Effect of thyme and/or garlic on growth performance, carcass traits, intestinal microbiota and health status in broilers

Abstract

The study determined the effect of addition of 2-3 % of garlic (AL2, Al.3) and its combination with 1 % of thyme (AL.2.Th, Al.3.Th) to iso-caloric and iso-nitrogenous diets for broiler chickens on the nutrient digestibility, production traits, slaughter analysis parameters, hematological and biochemical blood indices, and the weight of breast, drumstick meat, and chosen organs. Two hundred fifty one-day-old broiler chickens were assigned to five groups (treatments) with 5 replicates (10 birds per cage, 5 females and 5 males). The experiment lasted 6 weeks. Broiler chickens receiving diets supplemented with the herbs exhibited an increase (P = 0.025) in the average body weight (about 9,1 % vs C) and body weight grain (about 20,7 % vs C) in the finisher period and an increase (P < 0.05) in the organic matter digestibility of the diets (about average 6,9 % vs C). An improvement in the FCR (P = 0.019) index was noted in broiler chickens fed the herb-containing mixtures in each combination in the finishing period (even about 14,4 % vs C in Al.2.Th). Moreover, the best carcass quality with a high proportion of muscles and low abdominal fat content (P = 0.031) was noted in broilers feed 2 % of garlic + 1 % of thyme and 3 % of garlic + 1 % of thyme-enriched diets. The addition of the herbs into the diets contributed to an increase in the number of Lactobacillus and Enterococcus bacteria (P = 0.018) and a decrease in Escherichia coli counts in the small intestine, compared with the control treatment (respectively about 6,3 %↑ and 6,5 %↓ in Al2.Th and Al.3Th). No Salmonella sp. were isolated in any treatment. Additionally, an increase in the percentage of lymphocytes (about 17,1 %) and a simultaneous decrease in the granulocyte (about 17,9 %) count were observed in the blood of broiler chickens AL.3, AL2.Th and Al.3.Th treatments. In summary, incorporation of thyme and/or garlic into the diet for broiler chickens can effectively improve growth performance, carcass parameters, gut microbiota, and the defense potential in the organisms of broiler chickens.

Introduction

The major challenges in the poultry sector are posed by the need for maximization of production efficiency through optimization of environmental parameters that stimulate the growth and health of broiler chickens (Castro et al., 2023; Mohamed and Hassan, 2022).

However, the advancing intensification of production systems and genetic selection for rapid growth have increased the susceptibility of birds to environmental stressors (Ncho et al., 2025). As a result, these factors can lead to, oxidative stress, impaired body homeostasis, and reduced overall performance, ultimately leading to economic losses (Nordquist et al., 2017; Apalowo et al., 2024; Kim et al., 2024; Obianwuna et al., 2024). At the same time, restrictions on the use of antibiotic growth promoters (AGPs) in poultry feed have contributed to intensified research into safe and effective alternatives that will maintain bird health and high productivity in intensive farming conditions (Wickramasuriya et al., 2024). One of the solutions that can eliminate such undesirable phenomena in poultry production is a properly designed bird feeding strategy (Galli et al., 2020). Phytobiotics, which include diverse groups of plant-derived products, such as essential oils, oleoresins, and medicinal plants, have attracted particular interest. With their content of bioactive compounds, e.g. alkaloids, tannins, saponins, essential oils, and flavonoids, they have the potential to exhibit health-enhancing effects on the organism through a number of specific production- and health-related metabolic mechanisms (Kiczorowska et al., 2017; Arif et al., 2024). These effects result, among other things, from improved digestive processes and nutrient absorption, modulation of intestinal microflora, and strengthening of the animal's immune and antioxidant systems (Li et al., 2024; Urban et al., 2025). The inclusion of herbs in the feed ration can improve production parameters, e.g. increased feed intake and conversion (Deeb et al., 2024; Rehman et al., 2025;Lipiński et al., 2019), higher daily body weight gains (Arain et al., 2022; Alizadeh-Ghamsari et al., 2025; Dardouri et al., 2025), reduced bird mortality (Giannenas et al., 2018; Ullah et al., 2024; Kar et al., 2025), and modified biochemical (Olumide and Odunowo, 2019; Ekine et al., 2024; Quan et al., 2025) and hematological (Alagbe et al., 2018; Fathi et al., 2025; Nidaullah et al., 2025) parameters. The beneficial effects of phytobiotics depend largely on their concentration, form of administration, and possible synergistic or antagonistic interactions between their bioactive compounds (Rachwał and Gustaw, 2025; Obianwuna et al., 2024).

Garlic (Allium sativum L.) and thyme (Thymus vulgaris L.) are well-studied herbs with documented pharmacological and nutritional potential in animal production. Garlic is a source of many bioactive compounds, the main one being allicin (diallyl thiosulfate), a non-protein amino acid produced from allin by allinase, which can be converted into various sulfur compounds (e.g. diallylsulfide, γ–glutamyl-S-allyl-L-cysteine) which exhibit antifungal, antiatherosclerotic, hypolipidemic, antithrombotic, and anticancer activity (Chen et al., 2021; Melguizo-Rodríguez et al., 2022; Singh et al., 2025). Additionally, its high antimicrobial activity largely limits the growth of pathogenic microorganisms, thereby maintaining the intestinal microbiota balance and constituting a promising alternative to antibiotic growth promoters (Shittu et al., 2024; Deeb et al., 2025).

Thyme (Thymus vulgaris L.), a medicinal plant from the family Lamiaceae (Hassan and Awad, 2017) with a rich phytochemical composition dominated by thymol (40 %) and carvacrol (15 %) and comprising e.g. cymene, eugenol, and γ-terpinene, which contribute to its strong antibacterial, and anti-inflammatory and antioxidant properties (Kheiri et al., 2018; Dorojan et al., 2015 Ponnampalam et al., 2022; Waheed et al., 2024). In addition, these compounds strengthen the immune response, stabilize cell membranes, and support metabolic efficiency, which can translate into improved poultry health and performance (Ponnampalam et al., 2022; Aljabeili et al., 2018; Manuelian et al., 2021; Oni and Oke, 2025).

Previous studies have shown that garlic, thyme, and their combination may have beneficial effects on poultry (Attia et al., 2018; Yakob et al., 2024; Ashour et al., 2025; Tulasi et al., 2025). However, there is still a need for further research on the effects of simultaneously incorporating these ingredients into the diet of broilers, especially with regard to a comprehensive assessment of production performance and health-related parameters.

Therefore, the aim of the study was to analyze the impact of the addition of herbs: garlic (Allium sativum L.) and thyme (Thymus vulgaris) to diets for broiler chickens on the basic production parameters, nutrient digestibility, hematological and biochemical blood profile, intestinal bacteria in broiler chickens, and the weight of organs, breast muscle, and drumstick meat. The presented study hypothesized that the inclusion of two levels of garlic (2 % and 3 %) and the combination of garlic with thyme in broiler chickens mixtures would favorably affect the production efficiency of broiler chickens while maintaining their body homeostasis with respect to optimal health status.

Materials and methods

Herbs, experimental design, and management

Herbs: dry garlic (Allium sativum) and thyme (Thymus vulgaris) were purchased in a specialist store (Herbs Centre, Lublin, Poland) as a locally produced plant material originating from crop harvesting in 2023. As declared by the manufacturer, the garlic and the thyme were standardized for the alliin content (10 mg of alliin/1 g of garlic) and the thymol content (10 mg of thymol/1 g of thyme, 45 % essential oil), respectively. The chemical composition of the herbs is shown in Table 2. Herbs originating from four different places were subjected to chemical analysis. Three random samples were collected from each batch of herbs and analyzed in triplicate. The content of dry matter and basic nutrients in the dry herb samples (50 g) was determined according to standard AOAC procedures (AOAC 1990) described in chapter 2.3.1.

Table 2.

Basic chemical composition of herbal dry materials: garlic and thyme, g/kg.

Item	Herbs			Statistical parameters
	Garlic (Al)	Thyme (Th)		SEM 2	P-value 3
Dry matter	947		924	7.26	0.207
Crude ash	34.7b		115a	0.97	0.042
Crude protein 1	167a		93.1b	1.85	0.031
Ether extract	72.5		72.8	3.92	0.115
Crude fibre	89.3b		354a	2.43	0.017
Ca	0.84b		18.7a	1.57	0.029
P	4.29a		0.24b	0.98	0.018

Results of 9 samples in three replicates.

¹Calculated by Kjeldahl nitrogen N x 6.25;

²SEM - the standard error of the mean;

³P < 0.05; a, b … - statistical differences.

The experiment was carried out for 6 weeks on a poultry farm (Uścimów, Lubelskie Province, Poland) in accordance with the recommendations of the Second Local Ethics Committee at the University of Life Sciences in Lublin (No. 61/2023). 1-day-old (body weight 42.4 ± 0.1 g) Ross 308 broiler chickens (250 birds) were randomly assigned to 5 dietary treatments with 5 replicate cages per treatment, with 5 males and 5 females/cage (1-m² cages). The number of birds per experimental group (10 birds in 5 replicates) ensures repeatable results and valid statistical inference of the obtained data. The lighting scheme controlled the daylight schedule, temperature, and humidity of the air in the poultry house (Aviagen, 2014a) (Fig. 1). Vaccination and care protocols adhered to established guidelines for broiler chickens, specifically those mandated by Aviagen (2014a) and locally recommended by veterinary services. The vaccination regimen included: Newcastle Disease (ND): administered on day 1 and repeated on days 7 and 21; Marek's Disease: administered on day 1 and Infectious Bronchitis (IB): administered on day 1 and repeated between days 10 and 14.

Fig. 1.

The daylight schedule, temperature, and humidity of the air in the poultry house.

The experimental feed diets were based on cereal meal middlings (wheat and corn) and post-extraction soybean meal (Table 1). Three fattening periods: starter (days 0 to 20), grower (days 21 to 35), and finisher (days 36 to 42) were used in the experiment. The starter diet was administered in the crumble form, while the grower and finisher diets were granulated. From day 1 of the experiment, the broilers were fed according to the methodological design, with 2 and 3 % of garlic (Allium sativum) and 1 % of thyme (Thymus vulgaris) in the diets as an experimental factor. The experimental mixtures were iso-energetic and iso-nitrogenous and balanced in accordance with feeding recommendations for broiler chickens (Aviagen, 2014b). The birds were provided with continuous access to feed and water.

Table 1.

Dietary ingredients and nutrient content of experimental diets, g/kg.

Item		Diets1
		Starter (days 0 to 20)						Grower (days 21 to 35)					Finisher (days 36 to 42)
		C		Al.2	Al.3	Al.2.Th	Al.3.Th	C	Al.2	Al.3	Al.2.Th	Al.3.Th	C	Al.2	Al.3	Al.2.Th	Al.3.Th
Diet composition
Wheat	218.0			198.0	197.0	197.0	197.0	230.0	228.0	225.0	222.0	218.0	270.0	267.0	264.0	261.0	261.0
Soybean meal2	394.7			394.7	385.7	385.7	375.7	357.3	339.3	332.3	335.3	329.3	310.5	293.5	286.5	289.5	279.5
Maize	300.0			300.0	300.0	300.0	300.0	320.0	320.0	320.0	320.0	320.0	320.0	320.0	320.0	320.0	320.0
Soybean oil	60.0			60.0	60.0	60.0	60.0	70.0	70.0	70.0	70.0	70.0	80.0	80.0	80.0	80.0	80.0
Garlic	-			20.0	30.0	20.0	30.0	-	20.0	30.0	20.0	30.0	-	20.0	30.0	20.0	30.0
Thyme	-			-	-	10.0	10.0	-	-	-	10.0	10.0	-	-	-	10.0	10.0
Dicalcium phosphate	18.3			18.3	18.3	18.3	18.3	18.0	18.0	18.0	18.0	18.0	18.0	18.0	18.0	18.0	18.0
Limestone	12.0			12.0	12.0	12.0	12.0	10.0	10.0	10.0	10.0	10.0	7.0	7.0	7.0	7.0	7.0
NaCl	3.3			3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3
DL-Met 4	3.6			3.6	3.6	3.6	3.6	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3	3.3
L-Lys 5	3.4			3.4	3.4	3.4	3.4	3.6	3.6	3.6	3.6	3.6	3.6	3.4	3.4	3.4	3.4
Vitamin-mineral premix 6	5.0			5.0	5.0	5.0	5.0	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5
Chemical composition, g/kg
MEn (MJ/kg) 7			12.45		12.42	12.43	12.44	12.45	12.78	12.68	13.01	13.08	13.09	13.35	13.20	13.01	13.25
CP 3			221.3		221.1	221.2	220.9	221.3	219.2	216.2	218.1	223.1	217.8	209.5	215.4	216.7	295.1
Lys			14.29		14.30	14.28	14.33	14.29	13.75	13.84	14.43	14.28	12.82	13.57	13.16	12.54	12.75
Met + Cys			10.51		10.50	10.49	10.39	10.51	9.88	10.27	10.31	10.67	9.82	8.92	8.79	9.47	8.65
Ca			9.84		9.88	9.91	9.94	9.84	8.87	8.56	9.94	9.73	8.69	7.91	7.86	8.91	8.87
P			6.61		6.62	6.63	6.68	6.61	6.29	6.65	6.53	6.49	6.37	6.21	6.47	6.57	6.59

¹C – control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al.2.Th – with 2 % of garlic + 1 % of thyme; Al.3.Th – with 3 % of garlic + 1 % of thyme;

²46 % CP;

³CP – crude protein;

⁴Evonik Degussa Gmbh. Essen. Germany (per kilogram of 990 g methionine);

⁵Ajinomoto Eurolysine S.A.S., Amiens. France (per kilogram of 780 g lysine);

⁶Added minerals and vitamins per kg of the starter diet: Mn, 100 mg; I, 1 mg; Fe, 40 mg; Zn, 100 mg; Se, 0.15 mg; Cu, 10 mg; vitamin A, 15.000 IU; vitamin D3, 5.000 UI; vitamin E, 75 mg; vitamin K3, 4 mg; vitamin B1, 3 mg; vitamin B2, 8 mg; vitamin B6, 5 mg; vitamin B12, 0.016 mg; biotin, 0.2 mg; folic acid, 2 mg; nicotic acid, 60 mg; pantothenic acid, 18 mg; choline, 1.800 mg. Added minerals and vitamins per kg of the grower diet: Mn, 100 mg; I, 1 mg; Fe, 40 mg; Zn, 100 mg; Se, 0.15 mg; Cu, 10 mg; vitamin A, 12.000 IU; vitamin D3, 5.000 UI; vitamin E, 50 mg; vitamin K3, 3 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; vitamin B6, 4 mg; vitamin B12, 0.016 mg; biotin, 0.2 mg; folic acid, 1.75 mg; nicotic acid, 60 mg; pantothenic acid, 18 mg; choline, 1.600 mg. Added minerals and vitamins per kg of the finisher diet: Mn, 100 mg; I, 1 mg; Fe, 40 mg; Zn,100 mg; Se, 0.15 mg; Cu, 10 mg; vitamin A, 12.000 IU; vitamin D3, 5.000 UI; vitamin E, 50 mg; vitamin K3, 2 mg; vitamin B1, 2 mg; vitamin B2, 5 mg; vitamin B6, 3 mg; vitamin B12, 0.011 mg; biotin, 0.05 mg; folic acid, 1.5 mg; nicotic acid, 35 mg; pantothenic acid, 18 mg; choline, 1.600 mg;

⁷MEn = metabolizable energy in the mixtures corrected to zero nitrogen balance.

Growth performance and apparent digestibility of nutrients

The body weight (BW) of the broiler chickens and the average daily feed intake (ADFI) were recorded at 21, 35, and 42 d of life (for each cage). The body weight gains (BWG) and feed conversion ratio (FCR) were calculated in every experimental period. The mortality rates were recorded daily. The average weight gain, feed intake, and FCR were calculated.

Feed digestibility in the control and experimental mixtures was evaluated with acid-insoluble ash as an internal marker (Kussaibat and Leclercq, 1985). The birds were acclimated to the feed for 4 days. Their droppings were collected for the next 3 days. Each mixture was assessed by examination of four broiler chickens selected randomly from each cage at the final grower and finisher stage. The procedure for the digestibility analysis was described in detail in an earlier publication by the authors (Kiczorowska et al., 2016a, 2016b; Zając at al., 2021), where the chemical analysis of dry matter content (Method 925.09), crude ash (Method 923.03), ether extract (Method 920.39), and organic matter was carried out in accordance with the standard (AOAC 1990), the content of nitrogen was determined according to Krogdahl and Dalsgard (1981), and the nutrient digestibility coefficient and the content of nitrogen-corrected metabolizable energy (MEn) were calculated for experimental mixtures as recommended in the European Federation of Branches of the World’s Poultry Science Association (1986).

The selection of chickens for slaughter and the slaughter procedures (Ziołecki and Doruchowski, 1989) were carried out in accordance with the procedure described in detail in a previous publication by the authors (Kiczorowska et al., 2016a, 2016b, 2016c; Zając et al., 2021). After the slaughter, a simplified dissection analysis was performed. Liver, gizzard, heart, breast and drumstick muscles, and abdominal fat were collected, weighed, packed, and frozen at −25°C until chemical analyses.

Sample collection and chemical analyses

Herbs and diets

The contents of basic nutrients: dry matter (Method 925.09), crude ash (Method 923.03), crude protein (Method 920.87), ether extract (Method 920.39), and crude fiber (Method 962.09) in the herbs and diets were determined by AOAC (1990).

The amino acid contents in the experimental diets were determined using an automatic amino acid analyzer (AAA 400; Ingos, Prague, Czech Republic) after previous acid hydrolysis with 6 M HCl (method 994.12) (UE Low, Regulation (EU) No 1169/2011, 2011). Cysteine and methionine were determined after oxidative hydrolysis (Arnoldi, 2001).

The contents of Ca and P in the herbs as well as Ca and P in the diets were measured (three replicates of each sample) using flame atomic absorption spectrophotometry (FAAS) (Unicam 939/959AA-6300, Shimadzu Corp., Tokyo, Japan). Calcium was determined at λ=422.7 nm (Polish Standard, PN-EN ISO 6869, 2002). The total P content was determined colorimetrically (Polish Committee for Standardization, 2002). The individual stages of chemical determinations were described in previous articles (Al-Yasiry et al., 2017a, 2017b; Zając et al., 2022).

All the analyses were performed in triplicate and all data were expressed as means.

Microbiological analysis

Three samples of the small intestine from 10 chickens per treatment (two chickens per cage) collected during the dissection were weighed, diluted with phosphate buffered saline (PBS), and homogenized (Stomacher BagMixer 400; Interscience, Breda, the Netherlands). The detailed procedure for collecting and securing samples for microbiological analyses is described in the publication by Zając et al. (2021). Using the fluorescent in situ hybridization method, microorganisms were determined using group-specific probes (Thermo Scientific, Ulm, Germany) for the Bacteria domain, for Lactobacillus-Enterococcus spp., and for Bifidobacterium spp. as described previously (Kiczorowska et al., 2016a, 2016c). The count of Escherichia coli and Salmonella spp. in the small intestine was determined by culturing colonies as described previously (Kiczorowska et al., 2016a, 2016b).

Analysis of hematological and biochemical blood indices

The chickens selected for the slaughter were also assigned for blood sampling (two broiler chickens/cage – in total 10 birds per treatment). Ten hours before blood sampling, the chickens were not given any feed but were provided with continuous access to water. In the morning on day 42, blood was sampled before the slaughter from the ulnar vein (vena cutanea ulnaris). Blood samples for hematological analyses were collected in 2-ml Vacutest tubes with a K3EDTA anticoagulant (Vacutest Kima s.r.l., Arzergrande, PD, Italy). For biochemical assays, blood was sampled in 2-ml Vacutest tubes containing lithium heparin (Vacutest Kima s.r.l.).

Whole blood was analyzed within three hours after sampling. The red blood cell (RBC), mean cell volume (MCV), mean cell hemoglobin (MCH), packed cell volume (PCV), hemoglobin (HGB), white blood cell (WBC) counts, and leucogram were determined following the procedures described by Zając et al. (2022).

Plasma for the analyses of biochemical parameters was obtained by centrifugation of whole blood at 3000 rpm (603 x g) for 15 min in a laboratory centrifuge (MPW-350R, MPW Medical Instruments, Warsaw, Poland) at a temperature of 4°C. Plasma without hemolysis signs was analyzed within four hours after sampling, and the contents of total protein, albumin, creatinine, and glucose were determined along with the activity of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) enzymes, as described previously (Danek-Majewska et al., 2023).

Statistical analysis

The data were analyzed using the General Linear Model (GLM) procedure for one-way analysis of variance (ANOVA; P < 0.05). For the production data, each cage represented a statistical unit. Microbiological data and hematological and biochemical blood indices were analyzed at the level of an individual bird. The normality of data and homogeneity of variances were tested using the Shapiro–Wilk and Brown–Forsythe tests, respectively. Significant differences between the means were determined by Tukey's honestly significant difference (HSD) post hoc test.

$${\mathrm{Y}}_{\text{ij}}=\mu +{\mathrm{a}}_{\mathrm{i}}+{\mathrm{e}}_{\text{ij}}$$

where Y_ij is the measured variable, μ is an overall mean; a_i - the dietary inclusion of the herbs (treatment), and e_ij, - the random error.

Pre-planned orthogonal contrasts were also performed to partition the treatment variance. The following contrasts were applied:

• C1 (overall supplementation effect): difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th);

• C2 (thyme effect): difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th);

• C3 (garlic level effect): difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th);

• C4 (garlic x thyme interaction): assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme.

Statistica software (version 13.3; StatSoft, Tulsa, OK, US) was used for all calculations.

Results

Productivity parameters and nutrient digestibility

No significant differences in the analyzed broiler productivity parameters during the starter period (days 1–20) were found between the study groups (Table 3). The mean body weight (BW), average daily feed intake (ADFI), average periodic feed intake (APFI), body weight gains (BWG), and feed conversion ratio (FCR) were similar in all the experimental groups, regardless of the amount or type of the experimental factor. The inclusion of the 2 % dose of garlic with 1 % of thyme (Al.2.Th) to the experimental diets during the grower period (days 21–35) contributed to an approximately 6.4 % increase (P < 0.001) in BW, compared with the control (C). During the growing period, the applied herbal treatments (Al.2, AL.3, Al.2Th, and Al3.Th) significantly increased Body Weight Gain (BWG) by approximately 9.4 % compared to the control group (C) (C1:P < 0.001 in orthogonal contrast analyses). Concurrently, these treatments resulted in a reduction of the Feed Conversion Ratio (FCR) by roughly 9.2 % relative to the control group (C) (C1:P = 0.032). Furthermore, the inclusion of thyme (Th) in the mixtures enhanced the effect on feed intake, which led to a higher BWG (C4: P = 0.027) but simultaneously increased the FCR value (C4: P < 0.001).

Table 3.

Productivity parameters of broiler chicken fed mixtures with experimental herbs ¹.

Items	Treatments 2					Statistical parameters
						ANOVA		Contrasts, P-value
	C	Al.2	Al.3	A1.2.Th	A1.3.Th	SEM	P-value 4	C1	C2	C3	C4
days 1 - 20 (starter)
BW 5, g	785	779	773	768	759	45.3	0.137	0.120	0.214	0.158	0.215
ADFI 6, g/day	56.23	57.41	56.38	57.18	57.32	34.5	0.115	0.127	0.187	0.173	0.126
APFI total, g/day	1125	1148	1128	1144	1146	61.8	0.089	0.178	0.209	0.143	0.089
BWG 7, g/chicken	734	729	724	717	707	38.9	0.137	0.164	0.126	0.133	0.273
FCR 8, kg/kg	2.01	2.06	2.04	2.08	2.11	0.04	0.107	0.214	0.251	0.071	0.106
days 21 - 35 (grower)
BW 5, g	1756b	1812b	1827ab	1869a	1843ab	55.7	<0.001	0.250	0.104	0.137	0.165
ADFI 6, g/day	117.2	110.3	108.3	111.4	115.1	37.5	0.206	0.237	0.227	0.141	0.134
APFI total, g/day	1641	1544	1516	1560	1611	27.1	0.357	0.171	0.214	0.152	0.109
BWG 7, g/chicken	1022	1083	1103	1152	1136	43.5	0.151	<0.001	0.086	0.164	0.027
FCR 8, kg/kg	1.96	1.79	1.74	1.75	1.84	0.05	0.183	0.032	0.111	0.091	<0.001
days 36 - 42 (finisher period)
BW 5, g	2268b	2354b	2435ab	2462a	2486a	58.9	0.025	0.220	0.174	0.098	0.264
ADFI 6, g/day	256.1	254.5	243.8	248.4	251.8	87.5	0.129	0.177	0.134	0.213	0.136
APFI total, g/day	1793	1781	1707	1739	1763	67.2	0.270	0.143	0.217	0.156	0.129
BWG 7, g/chicken	512b	542b	608a	593ab	643a	40.3	0.013	0.034	0.206	0.073	0.018
FCR 8, kg/kg	1.94a	1.82ab	1.70b	1.66b	1.70b	0.08	0.019	<0.001	0.143	0.101	<0.001
Total rearing period
Mortality, %	3.04a	2.45b	2.51b	2.09b	2.39b	0.05	0.032	0.027	0.131	0.201	<0.001

¹Data represent the mean of 5 cages (10 broiler chicken/cage) per treatment;

²C - control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al.2.Th – with 2 % of garlic + 1 % of thyme; Al.3.Th – with 3 % of garlic + 1 % of thyme;

³ SEM - the standard error of the mean;

⁴P < 0.05, a, b … - statistical differences; C1 - difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th); C2 - difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th); C3 - difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th,); C4 (garlic x thyme interaction) - assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme;

⁶BW – average body weight;

⁷ADFI – average daily feed intake; APFI – average period feed intake.

⁸BWG – body weight gain

⁹ FCR – feed conversion ratio.

A significant effect of the experimental factors was recorded in the final rearing period (finisher). Chickens receiving the Al.2.Th and Al.3.T supplementation exhibited higher BW (P = 0.025) and BWG (P = 0.013) than the control birds (on average 9.1 % and 20.7 %, respectively). The incorporation of herbs into broiler feed mixtures resulted in an increase in Body Weight Gain (BWG) by approximately 15.6 % compared to the control group (C2: P = 0.034), with this effect being more pronounced when thyme was included (C4: P = 0,018). Concurrently, the Feed Conversion Ratio (FCR) decreased across all experimental groups by approximately 11.3 % compared to the control group (C1: P < 0.001). This reduction was particularly pronounced in the AL2.TH and AL3.TH treatments, where FCR was lowered by about 12.4 % (C4: P < 0.001).

Throughout the rearing period (days 1–42), an average 22.4 % reduction (P = 0.032) in the mortality rates was noted in all the groups of birds receiving the herbal supplementation. The beneficial effect of herbal supplementation in reducing mortality of broiler chickens (C2: P = 0.027) was more pronounced in the thyme treatments (Al2.Th and Al.3.Th), resulting in a reduction of up to 26.3 % compared to the control group (C). (C4: P < 0.001).

In the starter period, the inclusion of the herbs to the diets for the broiler chickens did not significantly affect the digestibility of basic nutrients and energy, with the exception of organic matter (OM) (P = 0.027) (Table 4). The Al.2.Th and Al.3.Th experimental groups exhibited higher OM digestibility than in the C treatment (approximately 9.3 % and 5.7 %, respectively). In the grower period, the positive effect of the herbal mixtures was evidenced by the better digestibility of dry matter (DM), crude protein (CP), and OM. The DM digestibility in the Al.3, Al.2.Th, and Al.3.Th treatments was approximately 8 % higher than that observed in the control chickens (P = 0.043). The CP digestibility increased by up to 7.5 % (P = 0.031) in all the experimental groups, compared with the control. The conducted in-depth contrast analysis (orthogonal) of the results obtained from the nutrient digestibility assay of the feed mixtures did not reveal any significant effect of the applied experimental factors: garlic and thyme.

Table 4.

Apparent nutrient and energy digestibility of broiler chicken mixtures, % ¹.

Items	Treatments 2					Statistical parameters
						ANOVA		Contrasts, P-value
	C	Al.2	Al.3	Al.2.Th	Al.3.Th	SEM 3	P-value 4	C1	C2	C3	C4
days 1 - 20 (starter)
Dry matter	74.6	79.7	76.1	81.7	75.8	0.13	0.241	0.157	0.104	0.156	0.135
Crude protein 5	74.3	75.3	75.6	76.4	76.1	0.26	0.133	0.163	0.142	0.134	0.121
Ether extract	76.5	71.8	74.6	73.2	76.8	0.19	0.208	0.132	0.261	0.145	0.109
Organic matter	83.6b	84.2ab	87.3a	92.9a	89.3a	0.15	0.027	0.204	0.106	0.213	0.143
Gross energy	87.3	85.8	87.6	90.3	87.9	0.09	0.253	0.079	0.125	0.201	0.093
days 21 - 35 (grower)
Dry matter	75.1b	76.7ab	81.1a	80.7a	81.7a	0.16	0.043	0.136	0.154	0.138	0.105
Crude protein 5	74.3b	78.9ab	77.7ab	79.9a	79.1a	0.34	0.031	0.147	0.181	0.163	0.206
Ether extract	77.3	76.4	74.9	78.5	77.9	0.13	0.154	0.151	0.139	0.151	0.142
Organic matter	85.6b	86.3ab	87.3ab	89.9a	88.2a	0.14	<0.001	0.138	0.122	0.135	0.133
Gross energy	87.8	85.9	88.1	89.3	88.4	0.09	0.217	0.104	0.141	0.081	0.201
days 36 - 42 (finisher)
Dry matter	85.3	82.4	84.8	85.5	86.1	0.32	0.181	0.216	0.155	0.135	0.111
Crude protein 5	78.1	77.3	78.94	76.3	79.2	0.09	0.242	0.134	0.211	0.131	0.212
Ether extract	75.4	76.6	74.3	77.2	75.6	0.31	0.118	0.143	0.135	0.149	0.135
Organic matter	83.3b	88.2a	89.1a	89.7a	89.4a	0.14	0.012	<0.001	0.162	0.235	0.013
Gross energy	84.3	85.2	85.5	86.7	86.3	0.18	0.149	0.214	0.132	0.106	0.143

¹Data represent the mean of 5 cages (4 broiler chicken/cage) per treatment;

²C - control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al.2.Th – with 2 % of garlic + 1 % of thyme; Al.3.Th – with 3 % of garlic + 1 % of thyme;

³SEM - the standard error of the mean;

⁴P < 0.05, a, b ... - statistical differences C1 - difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th); C2 - difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th); C3 - difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th,); C4 (garlic x thyme interaction) - assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme;

⁵Calculated by Kjeldahl nitrogen N x 6.25.

In the final rearing period, only the OM digestibility parameter differed significantly between the groups (P = 0.012), with the highest values recorded in the chickens from the Al.3, Al.2.Th and Al.3.Th treatments (about average 6,9 % vs C). The conducted orthogonal contrast analysis confirmed the beneficial effect of the herbs on Organic Matter (OM) digestibility in the experimental groups, which was recorded at a level approximately 7 % higher compared to the control group (C) (C1:P < 0.001). Specifically, the inclusion of thyme in the feed mixtures significantly increased (C4:P = 0.013) the efficiency of organic matter digestion by broiler chickens. (about 7,5 % vs C).

Analyses of the carcass traits of broiler chickens

In all the experimental groups, the percentage of abdominal fat in the carcasses decreased (P = 0.031), especially in the chickens from groups Al2.Th and Al3.Th (on average by 25 %, compared with the control) (Table 5). The in-depth contrast analysis also confirmed the beneficial (C1: P < 0.01) effect of the herbal components in the experimental broiler feed mixtures on the reduction of abdominal fat, which decreased by approximately 10 % compared to the control group (C). Furthermore, the inclusion of thyme intensified this dietary effect (C4: P < 0.01).

Table 5.

Carcass traits in broiler chickens fed mixtures with experimental herbs ¹.

Items	Treatments 2					Statistical parameters
						ANOVA		Contrasts, P-value
	C	Al.2	Al.3	Al.2.Th	Al.3.Th	SEM 3	P-value 4	C1	C2	C3	C4
Slaughter parameters
Dressing percentage, g/kg	77.6	78.1	77.9	79.2	78.3	3.51	0.156	0.136	0.145	0.095	0.101
Abdominal fat, g	18.9a	17.2ab	17.7ab	16.8b	16.5b	0.21	0.031	<0.001	0.101	0.157	<0.001
Muscle weight, g
Breast muscle, g	354b	361b	394a	415a	403a	2.38	0.019	0.016	0.009	0.205	0.013
Drumstick muscle, g	149b	161a	157ab	168a	153ab	7.81	0.024	<0.001	0.128	0.147	0.011
Organ weight, g
Liver	54.2	51.8	48.4	52.3	55.4	0.61	0.147	0.236	0.131	0.145	0.243
Stomach	32.3	35.4	35.8	33.8	34.1	0.35	0.234	0.084	0.125	0.132	0.107
Heart	13.6	13.3	12.3	12.8	13.2	0.11	0.109	0.211	0.135	0.153	0.231

¹Data represent the mean of 5 cages (4 broiler chicken/cage) per treatment;

²C - control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al.2.Th – with 2 % of garlic + 1 % of thyme; Al.3.Th – with 3 % of garlic + 1 % of thyme;

³SEM - the standard error of the mean;

⁴P < 0.05, a, b … - statistical differences C1 - difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th); C2 - difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th); C3 - difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th,); C4 (garlic x thyme interaction) - assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme.

Differences were also noted in the weight of the breast muscles of the experimental birds. The carcasses of chickens from groups Al.3, Al.2.Th, and Al.3.Th exhibited the highest weight (P = 0.019), i.e. it was approximately 14 % higher than the carcasses of the control birds. A similar relationship was observed in the case of drumstick muscles (P = 0.024). Their highest weight was observed in the carcasses of the Al.2 and Al.3 chickens; on average, the weight of these muscles was 10 % higher than the weight of the control birds. The positive effect of the herbs (Al.2, Al.3, Al.2Th and Al.3Th) on poultry tissue weight, observed in both the breast muscle and the drumstick (respectively about 11.8 and 7.2 % vs C; C1: P = 0,016 i P < 0.001), as well as the reinforced beneficial effect of thyme (Al.2Th and Al.3Th) (respectively about 15.5 and 7.7 % vs C;C4: P = 0,013 and P = 0.011), was confirmed by the contrast orthogonal analysis. The herbs included in the diet for the broiler chickens as an experimental factor did not induce changes in the size of the internal organs (liver, stomach, heart) of the birds.

Microbiological analyses

The herbal supplementation of the experimental diets did not affect the number of Bifidobacterium sp. (P = 0.156) and the total count (P = 0.232) of bacteria identified in the intestinal contents of broiler chickens on day 42 (Table 6). An increase in the bacterial population present in the intestinal contents of the experimental birds exposed to the Al2.Th and Al3.Th treatments, compared with the control group, was found only in the case of the genera Lactobacillus and Enterococcus (P = <0.001) (approximately 6.7 % and 5.8 %, respectively). There were also differences in the number of Escherichia sp. bacteria (P = 0.017). Their count in the intestines of the Al2.Th- and Al3.Th-treated birds was reduced by approximately 5.7 % and 8 %, compared with the control group. The inclusion of thyme, regardless of the dosage, further reduced the population size of Escherichia sp. (by about 6,8 % vs C; C4: P < 0.001). All the birds examined in the experiment were free of Salmonella sp.

Table 6.

Bacteria identified the intestinal contents of broiler chickens at d. 42. ¹.

Items	Treatments 2					Statistical parameters
						ANOVA		Contrasts, P-value
	C	Al.2	Al.3	Al.2.Th	Al.3.Th	SEM 3	P-value 4	C1	C2	C3	C4
Bacterial counts per g of intestinal contents (log10)
Lactobacillus and Enterococcus	8.10b	8.07b	8.09b	8.65a	8.57a	0.21	<0.001	0.106	0.215	0.137	0.203
Bifidobacterium sp.	8.24	8.31	8.18	8.15	8.10	0.18	0.156	0.201	0.126	0.192	0.117
Escherichia sp.	6.13a	6.08a	6.02ab	5.78b	5.64b	0.07	0.017	0.251	0.144	0.157	<0.001
Salmonella sp.	ND	ND	ND	ND	ND	-	-	-	-	-	-
Total count	10.82	10.21	10.43	10.78	10.53	0.31	0.232	0.123	0.114	0.146	0.138

¹Data represent the mean of 10 birds (2 broiler chicken/cage) per treatment;

²C - control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al.2.Th – with 2 % of garlic + 1 % of thyme; Al.3.Th – with 3 % of garlic + 1 % of thyme;

³SEM - the standard error of the mean;

⁴P < 0.05 - statistical differences C1 - difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th); C2 - difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th); C3 - difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th,); C4 (garlic x thyme interaction) - assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme; ND- not detected.

Hematological and biochemical blood indices in broiler chickens

The herbal supplementation had no effect (P > 0.05) on the level of red blood cells (RBCs), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), hematocrit (PCV), and total white blood cell count (WBC) (Table 7). The broiler group fed the diet supplemented with 3 % of garlic (Al.3) had a significantly higher value of MCV (107.38 fl oz) (P = 0.001), compared with the control group (C) and the Al.2 and Al2.Th groups. The Al3.Th group also exhibited a slightly higher but statistically insignificant value of this parameter of red blood cells.

Table 7.

Effect of dietary inclusion of herbs on hematological and biochemical indices in broiler chicken blood at d. 42¹.

Items	Treatments 2					Statistical parameters
						ANOVA		Contrasts, P-value
	C	Al.2	Al.3	Al.2.Th	Al.3.Th	SEM3	P-value4	C1	C2	C3	C4
Hematological indices5
RBC, 1012/l	2.96	3.00	2.88	2.88	3.18	0.03	0.067	0.092	0.127	0.259	0.004
HGB, mmol/l	8.19	8.32	8.45	8.46	8.46	0.06	0.534	0.536	0.751	0.998	0.448
MCH, pg	45.08	44.77	46.07	47.46	43.00	0.51	0.087	0.220	0.650	0.168	0.012
MCV, fl	100.42b	102.08b	107.38a	101.00b	102.66ab	0.57	0.001	0.340	0.089	0.013	0.012
PCV, l/l	0.24	0.25	0.22	0.23	0.29	0.005	0.052	0.208	0.059	0.056	0.061
WBC, 109/l	32.23	32.17	29.21	33.17	30.03	0.49	0.054	0.710	0.995	0.062	0.475
Leucogram, %
Lymphocytes	56.25b	60.39ab	66.02a	65.41a	66.29a	1.14	0.020	0.002	0.411	0.130	0.475
Monocytes	0.99	0.70	0.58	0.75	0.56	0.05	0.063	0.004	0.693	0.303	0.569
Granulocytes	41.74a	42.10a	34.80ab	34.81ab	33.15b	1.15	0.021	0.010	0.245	0.231	0.674
Biochemical indices in blood plasma
Total protein, g/l	39.95	40.40	39.73	38.70	38.23	0.41	0.096	0.951	0.136	0.522	0.758
Albumin, g/l	20.41	19.68	18.96	20.15	19.83	0.08	0.120	0.378	0.524	0.326	0.407
Creatinine, μmol/l	25.30	29.33	24.98	26.17	25.35	0.74	0.433	0.866	0.363	0.449	0.299
Glucose, mmol/l	15.51	15.55	15.13	15.09	15.75	0.13	0.556	0.854	0.957	0.919	0.251
Enzyme activity in blood plasma 6
AST, U/l	131.32	129.42	127.15	135.14	146.89	3.92	0.425	0.736	0.182	0.373	0.325
ALT, U/l	4.47	3.57	3.98	4.03	3.98	0.39	0.981	0.449	0.975	0.944	0.975
ALP, U/l	2174.00	2029.58	2502.88	2210.46	2377.88	55.53	0.166	0.792	0.372	0.392	0.976
LDH, U/l	2036.42	2147.58	2512.75	2411.53	1941.50	84.85	0.309	0.243	0.759	0.766	0.133

¹Data represent the mean of 10 birds (2 broiler chickens/cage) per treatment;

²C - control diet without herbs. Al.2 – diet with 2 % of garlic. Al.3 – with 3 % of garlic. Al2.Th – with 2 % of garlic + 1 % of thyme; Al3.Th – with 3 % of garlic + 1 % of thyme;

³SEM - standard error of the mean;

⁴P < 0.05, a,b … - statistical differences; C1 - difference between the control (C) and treatments with supplementation of herbs (Al.2, Al.3, Al2.Th, Al3.Th); C2 - difference between treatments with supplementation of only garlic (Al.2, Al.3) and garlic combined with thyme (Al2.Th, Al3.Th); C3 - difference between treatments with supplementation of 2 % of garlic (Al.2, Al2.Th) and 3 % garlic (Al.3, Al3.Th,); C4 (garlic x thyme interaction) - assessment of the interaction between the garlic level (2 % vs 3 %) and the presence of thyme;

⁵RBC - red blood cells, HGB – hemoglobin, MCH - mean corpuscular hemoglobin, MCV- mean corpuscular volume, PCV – packed cell volume, WBC – white blood cells;

⁶AST - aspartate aminotransferase; ALT – alanine aminotransferase; ALP - alkaline phosphatase; LDH - lactate dehydrogenase.

The herb treatments induced changes in the leucogram. The percentage of lymphocytes was higher (P = 0.020) in the group receiving only 3 % of garlic (Al.3) and in both groups supplemented with garlic and thyme (Al2.Th and Al3.Th), compared with the control group. An opposite trend (P = 0.021) was observed in the granulocyte count, which was lower in the group supplemented with 3 % of garlic and 1 % of thyme (Al3.Th), compared with the control group and the birds receiving the 2 % garlic addition.

A deeper statistical analysis using orthogonal contrasts provided further information on the source of these changes. It was confirmed that the overall effect of supplementation (C1) was the main reason for the changes in the leucogram, showing a highly significant effect on increasing the percentage of lymphocytes (P = 0.002) and decreasing the percentage of monocytes (P = 0.004) and granulocytes (P = 0.010). A significant main effect of garlic level (C3) observed for MCV (P = 0.013). This analysis also revealed relationships that were not apparent before. Most importantly, a statistically significant interaction (C4) was demonstrated between the garlic level and the addition of thyme for the red blood cell count (P = 0.004), MCH (P = 0.012), and MCV (P = 0.012).

No significant differences in the analyzed biochemical indices and enzyme activity in the blood plasma were found between the groups (P > 0.05).

Discussion

Medicinal plants are increasingly being used as natural growth promoters in broiler nutrition due to their multifaceted effects on the organisms of birds (Biswas et al., 2024; Ogbuewu et al., 2019; Giannenas et al., 2018). In the present study, the supplementation with garlic and its combination with thyme had no significant effect on the broiler rearing parameters, especially in the first rearing period (days 1-20). This may be explained by the immaturity of the gastrointestinal tract, characterized by low enzymatic activity and limited absorption surface, which reduces the utilization of nutrients and bioactive substances (Ravindran and Abdollahi, 2021). Furthermore, literature reports indicate that the full functional maturity of the intestines of birds, which are the main site of absorption of these compounds, is achieved around the third week of life, parallel to the maturation of the goblet cell population and the stabilization of gastrointestinal microbiota (Aruwa et al., 2021; Duangnumsawang et al., 2021). Low bioavailability of phytobiotics, especially in the case of monogastric animals, was reported by Sugiharto and Ayasan (2023). This finding may explain the absence of statistically significant productivity effects in the experimental birds during this period. This was also reported by Kikusato (2021), who reported poor availability of bioactive compounds in the small intestine of young birds, i.e. only at the level of 2 % with a potential increase to even 15 % during growth. The present study revealed increased body weight, higher daily gains, and improved FCR in the birds during the subsequent stages of rearing, especially after the addition of 2 % and 3 % of garlic combined with 1 % of thyme. The results obtained suggest improved nutrient utilization and enhanced digestive and immune functions in birds receiving herbal supplements. These effects can be explained by the multifaceted effects of the bioactive compounds contained in the herbal supplements. Both these raw materials exhibit strong health-promoting properties and are therefore effective natural growth stimulants. Garlic and thyme support defense mechanisms in the organism, especially in stressful and critical conditions, thereby ensuring better adaptation of birds to intensive production conditions (Abd El-Ghany, 2024). Enhanced immune resistance reduces energy losses resulting from the organism defense reactions, which is associated with higher intake of nutrients necessary for growth and development (Mylostуva et al., 2022). Additionally, the bioactive compounds contained in garlic (e.g. essential oils and organic sulfides) and thyme (mainly thymol and carvacrol) have a positive effect on digestive system functioning (Satyal et al., 2017; Salehi et al., 2018). They increase intestinal nutrient absorption through e.g. improvement of the intestinal mucosa integrity (Aydin and Yildiz, 2020; Ibrahim et al., 2021). Consequently, digestion and absorption processes are improved, which is directly reflected by higher feed conversion and growth rates (Adedokun and Olojede, 2019). These results are consistent with the findings reported by Abou-Elnaga et al. (2016), who found that supplementation of chicken diets with a combination of 1 % of garlic with 0.2 % of thyme and 2 % of garlic with 0.4 % of thyme resulted in higher body weight, higher body weight gain, reduced feed intake, and improved FCR, compared with the control group.

Bioactive compounds present in herbs may exert a positive effect on not only digestive system functioning but also the course and efficiency of digestive processes in farm animals (Metwally, 2023; Lei et al., 2018; Saleh et al., 2018; Kiczorowska et al., 2016). Nutrient digestibility determines the degree of decomposition and absorption of feed components in the gastrointestinal tract as well as the proper growth, development, and general condition of birds (Sugiharto, 2016; Adedokun and Olojede, 2019). The present study showed improvement in protein digestibility in the grower period and organic matter digestibility in the grower and finisher stages in the birds receiving feed rations supplemented with two doses of garlic (2 and 3 %) in combination with thyme (1 %). Due to the crucial importance of effective protein digestion for maximizing of poultry production, the results obtained are significant from a production point of view. However, the type of feed ingredients used and the activity of gastric and pancreatic enzymes contribute to significant variability in the digestibility of this nutrient (Kamel et al., 2015; Mahmood et al., 2017). Rapid passage of intestinal contents combined with the high demand of the organism for amino acids can limit the efficiency of digestive and absorption processes, leading to incomplete protein utilization (Dosković et al., 2013; Alfonso-Avila et al., 2022). According to Gholami-Ahangaran et al. (2022) and Mao et al. (2022), bioactive compounds contained in the analyzed medicinal plants, such as allicin and thymol, stimulate the secretion of digestive enzymes and bile and improve gastrointestinal motility. In addition, they support the development of intestinal villi, increasing the surface area for nutrient absorption, which may have contributed to the improvement in protein, dry matter, and organic matter digestibility observed in this study (Li et al., 2022;Abd El-Ghany, 2024). The present results are partially consistent with those reported by Mohamed et al. (2016), who supplemented broiler feed rations with 25 and 50 mg/kg of allicin and found an average 12.65 % increase in nutrient digestibility. Similarly, Mangisah et al. (2021) used a mixture of 1 %, 2 %, and 3 % of garlic powder with Lactobacillus casei and reported an average 11.60 % increase in the digestibility of crude fat, crude protein, and crude fiber. Thyme added to chicken feed rations improved the digestibility of nutrients, with an approximately 7 % increase in ether extract and a 23 % increase in crude ash (Abbasi et al., 2020; Malekzadeh et al., 2018).

Carcass slaughter characteristics are one of the key indicators of the efficiency of broiler chicken rearing. Rapid growth, increased broiler muscle weight, and the degree of fat deposition in the carcass are largely genetically determined (Liu et al., 2019). The deposition of abdominal fat is an unfavorable phenomenon in terms of the economic efficiency of poultry production and the dietary value of poultry meat (Lokaewmanee and Sirival, 2022). These parameters can be influenced to some extent by the quality of the diet. The present results confirmed the possibility of reducing the abdominal fat content in the carcasses of broiler chickens fed a diet containing the herbs, especially in the variants with 2 % and 3 % of garlic in combination with 1 % of thyme. Furthermore, an increase in the breast and drumstick muscle weight was noted in these birds, compared with the control group. The results obtained may be due to higher protein digestibility in the experimental chickens, which increases the availability of amino acids necessary for muscle protein synthesis (Zhao et al., 2022). As a result, a relatively smaller amount of energy remains available for lipogenesis, limiting abdominal fat deposition (Musigwa et al., 2024), which is reflected in the more favorable carcass structure of broilers from groups receiving the analyzed herbal components. These results are consistent with the findings reported by Brzóska et al. (2015), who observed an average 7.5 % reduction in the percentage of abdominal fat in carcasses of chickens fed a diet containing 1-1.5 ml/kg of garlic extract. Increased garlic extract supplementation (2.5 ml/kg of feed) resulted in an approximately 11 % and 6 % increase in breast and drumstick muscle weight, respectively, compared with the control group. A beneficial effect of thyme on the abdominal fat content was also demonstrated by Adam et al. (2020), who supplemented diets for birds with dried thyme at 2.5 and 5 g/kg of feed and found an average 30 % reduction in the analyzed parameter, compared with the control group. In turn, Belali et al. (2021), reported positive effects of thyme supplementation in broiler chicken diets (100 ppm of thyme extract + 150 mg/kg feed of dried thyme) on breast muscle weight gains, which reached even 15 %, compared with the control.

Intestinal microbiota forms a multilayered microbiological barrier that plays an important role in the organisms of birds, e.g. in digestion, absorption, and regulation of immune function (Zou et al., 2024). All factors, including herbs used in the diet influencing the composition and abundance of poultry gut microbiota, significantly contribute to the growth performance of these animals and thus determine the efficiency of poultry production (Meng et al., 2023; Liu et al., 2023; Rafeeq et al., 2022). The present study showed that the use of garlic alone (2 and 3 %) in the diet for broiler chickens did not induce changes in the bacterial count, in contrast to the combination of the herbs: garlic at the doses of 2 % and 3 % combined with thyme (1 %), which significantly increased the counts of Lactobacillus sp. and Enterococcus sp. bacteria in the small intestine of the broiler chickens. The effects obtained can largely be attributed to the strong antioxidant properties of the analyzed components, which may have prevented oxidative damage to epithelial cells and maintained the microbial balance in the intestines (Li et al., 2024). Furthermore, the lack of clear effects in the case of individual components indicates that appropriately selected combinations of medicinal plants can better support the maintenance of microbial balance in the intestines, which is a key element of preventive healthcare. The observed higher abundance of beneficial bacteria in the intestines of broiler chickens has a positive effect on the health of the birds. Bacteria of the genus Lactobacillus, belonging to lactic acid bacteria, inhibit pathogen growth, modulate immunity, and reinforce the intestinal structure (Broderick and Duong, 2016). In turn, Enterococcus sp. bacteria stabilize the intestinal microflora and support overall health (Wu et al., 2019). Both types of bacteria contribute to the sustenance of the beneficial microbiome and support intestinal integrity, which is reflected by higher feed conversion rates and improved production performance. Results of this study are slightly different results were reported by other researchers who used single herbs. In a study conducted by Elbaz et al. (2021), feed supplementation with 0.5 g/kg of garlic powder had only a slight effect on the Lactobacillus sp. population, which increased by 2.70 %, compared with the control group. However, the number of Enterococcus sp. bacteria decreased by approximately 3.5 % after the garlic supplementation. In turn, Placha et al. (2014) added 0.5 g/kg of thyme essential oil to the feed and observed a 19 % reduction in the number of Enterococcus sp. bacteria and a 14.8 % reduction in lactic acid bacteria, compared with the control group.

Hematological and biochemical blood indices were analyzed to determine the effect of the addition of garlic and thyme to the feed mixtures on the health status and well-being of the chickens. These indices help to assess the physiological and metabolic processes triggered in response to the nutritional strategy. In the present study, the values of blood indices corresponded to the reference ranges specified for this species and breeding line (Reece, 2015; Wakenell, 2010; Campbell, 2022; Zálešáková et al., 2025).

In the present study, a significant effect of the incorporation of the herbs into broiler chicken diets was the modulation of the leucogram, i.e., changes in the proportion of white blood cells (P < 0.05). The increase in the percentage of lymphocytes combined with the decrease in the granulocyte count in the groups receiving 3 % of garlic and the garlic and thyme combination is a beneficial phenomenon. These findings were corroborated and further elucidated by the contrast analysis, which identified the overall effect of supplementation (C1) as the primary statistical source of this variability. This rise suggests enhancement of immune defenses in the bird organism, which is one of the most frequently described and desirable effects of the use of such phytobiotics as garlic (Allium sativum) and thyme (Thymus vulgaris).

The study results are consistent with numerous scientific reports indicating the immunomodulatory properties of bioactive compounds contained in these plants: allicin in garlic and thymol and carvacrol in thyme (Kheiri et al., 2018; Melguizo-Rodríguez et al., 2022). Many studies have confirmed that these compounds can stimulate the production and activity of lymphocytes, which are crucial for the adaptive (specific) immune response. As demonstrated by Prasad et al. (2009) and Noruzi and Aziz-Aliabadi (2024), higher garlic levels in broiler chicken diets increased the percentage of lymphocytes, suggesting enhanced immune response. Thyme was found to improve immune parameters in broiler chickens as well. Hassan and Awad (2017) used powdered thyme (2, 5, or 8 g/kg) in feed mixtures and reported increased white blood cell and lymphocyte counts, indicating improved immunity in broiler chickens. However, the effect of phytobiotics used in broiler chicken diets as combined garlic and thyme supplementation on immune function has been addressed in only few studies. As shown by Ashour et al. (2025), a combination of garlic and thyme (1 g of powdered garlic + 1 g of powdered thyme per kg of feed mixture) in broiler chicken diets had a synergistic effect on immune function, supporting health and immune response. Their study showed that this combination significantly improved immune parameters, i.e. immunoglobulin levels, lymphocyte count, and the weight of immune organs.

In the present study, the evaluation of the red blood cell parameters revealed an influence of garlic level (C3) on MCV increase, alongside a significant interaction between garlic level and thyme supplementation (C4) in modifying the values of MCV, MCH, and RBC. This suggests that the impact of garlic level on erythrocyte morphology and count was strongly modulated by the addition of thyme. While the administration of 3 % garlic (Al.3) induced the formation of enlarged erythrocytes (increased MCV) accompanied by a relatively low RBC count, the combined use of thyme and garlic at the same level (Al3.Th) appeared to mitigate these morphological alterations (lower MCH and MCV). Simultaneously, this combination stimulated erythropoiesis, resulting in the highest red blood cell count (RBC) recorded in the study. This synergistic and beneficial influence of the garlic-thyme combination on red blood cell parameters is consistent with findings reported in other studies (Jameel et al., 2014). The beneficial effect of garlic on red blood cell parameters can be attributed to its antioxidant properties (Abd El-Ghany, 2024), which can protect erythrocyte cell membranes from damage, potentially extending their lifespan or influencing their morphology (Rehman and Munir, 2015). Noteworthy, the effect of garlic on red blood cell parameters described in the scientific literature is ambiguous. Some studies (Toghyani et al., 2011; Oleforuh-Okoleh et al., 2015) indicate improvement in such parameters as the red blood cell count, hemoglobin content, and hematocrit percentage, which is attributed to the antioxidant properties of garlic and potential stimulation of erythropoiesis.

Conversely, other studies have not confirmed changes in red blood cell parameters following garlic supplementation in broiler diets (Prasad et al., 2009). This discrepancy may be related to differences in the doses applied, supplementation methods (powder, extract, oil), and environmental conditions.

The existing literature also suggests the potential for positively modulating hematological indices through the use of various forms of thyme supplementation in broiler diets. Al-Kassie (2009) demonstrated that including an oil extract derived from thyme in broiler feeds increased red and white blood cell counts, hematocrit, and hemoglobin levels. Similarly, Attia et al. (2018) evaluated the effect of dried thyme powder supplementation (at 10, 20, or 30 g/kg feed) on the hematological parameters of broiler chickens. They reported an increase in hemoglobin and MCH at the 10 and 30 g supplementation levels. This beneficial effect is attributed to thyme's antioxidant properties, similar to those of garlic, and its established antimicrobial characteristics. Thyme also contains a significant amount of iron, which is essential for hemoglobin synthesis and the processes of erythropoiesis (Attia et al., 2018; Waheed et al., 2024).

In terms of the analyzed biochemical indices, the stable levels of total protein, albumin, creatinine, and glucose (P > 0.05) indicate that the addition of thyme and/or garlic, regardless of the dose, did not disrupt the basic metabolic processes or the nutritional status of the birds. The stable plasma levels of liver function markers, i.e., aspartate aminotransferase (AST) and alanine aminotransferase (ALT), suggest that the garlic and thyme additives did not cause hepatotoxicity at the doses and combinations used. This was also evidenced by the absence of an effect of these herbal supplements on the liver weight (P > 0.05). This is crucial for the safety of use of phytobiotics and confirms the results reported in other studies, which often indicate a protective (hepatoprotective) effect of these herbs (Gholami-Ahangaran et al., 2022; Javed et al., 2025). The present study also assessed the activity of two other enzymes, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), which provided additional information about the health status of the birds. Enhanced ALP activity is observed in bone growth and calcification disorders. The activity of LDH, which is present in all body cells, is increased in states of enhanced cell membrane permeability caused by e.g. the action of toxins (Reece, 2015; Scanes, 2015). The present experiment demonstrated no significant effect of the feed supplementation with the herbs on the activity of these two enzymes.

The effectiveness of bioactive substances is determined not only by their form but also by the quantity and quality of other feed ingredients, which can either strengthen or weaken their effect. The beneficial impact of the combined garlic and thyme supplementation observed in the present study, which was more potent than the effects of both components used alone and confirmed by orthogonal contrasts analysis, may have been a result of synergism (Giannenas et al., 2018; Karole et al., 2019). This phenomenon may have a pharmacodynamic nature, i.e. the compounds reinforce each other’s effects on the same biological targets, or a pharmacokinetic character, i.e. an impact on the processes of absorption, metabolism, and elimination, thus increasing the bioavailability of active substances (Zhou et al., 2016). The bioactive compounds in garlic and thyme, such as allicin, thymol, and carvacrol, can enhance each other’s activity, which results in improved digestibility and intestinal health as well as enhanced growth performance. Unlike the additive effect, which involves only the sum of the effects of individual components, synergism has a significantly stronger effect (Zhou et al., 2016). In nutritional practice, such interactions not only increase the effectiveness of the additives but also limit their potential toxicity (Malongane et al., 2017). Hence, a combination of natural plant ingredients can be an effective strategy for supporting the growth and health status of birds and serve as an alternative to synthetic stimulants. A greater abundance of beneficial bacteria in the intestines of broiler chickens supports birds’ health and improves production parameters.

Conclusions

The use of the 2 % of garlic + 1 % of thyme and 3 % of garlic + 1 % of thyme addition in the diets of broiler chickens at 21 to 42 d of age contributed to improved production performance (body weight, body weight gain, FCR in the finisher period), selected slaughter parameters (increased the abdominal fat content, while concurrently increasing the breast and drumstick muscle weights) without adversely affecting hematological and biochemical parameters. In addiction, the mixture with analyzed herbs promoted organic matter (day 1 to 42) and dry matter and crude protein (21 to 35 day) digestibility. beneficial changes in the intestinal microflora by increasing the number of Lactobacillus and Enterococcus and, reducing the number of Escherichia sp., and supported the birds’ immune system without causing signs of However, interpretation of these conclusions should take into account the limitations of the study, related to the use of a single bird genotype, housing system, constant levels, and fixed levels and forms of the components used. Therefore, further research is needed to confirm the effectiveness and clarify the mechanisms of action of garlic and thyme on broiler chicken under diverse production conditions.

CRediT authorship contribution statement

Szymon Milewski: Writing – original draft, Investigation, Formal analysis, Data curation. Bożena Kiczorowska: Writing – original draft, Supervision, Project administration, Methodology, Investigation, Formal analysis. Wioletta Samolińska: Writing – original draft, Methodology, Data curation. Renata Klebaniuk: Methodology, Formal analysis, Data curation. Piotr Kiczorowski: Writing – original draft, Formal analysis, Data curation. Julia Fabianowska: Formal analysis, Data curation.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.