Bioactive Essential Oil Blend in Broiler Diets: Effects on Performance and Intestinal Morphology

ABSTRACT

This study evaluated the effects of a commercial blend of cinnamon (Cinnamomum verum) and oregano (Origanum vulgare) essential oils on growth performance and intestinal health indicators of broilers. A total of 385 male chicks were allotted to five dietary treatments: a negative control diet without growth promoters, a positive control diet with an antibiotic growth promoter, and three diets supplemented with 0.05, 0.10 or 0.15 kg/t of the essential oil blend in a completely randomized design up to 42 days of age. Growth performance was recorded by phase, and gastrointestinal organ biometrics, serum biochemical profile and small-intestine histomorphometry were assessed at slaughter. Essential oil supplementation did not consistently affect body weight gain; however, the inclusion level of 0.10 kg/t improved feed conversion ratio and reduced cumulative feed intake compared with the antibiotic control during the grower phase and over the rearing period. At 42 days of age, increasing levels of the blend promoted a linear increase in villus height in the duodenum, jejunum and ileum, whereas gastrointestinal organ weights and serum biochemical variables remained within normal physiological ranges and were not influenced by the treatments. Under the present conditions, the cinnamon and oregano essential oil blend at 0.10 kg/t improved feed efficiency and intestinal villus development without adverse effects on health indicators, supporting its use as a phytogenic additive in broiler nutrition. Further studies including microbiota characterization, digestive enzyme activity, and inflammatory or oxidative stress markers are warranted to clarify mechanisms and to better position this blend within antibiotic-free feeding programs.

Keywords:
Broiler chickens; phytogenic additives; essential oils; intestinal morphology; feed conversion ratio; performance

INTRODUCTION

In recent years, the ban on antibiotic growth promoters (AGPs) in poultry production has created additional challenges for maintaining flock health and productivity. Although this policy is aimed at mitigating the risk of antimicrobial resistance in humans, many producers have reported difficulties in sustaining performance and controlling enteric disorders in the absence of AGPs (Rahman et al., 2022). Consequently, increasing attention has been directed to alternative strategies such as probiotics and natural feed additives. Nevertheless, their ability to consistently support growth and health is variable, and may be constrained by high costs, formulation limitations and inconsistent supply (Ghimpețeanu et al., 2022).

Since the prohibition of AGPs in the European Union in 2006, there has been an intensive search for feed additives capable of preserving or enhancing animal performance while supporting gut health (Zheng et al., 2025). Among the proposed alternatives, naturally occurring plant compounds have emerged as promising candidates (Fathima et al., 2022). Plants synthesize a wide variety of secondary metabolites with documented antioxidant, anti-inflammatory and antimicrobial properties, initially described in humans and rodent models and, more recently, in different livestock species (Soares et al., 2022). Polyphenols and other phytochemicals may modulate oxidative status, inflammatory responses and microbial populations in the gastrointestinal tract, thereby contributing to improved intestinal function and resilience to production stressors (Sumadja et al., 2025).

In this context, essential oils stand out as concentrated sources of bioactive phytochemicals. Essential oils obtained from aromatic plants contain complex mixtures of terpenes and phenolic compounds that have shown antimicrobial, antioxidant and anti-inflammatory activities, as well as the potential to modulate gut microbiota and epithelial integrity (Luna et al., 2025). Several studies have evaluated essential oils as feed additives in poultry nutrition, either as single compounds or in blends, with the aim of improving feed efficiency and supporting intestinal health in AGP-free systems (Ibeagha-Awemu et al., 2025).

In addition to conventional feed additives, increasing attention has been directed toward bioactive compounds capable of modulating key physiological pathways in poultry. These compounds may influence intestinal integrity, oxidative status, inflammatory responses, digestive functionality, and host-microbiota interactions, thereby contributing to gut health and productive efficiency. In this context, phytogenic additives have emerged as promising candidates because their effects extend beyond antimicrobial activity and may involve multiple complementary mechanisms. Among them, cinnamaldehyde, the major active compound of cinnamon (Cinnamomum spp.) essential oil, and thymol, a phenolic monoterpene present in oregano (Origanum spp.) essential oil, are among the most widely studied phytogenic molecules in broiler nutrition (Mostafavi et al., 2024). These compounds have also been discussed within the broader concept of molecular growth promoters because they may modulate biological pathways related to gut functionality, epithelial integrity, oxidative balance, inflammatory regulation, and host-microbiota interactions. Accordingly, cinnamaldehyde and thymol are considered promising feed additives not only because of their antimicrobial effects against pathogenic bacteria, but also because of their potential to modulate fermentative activity and improve digestive processes (Wang et al., 2025). However, responses in broilers have been inconsistent, and the combined use of cinnamaldehyde- and thymol-based essential oils, particularly under commercial-like conditions and in direct comparison with antibiotic growth promoters, remains insufficiently characterized (Puvača et al., 2022).

Therefore, zinc bacitracin was included in the present study as a positive control to provide a classical experimental benchmark for comparison with the phytogenic blend. Accordingly, this study was designed following a dual-endpoint approach to assess the effects of dietary supplementation with a commercial blend of cinnamon and oregano essential oils in broilers reared in an AGP-free context, focusing primarily on growth performance and intestinal health indicators. Gastrointestinal tract organ biometrics, serum biochemical parameters, and liver-related variables were also evaluated as secondary outcomes to provide a more comprehensive assessment of the birds’ physiological responses to the additive.

MATERIALS AND METHODS

The experiment was conducted in Rio Verde, Goiás, Brazil (17°48’34.5” S, 50°53’55.4” W). All procedures involving animals were approved by the institutional committee on animal use (protocol no. 8605090419). The trial lasted 42 days. The experimental design was structured to integrate productive performance assessment with intestinal health evaluation, while organ biometrics and biochemical variables were included as complementary physiological measures.

A total of 385 one-day-old male Cobb broiler chicks were used. Birds were randomly allotted to five dietary treatments, with seven replicates of 11 birds each, in a completely randomized design. Chicks were housed in galvanized wire cages (0.90 × 0.60 × 0.45 m) equipped with trough feeders and nipple drinkers. Feed and water were provided ad libitum throughout the experimental period.

Experimental diets were based on corn and soybean meal and were formulated to meet or exceed the nutritional requirements of broilers in each phase, according to recommendations previously described by Rostagno et al. (2017). Four feeding phases were adopted: pre-starter (1-7 days), starter (8-21 days), grower (22-35 days) and finisher (36-42 days). The ingredient composition and calculated nutrient content of the basal diets for each phase are presented in Table 1.

Table 1
Composition of experimental diet.

Ingredient (Kg)	Pre-starter (1-7 d)	Starter (8-21 d)	Grower (22-35 d)	Finisher (36-42 d)
Corn	55.30	56.02	61.40	67.00
Soybean meal, 45%	39.37	37.93	32.20	26.90
Soybean oil	0.80	1.70	2.90	2.80
Dicalcium phosphate	0.06	1.25	1.48	1.10
Premix	1.001	1.001	0.802	1.202
Common salt	0.50	0.49	0.48	0.45
DL-Methionine	0.26	0.50	0.29	0.20
L-Lysine	0.30	0.27	0.22	0.40
Limestone	2.2	1.20	0.19	0.20
L-Threonine	0.19	0.07	0.10	0.07
Total	100.00	100.00	100.00	100.00
Calculated levels
Metabolizable energy (Kcal/kg)	3000.00	3100.0	3147.54	3201.18
Crude protein (%)	25.31	24.50	20.64	18.68
Digestible lysine (%)	1.36	1.31	1.12	1.14
Digestible methionine (%)	0.55	0.53	0.58	0.46
Available P (%)	0.48	0.43	0.33	0.28
Ca (%)	1.01	0.84	0.75	0.66
Na (%)	0.23	0.21	0.20	0.19

• ¹
  Mineral-vitamin premix (per kg of product): methionine, 290 g; iron, 5,000 mg; copper, 1,500 mg; manganese, 14,000 mg; zinc, 12,000 mg; iodine, 28 mg; selenium, 70 mg; vitamin A, 1,500,000 IU; vitamin D3, 500,000 IU; vitamin E, 3,333 IU; vitamin K3, 250 mg; vitamin B1, 300 mg; vitamin B2, 1,000 mg; vitamin B6, 500 mg; vitamin B12, 3,333 mcg; niacin, 6,667 mg; calcium pantothenate, 2,000 mg; folic acid, 280 mg; biotin, 8.3 mg; choline chloride, 70 mg. Included at 1.001 kg/t of feed in the pre-starter and starter phases.
• ²
  Mineral-vitamin premix (per kg of product): methionine, 300 g; iron, 6,000 mg; copper, 1,850 mg; manganese, 16,800 mg; zinc, 14,500 mg; iodine, 330 mg; selenium, 84 mg; vitamin A, 1,500,000 IU; vitamin D3, 500,000 IU; vitamin E, 3,600 IU; vitamin K3, 240 mg; vitamin B1, 300 mg; vitamin B2, 1,100 mg; vitamin B6, 500 mg; vitamin B12, 3,600 mcg; niacin, 7,000 mg; calcium pantothenate, 2,000 mg; folic acid, 320 mg; biotin, 6 mg; choline chloride, 65 mg. Included at 0.802 and 1.202 kg/t of feed in the grower and finisher phases, respectively.

Five treatments were evaluated: a negative control diet (T1) containing no performance-enhancing additives; a positive control diet (T2) supplemented with zinc bacitracin as an antibiotic growth promoter at 0.25 g/t of active ingredients; and three diets containing the essential oil blend at 0.05, 0.10 and 0.15 kg/t (T3, T4 and T5, respectively). The essential oil product was an imported commercial blend recommended for use in broiler diets at 0.10 kg/t of feed. According to the manufacturer, the blend contains cinnamaldehyde from cinnamon (Cinnamomum verum) essential oil at 4.5 g/100 g of product and thymol from oregano (Origanum vulgare) essential oil at 13.5 g/100 g of product. Zinc bacitracin was included as the positive control because it is a classical and well-established antibiotic growth promoter in poultry experiments, providing a recognized reference for comparison with alternative feed additives. Although its commercial use has declined in several production systems, its inclusion remains relevant in experimental studies because it allows the effects of the phytogenic blend to be evaluated against a historically established growth-promoting standard.

The essential oil blend was thoroughly mixed into the respective diets at the predetermined inclusion levels. Throughout the experimental period and production phases, the environmental conditions inside the poultry house varied, with average maximum temperatures ranging from 27.83 to 31.05 °C, average minimum temperatures from 16.00 to 18.22 °C, and relative humidity from 50.00 to 69.52%.

Birds and feeders were weighed at 7, 14, 21 and 42 days of age to determine body weight, body weight gain, feed intake and feed conversion ratio by phase and for the overall period. Mortality was recorded daily, and performance calculations were adjusted for the number of live birds in each replicate.

An apparent nutrient digestibility trial was conducted during the grower phase. Birds remained on their respective experimental diets, and a 3-day adaptation period was followed by a 5-day total excreta collection period. Ferric oxide (1%) was added to the diets at the beginning and at the end of the collection period to mark the onset and conclusion of excreta collection. During the collection period, feed intake was recorded for each experimental unit, and care was taken to avoid feed wastage and contamination of excreta. Excreta were collected daily from trays placed under each cage, transferred to identified plastic bags and stored at −20 °C until analysis. At the end of the collection period, samples from each replicate were thawed, thoroughly homogenized and subsampled (up to 400 g per replicate).

Subsamples of excreta and representative samples of each experimental diet were pre-dried in a forced-ventilation oven at 55 ± 5 °C and then ground in a Wiley-type mill using a 2-mm screen. Ground samples were stored in labeled plastic containers and analyzed for dry matter, crude protein, ether extract and other components, following procedures previously described by Instituto Adolfo Lutz (2005). Apparent digestibility coefficients were calculated as the difference between nutrient intake and nutrient excretion, divided by nutrient intake. Apparent crude protein digestibility was also expressed relative to weight gain, considering the amount of crude protein ingested minus that excreted, divided by body weight gain. Nutrient retention was calculated according to the methodology described elsewhere by Silva & Queiroz (2002).

On days 7, 14, 21 and 42, one bird per replicate with body weight close to the replicate mean was selected, fasted for 8 h and used for blood and tissue sampling. Blood samples were collected by cardiac puncture, allowed to clot, and centrifuged to obtain serum (Minafra et al., 2010). Serum biochemical analyses were performed according to previously described procedures (Fathima et al., 2022) using commercial colorimetric kits. The following variables were determined: amylase (U/dL), cholesterol (mg/dL), calcium (mmol/dL), phosphorus (mmol/dL), glucose (mg/dL), total protein (g/dL), calcium:phosphorus ratio, triglycerides (mg/dL), aspartate aminotransferase (AST, U/L) and alanine aminotransferase (ALT, U/L).

For liver biochemistry, liver samples were collected at necropsy, homogenized at a ratio of 1 g tissue to 9 mL distilled water and centrifuged at 8,000 rpm for 10 min. The supernatant was used to quantify AST and ALT activities with the same analytical procedures applied to serum (Minafra et al., 2008). At necropsy, the gastrointestinal tract (GIT) and associated organs were carefully removed and weighed following procedures described previously (Gao et al., 2025). Total GIT weight was recorded from the esophageal insertion in the oropharynx to the junction of the large intestine and cloaca. The following components were weighed separately: proventriculus plus gizzard (with residual content), pancreas (after separation from the duodenal loop), small intestine (from the pyloric region to the ileocecal junction), large intestine (ceca, colon and rectum) and liver (without the gallbladder). Relative organ weights were calculated as:

$Relativeorganweight\left(\%\right)=\left(organweight/bodyweight\right)\times 100$

Segments of approximately 4 cm from representative regions of the small intestine were collected, gently flushed with distilled water, identified, and fixed in 10% buffered formalin. Histological processing was performed according to a methodology adapted from a previous study (Trevizan et al., 2016). Fixed tissues were dehydrated in an ascending alcohol series, cleared in xylene and embedded in paraffin. From each block, multiserial sections (6 µm thick) were obtained, and six sections per segment were mounted on glass slides, stained with hematoxylin and eosin, and coverslipped.

Morphometric analyses of the intestinal mucosa were performed using images captured at 4× magnification with an optical microscope coupled to the Leica imaging system (Image-Pro Plus, version 4.5.0.27). Image analysis was carried out using the AnatiQuanti software (Universidade Federal de Viçosa, Viçosa, MG, Brazil). The variables evaluated were villus height, crypt depth, and villus height:crypt depth ratio. Villus height was measured from the villus tip to the villus-crypt junction, whereas crypt depth was measured from the villus-crypt junction to the base of the crypt adjacent to the muscularis mucosa.

All data were subjected to analysis of variance. The effects of the essential oil blend were evaluated by regression analysis using the negative control (T1) and the diets containing increasing levels of essential oil (T3, T4 and T5). In addition, Dunnett’s test was applied to compare the positive control treatment (T2; antibiotic growth promoter) with the other treatments. In all analyses, a significance level of 5% (p≤0.05) was adopted. Statistical analyses were performed using the R software. The statistical model used was:

$Y_i{}_j=\mu +T_i+{\epsilon }_{ij}$

where Y _ij is the observed value for the j-th replicate of the i-th treatment, μ is the overall mean, T _i is the fixed effect of treatment, and ε_ij is the random error term.

RESULTS

Growth Performance

The growth performance of broilers fed diets containing increasing levels of the essential oil blend is summarized in Table 2.

Table 2
Performance of broilers fed diets supplemented with essential oil.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 7 days
WG (g)	55.00	56.00	57.00	55.00	52.00	4.88	0.267	0.000
FI (g)	73.00	71.00	76.00	75.00	74.00	5.98	0.595	0.001
FC	1.38	1.35	1.34	1.35	1.36	5.71	0.187	0.001
1 to 14 days
WG (g)	206.00	194.00	213.00	212.00	205.00	6.09	0.151	0.052
FI (g)	289.00	279.00	283.00	275.00	282.00	5.70	0.806	0.002
FC	1.41	1.40	1.35	1.28*	1.35	5.97	0.897	0.016
1 to 21 days
WG (g)	471.00	464.00	509.00	512.00	482.00	5.22	0.010a	0.005
FI (g)	760.00	797.00	744.00	741.00	722.00	4.55	0.003b	0.008
FC	1.59	1.69	1.47	1.43*	1.50	4.86	<0.001c	0.023
1 to 42 days
WG (g)	2.632	2.597	2.624	2.669	2.536	4.05	0.120	0.021
FI (g)	4.240	4.280	3.980	3.860*	3.890*	5.30	0.004d	0.049
FC	1.59	1.66	1.53	1.44*	1.53	5.48	0.001e	0.020

• PC - positive control; NC - negative control; WG - weight gain; FI - feed intake; FC - feed conversion; CV - coefficient of variation; SEM - standard error of the mean.
• a Quadratic effect y = 0.469 + 0.113x + (−0.07x²) (R² = 0.98).
• b Linear effect y = 0.785 + (−0.045x) (R² = 0.83).
• c Quadratic effect y = 1.691 + (0.554x) + 0.288x² (R² = 0.99).
• d Linear effect y = 4.203 + (−0.259x) (R² = 0.76).
• e Quadratic effect y = 1.671 + (−0.423x) + 0.220x² (R² = 0.96).
• *Differs from positive control treatment by Dunnett’s test at 5% probability.

From 1 to 7 days of age, body weight gain, feed intake and feed conversion ratio (FCR) were not affected by dietary treatments, and no significant responses were detected in the regression analysis. During the 1 to 14-day period, regression analysis likewise showed no effect of essential oil level on weight gain, feed intake or FCR. However, Dunnett’s test indicated that birds fed 0.10 kg/t of the essential oil blend had a lower FCR than those receiving the positive control diet with antibiotic, without changes in weight gain or feed intake.

From 1 to 21 days, all performance variables responded to the increasing inclusion of the essential oil blend. Weight gain showed a quadratic response, with the highest values occurring around 0.10 kg/t, whereas feed intake decreased linearly with increasing levels of the blend, indicating improved efficiency of nutrient use at higher inclusion levels. Feed conversion ratio also followed a quadratic pattern, and the 0.10 kg/t level yielded the lowest FCR among the essential oil treatments. According to the Dunnett’s test, only FCR differed from the positive control, with birds fed 0.10 kg/t exhibiting better feed efficiency than those receiving the antibiotic diet.

Over the entire period from 1 to 42 days, final body weight and cumulative weight gain were not affected by essential oil supplementation according to regression analysis. In contrast, cumulative feed intake decreased linearly with increasing levels of the essential oil blend, and the diets containing 0.10 and 0.15 kg/t produced the lowest feed intake values. Feed conversion ratio for the total period showed a quadratic response, with the best feed efficiency observed at 0.10 kg/t. In Dunnett’s comparisons, birds fed 0.10 kg/t of essential oil had lower cumulative feed intake and improved FCR compared with those fed the positive control diet, whereas the performance of birds receiving the antibiotic and negative control diets did not differ.

Apparent Nutrient Digestibility

Apparent digestibility coefficients of crude protein and ether extract at 42 days of age are presented in Table 3.

Table 3
Digestibility coefficients of protein and ether extract of 42-day-old broiler chickens fed diets with different essential oil levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
DCCP	70.06	72.99	69.99	71.28	70.99	8.30	0.160	0.080
DCEE	64.72	63.98	62.76	63.89	64.07	9.56	0.220	0.010

• PC - positive control; NC - negative control; DCCP - digestibility coefficient of crude protein; DCEE - digestibility coefficient of ether extract; CV - coefficient of variation; SEM - standard error of the mean.

Dietary treatments did not affect the digestibility coefficients of either nutrient, and no significant linear or quadratic effects were detected in the regression analysis. Likewise, Dunnett’s test indicated no differences between the positive control and the diets containing the essential oil blend, suggesting that the additive did not alter apparent nutrient digestibility under the conditions of this study.

Intestinal Histomorphometry

Histomorphometric measurements of the small intestine at 42 days are shown in Table 4.

Table 4
Histomorphometry of the small intestine of 42-day-old broiler chickens fed diets with essential oil supplementation.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
Duodenum
Villus (µm)	466.00	427.00	456.00	482.00	508.00	5.51	0.0241	11.090
Crypt (µm)	43.69	42.54	42.42	40.72	42.58	5.43	0.713	0.609
V:C	10.68	10.20	10.73	11.80	11.96	9.68	0.216	0.346
Ileum
Villus (µm)	318.00	286.00	312.00	339.00	347.00	6.76	0.0312	10.220
Crypt (µm)	44.34	41.89	43.35	42.24	42.42	3.44	0.667	0.599
V:C	7.16	7.04	7.20	8.04	8.19	7.19	0.075	0.288
Jejunum
Villus (µm)	426.00	369.00	400.00	417.00	459.00	7.32	0.0363	9.913
Crypt (µm)	42.25	43.69	42.44	41.08	42.45	2.64	0.114	0.391
V:C	10.07	8.44	9.43	10.18	10.83	9.11	0.051	0.345

• PC - positive control; NC - negative control; V:C - villus to crypt ratio; CV - coefficient of variation; SEM - standard error of the mean. ¹ Linear equation y = 428 + 53.92 (R² = 0.99). ² Linear equation y = 289 + 42.16x (R² = 0.95). ³ Linear equation y = 368 + 57.56x (R² = 0.97).

Villus height in the duodenum, jejunum and ileum increased linearly with increasing levels of the essential oil blend, indicating a positive response of mucosal development to the additive. In contrast, crypt depth and the villus height:crypt depth ratio were not significantly affected by dietary treatments in any of the intestinal segments evaluated. Although regression analysis evidenced a consistent linear improvement in villus height across segments, Dunnett’s test did not detect significant differences between birds fed essential oil diets and those receiving the positive control diet for any of the histomorphometric variables.

Gastrointestinal Tract Organ Biometrics

Biometrics of gastrointestinal tract organs in broilers at 7 and 14 days of age are presented in Table 5.

Table 5
Biometrics of the gastrointestinal tract organs of 7- and 14-days-old broiler chickens fed diets with different essential oil levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 7 days
BW	83.71	83.71	87.14	87.14	84.00	14.88	0.880	0.807
GITW	20.95	19.67	20.52	20.88	20.11	12.37	0.823	0.456
PR+GIZ	7.53	7.74	7.09	7.21	6.78	10.44	0.146	0.150
SI (g)	5.32	5.11	5.26	5.55	4.91	11.39	0.252	0.115
LI (g)	1.57	1.87	1.82	1.64	1.90	12.86	0.191	0.046
Pancreas (g)	0.52	0.53	0.55	0.57	0.54	13.15	0.862	0.013
Liver (g)	4.48	4.55	4.09	4.74	4.17	12.90	0.123	0.113
1 to 14 days
BW	228.00	218.00	229.00	225.00	230.00	6.31	0.387	2.703
GITW	15.99	17.39	17.32	17.08	16.68	7.22	0.698	0.226
PR+GIZ	5.64	5.94	5.60	5.70	5.20	10.47	0.155	0.117
SI (g)	3.77	5.26	5.33	5.04	4.90	12.83	0.610	0.122
LI (g)	1.33	1.29	1.26	1.42	1.33	13.78	0.433	0.034
Pancreas (g)	0.50	0.53	0.49	0.52	0.52	12.95	0.604	0.012
Liver (g)	3.80	3.59	3.69	3.59	3.76	9.36	0.749	0.063

• PC - positive control; NC - negative control; BW - body weight; GITW - gastrointestinal tract relative weight; PR+GIZ - proventriculus + gizzard; SI - small intestine; LI - large intestine; CV - coefficient of variation; SEM - standard error of the mean.

At these ages, neither body weight nor the absolute or relative weights of the total gastrointestinal tract, proventriculus plus gizzard, small intestine, large intestine, pancreas or liver were influenced by the inclusion of the essential oil blend. No significant effects were observed in the regression analysis, and Dunnett’s test showed that the positive control diet did not differ from the other treatments, indicating that the additive did not alter early GIT organ development.

Relative biometrics of gastrointestinal tract organs at 21 and 42 days of age are shown in Table 6.

Table 6
Relative biometrics of 21- and 42-days-old broiler chickens fed diets with different essential oil inclusion levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 21 days
BW	484.00	474.00	500.00	508.00	492.00	6.29	0.243	6.022
GITW	13.84	14.62	13.92	3.86	14.19	6.58	0.427	0.175
PR+GIZ	4.45	4.63	4.40	4.47	4.33	10.60	0.689	0.086
SI (g)	4.10	4.50	4.35	4.27	4.58	7.51	0.299	0.063
LI (g)	0.85	1.01	0.88	0.92	0.90	11.31	0.153	0.020
Pancreas (g)	0.45	0.41	0.44	0.43	0.45	13.81	0.591	0.011
Liver (g)	3.17	3.23	3.15	3.07	3.06	11.17	0.778	0.063
1 to 42 days
BW	2512.00	2475.00	2545.00	2575.00	2436.00	5.68	0.265	27.51
GITW	6.73	6.83	6.63	6.54	6.91	5.76	0.277	0.074
PR+GIZ	2.17	2.20	2.14	2.11	2.23	5.76	0.292	0.024
SI (g)	1.75	1.77	1.72	1.69	1.79	5.69	0.254	0.019
LI (g)	0.48	0.49	0.48	0.47	0.50	5.83	0.225	0.005
Pancreas (g)	0.18	0.19	0.18	0.18	0.19	5.37	0.219	0.002
Liver (g)	1.70	1.80	1.74	1.72	1.81	5.77	0.294	0.019

• PC - positive control; NC - negative control; BW - body weight; GITW - gastrointestinal tract relative weight; PR+GIZ - proventriculus + gizzard; SI - small intestine; LI - large intestine; CV - coefficient of variation; SEM - standard error of the mean.

At both time points, body weight and the relative weights of the total GIT, proventriculus plus gizzard, small intestine, large intestine, pancreas and liver were not affected by essential oil supplementation. The absence of significant effects in both regression analyses and Dunnett’s comparisons suggests that the essential oil blend did not modify gastrointestinal organ growth or relative development throughout the rearing period.

Serum Biochemical Profile

Serum biochemical variables of broilers at 7 and 14 days of age are presented in Table 7.

Table 7
Serum biochemical profile of 1- and 14-days-old broiler chickens fed diets with different essential oil levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 7 days
Amylase (U/dL)	809.00	793.00	742.00	777.00	795.00	10.2	0.583	14.69
Cholesterol (mg/dL)	160.00	161.00	165.00	164.00	150.00	4.43	0.095	2.55
Ca (mmol/dL)	7.62	7.80	7.69	7.99	7.34	10.68	0.528	0.153
P (mmol/dL)	3.16	4.03	3.35	3.99	3.69	12.76	0.219	0.128
Glucose (mg/dL)	255.00	248.00	265.00	276.00	260.00	5.49	0.234	4.78
TP (g/dL)	3.07	2.90	2.85	3.40	3.10	13.69	0.127	0.093
Ca:P (mmol/dL)	2.39	1.95	2.30	2.00	2.00	13.80	0.119	0.057
Triglycerides (mg/dL)	241.00	246.00	246.00	239.00	238.00	5.49	0.788	3.49
AST (U/L)	246.00	227.00	243.00	261.00	249.00	15.5	0.566	8.30
ALT (U/L)	33.43	28.7	30.8	30.2	28.5	13.06	0.802	0.891
1 to 14 days
Amylase (U/dL)	850.00	830.00	854.00	855.00	780.00	8.03	0.146	13.25
Cholesterol (mg/dL)	144.00	145.00	127.00	155.00	134.00	13.87	0.376	5.78
Ca (mmol/dL)	7.93	7.70	7.84	7.76	7.99	9.47	0.948	0.168
P (mmol/dL)	3.96	3.52	3.64	3.74	3.83	14.26	0.859	0.122
Glucose (mg/dL)	254.00	258.00	254.00	267.00	281.00	8.34	0.377	5.600
TP (g/dL)	2.55	2.11	2.45	2.39	2.60	13.37	0.060	0.071
Ca:P (mmol/dL)	2.04	1.97	2.29	2.02	2.00	13.83	0.395	0.072
Triglycerides (mg/dL)	232.00	231.00	231.00	207.00	187.00	9.28	0.071	7.410
AST (U/L)	279.00	279.00	269.00	278.00	274.00	6.76	0.802	3.930
ALT (U/L)	29.81	29.8	29.92	30.47	28.06	11.04	0.747	0.766

• PC - positive control; NC - negative control; TP - total protein; Ca:P - calcium to phosphorus ratio; Tri - triglycerides; AST - aspartate aminotransferase; ALT - alanine aminotransferase; CV - coefficient of variation; SEM - standard error of the mean.

At these ages, amylase, cholesterol, calcium, phosphorus, glucose, total protein, calcium:phosphorus ratio, triglycerides, AST and ALT activities were not influenced by dietary treatments. Neither the regression analysis nor Dunnett’s test indicated significant differences among the essential oil levels or between the essential oil treatments and the positive control diet, suggesting that early systemic metabolic status was not altered by the additive.

The serum biochemical profile of broilers at 21 and 42 days of age is shown in Table 8.

Table 8
Biochemical profile of 21- and 42-day-old broilers fed diet with essential oil levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 21 days
Amylase (U/dL)	835.00	910.00	854.00	872.00	826.00	10.35	0.622	21.500
Cholesterol (mg/dL)	188	148	161	182	142	15.94	0.294	7.760
Ca (mmol/dL)	7.19	7.13	6.88	7.28	7.60	11.53	0.675	0.198
P (mmol/dL)	3.52	3.81	3.60	3.78	3.78	12.05	0.898	0.103
Glucose (mg/dL)	262	281	290	272	249	9.27	0.171	6.920
TP (g/dL)	2.98	2.76	3.03	2.60	2.72	13.38	0.456	0.092
Ca:P (mmol/dL)	2.04	1.87	1.92	1.93	2.02	10.61	0.782	0.048
Triglycerides (mg/dL)	229.00	230.00	232.00	214.00	211.00	10.19	0.171	6.220
AST (U/L)	282.00	277.00	278.00	263.00	257.00	5.28	0.136	3.970
ALT (U/L)	20.23	20.61	21.65	17.16	18.53	12.40	0.851	0.704
1 to 42 days
Amylase (U/dL)	755.00	763.00	702.00	695.00	753.00	9.12	0.161	13.14
Cholesterol (mg/dL)	220.00	216.00	206.00	206.00	210.00	3.39	0.336	2.14
Ca (mmol/dL)	7.62	7.19	7.76	6.92	7.33	12.89	0.649	0.224
P (mmol/dL)	3.71	4.02	3.43	3.63	3.63	11.18	0.272	0.107
Glucose (mg/dL)	208.00	237.00	228.00	214.00	216.00	10.13	0.879	5.599
TP (g/dL)	2.98	2.79	3.30	3.18	3.20	14.29	0.178	0.087
Ca:P (mmol/dL)	2.28	1.78	2.27	1.94	2.01	14.46	0.161	0.079
Triglycerides (mg/dL)	239.00	239.00	236.00	235.00	238.00	2.22	0.806	1.37
AST (U/L)	310.00	310.00	259.00	311.00	288.00	18.74	0.421	12.24
ALT (U/L)	27.35	22.76	25.86	25.43	24.68	11.09	0.424	0.684

• PC - positive control; NC - negative control; TP - total protein; Ca:P - calcium to phosphorus ratio; Tri - triglycerides; AST - aspartate aminotransferase; ALT - alanine aminotransferase; CV - coefficient of variation; SEM - standard error of the mean.

As observed in the earlier phases, no significant effects of dietary treatments were detected on any of the evaluated variables, including amylase, cholesterol, calcium, phosphorus, glucose, total protein, calcium:phosphorus ratio, triglycerides, AST and ALT. Regression analyses showed no linear or quadratic responses to increasing essential oil levels, and Dunnett’s comparisons revealed no differences between birds fed the essential oil blend and those receiving the positive control diet. Overall, the data indicate that the essential oil blend did not affect the serum biochemical profile of broilers during the grower and finisher periods.

Liver Enzyme Activity in Liver Tissue

AST and ALT activities in liver tissue of broilers fed diets with different essential oil levels are presented in Table 9.

Table 9
Profile of AST and ALT in liver viscera of broiler chickens fed diets with different essential oil levels.

Parameter	PC	NC	Essential oil level (kg/t)			CV (%)	p-value	SEM
			0.05	0.10	0.15
1 to 7 days
AST (U/L)	311.00	303.00	272.00	267.00	299.00	22.14	0.794	14.74
ALT (U/L)	47.40	49.96	42.03	45.60	39.67	14.63	0.181	1.76
1 to 14 days
AST (U/L)	207.00	205.00	206.00	207.00	220.00	4.34	0.101	2.63
ALT (U/L)	42.39	27.51	32.55	32.42	32.03	15.44	0.420	1.20
1 to 21 days
AST (U/L)	222.00	206.00	220.00	226.00	233.00	5.88	0.068	3.87
ALT (U/L)	27.97	23.05	27.16	29.33	24.55	12.98	0.091	0.97
1 to 42 days
AST (U/L)	279.00	233.00	291.00	284.00	260.00	12.05	0.090	9.30
ALT (U/L)	37.32	37.26	41.04	39.56	37.15	5.4	0.060	1.00

• PC - positive control; NC - negative control; AST - aspartate aminotransferase; ALT - alanine aminotransferase; CV - coefficient of variation; SEM - standard error of the mean.

At 7, 14, 21 and 42 days of age, no significant differences were detected among treatments for either enzyme. Both regression analysis and Dunnett’s test indicated that increasing levels of the essential oil blend did not influence hepatic AST or ALT activities when compared with the control diets. These findings support the interpretation that the essential oil blend did not induce detectable alterations in liver enzyme activity or evidence of hepatic impairment under the conditions of this study.

DISCUSSION

Dietary supplementation with phytogenic additives has been widely investigated as a strategy to support broiler performance in antibiotic-free systems (Rahman et al., 2022), and several studies report that essential oils can partially or totally replace antibiotic growth promoters under specific conditions (Li et al., 2024). In the present trial, the commercial blend of cinnamon and oregano essential oils improved growth performance mainly by reducing feed intake and improving feed conversion ratio from 1 to 21 and from 1 to 42 days of age, particularly at the inclusion level of 0.10 kg/t (Table 2).

From a functional perspective, the comparison with zinc bacitracin is relevant because both treatments were associated with improved feed efficiency, although likely through different biological pathways. Zinc bacitracin was included as a positive control because it remains a classical reference growth promoter in poultry experiments, despite its reduced use in current commercial systems. Its inclusion, therefore, provided an experimental benchmark rather than evidence of mechanistic equivalence. Zinc bacitracin acts mainly through antimicrobial effects on susceptible intestinal bacteria, reducing microbial competition for nutrients and alleviating intestinal inflammatory pressure. In contrast, cinnamaldehyde- and thymol-based additives may combine antimicrobial activity with antioxidant, anti-inflammatory, and intestinal epithelial-supporting effects (Oliveira et al., 2024).

In the present study, this interpretation is supported by the linear increase in villus height observed in the small intestine, suggesting improved intestinal functionality and absorptive capacity, which may have contributed to the better feed conversion ratio observed at 0.10 kg/t. These findings agree with previous reports showing that oregano or mixed essential oils can improve feed efficiency and overall performance in poultry (Basiouni et al., 2023; Shehata et al., 2022), although the magnitude and consistency of the response depend on bird genotype, age, diet composition, and environmental conditions. Likewise, the performance pattern observed here, characterized by improved feed conversion without major changes in final body weight, suggests more efficient use of ingested nutrients rather than a marked anabolic effect. Similar responses have been reported in broilers and laying hens supplemented with oregano, thyme, or blends of cinnamon and other phytochemicals, in which feed efficiency improved while growth rate or egg production was maintained or only slightly increased compared with antibiotic-based programs (Latek et al., 2022; Zhang et al., 2023). Conversely, other studies have reported limited or no performance benefits from essential oils (Oni & Oke, 2025; Biswas et al., 2024), reinforcing that the response to phytogenic additives is highly context-dependent and influenced by the nutritional and sanitary status of the flock.

Despite the improvement in feed efficiency, the essential oil blend did not alter apparent digestibility coefficients of crude protein or ether extract at 42 days of age (Table 3). This contrasts with studies in which essential oils, alone or combined with organic acids, improved ileal digestibility of nutrients, apparent metabolizable energy, transporter expression and antioxidant status, and modulated gut microbiota (Bedford & Apajalahti, 2022; Elolimy et al., 2025). A plausible explanation is that, under the present experimental conditions, the basal corn-soybean diets already provided high nutrient availability and a relatively stable gut environment, leaving little room for further improvements in digestibility (Fathima et al., 2022; Gao et al., 2025). Moreover, the specific composition and dose of the commercial blend, the duration of supplementation, the age of the birds and the absence of severe enteric challenges may have limited its capacity to generate measurable changes in overall nutrient digestion and absorption (Latek et al., 2022; Kogut, 2022). Thus, in this study the beneficial effects of the additive appear to be more closely associated with subtle improvements in intestinal function and efficiency rather than with detectable changes in apparent nutrient digestibility.

Intestinal histomorphometry helps to clarify the functional response observed in the present study. At 42 days of age, villus height increased linearly in the duodenum, jejunum, and ileum with increasing dietary levels of the essential oil blend, whereas crypt depth and the villus:crypt ratio were not significantly affected (Table 4). In broilers, taller villi are generally associated with greater absorptive surface area, improved mucosal integrity, and more efficient nutrient uptake, whereas lower crypt activity may indicate reduced epithelial turnover and lower energy expenditure for tissue renewal (Wang et al., 2025). From a biological standpoint, these changes are relevant because improved intestinal architecture may enhance the functional use of ingested nutrients and help explain the better feed conversion ratio observed, particularly at 0.10 kg/t. Similar findings have been reported in studies with phytogenic additives, including blends of organic acids and oregano oil, in which increases in villus height and, in some cases, changes in crypt depth or goblet cell density were associated with favorable effects on mucosal architecture and barrier function (Behairy et al., 2023; Puvača et al., 2023; Li et al., 2024; Wang et al., 2025). Although the present experiment did not evaluate microbiota composition, mucin dynamics, or inflammatory markers, the linear increase in villus height suggests that the cinnamaldehyde- and thymol-based blend promoted a more developed and functionally efficient intestinal mucosa. This interpretation also strengthens the functional comparison with zinc bacitracin, since both treatments were associated with improved feed efficiency, although likely through different mechanisms. While zinc bacitracin acts mainly by reducing the proliferation of susceptible intestinal bacteria, the phytogenic blend may have contributed to better intestinal functionality through combined antimicrobial, anti-inflammatory, antioxidant, and epithelial-supporting effects, which is consistent with the improvement observed in feed efficiency.

In contrast with the clear effects on villus height, the essential oil blend did not influence the relative weights of gastrointestinal tract organs at any of the evaluated ages (Tables 5 and 6). The lack of changes in the relative weights of the total GIT, proventriculus plus gizzard, small and large intestines, pancreas and liver indicates that the additive did not induce hypertrophy or atrophy of these organs and that digestive development proceeded similarly across treatments. Previous studies have shown that some essential oils can modify organ weights, particularly in the liver and lymphoid organs, depending on dose, composition and administration route (El-Kasrawy et al., 2023; Movahedi et al., 2024). However, other reports using citrus or orange essential oils found no alterations in digestive organ biometrics or small-intestine length (Gore et al., 2024), which agrees with the present findings. Overall, the absence of effects on gastrointestinal organ weights suggests that the essential oil blend did not impose additional metabolic burden or structural changes on the digestive system beyond the subtle histomorphometric variations in villus architecture.

Serum biochemical variables and liver enzyme activities also remained within normal ranges and were not affected by essential oil supplementation, either in serum or in liver tissue (Tables 7-9). No significant differences were detected for glucose, total protein, calcium, phosphorus, triglycerides, cholesterol or liver-associated enzymes such as AST and ALT, regardless of age or dietary treatment. Increases in serum or tissue AST and ALT are commonly interpreted as indicators of hepatocellular damage, altered membrane integrity, or impaired liver function (Hong et al., 2023; Mousa et al., 2023). The stable values observed in the present study therefore support the interpretation that the cinnamaldehyde and thymol blend did not exert hepatotoxic effects at the tested inclusion levels. Other studies have reported reductions or favorable changes in liver enzyme activities and lipid profile when broilers were supplemented with specific essential oils, suggesting hepatoprotective, hypolipidemic, and antioxidant actions under certain conditions (Jachimowicz et al., 2022; Saad et al., 2024; Abd El-Hack et al., 2025; Elolimy et al., 2025). In the current experiment, the absence of changes in serum biochemistry and liver enzymes indicates that the essential oil blend maintained basic metabolic and hepatic function, comparable to that of birds receiving antibiotic or unsupplemented diets.

Taken together, the results of this study indicate that dietary supplementation with a commercial blend of cinnamon and oregano essential oils at 0.10 kg/t can improve feed conversion ratio and reduce cumulative feed intake in broilers without adversely affecting gastrointestinal organ development, serum biochemical profile, or liver enzyme activity. The linear increase in villus height throughout the small intestine suggests a positive influence on mucosal development and absorptive surface, which may partially explain the improved feed efficiency. However, the lack of effect on apparent nutrient digestibility, organ biometrics, or biochemical markers, as well as the predominance of non-significant differences for several variables, indicates that the physiological impact of the additive was moderate and likely dependent on the specific nutritional and sanitary context.

Future studies should include mechanistic endpoints and a broader range of nutritional and sanitary scenarios to clarify how cinnamaldehyde- and thymol-based blends interact with the intestinal environment and establish under which conditions they may consistently replace antibiotic growth promoters in commercial broiler production. Although the present findings support a favorable functional response to the phytogenic blend, the lack of microbiota characterization, challenge models, and inflammatory or oxidative markers does not allow definitive conclusions regarding mechanistic equivalence with zinc bacitracin.

CONCLUSION

Under the conditions of the present study, supplementation of broiler diets with a commercial blend of cinnamon and oregano essential oils at 0.10 kg/t improved feed conversion ratio and reduced cumulative feed intake during the grower and overall periods. This response was accompanied by a linear increase in villus height throughout the small intestine, suggesting improved intestinal functionality and absorptive capacity, which may have contributed to better feed efficiency. The absence of effects on apparent nutrient digestibility, gastrointestinal organ biometrics, and serum and hepatic biochemical indicators indicates that the additive was well tolerated at the tested inclusion levels. Therefore, the cinnamaldehyde- and thymol-based blend may be considered a promising phytogenic additive for improving feed efficiency in broilers reared without antibiotic growth promoters. Nevertheless, the lack of microbiota, challenge, and inflammatory or oxidative data does not allow definitive conclusions regarding mechanistic equivalence with zinc bacitracin.

ACKNOWLEDGEMENTS

The authors thank the Instituto Federal Goiano and the Universidade Federal de Goiás for providing the experimental facilities and laboratory infrastructure used in this study. We are also grateful to the technical staff and students of the poultry experimental unit for their assistance with bird management, sampling, and laboratory analyses. Finally, the authors acknowledge the support of Brazilian funding agencies that promote research in animal nutrition and poultry production.

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FUNDING

This research received no external funding.

DATA AVAILABILITY STATEMENT

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

DISCLAIMER/PUBLISHER’S NOTE

The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.

Corresponding author e-mail address

Júlia Silva Instituto Federal Goiano, Departamento de Zootecnia, Campus Rio Verde, Rodovia Sul Goiana, Km 01, Zona Rural, Rio Verde, 75901 970, Goiás, Brasil. Phone: [phone] Email:[e-mail]

CONFLICT OF INTEREST

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Section Editor:

Maria Fernanda Burbarelli

Publication Dates

• Publication in this collection
  26 Oct 2026
• Date of issue
  2026

History

• Received
  16 Jan 2026
• Accepted
  28 Apr 2026