Composition, Bioactivities, Microbiome, Safety Concerns, and Impact of Essential Oils on the Health Status of Domestic Animals

Featured Application

Featured Application: The literature survey may help develop essential oil-based supplements to improve the health status of animals and poultry.

Abstract

Essential oils (EOs) are highly concentrated and volatile blends of nonpolar substances that are derived from aromatic plant components and comprise terpenes, terpenoids, and phenylpropanoids, exhibiting diverse biological and pharmacological properties. The burgeoning pet industry is interested in EOs as a potential solution for common health issues in domestic animals, particularly in addressing antimicrobial resistance. The present literature review summarizes the composition, properties, benefits, safety considerations, and effects of EOs on domestic animals. The applications of EOs range from antimicrobial effects to antioxidant, anti-inflammatory, and anticancer activities, etc. The chemical constituents of EOs, exemplified by eucalyptus EO and rosemary EO, highlight their distinct aromatic profiles and potential benefits. Nevertheless, understanding the chemical makeup of EOs is fundamental to assessing their potential impacts on biological systems. The gut microbiota plays a crucial role in regulating various metabolic processes in the host, including energy homeostasis, glucose metabolism, and lipid metabolism. Safety considerations, including potential toxicity risk awareness, are essential when incorporating EOs into animal care routines. The feed additives incorporating EOs have shown promise in influencing gut microbiota balance, reducing inflammation, and acting as antioxidants. However, considering the potential risks associated with high doses or multiple administrations, cautious application is paramount. Preliminary studies suggest low toxicity levels, but further research is required to evaluate the safety of EOs. Though studies have reported the beneficial effects of EOs on pets and animals, further research is needed to validate the findings in real-world conditions. The paper also discussed the regulatory considerations and future perspectives on applying EOs in veterinary medicine.

1. Introduction

Essential oils (EOs) are being used in pet food as a new way to control microbial contamination and prevent pet health-associated issues [1]. Recently, EOs have become popular because of their significant biological [2] and pharmacological properties [3]. EOs have demonstrated antioxidant, low-haemolytic, antibacterial, and anticancer effects [4]. Moreover, EOs are used in various industries, including cosmetic products, detergents, soaps, cleansing gels, and fragrances, which hold substantial economic importance. Unlike esters of fatty acids, EOs are highly concentrated and volatile. They are obtained from the end products of primary metabolites found in various aromatic plant parts, including stems, leaves, bark, resin, flowers, fruit, seeds, and roots [5].

EOs are intricate and highly concentrated blends of volatile, nonpolar substances. They fall under the category of secondary metabolites in plants and play a crucial role in enabling plants to thrive in their respective environments [6,7,8]. These blends encompass various compounds, including terpenes, terpenoids, and phenylpropanoids, with potential applications in various pharmacological contexts [9,10,11,12].

Feed additives like probiotics, prebiotics, and EOs can be incorporated into pet food, individually or in combination [1,13]. These supplements can offer significant benefits to the animal, including acting as antioxidants, reducing inflammation, regulating the immune system, and influencing the balance of the gut microbiota [13].

The gut microbiota plays a crucial role in regulating various metabolic processes in the host, including energy homeostasis, glucose metabolism, and lipid metabolism [14]. The composition and function of the gut microbiota are affected by host genetics, dietary factors [15], antibiotic usage [16], etc. Moreover, the gut microbiota undergoes alterations during physiological stages, particularly gestation and lactation [17]. Compared to early gestation, there is a significant increase in Proteobacteria and Actinobacteria in the human gut at the later stage [18]. Changes in lipid metabolism and gut microbiota notably increase in the phylum Proteobacteria (Succinivibrio), Bacteriodetes (Bacteroides, Prevotella, and Parabacteroides), and the order Clostridiales that are involved in the degradation of carbohydrates during the progression of pregnancy in sows, which has clear characteristics associated with an increased risk of inflammation and energy loss [19]. Numerous studies have demonstrated that maternal gut microbiota can be transmitted to the fetus or offspring through the placenta or milk, highlighting their essential role in maternal and fetal or offspring performance [20,21,22].

The demand for EOs for pets could be attributed to several key factors. Firstly, pet owners have a growing preference for natural and holistic approaches to pet care. EOs, derived from plants, are seen as a more organic alternative to conventional pet products [23]. Secondly, EOs are known for their potential therapeutic benefits. They can address many common pet issues. However, research on plant-derived products’ safety and efficacy is required for pet owners to be aware of any possible adverse effects of plant-derived ingredients [23]. The natural approach aligns with the desire of many pet owners to minimize the use of synthetic chemicals in their pets’ care. The availability and accessibility of EOs have increased significantly in recent years, with a wide array of products tailored specifically for pets, which has made it more convenient for pet owners to incorporate EOs into their wellness routines.

The present review summarizes the composition, properties, safety concerns, beneficial effects of essential oils on pets, and the advantages and limitations of essential oils for pet healthcare.

The scientific literature was collected from the databases (Scopus, PubMed, and Google Scholar) using the keywords “Essential oil”, “Pets and essential oil”, “Essential oil and animal health”, and “Essential oil and poultry”. The relevant studies were selected without any time limit. The studies were reported in English, and unrestricted access was used for the preparation of the manuscript.

2. Eco-Friendly Extraction of Essential Oils

In the quest for sustainable practices in EO extraction, eco-friendly methods such as steam distillation, microwave-assisted hydro distillation, microwave hydro-diffusion and gravity, subcritical water treatment, and solvent-free microwave-assisted extraction have garnered considerable attention [24,25].

2.1. Steam Distillation

The steam distillation method of EO extraction has been illustrated in Figure 1. The conventional electric-powered steam distillation system consists of several components, including an electric boiler with a 2000 W rating that heats 40 L of water to produce steam, an extraction column made of food-grade stainless steel capable of holding 5 kg of raw material and 25 L of water, a 0.012 m drainage opening, and a 0.003 m sparging nozzle. A 0.005-meter perforated stainless-steel wire mesh inside the extractor supports the raw material during distillation. The separator, a 100-mL glass tube, separates extracted oil from condensate water. A stainless-steel shell and tube condenser condenses steam vapor back into liquid form, sealed with gaskets and silicone sealants to prevent leaks [26].

In contrast, the solar steam distillation system includes a 4 L solar tiny boiler designed to collect solar energy efficiently. Mounted on a two-axis tracking system, the Scheffler concentrator follows the sun’s movement throughout the day, maximizing sunlight capture with its 2.5 m² aperture area. This concentrated sunlight is directed onto the solar boiler at its focal point, integrated with the distillation assembly to extract oil [26].

2.2. Microwave-Assisted Hydrodistillation

Hydrodistillation is one of the traditional methods for extracting EOs from plant materials. Figure 2 shows a typical hydrodistillation method [24].

To improve the efficiency of the extraction process, microwave-assisted hydrodistillation could be a preferable option. For example, in the Rosmarinus officinalis EO extraction process using microwave-assisted techniques, ground, fresh, or dried plant samples were mixed with varying volumes of water. The mixture was heated inside a microwave oven operating at different power levels, typically 200 to 600 W, for various extraction times ranging from 20 to 90 min. During heating, the vapor mixture of water and EO was continuously condensed in an external cooling system connected to the microwave cavity, and the recovered distillate was collected in a Clevenger receiver. Excess condensed water was refluxed back into the extraction flask to maintain constant humidity throughout the extraction process. After collection, the EOs were treated with anhydrous sodium sulfate to remove any remaining water, weighed, and finally stored at 4 °C in dark vials [27].

2.3. Other Methods

Mukherjee et al. [28] reported a solvent-free microwave extraction protocol based on microwave hydro-diffusion and gravity to extract Tagetes erecta EO, which yielded tagetone-enriched oil rather than steam distillation.

The subcritical water treatment, combined with Soxhlet extraction, has been treated as an efficient method of producing EO from Canarium odontophyllum [29].

3. Chemical Constituents of Essential Oils

EOs are extremely concentrated aromatic constituents obtained from various plants and are renowned for their aromatic and therapeutic properties. Understanding the chemical constituents of EOs is crucial for comprehending their potential effects on biological systems. Recently, researchers have been interested in exploring the constituents of EOs due to market demand worldwide. The chemical compositions of EOs have been reported previously. The chemical structure of major compounds present in EOs is represented in Figure 3.

Eucalyptus EO is characterized by its high content of 1,8-cineole, which makes up about 63.1% of its composition. Additionally, it contains various monoterpene hydrocarbons, including p-cymene, α-pinene, α-limonene, γ-terpinene, β-pinene, and β-myrcene [30]. Lavandula angustifolia EO (L. angustifolia) is rich in linalyl acetate (27.5%) and linalool (24.1%), constituting 51.6% of the oil’s composition. Other significant compounds include E-β-ocimene, terpinen-4-ol, caryophyllene, carvacrol, lavandulyl acetate, (Z)-β-farnesene, and (Z)-β-ocimene. These constituents play a crucial role in determining the quality of the EO [31]. Rosemary EO is characterized by its key compounds like eucalyptol (9.48% to 12.58%). Lavender EO also contains significant amounts of eucalyptol (25.9% to 22.7%), borneol (12.43% to 14.09%), and camphor (9.61% to 5.76%). Ferulago contracta EO (F. contracta) is made up of bornyl ester (13.42% to 14.32%), trans-β-Ocimene (11.52% to 6.78%), and limonene (8.93% to 3.34%). These components are crucial in defining the aromatic profile of the respective oils [32].

Peppermint EO contains major components like menthol, menthone, 1,8-cineole, menthofuran, and isomenthyl acetate, making up a significant portion (72.4% of the entire EO) [33]. Cymbopogon spp. EO has α-elemol (ranging from 29.5% to 53.1%), geraniol (37.1%), and citral (90.4%) in different parts such as roots, root hair with stalk, and leaves [34]. Cymbopogon martinii EO (C. martinii) varies in composition between leaves and roots. In leaves, the major compounds are neral (36.1%) and geranial (53.1%), while in roots, they comprise α-elemol (31.5%), neral (16.6%), and geranial (25.0%) [34]. Lemongrass EO also shows a distinction between leaves and roots. In leaves, geraniol dominates (76.6%), followed by geranyl acetate (15.2%). In roots, geraniol is the major component (87.9%), with geranyl acetate constituting 4.4% [34].

Table 1. Major chemical compounds of the representative essential oils (EOs).

Phytocompounds	Source of EOs	References
(Concentration in %)
Geranial (35.07%)	Lippia alba	[1]
Neral (27.8%)
Trans-caryophyllene (6.72%)
Geranial/α-citral (42.88%)	Cymbopogon citratus
β-Citral (32.15%)
Myrcene (9.82%)
Carvacrol (18.97%)	Origanum vulgare
Trans-sabinene hydrate (17.75%)
Terpinen-4-ol (7.57%)
Thymol (48.9%)	Thymus vulgaris	[35]
p-Cymene (19.0%)
γ-Terpinene (4.1%)
Carvacrol (3.5%)
β-Caryophyllene (3.5%)
Carvacrol (12.8%)	Thymus tosevii
α-Terpinyl acetate (12.3%)
cis-Myrtanol (11.2%)
Thymol (10.4%)
Menthol (37.4%)	Mentha piperita
Menthyl acetate (17.4%)
Menthone (12.7%)
Limonene (6.9%)
Carvone (49.5%)	Mentha spicata
Menthone (21.9%)
Limonene (5.8%)
1,8-Cineole (26.54%)	Rosmarinus officinalis L.	[36]
α-Pinene (20.14%)
Camphor (12.88%)
Camphene (11.38%)
β-Pinene (6.95%)
cis-α-Santalol (39%)	Santalum album	[37]
cis-β-Santalol (17.38%)
β-Curcumen-12-ol (9.71%)
n-Hexadecanoic acid (78.25%)	Azadirachta indica	[38]
Tetradecanoic acid (7.24%)
Silane, triethylfluoro-(3.96%)
1,6-Octadien-3-ol, 3,7-dimethyl- (41.74%)	Lavandula angustifolia
Silane, triethylfluoro-(36.71%)
Bicyclo [2.2.1] heptan-2-one, 1,7,7-trimethyl-, (+)-(6.91%)
α-Zingiberene (36.78%)	Zingiber officinale	[39]
β-Sesquiphellandrene (10.25%)
ar-Curcumene (9.51%)
α-Farnesene (6.84%)
Camphene (3.80%)
β-Bisabolene (3.65%)
1,8-Sineol (24.38%)	Alpinia galanga	[40]
cis-β-Farnesene (12.19%)
β-Pinene (8.48%)
Phenol, 4-(2-propenyl)-, acetate (6.01%)
(S)-4-(1-Acetoxyallyl) phenyl acetate (5.66%)
Trans-Anethole *	Pimpinella anisum	[41]
γ-Himachalene *
Linalool *	Ocimum basilicum
1,8-Cineole *
Methyl eugenol *
Limonene *	Citrus bergamia
Linalyl acetate *
γ-Terpinene *
Linalool *
Eugenol *	Cinnamomum zeylanicum
Cinnamyl acetate *
Terpinen-4-ol *	Malaleuca alternifolia
α-Terpineol *
1,8-Cineole *
α-Terpinene *
γ-Terpinene *
Eugenol *	Syzygium aromaticum	[41]
β-Caryophyllene *
1,8-Cineole *	Eucalyptus globulus
α-Pinene *
Anethole *	Foeniculum vulgare
Fenchone *
Geranial *	Zingiber officinale
Neral *
β-Caryophyllene *	Hypericum perforatum
α-Pinene *
Linalyl acetate *	Lavandula angustifolia
Linalool *
Terpinen-4-ol *
Ocimene *
Geranial *	Cymbopogon citratus
Neral *
1,8-Cineole *	Thymus mastichina
Linalool *
Menthol *	Mentha piperita
Menthone *
α-Thuyone *	Rosmarinus officinalis
α-Pinene *
Camphene *
Camphor *
Lyratol *	Artemisia vulgaris
1,8-cineole *
α-Thuyone *	Salvia officinalis
Camphor *
1,8-Cineole *
α-Humulene *
Carvacrol *	Satureja montana
p-Cymene *
1,8-Cineole *	Thymus vulgaris
β-Phellandrene *
Camphor *
Terpinene (52.24%)	Melaleuca alternifolia	[42]
Dihydro-α-terpineol (5.97)
Diterpene (2.87%)
(L)-alpha-terpineol (18.32%)	Citrus limon
Alpha-terpinol (13.43%)
Trans-4-thujanol (9.64%)
α- Terpinolene (5.81%)
Citral propylene glycol acetal (5.73%)
Geranial propylene glycol acetal (4.00%)
α-Terpineol acetate (3.60%)
(E)-Cinnamaldehyde (69.0%)	Cinnamomum verum J. Presl	[43]
Eugenol (6.43%)	(Bark oil)
β-Caryophyllene (6.33%)
Linalool (5.02%)
Eugenol (79.0%)	Cinnamomum verum J. Presl
Eugenyl acetate (2.71%)	(Leaf oil)
Benzyl benzoate (3.54%)
(E)-Cinnamaldehyde (0.86%)
Sabinene *	Myristica fragrans Houtt.	[44]
α-Pinene (pin-2(3)-ene) *
Myristicin *
β-Pinene (pin-2(10) ene) *
4-Terpineno *
Limonene *
γ-Terpinene *

* Major compounds of the plant and percentage are not available.

lists the representative constituents, and Supplementary Table S1 lists the constituents of the EOs.

4. Some of the Bioactive Properties of EOs

EOs are blends of various compounds, each with distinct pharmacological effects. Studies demonstrate their abilities to combat bacteria, parasites, and viruses and regulate various metabolic functions [45]. The distinct elements of EOs have been applied independently and demonstrated various biological effects, including antimicrobial, antioxidant, anti-inflammatory, and insecticidal properties [46] (Figure 4).

4.1. Antimicrobial Activity

Cinnamon EO exhibited the most promising minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against the strains obtained from dairy cows with clinical endometritis, Escherichia coli (E. coli) (MIC: 2048 µg/mL; MBC: 32768 µg/mL), and Trueperella pyogenes (T. pyogenes) (MIC: 512 µg/mL; MBC: 16384 µg/mL). Cinnamon EO and other EOs also showed synergistic inhibitory activity against T. pyogenes and E. coli [47].

EOs from Cinnamomum zeylanicum (C. zeylanicum) (MIC: 2.52 mg/mL), Cymbopogon citratus (C. citratus) (MIC: 1.118 mg/mL), Litsea cubeba (L. cubeba) (MIC: 1.106 mg/mL), Mentha piperita (M. piperita) (MIC: 1.14 mg/mL), Syzygium aromaticum (S. aromaticum) (MIC: 1.318 mg/mL), Ocimum basilicum (O. basilicum) (MIC: 9.15 mg/mL), and Pelargonium graveolens (P. graveolens) (MIC: 17.8 mg/mL) showed inhibitory activity against E. coli [48]. C. zeylanicum EO (MIC: 1.26 mg/mL to 0.63 mg/mL), S. aromaticum EO (MIC: 2.637 mg/mL to 0.164 mg/mL), and their combination (MIC: 1.289 mg/mL to 0.322 mg/mL) exhibited significant inhibitory activity against Salmonella enterica (S. enterica) serotype Enteritidis and S. enterica serotype Typhimurium strains [49]. In addressing pathogens accountable for otitis externa in dogs and cats, the EO from Salvia sclarea (S. sclarea) showed inhibitory activity against Staphylococcus pseudointermedius (S. pseudointermedius), with an MIC value of 2.23 µg/µL. Origanum vulgare (O. vulgare) EO inhibited the growth of both Staphylococcus aureus (S. aureus) and S. pseudointermedius, with MIC values of 2.36 µg/µL and 1.18 µg/µL, respectively. Conversely, no discernible activity was observed against Pseudomonas aeruginosa (P. aeruginosa) [50]. Thymus vulgaris (T. vulgaris) and O. vulgare EOs showed antimicrobial activity against Candida albicans (C. albicans), Candida famata (C. famata), E. coli, and Enterococcus spp. [51]. Similarly, the EOs from O. vulgare and Rosmarinus officinalis (R. officinalis) exhibited the lowest MIC values when combating fungi. S. sclarea, R. officinalis, and T. vulgaris demonstrated MIC values below <0.3 µg/µL against Candida tropicalis (C. tropicalis), while O. vulgare displayed a potential anti-mycological effect against Aspergillus. Trichosporon sp. showed sensitivity exclusively to R. officinalis, whereas both R. officinalis and O. vulgare inhibited Rhodotorula sp. [50].

The antidermatophytic activity of EOs from Curcuma longa (C. longa) and Zingiber officinale (Z. officinale) was investigated against Trichophyton verrucosum (T. verrucosum) and Microsporum canis (M. canis). C. longa and Z. officinale Eos combination demonstrated robust antifungal effects against T. verrucosum and M. canis, comparable to standard antifungal drugs’ efficacy [52].

The effects of O. vulgare EO (OVEO) with its phenolic constituents, thymol and carvacrol, against four clinical strains of Candida, including C. albicans, Candida glabrata (C. glabrata), Candida krusei (C. krusei), and Candida tropicalis (C. tropicalis), had been demonstrated. The MIC and minimum fungicidal concentration (MFC) values were determined for each strain. For all Candida species, the MIC values of OVEO ranged from 780 to 1560 µg/mL, and the MFC values ranged from 780 to 3120 µg/mL. Carvacrol exhibited MIC and MFC values between 97.5 and 195 µg/mL. Thymol demonstrated MIC values between 195 and 390 µg/mL, and MFC values ranged from 390 to 780 µg/mL. C. glabrata, C. krusei, and C. tropicalis displayed resistance to fluconazole. In contrast, C. albicans displayed susceptibility to fluconazole, with MIC values of 0.5 µg/mL. The MFC for fluconazole in all tested strains fell within 64 to >128 µg/mL [53].

Similarly, the mycelial growth and spore germination of Botrytis cinerea (B. cinerea) were significantly inhibited by the EO of O. vulgare and its primary components, thymol and carvacrol, when tested in vitro. In vivo experiments, EO, at a concentration of 250 mg/L, exhibited strong antifungal effects, reducing the decay in cherry tomatoes by 96.39%. Additionally, thymol and carvacrol completely inhibited gray mold. In postharvest scenarios, O. vulgare EO could be an environmentally friendly and non-toxic botanical fungicide controlling gray mold [54].

The diluted lemongrass EO was most active against the clinical isolates of Malassezia pachydermatis (M. pachydermatis). Diluted oregano, palmarosa, winter savory, cinnamon leaf, clove, Indian melissa, manuka, and rose geranium EOs also exhibited bioactivity against M. pachydermatis [55].

Loizzo et al. [56] demonstrated that Laurus nobilis (L. nobilis) berries EO had an IC50 value of 120 µg/mL against severe acute respiratory syndrome coronavirus (SARS-CoV), with a selectivity index (SI; TC50/IC50) of 4.2. The oil also showed noteworthy activity against herpes simplex virus (HSV)-1, with an IC50 value of 60 mg/mL [56]. Senthil Kumar et al. [57] reported the angiotensin-converting enzyme 2 (ACE2) inhibitory effects of ten EOs. Geranium and lemon EOs exhibited significant ACE2-inhibitory effects in vitro. Immunoblotting and qPCR analysis further confirmed the potent ACE2 inhibitory effects of geranium and lemon EOs. The major compounds of geranium EO were citronellol, geraniol, and neryl acetate, while limonene was the predominant compound in lemon EO. The findings suggest that geranium and lemon essential EOs could serve as valuable natural anti-viral agents, potentially preventing SARS-CoV-2/COVID-19 invasion into the human body [57]. Four EOs [lemon-Citrus limon (C. limon), sweet orange—Citrus sinensis (C. sinensis), grapefruit—Citrus paradisi (C. paradisi), and rosemary cineole (R. officinalis chemotype 1.8 cineole)] effectively reduced the viral loads of hepatitis A virus (HAV) [58].

The in vitro study on the anthelmintic activities of Lavandula officinalis (L. officinalis), Anthemis nobile (A. nobile), and Citrus aurantifolia (C. aurantifolia) EOs against Haemonchus contortus (H. contortus) showed significant inhibitory effects on egg hatching in the egg hatch test. The dose-dependent response profile was observed for all oils in inhibiting egg hatching. Additionally, the anthelmintic activity of these oils was confirmed in the larval development test. C. aurantifolia, A. nobile, and L. officinalis EOs exhibited a larval development inhibition rate greater than 85%, with IC50 values of 0.187, 0.375, and 1.5 mg/mL, respectively [59].

The reduction in bacterial levels in the feces of dogs in the natural antioxidant feed (NAT) group may be attributed to the antimicrobial properties of EOs [60]. The finding aligns with the de Oliveira et al. [61] report, which investigated the antimicrobial effects of EOs (specifically cloves and lemongrass) against pathogenic bacteria isolated from swine, cattle, and poultry feces. They observed that these oils demonstrated effectiveness against both Gram-negative and Gram-positive bacteria.

EOs Mechanism of Antibacterial Action

The activity of EOs is influenced by various factors, such as their composition, the presence of functional groups in their active components, and how these components interact synergistically [62]. Gram-positive bacteria are generally more susceptible to EOs than Gram-negative bacteria [63,64]. Gram-negative bacteria pose a greater challenge to EOs due to their outer membrane’s complexity, rigidity, and high content of lipopolysaccharides. These characteristics restrict the diffusion of hydrophobic compounds through the membrane [65]. Conversely, Gram-positive bacteria lack this outer membrane complexity and instead have a thick peptidoglycan wall that is less resistant to small antimicrobial molecules. This makes it easier for antimicrobial compounds to penetrate through the cell membrane. Furthermore, the presence of lipophilic ends in lipoteichoic acid, a component of Gram-positive bacterial cell membranes, may enhance the penetration of hydrophobic components present in EOs [66].

Several studies have demonstrated that the active components present in EOs initially adhere to the surface of bacterial cells before penetrating the phospholipid bilayer of the cell membrane. This accumulation disrupts the structural integrity of the cell membrane, leading to detrimental effects on cell metabolism and ultimately culminating in cell death [67,68]. Moreover, EOs have been found to alter the permeability of the cell membrane, resulting in the loss of essential intracellular components such as proteins, reducing sugars, ATP, and DNA. This disruption also inhibits the generation of energy (ATP) and related enzymes, leading to cell breakdown and electrolyte leakage [69]. Consequently, a cascade of events affecting the entire bacterial cell is believed to underlie the antimicrobial activity of EOs [70]. Figure 5 illustrates the mechanisms by which EOs exert their toxic effects on bacterial cells.

4.2. Antioxidant Properties

Zhang et al. [71] demonstrated the impact of natural oregano EO (NOEO) on antioxidant properties in the serum of 21-day-old broilers. In comparison to the control group, broilers receiving antibiotics, NOEO, and synthetic oregano EO (SOEO) demonstrated significantly higher concentrations of serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Furthermore, dietary supplementation with NOEO and SOEO increased serum glutathione reductase (GR) concentration. The malondialdehyde (MDA) level decreased with dietary antibiotics and NOEO supplementation, but it was not affected by dietary SOEO supplementation on day 21. On the 42nd day, the serum MDA proportion did not vary among the four therapies. Though SOEO and NOEO supplementation raised serum proportions of GR and SOD compared to the control, birds nourished with antibiotics, SOEO, and NOEO exhibited elevated serum concentrations of GSH-Px and total antioxidant capacity (TAC) compared to those fed with a control diet [71].

The laying hens in the experimental group exhibited significantly elevated levels of TAC (9.8 ± 0.8 mmol/L) compared to the basal diet feed control group (CON). Conversely, MDA levels (10.7 ± 1.4 nmol/mg) were notably lower in the experimental group than in the CON group. Birds supplemented with oregano EO showed consistently higher concentrations of GSH-Px. Additionally, the experimental group exhibited significantly lower alanine and aspartate transferase concentrations [72].

Broilers that were provided with thyme EO (TEO) and rosemary EO (REO) in their diets exhibited numerically lower levels of total antioxidant status (TAS) and total oxidant status (TOS). In contrast, all groups (3-day-old male broiler chicks (Ross 308) were arbitrarily separated into five groups of 80 animals per treatment in a total of four hundred chicks) receiving TEO and REO demonstrated significantly elevated SOD activity and glutathione (GSH) levels, along with reduced MDA in the breast muscle. Conversely, catalase (CAT) activity in the breast muscle of the broilers was statistically similar across all groups (p > 0.05). Furthermore, it determined that dietetical supplementation of REO and TEO elevated the oxidative stress index (OSI) and TOS levels while decreasing TAS levels (p < 0.05). The investigated doses (Control: Basal diet alone, Thyme-1: Basal diet + 0.15 g kg⁻¹ of thyme EO (TEO), Thyme-2: Basal diet + 0.30 g kg⁻¹ of TEO, Rosemary-1: Basal diet + 0.10 g kg⁻¹ of REO, and Rosemary-2: Basal diet + 0.20 g kg⁻¹ of REO) of dietary TEO and REO enhanced intestinal morphology and boosted antioxidant metabolism, particularly in the breast, drumstick, and liver muscles in broilers [73].

The EO from Thymus algeriensis (T. algeriensis) (TAEO) has reduced 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS •+) radicals, with a mean IC₅₀ value of 2.7 mg/mL. TAEO also exhibited a scavenging hydrogen peroxide (H₂O₂) capacity, with an IC₅₀ value of 512 µg/mL. The involvement of electron transfer mechanisms was identified in TAEO, supported by its reducing power in the TAC assay (313 mg ascorbic acid equivalent (AAE)/g of EO) and ferrous iron reduction (25.4 µg AAE/g TAEO). The lipophilic nature of TAEO components significantly inhibited oxidation reactions in lipidic systems, as evidenced by the β-carotene bleaching assay (55.5% at 5 mg/mL). However, within the tested concentration level (1–10 mg/mL), TAEO could not bind with ferrous ions [74].

The observed rise in SOD activity following EO blend (EOB) (100 mg EOB/kg diet (EO100); 200 mg EOB/kg diet (EO200); 400 mg EOB/kg diet (EO400)) supplementation to pigs is considered advantageous, as GSH levels showed no decline. EOB effectively triggers the antioxidant defense system without elevating reactive oxygen species (ROS) levels. If ROS had increased, GSH levels would have decreased due to its involvement in neutralizing H2O2, underscoring the significance of investigating these compounds to comprehend their efficacy in the diet of weaned pigs. The addition of 200 and 400 mg EOB/kg diet led to a decrease in diarrhea occurrence. It proved beneficial for blood biochemical and hematological profiles and the liver status of antioxidants in nursery pigs. EO supplementation did not adversely affect the pigs’ growth performance or gastrointestinal health [75].

4.3. Anti-Inflammatory Activity

A combination of Eucalyptus globulus (E. globulus) EO with a drug (flurbiprofen) showed significantly better membrane stabilization effects than groups treated with only E. globulus EO or drug. When comparing the combination (EO + drug)-treated group with the drug-treated group, the combination of EO and drug demonstrated significantly better anti-inflammatory and antipyretic effects, with non-significant differences in the analgesic model [76].

The EOs derived from Eucalyptus citriodora (E. citriodora) (EC), Eucalyptus tereticornis (E. tereticornis) (ET), and E. globulus (EG) at a dose of 100 mg/kg, but not at 10 mg/kg, significantly reduced swelling in rat hind paws caused by carrageenan and dextran within 1–4 h after administration compared to control. While there wasn’t a clear distinction in the effectiveness of the three EOs in most experiments (Control, EC, ET, and EG for 1 h, 2 h, 3 h, and 4 h), ET oil extracts showed greater efficacy against dextran-induced paw swelling at the 100 mg/kg dose. Compared to the positive control, dexamethasone (1 mg/kg), which nearly completely inhibited neutrophil migration, the EOs demonstrated 70–80% inhibitions compared to the control [77].

The EO from TAEO effectively suppressed acute inflammation induced by xylene application to mice’s ears. The reduced ear inflammation showed a pattern of dose dependence in the examined groups, ranging from 11.6 mg at 200 mg of TAEO/kg body weight to 9.2 mg at 600 mg of TAEO/kg body weight, compared to the control group. The excessive dose, 600 mg of TAEO/kg body weight, showed a 58% decrease in xylene-mediated ear inflammation [74].

The repeated inhalation of peppermint EO significantly decreased heightened levels of MDA in the skeletal muscles induced by a two-week exhaustive swimming regimen in rats. MDA is a marker for lipid peroxidation, a process triggered by free radicals damaging cell membranes and generating ROS. Inflammation is also believed to contribute to ROS formation and lipid peroxidation. Peppermint EO, known for its free radical-scavenging and anti-inflammatory properties, could potentially counteract ROS, reduce inflammation-mediated ROS generation, and consequently lower MDA levels [78].

4.4. Insecticidal Activity

According to the findings of Medeiros et al. [79], both T. vulgaris (commonly known as thyme) and thymol exhibited significant insecticidal effects against Cochliomyia hominivorax (C. hominivorax), specifically on third-stage larvae. It suggests the potential for enhancing novel formulations to control myiasis. C. hominivorax is prevalent in tropical and subtropical regions of the Caribbean and South America, excluding Chile, and is accountable for 64.3% of reported myiasis occurrences in domesticated populations of animals [80]. T. vulgaris EO’s positive effects mainly rely on the key component thymol. It has been reported as a potential insecticide, insect repellent, acaricide, and fungicide [81]. The EO of O. vulgare, where thymol is the major component, demonstrated the LC50, which is 3.07 times lower than that of T. vulgaris EO after 48 h of exposure. However, validating in vitro findings with in vivo experiments is crucial, considering factors like quantity and persistence and ensuring the interaction with larvae in real-world environments. Additionally, the topical application of EO must be thoroughly evaluated for safety and concern [79].

The in vitro effectiveness, LC50, and harmfulness of EOs derived from Alpinia zerumbet (A. zerumbet) (Pers.) B. L. Burtt & R. M. Sm, Laurus nobilis L. (L. nobilis), Cinnamomum spp., Mentha spicata L. (M. spicata), Cymbopogon nardus (L.) Rendle (C. nardus), and Ocimum gratissimum L. (O. gratissimum) were reported against infantile stages and adult forms of Ctenocephalides felis (C. felis) (cat fleas). O. gratissimum EO demonstrated the highest efficacy in the in vitro assays against all the stages of the flea, exhibiting adulticidal (LC50 = 5.85 μg cm⁻²), ovicidal (LC50 = 1.79 μg cm⁻²), and larvicidal (LC50 = 1.21 μg cm⁻²) activity at low doses. The findings could enhance toxic-free products to control fleas in cats and dogs [82].

The toxicity of each component in Ajwain (Carum copticum L.; C. copticum) EO (AEO) and their combined effects on Chilo suppressalis (C. suppressalis) larvae. AEO and thymol exhibited significant insecticidal activity with LD50s of 13.10 and 17.11 μg/larvae, respectively. The effectiveness of insecticidal activity was further enhanced by combining thymol with γ-terpinene or p-cymene. Among these compounds, thymol appeared as a promising biopesticide for controlling C. suppressalis populations, demonstrating insecticidal properties and compatibility with other components possessing high acetylcholinesterase inhibition capabilities [83].

The R. officinalis (REO) EO induces mortality, diminishes geotaxis (climbing ability), and has a repellent effect comparable to conventional repellents against the Drosophila melanogaster (D. melanogaster) model. The study revealed that EO could induce oxidative damage and disrupt the antioxidant defenses in adult fruit flies, leading to significant larvicidal effects that result in mortality and hinder larval development. Furthermore, when the EO (at 3.2 μg/mL) is combined with paraquat, there is a synergistic impact on survival and geotaxis. The study suggested the prooxidant mechanism of REO, linked to oxidative damage and impairment of enzymatic and nonenzymatic systems. REO demonstrates dual potential as a bio-insecticide and larvicide against adult and third-instar larvae of D. melanogaster, with approximate LC50 values of 6.9 μg/mL and 1.81%, respectively [84].

5. Effects of EO on the Health Status of Pets and Animals

In treating Malassezia otitis (M. otitis) externa in atopic dogs, a combination of EOs was used. T. vulgaris exhibited the lowest MIC at 0.05%, whereas R. officinalis, S. sclarea, and Lavandula hybrida (L. hybrida) indicated higher MIC levels (>2%). The MIC for ketoconazole was less than 0.03 μg/mL. The EOs combinations 1 (0.5% C. paradisi, 0.5% S. sclarea, 0.5% O. basilicum, and 1% R. officinalis) and 3 (1% S. sclarea, 1% R. officinalis, and 1% L. hybrida) showed the MIC at 50% concentration. In contrast, mixtures 4 (1% C. limon, 0.5% R. officinalis, 1% C. paradisi, and 0.5% Anthemis nobilis (A. nobilis)) and 5 (1% A. nobilis, 0.5% C. paradisi, 0.5% T. vulgaris, and 1% L. hybrida 1%) inhibited fungal growth at a 75% dilution. Mixture 2 (1% C. limon, 0.5% S. sclarea, 1% R. officinalis, and 0.5% A. nobilis) showed MIC at 25% concentration. The synergic activity of EOs showed significant results in in vivo studies, even at sub-MIC concentrations. Mixture 2 showed better results in in vivo studies, whereas Mixture 5 showed insignificant results. Some dogs showed adverse effects like swollen erythema against mixture 4 [85].

A recent study investigated the combined impact of yeast cell wall and oregano EO on dogs’s digestion, taste preference, intestinal fermentation byproducts, and gut microbiota. Including this blend in the dogs’ diet decreased the apparent total tract digestibility of dry matter and intake ratio compared to the control diet. It also positively influenced beneficial microorganisms in the fecal matter, resulting in greater bacterial diversity and low concentrations of ammonia, histamine, and phenol [86].

Graham et al. [84] reported the olfactory-stimulating properties of EOs (lavender, chamomile, rosemary, and peppermint) in dogs housed in a rescue shelter. The dogs were exposed to each olfactory stimulant, facilitated by the diffusion of EOs, for 4 h per day over 5 days, with a 2-day interval between each exposure. The study revealed that prolonged exposure to EOs had a noticeable effect on the dog’s behavior, without any clear signs of habituation to the smells. Interestingly, stimulating odors (rosemary and peppermint EOs) increased activity levels among the sheltered dogs. In contrast, stress-reducing odorants (lavender and chamomile EOs) did not produce a similar effect despite introducing all odors for the same duration. Notably, the dogs did not habituate to any odors, even those known for stress reduction, such as lavender and chamomile. However, the possibility of eventual habituation with prolonged exposure remained uncertain. The study suggested that the observed behavioral changes were likely attributable to the introduced odors rather than other environmental influences. The findings underscored the importance of carefully selecting odors, as introducing scents that induce agitation or stress could have detrimental effects. It was highlighted that stimulating odors might not be suitable for dogs already displaying hyperactivity or abnormal behavior. In contrast, calming scents (lavender and chamomile EOs) could contribute to the improved well-being of dogs [87].

A topical formulation containing polyunsaturated fatty acids (PUFAs) and EOs impacts the itching and skin lesions associated with canine atopic dermatitis (CAD). The dogs were treated with a test formula or placebo on the upper part of the neck once a week. The degree of CAD and severity, measured by the Canine Atopic Dermatitis Extent and Severity Index-03 (CADESI-03), as well as pruritus scores, were assessed by veterinarians and owners before and after the study. The results showed a significant improvement in CADESI-03 and pruritus scores in the treatment group compared to the placebo group. Furthermore, more dogs in the treatment group exhibited at least a 50% improvement in CADESI-03 and pruritus scores compared to the placebo group. The topical preparation containing PUFAs and EOs proved to be a safe and beneficial treatment for alleviating the clinical signs of CAD [88].

In a blinded crossover clinical trial, dogs underwent a dental cleaning and examination, which included a periodontal assessment involving probing and evaluations of plaque, calculus, and gingivitis. Later, pet owners administered a gel (either active or a placebo) to the soft tissues inside their dogs’ mouths twice daily for 4 weeks. Subsequently, the dogs’ teeth underwent another cleaning, and the owners then applied the alternative gel for an additional 4 weeks. Clinicians evaluated bad breath immediately after the initial cleaning and at the 4th and 8th weeks, while owners provided weekly scores for bad breath. In the group that switched from placebo to active gel, bad breath reduced during the placebo application and continued to decrease during the application of the active gel. Seven out of nine owners reported reduced bad breath when using the active gel. Applying the topical gel containing menthol, thymol EO, and polyphenolic antioxidants (phloretin and ferulic acid) reduced oral malodor in dogs [89].

Goode et al. [87] aimed to assess the impact of various EOs or active ingredients in commercially available repellents on Ixodes ricinus (tick) attachment to dogs. Turmeric EO prevented the climbing response by a tick and had a longer residual activity than other EOs. A subsequent blanket drag field assay compared tick attachment on blankets treated with turmeric EO, orange EO, N, N-Diethyl-3-methylbenzamide (positive control), or 1% coco glucoside excipient solution (negative control). The results, based on the counting of 899 ticks, showed an average of 23.3 ± 21.3, 26.9 ± 28.6, 2.6 ± 2.0, and 3.4 ± 3.7 ticks per blanket were found in the negative control, orange EO treatment, turmeric EO treatment, and positive control groups, respectively. Dogs sprayed with 2.5% turmeric EO (n = 24) and 2.5% orange EO (n = 16), control (n = 15), were allowed to walk in known tick-infested areas. Dogs sprayed with turmeric EO had a significantly lower percentage of ticks attached to their legs or belly than dogs sprayed with orange EO and control dogs. These findings suggest that turmeric EO may be useful in a tick management program for domestic dogs [90].

In the quest for effective and safe control of Rhipicephalus sanguineus Sensu Lato (R. sanguineus) (brown dog tick) on dogs, a microemulsion formulation containing thymol and eugenol was developed and assessed for its safety and physical characteristics. Microemulsions demonstrate strong physical stability. Treatment with microemulsion led to a significant decrease in larvae infesting the dogs on the first day, while the numbers of nymphs and adults did not show a notable reduction. When assessing the reproductive biology of engorged females, there was a notable impact on larval hatchability, resulting in an impressive control rate of 85.5%. In conclusion, the microemulsion effectively reduces the number of larvae and affects the reproductive parameters of engorged female brown dog ticks. Still, it is also safe for dogs and maintains physical stability for two years [91].

The dog feed enriched with natural antioxidants (a blend of EOs and vitamin E) was assessed for its impact on food preservation and canine health. The experimental group receiving the natural antioxidant feed showed a notable reduction in ROS levels, signifying a mitigation of oxidative stress. Also, an increase in non-protein thiol and glutathione S-transferase levels was noted, potentially elucidating the observed decline in ROS levels and concluding that incorporating natural antioxidants into dog feed not only enhances food preservation but also elevates systemic antioxidant levels, effectively minimizing the adverse effects induced by free radicals in the dogs [60].

Batista et al. [89] revealed the chemical constituents of extracts and EO obtained from Schinus molle (S. molle). They evaluated their effectiveness against adults of C. felis and its eggs, a prevalent flea species infesting dogs and cats in Brazil. The non-polar (n-hexane) extract exhibited 100% effectiveness (800 µg cm⁻²; LD50 = 524·80 µg cm⁻²). Lupenone was the major compound in the extract. EOs extracted from fruits and leaves demonstrated 100% efficacy against adult fleas at concentrations of 800 µg cm⁻² (LD50 = 353·95 µg cm⁻²) and 50 µg cm⁻² (LD50 = 12·02 µg cm⁻²), respectively [92].

A study was conducted on fourteen cats with symptoms of spontaneous dermatophytosis caused by M. canis. In group 1, cats were administered orally with itraconazole at a 5 mg/kg/day dose for one week, with at least 6 weeks of total treatment duration. Natural shampoo, about 5 mL containing Thymus serpyllum (T. serpyllum) (2%), O. vulgare (5%), and R. officinalis (5%) EOs, was used to wash twice a week. In group 2, cats received the same oral itraconazole dose and 2% miconazole/2% chlorhexidine shampoo twice a week during treatment. By week 3 after treatment, two cats in group 1 were clinically healthy, and all others achieved clinical health by week 11. At the trial’s conclusion, all cats in this group tested negative for fungal cultures. The mean time to clinical cure and mycological cure for group 1 cats was 6 weeks (median 4 weeks, range 3 to 11 weeks) and 15 weeks (median 14 weeks, range 7 to 42 weeks), respectively. In group 2, one cat was dermatologically normal by week 3, and all cats achieved clinical cure by week 10. By the end of the study, 6 out of 7 cats in this group were negative about fungal culture. The mean time to clinical cure and mycological cure for group 2 cats was 5.9 weeks (median 6 weeks, range 3–10 weeks) and 12.8 weeks (median 6 weeks, range 7–21 weeks), respectively. The study suggested that T. serpyllum, O. vulgare, and R. officinalis EOs containing shampoo could be an alternative to conventional cat dermal treatment [93].

The antifungal efficacy of EOs derived from C. limon, T. serpillum, R. officinalis, O. vulgare, and Illicium verum (I. verum) was investigated against eleven clinical isolates of M. canis. Among the tested EOs, T. serpillum and O. vulgare exhibited the lowest MICs, followed by I. verum, R. officinalis, and C. limon. A formulated mixture comprising 5% O. vulgare, 5% R. officinalis, and 2% T. serpillum in sweet almond oil demonstrated enhanced antimycotic activity compared to individual components. Subsequently, this mixture was treated in seven symptomatic M. canis-infected cats. Recovery both clinically and culturally was observed in four of the seven treated cats, highlighting the potent antifungal activity of T. serpillum and O. vulgare EOs [94].

One hundred and fifty cats were exposed to various odor conditions: control (no smell), biologically relevant odor (rabbit scent-prey scent), and biologically non-relevant odors (lavender and catnip scents). The cats were exposed to the odors for 3 h a day over five successive days. Cats’ behaviors were recorded every 5 min on days one, three, and five. The cats showed limited interest in scented clothes, spending slightly over 6% of the observation time. Particularly, cats exposed to catnip-scented cloths displayed significantly more interest, spending an average of 11.14% of the observation time. Across all conditions, interest in clothes decreased in the second and third hours, indicating habituation. Olfactory stimulation influenced certain aspects of the cats’ behavior. Both catnip and prey scents led to a higher frequency of behaviors associated with lowered activity. Catnip also triggered a specific play-like behavior called the ‘catnip response’ [95].

The anthelmintic efficacy of peppermint EO (M. piperita L.) has been reported. In the egg hatch test in vitro, the ovicidal activity ranged from 21.0 to 90.3%, depending on the concentration of the EO. The in vivo fecal egg count reduction test was conducted using the mean dose of EO (150 mg/kg), which demonstrated a certain level of anthelmintic efficacy, with an average reduction in nematode eggs of 26.9 and 46.0% at days 7 and 14 after treatment, respectively [96].

Anti-toxic effects of O. vulgare EO (OEO) against aflatoxin B1 (AFB1) have been reported. Forty-eight New Zealand white-growing rabbits aged four weeks were randomly divided into four groups with four replicates, each containing three animals: the control group (only basal diet), the AFB1 group (0.3 mg AFB1/kg diet), the OEO group (1 g OEO/kg diet), and the co-exposed group (1 g OEO/kg + 0.3 mg AFB1/kg diet). The results indicated that OEO significantly alleviated the toxic effects of AFB1 on rabbit kidneys by reducing cystatin C levels. Additionally, OEO mitigated oxidative stress and lipid peroxidation in the co-exposed group. Furthermore, OEO mitigated DNA damage and inflammatory responses, along with the downregulation of genes encoding stress and inflammatory cytokines. Moreover, OEO preserved the cytoarchitecture of rabbit kidneys treated with AFB1 [97] (

Table 2. Summary of essential oils for animals and poultry healthcare.

Study Subjects	Essential Oil	Dose and Duration	Results	References
Dogs	A mixture of EOs of 6% clove, 2% rosemary, 1% oregano, 3.3% vitamin E, and 87.7% soybean oil (vehicle).	1% of test mixture in dry feed. The control and test groups had 380 g of feed daily for 28 days. Then, the animals were swapped with a 15-day washout period.	Improved the antioxidant status of the study subjects.	[60]
Dogs with otitis	Mixture 1: Citrus paradisi (0.5%), Salvia sclarea (0.5%), Ocimum basilicum (0.5%), Rosmarinus officinalis (1%). Mixture 2: Citrus limon (1%), R. officinalis (1%), Anthemis nobilis (0.5%), S. sclarea (0.5%). Mixture 3: S. sclarea (1%), Lavandula hybrida (1%), R. officinalis (1%). Mixture 4: C. limon (1%), R. officinalis (0.5%), C. paradisi (1%), A. nobilis (0.5%). Mixture 5: Thymus vulgaris (0.5%), A. nobilis (1%), C. paradisi (0.5%), L. hybrida (1%).	200 μL of oil mix per ear once daily for 2 weeks	Mixture 2 showed better improvement in canine otitis.	[85]
Dogs	Diet 1: Control; Diet 2: 1.5 kg/ton of yeast cell wall and oregano EO (1.5 YCO); Diet 3: 3.0 kg/ton of yeast cell wall and oregano EO (3.0 YCO).	1.5 kg/ton YCO or 3.0 kg/ton YCO twice a day for 20 days	Dogs treated with the YCO blend showed signs of enhanced intestinal function. Beneficial bacterial diversity was increased. The concentrations of histamine, phenol, and ammonia were reduced.	[86]
Dogs	EOs of Lavandula angustifolia, Anthennis nobilis, Cymbopogon citrates and Mentha piperita.	* EO was diffused using an oil burner into the dogs’ places for 4 h per day for five consecutive days. After 2 days of break, the next EO was used.	L. angustifolia and A. nobilis EOs improved the behaviors and relaxation of dogs in the rescue shelter.	[87]
Dogs with CAD	PUFAs: (6 mg/mL of α-linolenic and 30 mg/mL of linoleic acid); EOs (neem oil, rosemary extract, lavender oil, clove oil, tea tree oil, oregano extract, peppermint extract and cedar bark extract) *	Dogs < 10, 10 to 20, and 20 to 40 kg received 0.6, 1.2, and 2.4 mL, respectively. once a week for 8 weeks.	The topical preparation containing PUFAs and EOs ameliorates the clinical signs of CAD and is safe for dogs.	[88]
Dogs	Placebo or active gel ** containing EO compounds (menthol and thymol) and polyphenolic antioxidants (phloretin and ferulic acid).	12 mm length (and 0.75 mm width) of gel/each side of the mouth. Twice daily for 4 weeks.	A daily application of tested formulation following an initial dental cleaning reduced halitosis in dogs.	[89]
Dogs	EOs of turmeric or orange; excipient only (negative control); N,N-Diethyl-3-methylbenzamide (DEET) (positive control)	2.5% (v/v) of turmeric or orange EO diluted in water with a 1% coco glucoside excipient. Ten sprays per day for 28 days.	Dogs treated with turmeric EO showed a significantly reduced percentage of ticks attached to their legs or bellies compared to controls.	[90]
Dogs infested with ticks	Microemulsion containing 0.5 mg/mL of thymol and 0.5 mg/ mL of eugenol.	After ticks infestation, each dog of the treatment goup was sprayed with 10 mL of freshly prepared microemulsion/kg on the same day.	The microemulsion reduced the number of tick larvae in dogs and reduced the larval hatching. The microemulsion was stable and safe.	[91]
Cats infected with Microsporum canis (dermatophytosis)	EO treatment: oral itraconazole + Shampoo containing Thymus serpyllum (2%), O. vulgare (5%), and R. officinalis (5%), EOs. Conventional treatment: (oral itraconazole + 2% miconazole/ 2% chlorhexidine shampoo	Oral itraconazole (5 mg/kg/day) for 1 week, every 2 weeks for at least 6 weeks. Washed twice a week using 5 mL of shampoo during treatment.	The treatment was effective, and EO shampoo could be natural alternative cat dermatophytosis treatment.	[93]
Cats infected with Microsporum canis (dermatophytosis)	EO mixture containing 2% Thymus serpillum, 5% O. vulgare, and 5% R. officinalis in sweet almond oil	EO mixture was applied to the lesion for one month. Itrafungol® (5 mg/kg/day) for 1 week, washout period for 1 week (3 cycles)-served as efficacy control.	Four out of seven cats that received EO treatment showed recovery on both clinically and culturally. No adverse effects were observed in any of the treated cats.	[94]
Sheep	EO of Mentha piperita diluted in sunflower oil at 1: 4.5 ratio.	EO treatment: Regular diet (barley and maize grains) with 150 mg/kg EO. Diet and 3.8 mg/kg of albendazole (Positive control). Diet and 50 mL of sunflower oil/ animal (negative control). For 14 days.	EO of M. piperita has potent anthelmintic efficacy. It could be used to control gastrointestinal nematodes in sheep.	[96]
Rabbit	O. vulgare EO	Control, AFB1 group (0.3 mg AFB1/kg diet), OEO group (1 g OEO/kg diet), and Combination group (1 g OEO/kg + 0.3 mg AFB1/kg diet) for 8 weeks.	OEO supplementation improved the harmful effects of AFB1. Improved the antioxidant levels, Decreased the inflammation, and reversed oxidative DNA damage in rabbits.	[97]

* Concentration is not available; ** PerioSciences LLC with exclusive technology licensing to Tooth To Tail Animal Inc., Dallas, USA; PUFAs: Polyunsaturated fatty acids; EOs: Essential oils; YCO: Yeast cell wall and oregano EO; CAD: Canine atopic dermatitis; AFB1: Aflatoxin B1; OEO: O. vulgare EO.

).

Male broiler chicks (Cobb) were supplemented with or without oregano essential oil (OEO) or Bacillus subtilis via water for 28 days. Then, changes in the hematological parameters, phagocytosis, lymphocyte proliferation, and antibody responses were studied at different study points (6, 18, and 28 days). OEO and B. subtilis supplementation significantly improved growth performance, as evidenced by increased weight gain and a reduced feed conversion ratio. OEO and B. subtilis-supplemented groups notably enhanced hematological parameters and phagocytic activity. OEO supplementation significantly increased lymphocyte proliferation. OEO and B. subtilis supplementations boosted serum antibody levels at 18 and 28 days, respectively. Histological examination revealed improvements in intestinal morphology. Overall, supplementation with B. subtilis and OEO showed beneficial effects on growth and immunity, suggesting potential alternatives to antibiotics in poultry production [98].

Holstein dairy cows supplemented with Digestarom™ Dairy^®, an essential oil blend (3 g/day), showed improvement in their immune status and energy metabolism during the transition period without affecting the health of the newborn calves [99].

Hy-line Brown laying hens were supplemented with different concentrations (100, 300, and 500 mg/kg) of microencapsulated essential oils (MEO) for 56 days (supplements for one week on and one week off). MEO supplementation (300 and 500 mg/kg) improved egg production and feed conversion ratios compared to the control group and decreased the egg-breaking ratio. Additionally, shell thickness and Haugh unit values increased significantly. Immunoglobulin and cytokine levels in serum and parameters related to intestinal morphometry were higher in the MEO300 and MEO500 groups than in the control. Furthermore, supplementation with MEO enhanced the plasma antioxidant capacity. Overall, intermittent feeding of MEO improved egg production, antioxidative processes, immune functions, and intestinal morphology, enhancing egg quality in laying hens. The study suggests feeding 300 mg/kg of MEO can significantly benefit animal health and egg quality [100].

6. Essential Oil, Gut Microbiota, and Host Health

The gut microbiota plays a crucial role in regulating the health and physiology of the host [101]. It is also closely associated with several diseases and disorders [102]. Supplementing phytocompounds and natural products could improve the composition and diversity of the microbiome [13,103].

Dietary EO supplementation could increase the abundance of beneficial bacteria, promoting the growth of beneficial microbes in the colon [104]. Importantly, the gut microbiota significantly influences the microbiota-gut-brain axis, which is the bidirectional communication between the gastrointestinal and brain systems [102]. A healthy gut microbiota is more important for the intestinal integrity, immunity, feed utilization, and growth of nursery pigs [105,106,107].

Dietary supplementation of cinnamaldehyde and carvacrol enhances performance and appetite in nursery pigs by modulating intestinal health and microbiota. In detail, supplementing cinnamaldehyde (100 mg/kg), carvacrol (100 mg/kg), or a combination improved body weight, average daily weight gain, feed intake, and diarrhea rates. Also, the supplementation increased intestinal absorption area, tight junction protein abundance, and intestinal development, decreasing intestinal permeability and local inflammation. Goblet cell numbers were also increased, indicating enhanced mucus barrier function. Furthermore, piglets in the supplemented group showed upregulated transporters and digestive enzyme levels in the intestine, which correlated significantly with daily weight gain and feed utilization. The abundance of Lactobacillus spp., Prevotella spp., Megamonas spp., Megasphaera spp., and Blautia spp. increased after the supplementation. Dietary cinnamaldehyde and carvacrol improve intestinal barrier function and development, increase digestive enzyme activity and absorption capacity and microbial communities, and enhance appetite, leading to improved performance and reduced diarrhea in nursery pigs [101] (Figure 6).

Healthy white × landrace sows were supplemented with or without 500 g/t Meriden-Stim^® (which contains 5% OEO (extracted from Origanum vulgare subsp. hirtum) and 95% natural feed grade inert carrier) from day 100 of gestation until day 21 of lactation. OEO caused a notable decline in the phylum Proteobacteria and Actinobacteria (p < 0.05). Additionally, OEO significantly increased the relative abundance of Lactobacillus spp. and Prevotellaceae UCG 003 and UCG 005 and reduced Escherichia spp. and Shigella spp. abundances. Overall, supplementing maternal diets with OEO during late gestation and lactation improved serum metabolites and antioxidant capacity. It balanced the intestinal flora of sows, potentially resulting in increased piglet weight at weaning [105]. Notably, an increased prevalence of Proteobacteria signifies microbial community instability and could serve as a disease diagnostic marker [106]. At the phylum level, a notable decline in relative abundance was observed in the OEO group, suggesting potential energy conservation in sows for enhanced milk quantity and quality [108].

Likewise, Escherichia and Shigella species are closely linked with gut dysbiosis [109] and demonstrated reduced abundance in the OEO-treated group. E. coli O157:H7, a zoonotic pathogen, represents a significant serotype of Shiga toxin-producing E. coli [108]. A study indicates that E. coli O157:H7 triggered notable increases in serum creatinine and urea levels and reported Thymus vulgaris EO mitigating hepatorenal dysfunction induced by E. coli O157:H7 [110]. Another study revealed how carvacrol inhibited flagellin production and subsequent flagellar development, rendering E. coli O157:H7 immobile through protein heat shock [111].

Prevotella spp. possess a substantial repertoire of carbohydrate-degrading enzymes. They are recognized for producing short-chain fatty acids that regulate long-term energy metabolism. It is important to underscore the need for increased research on the relationship between Prevotella spp. and nutrition, as multiple studies have demonstrated conflicting results regarding their association with dietary patterns [112]. Furthermore, high Lacticaseibacillus spp. abundance is beneficial for preserving intestinal flora equilibrium and inhibiting the proliferation of harmful bacteria. Previous research has indicated that Lacticaseibacillus spp. competes with intestinal pathogens for binding sites in the mucosal layer, thus suppressing bacterial infections [113].

In EO-treated calves, Firmicutes decreased initially but rebounded after two weeks, while Proteobacteria and Bacteroidota declined and Actinobacteriota increased. Moraxellaceae (36.9%), Burkholderiaceae (14.7%), Microbacteriaceae (10.6%), Mycoplasmataceae (8.8%), and Pasteurellaceae (5.9%) are the dominant families. Moraxella, Ralstonia, Mycoplasma, Mannheimia, Filobacterium, Pseudomonas, and Pasteurella are the major genera found in the EO-treated group. EO-treated calves exhibited reduced Mannheimia spp. and Filobacterium spp. abundances on day 2, while Corynebacterium decreased on day 1 post-treatment. EO administration altered the nasopharyngeal microbiota composition and diversity, particularly within the first 24 to 48 h, with notable decreases in Mannheimia spp. abundance [114].

The impact of dietary supplementation with Lippia sidoides EO (LSEO) on the microbiota and intestinal structure of Danio rerio has been reported. D. rerio was supplemented with varying concentrations of LSEO (0.25%, 0.50%, 0.75%, 1.00%, and 1.25%). Microbiological counts did not significantly differ among groups, but microbiome diversity was higher in the 0.25% LSEO-supplemented group compared to the control. LSEO demonstrated inhibitory effects on potential pathogenic bacterial growth. Fish receiving 0.25% LSEO exhibited improved intestinal histomorphometric indices, indicating enhanced intestinal integrity. Thus, incorporating 0.25% LSEO in the diet of D. rerio could improve the microbiota and intestinal health [115].

He et al. [116] noted increased species richness and diversity in Litopenaeus vannamei’s intestinal microbiota when fed a diet supplemented with organic acid and EO. Huyben et al. [117] found no significant differences in rainbow trout (Oncorhynchus mykiss) microbiota operational taxonomic units between groups receiving EO-supplemented diets and the control. It has been proposed that organic acid and EO contribute to midgut health by modulating microbial communities [117].

The impact of early OEO supplementation in milk replacers on growth, immune responses, microbiota, and metabolomes in dairy calves during preweaning and adulthood was reported. The EO group exhibited elevated average CD14+ population values and decreased abundance of Ruminococcaceae UCG-014, Faecalibacterium, Blautia species, and Alloprevotella, while Allistipes and Akkermansia species abundances increased. Changes in plasma metabolites like butyric acid, 3-indole-propionic acid, and succinic acid, especially at 45 days, were correlated with alterations in the intestinal microbiota. Early EO supplementation improved feed efficiency only during the suckling period, accompanied by significant shifts in the microbiota and plasma metabolome. However, these changes may not be favorable for gut health [118].

7. Safety Considerations of Essential Oils

While EOs have gained popularity, a major concern lies in the limited studies on their toxicity [119]. The potentially harmful effects of EOs and their components are typically assessed in lab animals like rodents. Initial tests on rats have shown that most EOs have low toxicity, with an LD50 range of 1–20 g/kg [120]. However, studies examining their effects on the host are scarce.

Preliminary toxicity tests on peppermint EO (M. piperita) in sheep demonstrated no adverse effects on behavior, blood parameters, or kidney and liver functions. It indicates the safety of using the formulation on sheep, especially in the short term [96]. Similar results were observed for lemongrass EO Cymbopogon schoenanthus L. (C. schoenanthus) applied to sheep at doses of 180 and 360 mg/kg orally [121] and for the encapsulated combination of anethole and carvone applied to lambs at doses of 20 and 50 mg/kg orally [122]. In both cases, there were no toxic effects on animal behavior or liver and kidney functions, affirming their safety [121,122].

Studies demonstrate that pesticides classified as having minimal risk and not registered with the Environmental Protection Agency (EPA) can have notable detrimental effects on dogs and cats. Among the exposed animals, most (92%) exhibited symptoms after using naturally derived plant flea products. It’s worth noting that even when labeled as “natural” and applied according to the instructions, these minimal-risk pesticides may still have adverse effects on animals [23]. Furthermore, individual animals may display varying sensitivity to EOs due to their distinct host characteristics [23,123].

In a study, pets infested with fleas were thoroughly treated with an infusion derived from Melissa officinalis (M. officinalis) (prepared as a decoction) for 30 min. The animals’ coats were allowed to air dry after this treatment. Additionally, alternative formulations, including a spray made from Citrus spp. (prepared as a decoction), L. officinalis (also prepared as a decoction), and EOs from Thuja plicata (T. plicata) and Juniperus communis (J. communis), were employed against the fleas [124]. In all trials, effective repellence was observed 24 h after treatment. However, the study did not provide information regarding the percentage of infestation post-treatment, which consequently hindered the determination of overall efficacy [124].

Utilizing botanical anthelmintics presents various benefits. These include their diverse chemical composition, comprising compounds from different chemical groups, which reduces the likelihood of resistance development. Additionally, their natural origin leads to fewer residues in animal products and the environment, making them an economically viable option [59,125,126,127]. Furthermore, employing an encapsulation technique can enhance the in vivo effectiveness of EO by safeguarding its active components from degradation, thus increasing their availability [128].

Alternatively, better outcomes can be attained by augmenting the dosage or administering multiple doses over consecutive days instead of a single application. However, assessing the potential toxicity when employing higher doses or multiple administrations is imperative [129]. Alternative application methods like lick blocks containing plant-based compounds [130] can also enhance efficiency and facilitate controlled release, allowing for prolonged usage [129].

8. Limitations in the Use of Essential Oils

• Comprehensive studies are scarce on the toxicity of EOs, particularly concerning pets and animals, leading to a lack of robust evidence on their potential risks and benefits.
• The effects of EOs can vary significantly among different species of animals. This variability introduces complexity in establishing standardized dosages and safety guidelines applicable across diverse animal groups.
• Determining the appropriate dosage and application methods of EOs for animals is challenging due to factors like body weight, metabolism, and individual sensitivities.
• The chemical composition of EOs could vary based on factors like plant source, extraction method, and storage conditions. This lack of standardization poses challenges in predicting their precise effects on animals and requires careful consideration of each oil’s unique properties.
• Some EOs, even those derived from plants, can pose risks to animals. For instance, ingestion of tea tree oil has led to intoxication in both humans and animals, demonstrating the importance of informed usage.

9. Future Perspectives

The future of EO applications in veterinary care is poised for exciting advancements. Through cutting-edge research and personalized guidance, we anticipate a transformative approach to using EOs for animal health. Innovations in technology and controlled clinical trials will enhance the effectiveness and safety of these interventions. Additionally, a deeper understanding of the gut microbiome and regulatory enhancements will lead to a more refined and holistic approach to incorporating EOs in animal healthcare. These strides not only signify progress in veterinary medicine but also underscore a dedicated commitment to the well-being of our cherished animal companions.

10. Conclusions

The utilization of EOs in pet and animal care holds promise for addressing a range of health concerns. The diverse biological and pharmacological properties of EOs, encompassing antimicrobial, antioxidant, and anti-inflammatory effects, offer potential benefits for domestic and exotic species. However, caution must be exercised to ensure safe and effective application. Understanding the chemical composition of EOs is crucial in evaluating their potential impacts on biological systems, while preliminary toxicity studies provide important insights into their safety profiles. In insecticidal activity, thymol and thyme EOs demonstrate notable efficacy against the larvae of C. hominivorax, suggesting potential applications in myiasis control. These findings present an important avenue for further research and development in combating myiasis, particularly in regions where the parasite poses a significant threat to animal health. Dietary supplementation with EOs, such as cinnamaldehyde and carvacrol, demonstrates promising potential in enhancing intestinal health, microbial diversity, and performance outcomes in nursery pigs, underscoring the importance of exploring natural additives for improving animal welfare and productivity.

While EOs offer promising therapeutic benefits, their mode of action and potential risks remain subjects of ongoing investigation. It is imperative to continue research in this area, focusing on understanding the mechanisms underlying their effects and conducting rigorous safety assessments. Regulatory frameworks warrant refinement for tailored veterinary guidelines. As research advances, personalized protocols and specialized formulations hold the potential to revolutionize animal healthcare. As the interest in natural and holistic approaches to pet care continues to grow, EOs represent a valuable area of exploration in veterinary medicine. With careful consideration and further study, EOs may emerge as valuable tools for enhancing the well-being of pets and animals.