Growth Performance, Mortality, and Blood Biochemistry of Broiler Chickens Supplemented with Cypress and Juniper Essential Oils

INTRODUCTION

For decades, antibiotics have been widely used as growth promoters in poultry diets to enhance productivity, improve feed efficiency, and maintain flock health. This practice has ultimately been reshaped worldwide in response to growing concerns over antimicrobial resistance, which is driven by antibiotic residues in animal-derived food products, as well as the proven link between antimicrobial use in food-producing animals and the emergence of resistant bacteria transmissible to humans (Abate et al., 2025; Theodoridou Oxinou et al., 2025). Consequently, antibiotic growth promoters (AGPs) have been progressively restricted and eventually banned in many regions, beginning with a complete ban in the European Union in 2006 (Beber et al., 2025), with similar restrictions implemented in the United States in 2017 (Gens et al., 2022), followed by China in 2020 (Wen et al., 2022). These regulatory changes have pushed the poultry industry to develop effective non-antibiotic approaches capable of sustaining performance, ensuring food safety, and satisfying increasing consumer expectations for antibiotic-free products.

The withdrawal of AGPs has created a production environment in which broilers are more susceptible to intestinal imbalance, subclinical health challenges, and performance losses, particularly under intensive, high-density rearing conditions (Fernández Miyakawa et al., 2024; Maria Cardinal et al., 2019; Selim et al., 2024). In response to these challenges, phytogenic feed additives (PFAs), commonly termed phytobiotics or botanicals (Windisch et al., 2008), have attracted considerable attention. These additives comprise plant-derived bioactive compounds, including herbs, spices, plant extracts, and essential oils (EOs), and are characterized by a high content of secondary metabolites such as flavonoids, alkaloids, tannins, saponins, and terpenoids (Adetunji et al., 2025; Aminullah et al., 2025). Among PFAs, EOs have received particular interest due to their broad biological activities, including antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory effects (Mirković et al., 2025; Pezantes-Orellana et al., 2024; Varijakzhan et al., 2021).

A growing body of literature confirms that EOs from diverse botanical sources can act as effective growth-promoting additives in broiler diets. Supplementation with EO blends has been shown to improve body weight, feed conversion ratio (Abd El-Hack et al., 2022; Khan et al., 2024), intestinal morphology, and digestive enzyme activity (Johnson et al., 2022; Su et al., 2021), as well as to beneficially alter serum lipid profiles and antioxidant status (Elbaz et al., 2022b; Yarmohammadi Barbarestani et al., 2020), often under both standard and challenge conditions such as necrotic enteritis (Gharaibeh et al., 2021; Huang et al., 2025). Meta-analyses and controlled trials consistently report that EO supplementation can reduce cecal pathogenic bacteria, enhance beneficial microbiota (Akan et al., 2025; Irawan et al., 2021), and improve carcass and organ traits (Islam et al., 2025; Obeidat et al., 2022), thereby providing a functional alternative to AGPs in modern broiler production.

The biological efficacy of EOs is largely attributed to their dominant terpenoid constituents, of which alpha-pinene is one of the most abundant and widely distributed monoterpenes in nature (Bagchi et al., 2020). Alpha-pinene is a major component of turpentine and of EOs derived from coniferous species (Al-Tel et al., 2020). This compound has been reported to possess broad-spectrum antimicrobial activity (Allenspach et al., 2021; Borges et al., 2022), antiviral effects (Başer et al., 2023), and notable anti-inflammatory and antioxidant properties (Allenspach et al., 2021; Rahimi et al., 2023). Given these bioactivities, alpha-pinene represents a promising target for the development of functional feed additives aimed at enhancing gut health, immune resilience, and overall performance in broilers. Among conifer-derived EOs, those obtained from cypress (Cupressus sempervirens L.) and juniper (Juniperus communis L.) are particularly rich in alpha-pinene (Ložienė et al., 2016; Patgar et al., 2021). Yet, despite their long-standing use in traditional medicine (Galovičová et al., 2023; Tufail et al., 2023), these oils have received limited attention as feed additives in poultry nutrition. So far, the effects of these oils in broiler chickens remain unexplored, as research on EOs in poultry has mostly focused on a relatively limited set of botanical sources, such as oregano, thyme, rosemary, and eucalyptus species.

Therefore, the present study addresses the knowledge gap concerning alpha-pinene-rich EOs from cypress and juniper by evaluating their individual and combined effects on broiler growth performance and selected serum biochemical markers. It is hypothesized that supplementation with either cypress or juniper EOs will improve broiler performance and favorably modulate serum biochemical markers compared with non-supplemented controls, while concurrent administration of both oils may exert additive or synergistic effects on performance and health-related parameters, thereby representing a promising natural alternative to AGPs and supporting more sustainable poultry production.

MATERIALS AND METHODS

Study location and environment: This study was conducted at the Salem Poultry Group facility in Biskra, Algeria (Latitude: 34°50′13″ N, Longitude: 5°53′09″ E, Height: 101 m). The broiler production site includes 12 houses, each with a capacity of 50,000 caged birds. The houses were equipped with Big Dutchman’s climate controller system, Viper Touch® (software version: 5.3.4 Build 14).

Essential oils: Juniper (Juniperus communis) and cypress (Cupressus sempervirens) essential oils (EOs) were purchased from local suppliers, who also provided the main components detected by GSMS (Table 1). To prepare the EOs solutions for administration in drinking water, Tween 20 and propylene glycol were used as emulsifying agents to ensure solubility. The juniper (JEO) and cypress (CEO) solutions contained 25% EO, 25% Tween 20, and 50% propylene glycol. Additionally, a mixture of the two oils (MEO) was prepared containing 12.5% of each oil, 25% Tween 20, and 50% propylene glycol.

Table 1: Main compounds of the essential oil of C. sempervirens and J. communis

CEO: Essential oil of Cupressus sempervirens.
JEO: Essential oil of Juniperus communis.

Broiler chickens, experimental design, and diets: The experiment was conducted on 480 one-day-old, non-sexed Arbor Acres Plus chicks (mean body weight of 49.02 ± 0.91 g), obtained from a commercial hatchery belonging to the same poultry production group. Hatching eggs from a 54-week-old breeding flock were stored for 4-8 days at 16 °C and approximately 75% relative humidity, with automatic turning every hour. Short periods of incubation during egg storage (SPIDES) were not performed.  At hatching, chicks received two vaccine injections: TRANSMUNE^® for Infectious Bursal Disease (IBD), and NEWFLEND^® ND H9 for Newcastle disease virus (NDV) and low-pathogenic avian influenza virus (LPAIV) subtype H9. Additionally, live vaccines were administered by spraying: CEVAC^® VITABRON L for NDV and infectious bronchitis virus (IBV), and CEVAC^® IBIRD for an IBV variant.

After a 4-hour processing time at the hatchery, chicks were transported in the early afternoon to a broiler house located at the same facility. Upon arrival, chick quality was assessed using the Pasgar© scoring system, resulting in an average score of 9.72, while mean chick length and cloacal temperature were 19.5 ± 0.41 cm and 39.79 ± 0.37 °C, respectively. After quality assessment and individual weighing, the birds were randomly allocated to five treatment groups following a completely randomized design (CRD), with each treatment comprising eight replicate pens of 12 birds each.

The study was conducted under standard commercial farming conditions concurrently with an ongoing production cycle. The negative control (NC) group received a corn–soybean meal diet without antibiotic growth promoters (AGPs) and plain drinking water. The positive control (PC) group followed the standard management program applied in the broiler facility, including continuous in-feed monensin (80 mg/kg) as AGP, and a preventive course of tylosin (100% active substance) on day 8 for 5 days, as part of the farm's established prophylactic protocol. A therapeutic course of tylosin combined with enrofloxacin (200 mg/ml) was also administered to the PC group via drinking water. The Juniper essential oil (JEO), Cypress essential oil (CEO), and mixed essential oil (MEO) group (a mixture of the two EOs), received their respective EO solutions in drinking water at 2.5 ml/L of 25% stock solution, delivering a final concentration of 0.625 ml/L (625 ppm) of pure EO, for 3 days followed by 3 days of plain water. This administration cycle was repeated until the end of the experiment. Since the chicks were initially vaccinated at the hatchery, they only received a booster vaccination against NDV via spray on day 12 using CEVAC^® NEW L vaccine. The physicochemical characteristics of drinking water are shown in Table 2.

Table 2: Physicochemical composition of the water sample.

SP: Spectrophotometry.
¹ Algerian National Standard for water quality (1992 version).

Broilers had ad libitum access to the following diets: starter (1–19 days of age), grower (20–35 days of age), and finisher (36–42 days of age). The feed ingredients and nutrient compositions of the basal diets are presented in Table 3.

Table 3: Ingredient (%) and nutrient composition of the basal diet (as-fed basis).

Vitamin and mineral premix supplied per kg of diet

¹ Starter phase : 833,333 IU of vitamin A; 250,000 IU of vitamin D3; 3,750 mg of vitamin E; 191.67 mg of vitamin K3; 150 mg of thiamine; 450 mg of riboflavin; 3,750 mg of niacin; 1,225 mg of pantothenic acid; 375 mg of pyridoxine; 116.67 mg of folic acid; 2.25 mg of cyanocobalamin; 8.33 mg of biotin; 33,600 mg of choline chloride; 4583 mg of Fe; 1,167 mg of Cu; 4,583 mg of Zn; 5,250 mg of Mn; 167 mg of I; 16.67 mg of Se.

² Grower phase: 666,666 IU of vitamin A; 200,000 IU of vitamin D3; 3,000 mg of vitamin E; 153.33 mg of vitamin K3; 120 mg of thiamine; 360 mg of riboflavin; 3,000 mg of niacin; 980 mg of pantothenic acid; 300 mg of pyridoxine; 93.33 mg of folic acid; 1.8 mg of cyanocobalamin; 6.67 mg of biotin; 16,800 mg of choline chloride; 3667 mg of Fe; 933 mg of Cu; 3,667 mg of Zn; 4,200 mg of Mn; 133.33 mg of I; 13.33 mg of Se.

³ Finisher phase: 623,338 IU of vitamin A; 187,001 IU of vitamin D3; 2,805 mg of vitamin E; 143.37 mg of vitamin K3; 112.2 mg of thiamine; 336.6 mg of riboflavin; 2,805 mg of niacin; 916.31 mg of pantothenic acid; 280.5 mg of pyridoxine; 87.27 mg of folic acid; 1.68 mg of cyanocobalamin; 6.23 mg of biotin; 23,400 mg of choline chloride; 3667 mg of Fe; 933 mg of Cu; 3,667 mg of Zn; 4,200 mg of Mn; 133.33 mg of I; 13.33 mg of Se.

⁴ Values were determined using NIR spectroscopy.

⁵ Values were calculated from data provided by the INRA CIRAD AFZ Feed Tables (2020 edition, as-fed).

Health challenge and interventions: A disease outbreak occurred during the experimental period, with the first clinical signs, including depression and ruffled feathers, observed at 28 days of age. To investigate the etiology of the outbreak, 10 clinically affected birds, selected across the experimental groups, were subjected to necropsy and submitted to the Veterinary Hospital Center “Le Refuge”, Ayoun El Assafir, Batna, Algeria. Gross pathological examination revealed hepatomegaly with fibrinous perihepatitis, lesions consistent with colibacillosis. Liver and heart samples were aseptically collected and cultured on MacConkey agar and blood agar, followed by aerobic incubation at 37 °C for 24–48 h. Bacterial isolates were identified as Escherichia coli based on Gram stain, colony morphology and confirmation using API 20E biochemical tests system, after that, serotyping by agglutination has confirmed belonging to Serotype O1, which is highly implicated in avian colibacillosis. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar.

Following laboratory confirmation of E. coli infection and demonstration of in vitro susceptibility to enrofloxacin, a therapeutic antibiotic intervention was initiated in the PC group on day 29 and administered for five consecutive days. Enrofloxacin was selected based on the antibiogram results, while tylosin was co-administered to address the potential involvement of concurrent bacterial respiratory pathogens commonly associated with field outbreaks. No therapeutic antibiotic intervention was administered to the NC or EO-treated groups during the outbreak.

Management and rearing of birds: Broiler chickens were placed in a 240 × 160 × 50 cm cage divided into four compartments, each comprising a pen, resulting in a stocking density of approximately 12 birds per square meter. The drinking nipples and automatic feeding systems were disabled, and manual feeders (6 kg) and water reservoir tanks (3 L) were used instead. Feed and water quantities were manually measured and recorded daily to ensure precise intake control.

Lighting was provided using LED tube lamps installed in each cage. White light intensity was adjusted according to the rearing phase, decreasing from 50 lux during the starter period to 20 lux during the finisher phase. The light color was temporarily switched to blue at an intensity of 5 lux during stressful handling procedures, such as vaccination and weighing. Continuous light was given for the first two days (24 h), followed by 23 h of light and 1 h of darkness until day 9. From day 9 onwards, the light cycle was 18 h of light followed by 6 h of continuous darkness.

The house temperature was carefully controlled using an automated climate control system, starting at 33 °C and gradually decreasing to 21 °C. Relative humidity (RH) averaged 48.4 ± 6.23%, and the mean temperature-humidity index (THI) was 72.9 ± 5.04. The THI was calculated according to NRC (1971) using the formula:

THI = (1.8 × T + 32) − [(0.55 − 0.0055 × RH) × (1.8 × T − 26)]

where T is dry-bulb temperature (°C) and RH is relative humidity (%).

Adequate ventilation and climatic parameters were maintained throughout the study to ensure optimal conditions for birds. The evolution of temperature, RH and THI over the study period are shown in Figure 1.

Figure 1: Air temperature, relative humidity and average temperature-humidity index during experimental period.

Traits Evaluated

Growth performance: During the rearing period, body weight (BW) was calculated weekly by weighing all the birds individually. Feed intake (FI) and water intake (WI) were recorded daily. Feed conversion ratio (FCR) and body weight gain (BWG) were then calculated. At the end of the study, the European production efficiency factor (EPEF) was calculated according to the following formula: (BW (kg) × livability (%) / age (days) × FCR) x 100 (Huff et al., 2013).

Collection of blood samples for serum biochemical analyses: On day 42 of the experiment and after 8 h of fasting, blood samples were collected from the left-wing vein of one randomly selected chicken per pen (eight birds per treatment group). Immediately after collection, the blood samples were transported to a private medical laboratory for serum biochemical analyses. The samples were analyzed for various biochemical parameters including blood glucose, uric acid, creatinine, total protein, total cholesterol, HDL and LDL cholesterol, and triglyceride concentrations.

Statistical analysis: Prior to analysis, data collected and calculated during the experiment were tested for normality with the Shapiro–Wilk test and for homogeneity of variances using Levene’s test. One-way analysis of variance (ANOVA) was performed to evaluate statistical differences between the treatment groups in a completely randomized design using IBM SPSS software (version 26). The following mathematical model was applied:

Y_ij = µ + T_i + e_ij

In which Y_ij is the j-th observation in the i-th treatment group, µ is the overall mean, T_i is the fixed effect of the i-th treatment group, and e_ij is the random error. When the ANOVA indicated significant effects, treatment means were compared using Tukey’s honestly significant difference (HSD) test. Differences were considered statistically significant at p ≤ 0.05.

RESULTS

Table 4: Effects of different treatments on body weight during the experiment.

^{a, b} Means within the same row carrying different superscripts are significantly different (p ≤ 0.05).
NC: Negative control; PC: Positive control; CEO: Cypress essential oil; JEO: Juniper essential oil; MEO: Mixed essential oils; SEM: Standard error of the mean.

The effects of essential oils (EOs) on body weight (BW) are summarized in Table 4. Significant differences in BW between the treatment groups were observed only on day 7 (p = 0.022), with the NC group recording the highest weight. From day 14 onward, BW did not differ significantly among treatments. However, from day 35 onward, EO-supplemented groups exhibited numerically higher BW than the controls, with the CEO group achieving the greatest increase (8.6% vs NC), followed by MEO (3.9%) and JEO (3.5%). This numerical trend persisted to day 42, where final BW was highest in the CEO group (6.8% relative to NC), followed by JEO (4.8%) and MEO (3.1%). Additionally, the PC group also outperformed the NC group by 3.9% and showed final BW values comparable to those of the JEO and MEO groups.

Table 5: Effects of different treatments on cumulative feed conversion ratio during the experiment.

^{a, b} Means within the same row carrying different superscripts are significantly different (p ≤ 0.05).
NC: Negative control; PC: Positive control; CEO: Cypress essential oil; JEO: Juniper essential oil; MEO: Mixed essential oils; SEM: Standard error of the mean.

Feed conversion ratio (FCR) did not differ among treatments at 7 days of age (p = 0.223). Subsequently, significant differences were observed, with the PC group exhibiting a significantly lower FCR at 14 and 21 days of age (p < 0.001). This pattern persisted until day 28 (P = 0.006), with the PC group maintaining the lowest FCR, while EO-supplemented groups showed values comparable to the NC group.

At day 35, the CEO group exhibited a significantly lower FCR compared to the NC group (P = 0.008), while the PC group and the other EO-treated groups showed intermediate values. By the end of the trial (day 42), this difference was no longer statistically significant; however, the CEO group maintained a numerically lower FCR (8.1% relative to NC) and remained comparable to the PC group.

Table 6: Effects of different treatments on the zootechnical parameters of broilers.

^{a, b} Means within the same row carrying different superscripts are significantly different (p ≤ 0.05).
NC: Negative control; PC: Positive control; CEO: Cypress essential oil; JEO: Juniper essential oil; MEO: Mixed essential oils; SEM: Standard error of the mean; Mort: Mortality; FI: Feed intake; WI: Water intake; ADG: Average daily gain; EPEF: European production efficiency factor.

Significant differences were observed between treatments for mortality (p = 0.001) and the European Production Efficiency Factor (EPEF) (p ≤ 0.001). The CEO group recorded the lowest mortality rate (54.6% lower relative to NC), whereas the PC group showed the highest mortality (22.7% higher relative to NC). Weekly mortality rates are presented in Figure 2, showing a pronounced peak around week 5. Accordingly, the PC group received additional therapeutic interventions during the trial and was therefore not considered a pure AGP control or used as a benchmark for growth performance comparisons.

EO-supplemented groups achieved higher EPEF values than the NC group, with the CEO differing significantly compared to the control groups, exhibiting the greatest increase (29.8% relative to NC), followed by JEO (13.4%) and MEO (8.2%). No significant differences were found between the treatment groups in terms of feed intake (FI) (p = 0.489), water intake (WI) (p = 0.076), or average daily gain (ADG) (p = 0.220). Nevertheless, CEO group exhibited numerically higher ADG than the NC group (6.8%).

Figure 2: Weekly (non-cumulative) mortality rates during the experimental period.

Table 7: Effects of different treatments on the blood serum parameters of broilers.

^{a, b} Means within the same row carrying different superscripts are significantly different (p ≤ 0.05).
NC: Negative control; PC: Positive control; CEO: Cypress essential oil; JEO: Juniper essential oil; MEO: Mixed essential oils; SEM: Standard error of the mean; Glu: Glucose; TG: Triglycerides; CHOL: Cholesterol; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; Crea: Creatinine; UA: Uric acid; TP: Total protein.

Glycaemia levels were similar across all treatment groups, with no significant differences observed (p = 0.899), indicating that the inclusion of EOs did not significantly impact blood glucose levels compared to the control groups.

Triglyceride concentrations varied significantly between the groups (p ≤ 0.001), with the MEO group having the lowest value. Total cholesterol levels also differed (p = 0.008) with the JEO group had the lowest value, significantly lower than those of the NC and MEO groups, whereas the PC and CEO groups showed intermediate values. Dietary supplementation with CEO significantly reduced HDL concentrations (p = 0.012) compared to the other treatment groups. Significant differences were also observed in LDL levels (p = 0.020), with the JEO group recording the lowest value, significantly lower than those in the NC and MEO groups.

Serum creatinine concentrations varied significantly among the treatment groups (p ≤ 0.001), with the MEO group having the lowest value. Uric acid levels also varied significantly (p = 0.009); the NC group had the lowest concentration, significantly lower than those in the CEO and JEO groups.

No significant differences were observed in serum total protein levels between the treatment groups (p = 0.616), indicating that the EOs did not significantly affect the total protein compared to the control groups.

DISCUSSION

Alpha-pinene, a major monoterpene, is widely found in the essential oils (EOs) of various plant species, particularly in conifers of the Pinaceae family (Bakó et al., 2024; Dudek et al., 2025), as well as in some herbs of the Lamiaceae family (Spréa et al., 2024). It is known for its distinctive pine aroma and its diverse biological activities have been extensively studied (Silva et al., 2012; Park et al., 2021). Numerous studies have explored the effects of EOs containing alpha-pinene in broiler chickens, highlighting their potential as natural growth promoters (Hairui et al., 2024; Hesabi Nameghi et al., 2019; Tekce et al., 2020; Yesilbag et al., 2011). Nevertheless, this study is the first to investigate the EOs of juniper (Juniperus communis) and cypress (Cupressus sempervirens), both of which are rich in alpha-pinene (Zheljazkov et al., 2021; Medini et al., 2025).

The current study’s trial was conducted under routine commercial field conditions in an intensive broiler production facility, following standard industry management practices. During the late grower phase, a natural outbreak of colibacillosis occurred, causing clinical signs and characteristic post-mortem lesions. As a result, the study was effectively transformed from a standard growth performance evaluation into a disease challenge model, providing a realistic context to assess the response of EO-supplemented groups under bacterial stress. The positive control (PC) group, which received an in-feed antibiotic growth promoter (AGP) along with preventive and therapeutic antibiotic courses, does not represent a pure AGP control and should not be considered a valid benchmark for growth performance under these conditions.

Overall, the core growth parameters showed non-uniform temporal patterns, with statistically significant differences appearing sporadically, rather than persisting throughout the experimental period. Such transient responses are consistent with the widely reported variability in the efficacy of EOs, which tend to exert more pronounced benefits under challenging conditions, such as environmental heat stress or increased pathogenic pressure (Placha et al., 2019; Abdelli et al., 2021; Hosseinzadeh et al., 2023). The spontaneous infection in the current study may have interacted with EOs supplementation to drive the observed transient improvements in performance and health parameters in the treated groups. For instance, although differences were not statistically significant, numerical improvements were observed in the cypress EO (CEO) group, with body weight (BW) approximately 7% higher than the negative control (NC) group on day 42, and 9% higher on day 35. Similar responses under disease challenge conditions have been reported by Elbaz et al. (2024) who observed improvements in BW and body weight gain (BWG) under coccidiosis challenge conditions following supplementation with a mixture of oregano EO and probiotics. Similarly, broiler chickens facing an intestinal challenge involving Eimeria spp. and Clostridium perfringens showed improvements in feed intake (FI), BWG, and feed conversion ratio (FCR) when supplemented with a protected blend of organic acids and EOs (Stefanello et al., 2020). Although immune parameters, anti-inflammatory markers, and gut health metrics were not measured in the present study,  the numerical and transient improvements may be attributed, at least in part, to the immunomodulatory effects exerted by the EO and their bioactive components (Ibrahim et al., 2022; Zaazaa et al., 2022). EO supplementation has been shown to attenuate inflammatory responses, support intestinal barrier integrity, and enhance overall immune competence (Movahedi et al., 2024; Oni et al., 2025; Sampath et al., 2025), thereby facilitating compensatory growth and performance recovery in challenged broiler chickens (Zhang et al., 2023; Mountzouris et al., 2024). However, under E. coli challenge conditions, average daily gain (ADG) and FI remained unaffected across all treatments potentially due to nutrient repartitioning toward immune function (Remus et al., 2014). This phenomenon likely limited the potential of the EOs to enhance FI and BWG in the present study, resulting in the final BW of the best-performing group (CEO) fell short of the breed’s performance objectives (Aviagen, 2022) by approximately 9.4%. Similarly, Gordillo Jaramillo et al. (2021) found no significant effects of oregano and citrus EOs on FI or BWG in broiler chickens orally challenged with a coccidiosis vaccine at 25 times the recommended dose. In line with these findings, under non-challenged conditions, broilers fed diets supplemented with rosemary, thyme and oregano powder showed no significant changes in FI or BWG (Khatun et al., 2025), nor did those receiving thyme and oregano EOs, alone or combined (Zaazaa et al., 2022). Furthermore, several studies have indicated limited EO impacts on growth: for example, Gumus and Gelen (2023) found no effect of thyme and rosemary EOs on BW, BWG or FCR; Noruzi et al. (2022) reported no significant changes in BW following dietary supplementation with a blend of nine EOs;  and dietary inclusion of different levels of cinnamon EO in Arbor Acres commercial male broilers did not affect performance parameters, including BW and BWG (Yang et al., 2019).  In contrast, certain EO formulations have demonstrated benefits, such as 400 mg/kg fenugreek EO enhancing FI and ADG (Fawaz et al., 2025), or a blend of EOs containing 200 mg/kg cinnamaldehyde, 200 mg/kg carvacrol, and 100 mg/kg thymol significantly improving these metrics (Malhi et al., 2025).

In terms of FCR, the CEO group showed lower values compared to the control groups on day 35, indicating improved feed efficiency at the end of the grower period. This improvement may be related to the beneficial effects of EOs on gut health and inflammatory status (Adil et al., 2025). In this regard, Pham et al. (2023) reported that dietary supplementation with a coated EO and organic acids improved FCR in broilers challenged with E. coli, an effect associated with reduced intestinal damage, lower bacterial load, and modulation of inflammatory responses. Such mechanisms may partially explain the improved feed efficiency observed, particularly under the natural challenge condition of the present study. The overall trend of improved FCR with cypress EO aligns with previous studies conducted under standard rearing conditions, in which supplementation with lavender, thyme, and clove EOs resulted in reduced FCR (Yarmohammadi Barbarestani et al., 2020; Amouei et al., 2021; Elbaz et al., 2022a).

Previous studies have shown that phytogenic supplements administered in drinking water can influence both water intake (WI) and productive responses in broilers. For example, Ashour et al. (2025) reported a decrease in WI following water supplementation with Echinacea extracts, yet this was accompanied by improvements in physiological and productive parameters. In contrast, the present study found no significant differences in WI between treatments, indicating that EO supplementation did not adversely affect water palatability. This stability in water consumption highlights a key advantage of administering EOs via drinking water over conventional in-feed supplementation (Rocha et al., 2024), particularly under health-challenge conditions in which FI may be compromised while water consumption remains relatively stable, thereby ensuring sustained delivery of EO bioactive compounds.

The significant difference in BW on day 7, with the NC group exhibiting the highest values, indicates that neither the EOs nor the AGP provided a growth advantage during the initial starter phase. Although EOs can affect water intake in young chicks due to their strong aroma, no such effects were observed in this study. Instead, the superior performance of the NC group likely reflects the absence of unnecessary interventions in a well-managed, low-stress environment, where birds  perform near their genetic potential; therefore, growth promoters tend to have less favorable effects (Attia et al., 2016; Attia et al., 2017; Pourmahmoud et al., 2013). Furthermore, early AGP use can suppress beneficial gut bacteria and induce dysbiosis (Abbas et al., 2024), while the EO dose employed, though beneficial later, may have been relatively high for immature chicks, potentially transiently impairing organ function or nutrient use (Aguilar et al., 2013).

Mortality rates increased across all experimental groups, peaking in the fifth week after the colibacillosis outbreak. Control groups experienced higher mortality, whereas EO-supplemented groups, especially the CEO group, showed significantly lower rates. These findings align with necrotic enteritis challenge studies, in which EO blends resulted in zero mortality compared to 10% in untreated groups, accompanied by a significant reduction in intestinal lesion scores and modulation of the immune response toward an anti-inflammatory pathway (Gharaibeh et al., 2021). As noted previously, the absence of physiological assessments limits direct causal links; nevertheless, the protective effects of the studied EOs are consistent with their well-documented antioxidant, antibacterial, and anti-inflammatory properties (Patgar et al., 2021; Albrecht et al., 2022; Tufail et al., 2023; Maral et al., 2024; Mirković et al., 2025), which are mainly linked to their major active components, including alpha-pinene (Borges et al., 2022; Bomfim De Barros et al., 2023; Hoosen et al., 2026). In vitro studies of cypress and juniper EOs have demonstrated notable antimicrobial activity against poultry-relevant pathogens. For example, cypress EO exhibited antibiofilm activity against Salmonella enterica isolated from chicken samples (Galovičová et al., 2023), while juniper EO inhibited the growth of Campylobacter jejuni (Klančnik et al., 2018), and extracts from Juniperus species showed broad-spectrum activity against both Staphylococcus aureus and E. coli (Darwish et al., 2020). Additionally, EO derived from Sideritis species, in which alpha-pinene was identified as the major component, demonstrated antimicrobial activity similar to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) and other multidrug-resistant bacteria (Kose et al., 2010).

However, alpha-pinene itself showed moderate to low antimicrobial action against E. coli and multi-resistant E. coli (Dorman et al., 2000; Leite-Sampaio et al., 2022), suggesting that the observed reductions in bacterial burden and fibrin deposition in EO-treated groups are likely associated with synergistic interactions among EO constituents. Such synergistic interactions have been documented for combinations such as peppermint and thyme or citrus EOs, resulting in enhanced activity against E. coli (Angane et al., 2024; Ellouze et al., 2024).

Beyond direct pathogen inhibition, EOs can modulate host immunity and intestinal microbiota, thereby enhancing systemic immune responses (Ge et al., 2024). For instance, dietary supplementation with a blend of peppermint and clove EOs enhanced digestive enzyme activity and improved immune organ development (Reda et al., 2025). Additionally, supplementation with basil, thyme, and sage EOs restored Lactobacillus populations and suppressed enteric pathogens (Vlaicu et al., 2023). Similarly, clove EO has been shown to boost intestinal IgA and Lactobacillus counts and reduced E. coli populations (Elbaz et al., 2022a), while oregano EO has been reported to improve gut morphology (villus height and crypt depth) and support lymphocyte proliferation and phagocytic activity (El-Sayed et al., 2024). Collectively, these immunomodulatory and microbiota‑stabilizing actions may help explain the enhanced resilience observed in the EO‑supplemented groups.

The higher mortality in the PC group highlights a limitation in mechanistic interpretation inherent to the study design. The PC group likely included birds with more severe initial disease presentations; however, because disease severity was not objectively assessed, and the protocol involved administration of therapeutic antibiotics during a disease episode, in addition to baseline AGP supplementation, multiple confounding variables are introduced. Furthermore, the timing and dosage of the therapeutic intervention may have been insufficient or delayed to fully mitigate the disease impact, complicating the determination of individual factors to the observed mortality.

The European Production Efficiency Factor (EPEF), which combines several zootechnical parameters, showed a highly significant improvement in the CEO group. This supports the notion that EOs can enhance the overall production efficiency, as reported by İpçak et al. (2024), who found that the addition of fennel seeds EO at different doses significantly improved both FCR and EPEF. Another study indicated that broiler chickens fed diets supplemented with EOs from basil, thyme, and sage showed enhanced EPEF and European Broiler Index (EBI) (Vlaicu et al., 2023). In contrast, Ghiasvand et al. (2021) found that fennel EO reduced BWG during the finisher period and lowered the EPEF. In the present experiment, the improvement in EPEF observed in the CEO group was primarily driven by the significantly lower mortality rate, as differences in BW and FCR were only numerical. Thus, the higher EPEF recorded in the CEO group can be directly attributed to improved survivability rather than to changes in growth performance parameters.

The results of the present study showed no significant differences in blood glucose levels between the treatment groups, suggesting that EOs did not interfere with carbohydrate metabolism. This is consistent with previous reports by Santos et al. (2019), who similarly reported no significant differences in glycemia, total cholesterol, or total protein levels between the control groups and those receiving diets supplemented with lemongrass EO. The JEO group showed significantly lower cholesterol and LDL levels, and MEO significantly reduced triglycerides levels, in contrast to the study of Khan et al. (2024), who found that adding different levels of a blend of EOs containing eucalyptus and citrus to broiler diets did not significantly affect cholesterol, triglycerides, LDL, or HDL levels. The plasma lipid-lowering effects observed with JEO and MEO are consistent with previous findings; for example, dietary supplementation with a commercial blend of EOs has shown similar effects (Kahiel et al., 2025). Similarly, Chowdhury et al. (2018) reported lower cholesterol concentrations in diets supplemented with cinnamon and clove EO compared to the control and AGP groups. Furthermore, a linear reduction in cholesterol levels was observed in laying hens as dietary inclusion of clove bud powder increased (Rahman Alizadeh et al., 2017). This effect could be attributed to the lipid-lowering activity of specific bioactive compounds, particularly those found in EOs, which may interfere with cholesterol synthesis or enhance lipid metabolism (Ahmad Firdaus B et al., 2020; Zhu et al., 2023). In addition, it has been reported that the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA) is the rate-limiting enzyme involved in cholesterol synthesis (Baskaran et al., 2015), thus, it was suggested that the decrease in cholesterol content could be due to the inhibiting effects of geraniol and carvacrol on HMG-CoA reductase (Almalki et al., 2024).

In the current study, no significant differences were observed between treatments in total protein levels, in contrast to the studies of Hosseinzadeh et al. (2023), who reported an increase in total protein in broilers fed 200 mg/kg of Indian borage EO, while uric acid, glucose, and triglycerides concentrations remained unchanged. Furthermore, supplementation with different levels of eucalyptus EO did not significantly affect glucose, total protein, cholesterol, triglyceride, and HDL levels (Mohebodini et al., 2021). Significant differences were observed in blood creatinine levels, with the MEO group showing the lowest values. This is consistent with the findings of  Abo Ghanima et al. (2021), who reported that dietary supplementation with tea tree EO significantly reduced creatinine and uric acid concentrations. The lower creatinine levels observed in the MEO group may  indicate enhanced renal function and reduced muscle catabolism, as elevated creatinine concentrations are typically associated with significant muscle damage (Ritchie et al., 1994).

Although the precise modes of action of EOs remain incompletely defined, the present findings indicate that their beneficial effects are more pronounced under health-challenging conditions. In this study, the improved performance of the CEO group, particularly in survivability and overall production efficiency, appears to reflect enhanced physiological resilience rather than a sustained improvements in growth performance. Meanwhile, juniper and the combined EO treatments mainly contributed to maintaining stable performance relative to the control groups.

A key limitation of the present study is that mechanistic effects of cypress and juniper EOs were not directly assessed. Parameters such as gut morphology, microbiota composition, anti-inflammatory function, and immune markers were not measured; thus, the suggested benefits remain hypothetical and based on previous literature. Another limitation concerns the design of the PC group, which received additional antibiotic treatments in combination with the in-feed AGP. Consequently, this group does not represent a strict AGP-only control, and comparisons of growth performance involving this group should be viewed in light of the potential confounding effects of the additional therapeutics. Furthermore, weekly growth parameters (BW and FCR) were analyzed independently at each time point, using repeated-measures model could have provided a more robust analysis and allowed assessment of treatment and time interactions.

Conclusion: Within the constraints of the current study, the tested EOs exerted their primary benefits by improving resilience and survivability under conditions of compromised flock health. The EOs favorably modulated lipid profiles and creatinine concentrations, while CEO in particular significantly reduced mortality and improved EPEF.

The observed variability in responses may be related to pulsed administration regimen or dosage optimization, highlighting the need to explore different concentrations and continuous supplementation. Further limitations include the absence of direct assessment of nutrient digestibility, gut microbiota, and immune responses which limit physiological interpretation of the observed effects. Future studies should therefore focus on alpha-pinene-rich EOs, with particular emphasis on gut health and immune function, to better define their role in poultry nutrition.

Acknowledgments: The authors would like to thank Groupe Salem Avicole (Biskra, Algeria) for providing the experimental animals and facilities necessary for conducting this study. We are also grateful to the Laboratory of Biology, Environment and Health (LBEH) at the University of El Oued, Algeria, for supporting the laboratory analyses. This study is part of the PhD research of the first author at the University of El Oued.

REFERENCES

Abate, T. A. and A. G. Birhanu (2025). Antibiotic use in livestock and environmental antibiotic resistance: A Narrative Review. Environmental Health Insights, 19(1): 1-12.  https://doi.org/10.1177/11786302251357775

Abbas, W., R. Bi,  M. D. Hussain, A. Tajdar, F. Guo, Y. Guo and Z. Wang (2024). Antibiotic cocktail effects on intestinal microbial community, barrier function, and immune function in early broiler chickens. Antibiotics, 13(5): 413.  https://doi.org/10.3390/antibiotics13050413

Abd El-Hack, M. E., M. T. El-Saadony, A. M. Saad, H. M. Salem, N. M. Ashry, M. M. Abo Ghanima, M. Shukry, A. A. Swelum, A. E. Taha, A. M. El-Tahan, S. F. Abu Qamar and K. A. El-Tarabily (2022). Essential oils and their nanoemulsions as green alternatives to antibiotics in poultry nutrition: a comprehensive review. Poultry Science, 101(2): 101584.  https://doi.org/10.1016/j.psj.2021.101584

Abdelli, N., D. Solà-Oriol and J. F. Pérez (2021). Phytogenic feed additives in poultry: achievements, prospective and challenges. Animals, 11(12): 3471.  https://doi.org/10.3390/ani11123471

Abo Ghanima, M. M., A. A. Swelum, M. Shukry, S. A. Ibrahim, M. E. Abd El-Hack, A. F. Khafaga, A. R. Alhimaidi, A. A. Ammari, K. A. El-Tarabily and M. E. M. Younis (2021). Impacts of tea tree or lemongrass essential oils supplementation on growth, immunity, carcass traits, and blood biochemical parameters of broilers reared under different stocking densities. Poultry Science, 100(11): 101443. https://doi.org/10.1016/j.psj.2021.101443

Adetunji, A. O., J. Price, H. Owusu, E. F. Adewale, P. A. Adesina, T. P. Saliu, Z. Zhu, C. Xedzro, E. Asiamah and S. Islam (2025). Mechanisms by which phytogenic extracts enhance livestock reproductive health: current insights and future directions. Frontiers in Veterinary Science, 12: 1568577. https://doi.org/10.3389/fvets.2025.1568577

Adil, S., M. T. Banday, S. A. Hussain, M. A. Wani, A. A. Khan, I. U. Sheikh, Y. Afzal, Z. Kashoo, S. Shah, S. A. R. Shilbayeh and G. A. El-Shaboury (2025). Modulation of performance, meat quality and gut health of broiler chicken in response to dietary nanoencapsulated lavender essential oil. Scientific Reports, 15(1): 40036. https://doi.org/10.1038/s41598-025-23938-4

Aguilar, C. A. L., K. R. D. S. Lima, M. C. Manno, F. B. Tavares, V. P. D. Souza and D. L. Fernandes Neto (2013). Effect of copaiba essential oil on broiler chickens’ performance. Acta Scientiarum. Animal Sciences, 35(2): 145–151. https://doi.org/10.4025/actascianimsci.v35i2.15376

Ahmad Firdaus B, L. and I. Noor Hanis (2020). Therapeutic application of herbal essential oil and its bioactive compounds as complementary and alternative medicine in cardiovascular-associated diseases. Insights on the Depression and Anxiety, 4(1): 025–036. https://doi.org/10.29328/journal.ida.1001016

Akan, M., K. S. Di̇Ker, S. Özlü, M. Çelebi̇, S. Sariçam İNce, B. Doğan and O. Eli̇Bol (2025). Effects of a diet supplemented with a blend of protected organic acids and essential oils on growth performance and cecal microbiota composition in broilers. Turkish Journal of Veterinary and Animal Sciences, 49(1): 11–18. https://doi.org/10.55730/1300-0128.4365

Albrecht, U. W. and A. Madisch (2022). Therapeutic potentials associated with biological properties of Juniper berry oil (Juniperus communis L.) and its therapeutic use in several diseases – A review. Bioactive Compounds in Health and Disease, 5(9): 174. https://doi.org/10.31989/bchd.v5i9.999

Allenspach, M. and C. Steuer (2021). α-Pinene: A never-ending story. Phytochemistry, 190: 112857. https://doi.org/10.1016/j.phytochem.2021.112857

Almalki, S. G., M. Alsaweed, H. Muteb Albadrani, Y. E. Alqurashi, M. A. Bazuhair, H. H. Ahmed, P. Ahmad, A. Alfahed, A. Al Othaim and D. Iqbal (2024). A molecular informatics and in-vitro approach to evaluate the HMG-CoA reductase inhibitory efficacy of monoterpenes, carvacrol and geraniol. Journal of Taibah University of Science, 18(1): 2297456. https://doi.org/10.1080/16583655.2023.2297456

Al-Tel, T. H., H. Tarazi, L. O. Aloum, D. E. Lorke and G. A. Petroianu (2020). Possible metabolic conversion of pinene to ionone. Die Pharmazie, 75(7): 360–363. https://doi.org/10.1691/ph.2020.9925

Aminullah, N., A. Mostamand, A. Zahir, O. Mahaq and M. N. Azizi (2025). Phytogenic feed additives as alternatives to antibiotics in poultry production: A review. Veterinary World, 18(1): 141–154. https://doi.org/10.14202/vetworld.2025.141-154

Amouei, H., G. Ferronato, A. A. A. Qotbi, M. Bouyeh, P. G. Dunne, A. Prandini and A. Seidavi (2021). Effect of essential oil of thyme (Thymus vulgaris L.) or increasing levels of a commercial prebiotic (TechnoMOS®) on growth performance and carcass characteristics of male broilers. Animals, 11(11): 3330. https://doi.org/10.3390/ani11113330

Angane, M., S. Swift, K. Huang, J. Perera, X. Chen, C. A. Butts and S. Y. Quek (2024). Synergistic antimicrobial interaction of plant essential oils and extracts against foodborne pathogens. Food Science and Nutrition, 12(2): 1189–1206. https://doi.org/10.1002/fsn3.3834

Ashour, E. A., A. K. Aldhalmi, I. E. Ismail, M. Kamal, A. A. Elolimy, A. A. Swelum and M. E. Abd El-Hack (2025). The effect of using Echinacea extract as an immune system stimulant and antioxidant on blood indicators, growth efficiency, and carcass characteristics in broiler chickens to produce a healthy product. Poultry Science, 104(1): 104392. https://doi.org/10.1016/j.psj.2024.104392

Attia, Y. A., F. Bovera, A. E. Abd El‐Hamid, A. F. Tag El‐Din, M. A. Al‐Harthi and A. S. El‐Shafy (2016). Effect of zinc bacitracin and phytase on growth performance, nutrient digestibility, carcass and meat traits of broilers. Journal of Animal Physiology and Animal Nutrition, 100(3): 485–491. https://doi.org/10.1111/jpn.12397

Attia, Y. A., A. A. Bakhashwain and N. K. Bertu (2017). Thyme oil (Thyme vulgaris L.) as a natural growth promoter for broiler chickens reared under hot climate. Italian Journal of Animal Science, 16(2): 275–282. https://doi.org/10.1080/1828051X.2016.1245594

Aviagen (2022). Arbor Acres Broiler Performance Objectives: 13

Bagchi, A., Y. Yu, J.-H. Huang, C.-C. Tsai, W.-P. Hu and C. C. Wang (2020). Evidence and evolution of Criegee intermediates, hydroperoxides and secondary organic aerosols formed via ozonolysis of α-pinene. Physical Chemistry Chemical Physics, 22(12): 6528–6537. https://doi.org/10.1039/C9CP06306D

Bakó, E., A. Böszörményi, B. Vargáné Szabó, M. A. Engh, P. Hegyi, A. Ványolós and D. Csupor (2024). Chemometric analysis of monoterpenes and sesquiterpenes of conifers. Frontiers in Plant Science, 15: 1392539. https://doi.org/10.3389/fpls.2024.1392539

Başer, K. H. C., A. E. Karadağ, S. N. Biltekin, M. Ertürk and F. Demirci (2023). In vitro antiviral evaluations of Coldmix®: An essential oil blend against SARS-CoV-2. Current Issues in Molecular Biology, 45(1): 677–684. https://doi.org/10.3390/cimb45010045

Baskaran, G., M. Y. Shukor, S. Salvamani, S. A. Ahmad, N. A. Shaharuddin and P. D. Pattiram (2015). HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia. Drug Design, Development and Therapy, 9: 509-517. https://doi.org/10.2147/DDDT.S75056

Beber, C. L., M. Aragrande and M. Canali (2025). Policies and strategies to control antimicrobial resistance in livestock production: A comparative analysis of national action plans in European Union member states. Health Policy, 152: 105238. https://doi.org/10.1016/j.healthpol.2024.105238

Bomfim De Barros, D., L. De Oliveira E Lima, L. Alves Da Silva, M. Cavalcante Fonseca, R. C. Ferreira, H. Diniz Neto, D. Da Nóbrega Alves, W. P. Da Silva Rocha, L. Scotti, E. De Oliveira Lima, M. Vieira Sobral, L. R. Cançado Castellano, J. Moura-Mendes, F. Queiroga Sarmento Guerra and M. V. Da Silva (2023). α-Pinene: Docking study, cytotoxicity, mechanism of action, and anti-biofilm effect against Candida albicans. Antibiotics, 12(3): 480. https://doi.org/10.3390/antibiotics12030480

Borges, M. F. D. A., R. D. S. Lacerda, J. P. D. A. Correia, T. R. De Melo  and S. B. Ferreira (2022). Potential antibacterial action of α-Pinene. The 2nd international electronic conference on antibiotics-drugs for superbugs: Antibiotic discovery, modes of action and mechanisms of resistance, 12(1): 11. https://doi.org/10.3390/eca2022-12709

Chowdhury, S., G. P. Mandal, A. K. Patra, P. Kumar, I. Samanta, S. Pradhan and A. K. Samanta (2018). Different essential oils in diets of broiler chickens: 2. Gut microbes and morphology, immune response, and some blood profile and antioxidant enzymes. Animal Feed Science and Technology, 236: 39–47. https://doi.org/10.1016/j.anifeedsci.2017.12.003

Darwish, R. S., H. M. Hammoda, D. A. Ghareeb, A. S. A. Abdelhamid, E. M. Bellah El Naggar, F. M. Harraz and E. Shawky (2020). Efficacy-directed discrimination of the essential oils of three Juniperus species based on their in-vitro antimicrobial and anti-inflammatory activities. Journal of Ethnopharmacology, 259: 112971. https://doi.org/10.1016/j.jep.2020.112971

Dorman, H. J. D. and S. G. Deans (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88(2): 308–316. https://doi.org/10.1046/j.1365-2672.2000.00969.x

Dudek, T. and M. Marć (2025). Terpene content in the air of younger and older forests in southeastern Poland: Implications for forest therapy. Scientific Reports, 15(1): 42174. https://doi.org/10.1038/s41598-025-26137-3

Elbaz, A., E. Ashmawy, M. Farahat, S. Ali, S. Amin, H. Thabet and G. Gad (2022a). Effect of different levels of clove essential oil on the growth performance, lipid metabolism, immunity, and intestinal microbial structure of broiler chickens. Egyptian Journal of Nutrition and Feeds, 25(3): 361–368. https://doi.org/10.21608/ejnf.2022.286670

Elbaz, A. M., E. S. Ashmawy, A. A. Salama, A.-M. E. Abdel-Moneim, F. B. Badri and H. A. Thabet (2022b). Effects of garlic and lemon essential oils on performance, digestibility, plasma metabolite, and intestinal health in broilers under environmental heat stress. BMC Veterinary Research, 18(1): 430. https://doi.org/10.1186/s12917-022-03530-y

Elbaz, A., E. S. Ashmawy, D. M. Mourad, S. A. Amin, E. Kamel M. Khalfallah and Z. S. Mohamed (2024). Effect of oregano essential oils and probiotics supplementation on growth performance, immunity, antioxidant status, intestinal microbiota, and gene expression in broilers experimentally infected with Eimeria. Livestock Science, 291: 105622. https://doi.org/10.1016/j.livsci.2024.105622

Ellouze, I., B. Ben Akacha, I. G. Mekinić, R. Ben Saad, M. Kačániová, M. I. Kluz, W. Mnif, S. Garzoli and A. Ben Hsouna (2024). Enhancing antibacterial efficacy: Synergistic effects of Citrus aurantium essential oil mixtures against Escherichia coli for food preservation. Foods, 13(19): 3093. https://doi.org/10.3390/foods13193093

El-Sayed, Y., W. Khalil, N. Fayez and A.-F. Mohamed Abdel-Fattah (2024). Enhancing effect of oregano essential oil and Bacillus subtilis on broiler immune function, intestinal morphology and growth performance. BMC Veterinary Research, 20(1): 112. https://doi.org/10.1186/s12917-024-03960-w

Fawaz, M. A., A. A. M. Abd El-Hafez, Y. A. Abdelmoati, A. H. H. Ali and H. A. Hassan (2025). Physiological, nutritional, and productive responses of broilers supplemented with lemon, fenugreek, and sesame oils as feed additives. Scientific Reports, 15(1): 24993. https://doi.org/10.1038/s41598-025-09252-z

Fernández Miyakawa, M. E., N. A. Casanova  and M. H. Kogut (2024). How did antibiotic growth promoters increase growth and feed efficiency in poultry? Poultry Science, 103(2): 103278. https://doi.org/10.1016/j.psj.2023.103278

Galovičová, L., N. Čmiková, M. Schwarzová, M. D. Vukic, N. L. Vukovic, P. L. Kowalczewski, L. Bakay, M. I. Kluz, C. Puchalski, A. D. Obradovic, M. M. Matić and M. Kačániová (2023). Biological activity of Cupressus sempervirens essential oil. Plants, 12(5): 1097. https://doi.org/10.3390/plants12051097

Ge, C., X. Luo, Y. Lv, L. Wu, Z. Hu, W. Huang, S. Zhan, X. Shen, C. Hui, D. Yu and B. Liu (2024). Essential oils ameliorate the intestinal damages induced by nonylphenol exposure by modulating tryptophan metabolism and activating aryl hydrocarbon receptor via gut microbiota regulation. Chemosphere, 362: 142571. https://doi.org/10.1016/j.chemosphere.2024.142571

Gens, K. D., R. S. Singer, T. J. Dilworth, E. L. Heil and A. L. Beaudoin (2022). Antimicrobials in animal agriculture in the united states: A multidisciplinary overview of regulation and utilization to foster collaboration: On behalf of the society of infectious diseases pharmacists. Open Forum Infectious Disease, 9(11): ofac542. https://doi.org/10.1093/ofid/ofac542

Gharaibeh, M. H., M. S. Khalifeh, A. N. Nawasreh, W. M. Hananeh and M. S. Awawdeh (2021). Assessment of immune response and efficacy of essential oils application on controlling necrotic enteritis induced by Clostridium perfringens in broiler chickens. Molecules, 26(15): 4527. https://doi.org/10.3390/molecules26154527

Ghiasvand, A. R., A. Khatibjoo, Y. Mohammadi, M. Akbari Gharaei and H. Shirzadi (2021). Effect of fennel essential oil on performance, serum biochemistry, immunity, ileum morphology and microbial population, and meat quality of broiler chickens fed corn or wheat-based diet. British Poultry Science, 62(4): 562–572. https://doi.org/10.1080/00071668.2021.1883551

Gordillo Jaramillo, F. X., D.-H. Kim, S. H. Lee, S.-K. Kwon, R. Jha and K.-W. Lee (2021). Role of oregano and Citrus species-based essential oil preparation for the control of coccidiosis in broiler chickens. Journal of Animal Science and Biotechnology, 12(1): 47. https://doi.org/10.1186/s40104-021-00569-z

Gumus, R. and S. U. Gelen (2023). Effects of dietary thyme and rosemary essential oils on performance parameters with lipid oxidation, water activity, pH, color and microbial quality of breast and drumstick meats in broiler chickens. Archives Animal Breeding, 66(1): 17–29. https://doi.org/10.5194/aab-66-17-2023

Hairui, Y., R. Abdur, U. Fahad and W. Muhammad (2024). Effect of supplementing a blend of essential oils on the growth performance, carcass characteristics, meat quality, serological parameters and gut health in broiler chickens. The Pakistan Veterinary Journal, 44(4): 1329-1337. https://doi.org/10.29261/pakvetj/2024.301

Hesabi Nameghi, A., O. Edalatian and R. Bakhshalinejad (2019). Effects of a Blend of Thyme, Peppermint and Eucalyptus Essential Oils on Growth Performance, Serum lipid and hepatic enzyme indices, immune response and ileal morphology and microflora in broilers. Journal of Animal Physiology and Animal Nutrition, 103(5): 1388–1398. https://doi.org/10.1111/jpn.13122

Hoosen, N., A. Viljoen and S. Van Vuuren (2026). Stereoisomeric interactions of α- and β-pinene with essential oil compounds. Fitoterapia, 188: 106989. https://doi.org/10.1016/j.fitote.2025.106989

Hosseinzadeh, S., F. Shariatmadari, M. A. Karimi Torshizi, H. Ahmadi and D. Scholey (2023). Plectranthus amboinicus and rosemary (Rosmarinus officinalis L.) essential oils effects on performance, antioxidant activity, intestinal health, immune response, and plasma biochemistry in broiler chickens. Food Science and Nutrition, 11(7): 3939–3948. https://doi.org/10.1002/fsn3.3380

Huang, J., T. Xu, F. Guo, R. Bi, H. Lu, P. Li, W. Abbas, Z. Hu, L. Liu, M. J. Sengers, X. Xie, T. Cheng, Y. Guo and Z. Wang (2025). Effects of drinking water supplemented with essential oils and organic acids mixtures on growth performance and intestinal health of broilers challenged with necrotic enteritis. Poultry Science, 104(2): 104712. https://doi.org/10.1016/j.psj.2024.104712

Huff, G. R., W. E. Huff, S. Jalukar, J. Oppy, N. C. Rath and B. Packialakshmi (2013). The effects of yeast feed supplementation on turkey performance and pathogen colonization in a transport stress/Escherichia coli challenge. Poultry Science, 92(3): 655–662. https://doi.org/10.3382/ps.2012-02787

Ibrahim, D., F. Eldemery, A. Sh. Metwally, E. M. Abd-Allah, D. T. Mohamed, T. A. Ismail, T. A. Hamed, G. M. Al Sadik, A. N. F. Neamat-Allah and M. I. Abd El-Hamid (2022). Dietary eugenol nanoemulsion potentiated performance of broiler chickens: orchestration of digestive enzymes, intestinal barrier functions and cytokines related gene expression with a consequence of attenuating the severity of E. coli O78 infection. Frontiers in Veterinary Science, 9: 847580. https://doi.org/10.3389/fvets.2022.847580

INRAE; CIRAD; AFZ (2020). INRAE-CIRAD-AFZ Feed Tables.

İpçak, H. H., A. Alçiçek and M. Denli (2024). Dietary encapsulated fennel seed (Foeniculum vulgare Mill.) essential oil supplementation improves performance, modifies the intestinal microflora, morphology, and transcriptome profile of broiler chickens. Journal of Animal Science, 102: skae035. https://doi.org/10.1093/jas/skae035

Irawan, A., C. Hidayat, A. Jayanegara and A. Ratriyanto (2021). Essential oils as growth-promoting additives on performance, nutrient digestibility, cecal microbes, and serum metabolites of broiler chickens: A meta-analysis. Animal Bioscience, 34(9): 1499–1513. https://doi.org/10.5713/ab.20.0668

Islam, M. S., D. A. Mir, M. E. Nazir, S. Islam, S. M. I. Hossain, M. I. Hasan, S. Zaman, D. K. Swar and S. Sultana (2025). Effects of supplementation of oregano essential oil on the growth efficiency and blood biochemical parameters of broiler chickens. Journal of World’s Poultry Research, 15(2): 184-193. https://doi.org/10.36380/jwpr.2025.19

Johnson, A. M., G. Anderson, M. Arguelles-Ramos and A. A. B. Ali (2022). Effect of dietary essential oil of oregano on performance parameters, gastrointestinal traits, blood lipid profile, and antioxidant capacity of laying hens during the pullet phase. Frontiers in Animal Science, 3: 1072712. https://doi.org/10.3389/fanim.2022.1072712

Kahiel, M., K. Wang, H. Xu, J. Du, S. Li, D. Shen and C. Li (2025). Effect of supplemental essential oils blend on broiler meat quality, fatty acid profile, and lipid quality. Animals, 15(7): 929. https://doi.org/10.3390/ani15070929

Khan, T., M. Rehman, M. Arslan, A. Javaid, U. Farooq, T. Asad, Q. Nisa, M. Azhar, E. Bughio, S. Liaqat, S. Aslam, F. M. Ali, H. Ali, F. Wadood, M. Gondal, M. Rauf, D. Fouad, F. Ataya, M. Safdar and M. Qumar (2024). Effect of blend of essential oils on growth performance, carcass characteristics, meat quality, intestinal morphology, serum biochemistry, and immune response of broiler chickens. Brazilian Journal of Poultry Science, 26(1): 01–10. https://doi.org/10.1590/1806-9061-2023-1853

Khatun, A., A. Lahiry, N. J. Nishat, M. Begum, B. C. Roy and S. C. Das (2025). Effects of feeding thyme, rosemary, and oregano powder supplementation on broiler chickens: Performance, meat quality, hematology and serum biochemistry. Advances in Animal and Veterinary Sciences, 13(4): 752-763. https://doi.org/10.17582/journal.aavs/2025/13.4.752.763

Klančnik, A., Š. Zorko, N. Toplak, M. Kovač, F. Bucar, B. Jeršek and S. Smole Možina (2018). Antiadhesion activity of juniper (Juniperus communis L.) preparations against Campylobacter jejuni evaluated with PCR‐based methods. Phytotherapy Research, 32(3): 542–550. https://doi.org/10.1002/ptr.6005

Kose, E., T. G. Deniz, C. Sarıkürkcü, Ö. Aktaş and M. Yavuz (2010). Chemical composition, antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha Boiss. and Heldr. (var. erythrantha and var. cedretorum P.H. Davis) endemic in Turkey. Food and Chemical Toxicology, 48(10): 2960–2965. https://doi.org/10.1016/j.fct.2010.07.033

Leite-Sampaio, N. F., C. N. F. L. Gondim, C. E. S. De Souza and H. D. M. Coutinho (2022). Antibiotic potentiating action of α-pinene and borneol against EPEC and ETEC sorotypes. Microbial Pathogenesis, 162: 105371. https://doi.org/10.1016/j.micpath.2021.105371

Ložienė, K. and P. R. Venskutonis (2016). Juniper (Juniperus Communis L.) oils. In: Preedy, V.R. (Ed.) Essential oils in food preservation, flavor and safety (pp. 495-500). Academic Press; San Diego, USA. https://doi.org/10.1016/B978-0-12-416641-7.00056-0

Malhi, K. K., J. Chen, T.-H. Wang, M.-Y. Huang, K.-P. Xing, H.-B. Xing, M. Song, C. Kumar, X.-H. Zou and J.-L. Li (2025). Dietary supplementation with blended essential oils improves meat quality of broilers through SCFA-mediated gut-muscle axis. Poultry Science, 104(12): 105911. https://doi.org/10.1016/j.psj.2025.105911

Maral, H., Ö. Çeçen, A. Türk Baydır and M. Türkmen (2024). Determination of essential oil constituents and antioxidant activities of hybrid species Salvia × karamanensis Celep and B. T. Drew and its parents (Salvia aucheri Benth. subsp. canescens (Boiss. and Heldr.) Celep, Kahraman and Dogan × Salvia heldreichiana Boiss. ex Benth.). Biochemical Systematics and Ecology, 113: 104795. https://doi.org/10.1016/j.bse.2024.104795

Maria Cardinal, K., M. Kipper, I. Andretta and A. Machado Leal Ribeiro (2019). Withdrawal of antibiotic growth promoters from broiler diets: performance indexes and economic impact. Poultry Science, 98(12): 6659–6667. https://doi.org/10.3382/ps/pez536

Medini, H., M. Khouja, M. L. Khouja, S. Banfi and A. Elaissi (2025). Chemotaxonomy and bioactivity of Cupressus sempervirens varieties essential oils: Insights into their antioxidant and enzyme inhibitory potential. Chemistry and Biodiversity, 22(12): e01650. https://doi.org/10.1002/cbdv.202501650

Mirković, S., M. Martinović, V. M. Tadić, I. Nešić, A. S. Jovanović and A. Žugić (2025). Antimicrobial and antioxidant activity of essential oils from selected Pinus species from Bosnia and Herzegovina. Antibiotics, 14(7): 677. https://doi.org/10.3390/antibiotics14070677

Mohebodini, H., V. Jazi, A. Ashayerizadeh, M. Toghyani and G. Tellez-Isaias (2021). Productive parameters, cecal microflora, nutrient digestibility, antioxidant status, and thigh muscle fatty acid profile in broiler chickens fed with Eucalyptus globulus essential oil. Poultry Science, 100(3): 100922. https://doi.org/10.1016/j.psj.2020.12.020

Mountzouris, K. C. and I. Brouklogiannis (2024). Phytogenics as natural gut health management tools for sustainable poultry production. Livestock Science, 286: 105525. https://doi.org/10.1016/j.livsci.2024.105525

Movahedi, F., N. Nirmal, P. Wang, H. Jin, L. Grøndahl and L. Li (2024). Recent advances in essential oils and their nanoformulations for poultry feed. Journal of Animal Science and Biotechnology, 15(1): 110. https://doi.org/10.1186/s40104-024-01067-8

National Research Council (NRC) (1971). A guide to environmental research on animals. Washington, DC: National Academy of Sciences. https://doi.org/10.17226/20608

Noruzi, S., M. Torki and H. Mohammadi (2022). Effects of supplementing diet with Thyme (Thymuas vulgaris L.) essential oil and/or selenium yeast on production performance and blood variables of broiler chickens. Veterinary Medicine and Science, 8(3): 1137–1145. https://doi.org/10.1002/vms3.736

Obeidat, M. D., B. M. Nusairat and B. S. Obeidat (2022). Growth performance and carcass traits of two commercial broiler strains fed diet supplemented with essential oils. Heliyon, 8(12): e12094. https://doi.org/10.1016/j.heliyon.2022.e12094

Oni, A. I. and O. E. Oke (2025). Gut health modulation through phytogenics in poultry: Mechanisms, benefits, and applications. Frontiers in Veterinary Science, 12: 1616734. https://doi.org/10.3389/fvets.2025.1616734

Park, B. B., J. Y. An and S.U. Park (2021). Recent studies on pinene and its biological and pharmacological activities. EXCLI Journal, 20: 812-818. https://doi.org/10.17179/EXCLI2021-3714

Patgar, B. G., S. Sathish and A. R. Shabaraya (2021). Essential oil of Cupressus sempervirens: A brief review. World Journal of Pharmacy and Pharmaceutical Science, 10(4): 864-872. https://doi.org/10.20959/wjpps20214-18713

Pezantes-Orellana, C., F. German Bermúdez, C. Matías De La Cruz, J. L. Montalvo and A. Orellana-Manzano (2024). Essential oils: A systematic review on revolutionizing health, nutrition, and omics for optimal well-being. Frontiers in Medicine, 11: 1337785. https://doi.org/10.3389/fmed.2024.1337785

Pham, V. H., W. Abbas, J. Huang, F. Guo, K. Zhang, L. Kong, W. Zhen, Y. Guo and Z. Wang (2023). Dietary coated essential oil and organic acid mixture supplementation improves health of broilers infected with avian pathogenic Escherichia coli. Animal Nutrition, 12: 245–262. https://doi.org/10.1016/j.aninu.2022.09.010

Placha, I., V. Ocelova, R. Chizzola, G. Battelli, F. Gai, K. Bacova and S. Faix (2019). Effect of thymol on the broiler chicken antioxidative defense system after sustained dietary thyme oil application. British Poultry Science, 60(5): 589–596. https://doi.org/10.1080/00071668.2019.1631445

Pourmahmoud, B., A. M. Aghazadeh and N. M. Sis (2013). The effect of thyme extract on growth performance, digestive organ weights and serum lipoproteins of broilers fed wheat-based diets. Italian Journal of Animal Science, 12(3): e53. https://doi.org/10.4081/ijas.2013.e53

Rahimi, K., M. Zalaghi, E. G. Shehnizad, G. Salari, F. Baghdezfoli and A. Ebrahimifar (2023). The effects of alpha-pinene on inflammatory responses and oxidative stress in the formalin test. Brain Research Bulletin, 203: 110774. https://doi.org/10.1016/j.brainresbull.2023.110774

Rahman Alizadeh, M., A. H. Mahdavi, H. R. Rahmani and E. Jahanian (2017). Clove bud (Syzygium aromaticum) improved blood and hepatic antioxidant indices in laying hens receiving low n‐6 to n‐3 ratios. Journal of Animal Physiology and Animal Nutrition, 101(5): 881–892. https://doi.org/10.1111/jpn.12502

Reda, F. M., M. Kamal, H. K. Mahmoud, H. A. Rudayni, A. A. Allam, A. F. Khafaga, M. M. H. Khan and R. S. Mohamed (2025). Effect of a mixture of peppermint and clove oils on growth performance, carcass traits, blood parameters, and gut microbiota in broiler chickens. Poultry Science, 104(11): 105718. https://doi.org/10.1016/j.psj.2025.105718

Remus, A., L. Hauschild, I. Andretta, M. Kipper, C. R. Lehnen and N. K. Sakomura (2014). A meta-analysis of the feed intake and growth performance of broiler chickens challenged by bacteria. Poultry Science, 93(5): 1149–1158. https://doi.org/10.3382/ps.2013-03540

Ritchie, B. W., G. J. Harrison and L. R. Harrison (1994). Avian medicine: Principles and application. Wingers Publishing; Lake Worth, Florida, USA, pp. 223-245.

Rocha, J. J. and G. S. Archer (2024). The effect of supplementation with organic acid and oregano oils in drinking water on pekin duck growth and welfare. Poultry, 3(2): 95–106. https://doi.org/10.3390/poultry3020009

Rodier, J., B. Legube, N. Merlet, C. Alary and A. Belles (2016). L’analyse de l’eau: Contrôle et interprétation. 10th Edition. Dunod; Paris.

Sampath, V., L. Pineda, E. Hambrecht and I. H. Kim (2025). Synergistic blend of natural essential oils improved growth performance and gut barrier integrity in broilers by alleviating intestinal inflammation. Poultry Science, 104(11): 105783. https://doi.org/10.1016/j.psj.2025.105783

Santos, V. K. F. D. R., W. C. L. Nogueira, R. D. L.Santos, N. J. F. D. Oliveira, E. R. Martins, I. L. Azevedo, T. F. D. Carvalho and A. C. D. Almeida (2019). Blood parameters and hepatic histopathology of broilers fed rations supplemented with essential oils. Revista Brasileira de Zootecnia, 48: e20180254. https://doi.org/10.1590/rbz4820180254

Selim, S., N. S. Abdel-Megeid, R. A. Alhotan, A. Ebrahim and E. Hussein (2024). Nutraceuticals vs. antibiotic growth promoters: Differential impacts on performance, meat quality, blood lipids, cecal microbiota, and organ histomorphology of broiler chicken. Poultry Science, 103(9): 103971. https://doi.org/10.1016/j.psj.2024.103971

Silva, A. C. R. D., P. M. Lopes, M. M. B. D. Azevedo, D. C. M. Costa, C. S. Alviano and D. S. Alviano (2012). Biological activities of α-Pinene and β-Pinene enantiomers. Molecules, 17(6): 6305–6316. https://doi.org/10.3390/molecules17066305

Spréa, R. M., C. Caleja, T. C. Finimundy, R. C. Calhelha, T. C. S. P. Pires, J. S. Amaral, M. A. Prieto, I. C. F. R. Ferreira, E. Pereira and L. Barros (2024). Chemical and bioactive evaluation of essential oils from edible and aromatic mediterranean Lamiaceae plants. Molecules, 29(12): 2827. https://doi.org/10.3390/molecules29122827

Stefanello, C., D. P. Rosa, Y. K. Dalmoro, A. L. Segatto, M. S. Vieira, M. L. Moraes and E. Santin (2020). Protected blend of organic acids and essential oils improves growth performance, nutrient digestibility, and intestinal health of broiler chickens undergoing an intestinal challenge. Frontiers in Veterinary Science, 6: 491. https://doi.org/10.3389/fvets.2019.00491

Su, G., L. Wang, X. Zhou, X. Wu, D. Chen, B. Yu, W. Huang, Y. Luo, X. Mao, P. Zheng, J. Yu, J. Luo and J. He (2021). Effects of essential oil on growth performance, digestibility, immunity, and intestinal health in broilers. Poultry Science, 100(8): 101242. https://doi.org/10.1016/j.psj.2021.101242

Tekce, E., K. Çınar, B. Bayraktar, Ç. Takma and M. Gül (2020). Effects of an essential oil mixture added to drinking water for temperature-stressed broilers: Performance, meat quality, and thiobarbituric acid-reactive substances. Journal of Applied Poultry Research, 29(1): 77–84. https://doi.org/10.3382/japr/pfz030

Theodoridou Oxinou, D., D. Lamnisos, C. Filippou, N. Spernovasilis and C. Tsioutis (2025). Antimicrobial use and antimicrobial resistance in food-producing animals: Cross-sectional study on knowledge, attitudes, and practices among veterinarians and operators of establishments in the republic of Cyprus. Antibiotics, 14(3): 251. https://doi.org/10.3390/antibiotics14030251

Tufail, T., H. B. U. Ain, A. Saeed, M. Imran, S. Basharat and G. A. Nayik (2023). Juniper essential oil: An overview of bioactive compounds and functional aspects. In: Nayik, G.A. and Gull, A. (Eds.), Essential Oils (pp. 415–427). Academic Press; London. https://doi.org/10.1016/B978-0-323-91740-7.00020-7

Varijakzhan, D., S.-K. Yang, C. M. Chong, R. Akseer, M. S. Alhosani, W. Thomas, K. S. Lai and S. H. E. Lim (2021). Essential oils as potential antimicrobial agents. Sustainable Agriculture Reviews, 49: 93–122. https://doi.org/10.1007/978-3-030-58259-3_4

Vlaicu, P., A. Untea, T. Panaite, M. Saracila, R. Turcu and M. Dumitru (2023). Effect of basil, thyme and sage essential oils as phytogenic feed additives on production performances, meat quality and intestinal microbiota in broiler chickens. Agriculture, 13(4): 874. https://doi.org/10.3390/agriculture13040874

Wen, R., C. Li, M. Zhao, H. Wang and Y. Tang (2022). Withdrawal of antibiotic growth promoters in China and its impact on the foodborne pathogen Campylobacter coli of swine origin. Frontiers in Microbiology, 13: 1004725. https://doi.org/10.3389/fmicb.2022.1004725

Windisch, W., K. Schedle, C. Plitzner and A. Kroismayr (2008). Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science, 86(14): E140–E148. https://doi.org/10.2527/jas.2007-0459

Yang, Y., L. Zhao, Y. Shao, X. Liao, L. Zhang, L. Lu and X. Luo (2019). Effects of dietary graded levels of cinnamon essential oil and its combination with bamboo leaf flavonoid on immune function, antioxidative ability and intestinal microbiota of broilers. Journal of Integrative Agriculture, 18(9): 2123–2132. https://doi.org/10.1016/S2095-3119(19)62566-9

Yarmohammadi Barbarestani, S., V. Jazi, H. Mohebodini, A. Ashayerizadeh, A. Shabani and M. Toghyani (2020). Effects of dietary lavender essential oil on growth performance, intestinal function, and antioxidant status of broiler chickens. Livestock Science, 233: 103958. https://doi.org/10.1016/j.livsci.2020.103958

Yesilbag, D., M. Eren, H. Agel, A. Kovanlikaya and F. Balci (2011). Effects of dietary rosemary, rosemary volatile oil and vitamin e on broiler performance, meat quality and serum SOD activity. British Poultry Science, 52(4): 472–482. https://doi.org/10.1080/00071668.2011.599026

Zaazaa, A., S. Mudalal, I. Alzuheir, M. Samara, N. Jalboush, A. Fayyad and M. Petracci (2022). The impact of thyme and oregano essential oils dietary supplementation on broiler health, growth performance, and prevalence of growth-related breast muscle abnormalities. Animals, 12(21): 3065. https://doi.org/10.3390/ani12213065

Zhang, L., X. Wang, S. Huang, Y. Huang, H. Shi and X. Bai (2023). Effects of dietary essential oil supplementation on growth performance, carcass yield, meat quality, and intestinal tight junctions of broilers with or without Eimeria challenge. Poultry Science, 102(9): 102874. https://doi.org/10.1016/j.psj.2023.102874

Zheljazkov, V. D., C. L. Cantrell, I. Semerdjieva, T. Radoukova, A. Stoyanova, V. Maneva, M. Kačániová, T. Astatkie, D. Borisova, I. Dincheva and I. Salamon (2021). Essential oil composition and bioactivity of two juniper species from Bulgaria and Slovakia. Molecules, 26(12): 3659. https://doi.org/10.3390/molecules26123659

Zhu, Q., G. Li, L. Ma, B. Chen, D. Zhang, J. Gao, S. Deng and Y. Chen (2023). Virgin camellia seed oil improves glycolipid metabolism in the kidney of high fat-fed rats through AMPK-SREBP pathway. Nutrients, 15(23): 4888. https://doi.org/10.3390/nu15234888