAs/n-3PUFAs is associated with a reduction in cardiovascular disease and inflammation in humans (Qaid et al. 2023).

Conclusion

Based on our findings, it is concluded that there is a significant difference in broiler response to the control diet and diets containing various levels of BMEOs. Dietary supplementation of the BMEOs-900 increased ADBWG and decreased FCR, which are reflected in a greater economic index, including EPEF and EBI, throughout the experimental period. Significantly higher relative weight of the immune organs, proventriculus, and pancreas, as well as significantly lower relative weight of the abdominal fat, was achieved by BMEOs-900 treatment. In addition, dietary BMEOs-900 supplementation increases the levels of ΣPUFA, Σn-6 PUFA, Σn-3 PUFA, and the ratio of PUFA/SFA, as well as decreases the levels of ΣSFA and the Σn-6 PUFA/Σn-3 PUFA ratio in thigh and breast muscle. Our results suggest that the MEOs-900 should be sought after by the poultry feed industry as a new strategy to achieve an effective and valuable feed additive for promoting growth performance, improving carcass characteristics, and the immune organs index, as well as enhancing the quality of poultry meat in the poultry diet.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

Hassan Safari (Writing—review & editing, Writing—original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization), Fatemeh Mohammadpour (Writing—review & editing, Writing—original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization), and Omid Jafaryani (Visualization, Validation, Supervision, Resources, Methodology, Investigation, Data curation)

Conflicts of interest

The authors declare that there are no conflicts of interest.

Data Availability

All data obtained and analyzed during the current study are included in this published article. Raw data are available from the corresponding author upon reasonable request.