Gastrointestinal nematodes (GINs) are one of the most important endoparasites in grazing animals, especially ruminants (1). In sheep, these parasites may cause various negative effects including subclinical disease with weight loss and reduced animal production, or clinically manifested disease with signs of anemia, diarrhea, protein loss, anorexia, decreased immunity, and fertility (2–4). In the case of a high worm burden, a possibly fatal outcome may occur (5). Therefore, due to decreased productivity, high treatment costs, and possible death of the animals, GIN parasitism can have a huge economic impact (6). Commercially available anthelmintics have been used successfully for decades to control these parasites (7). However, as a result of their irrational use, anthelmintic resistance (AR) has developed, whereby the following are considered high-risk factors: underdosing, overfrequent treatments, mass treatment, and single-drug regimens (8, 9).

Thus, increasing resistance of nematodes to the well-known group of anthelmintics such as benzimidazoles, macrocyclic lactones, and imidazothiazoles has been reported, sometimes simultaneously to several different classes (10, 11). The problem is also present in novel anthelmintic groups such as monepantel, an amino-acetonitrile derivative (12). Annual economic losses resulting from the development of AR are estimated at €38 million in Europe, with expected growth in the future (10). Other problems associated with the use of commercial anthelmintics are residues present in animal products such as meat and milk or in the ecosystem and biodiversity. This represents a serious problem associated with many currently available chemotherapeutic drugs, along with the increasing price (13, 14). All the above-mentioned issues suggest the urgent need for developing novel strategies for future treatments (4).

As a possible solution for achieving sustainable control of sheep GINs, an integrated approach has been proposed by many researchers (1, 15–19). This refers to the rational use of commercial anthelmintics, along with the use of alternatives. On the one hand, the rational use of anthelmintics should be based on refugia strategies such as targeted treatments (TT) or targeted selective treatments (TST), which imply only the treatment of herds or animals that require it due to clinical or economic reasons (19–21). Besides, the rational use of commercial drugs may refer to the combination or rotation of anthelmintics from different chemical classes in order to avoid the excessive use of only one drug (5, 7). On the other hand, integrated control implies the incorporation of various alternative strategies into the practice, such as genetic selection of sheep naturally resistant to GINs, pasture management, dietary manipulation, vaccine development, biological control (direct–use of nematophagous fungi, bacteria, and predatory nematodes or indirect–use of earthworms or dung beetles), or the use of plant-based anthelmintics (condensed tannins, various extracts, or essential oils) (6, 15, 19, 22, 23).

As aromatic, complex, and concentrated mixtures of volatile, nonpolar compounds, essential oils (EOs) belong to the group of plant secondary metabolites that are responsible for enabling plants to be competitive in their own environment (24, 25). Thus, these mixtures contain various compounds such as terpenes, terpenoids, and phenylpropanoids that can be used for various pharmacological purposes (26, 27). EOs and their compounds can be obtained from plants by various extraction methods, and along with their extracts, they are increasingly being used in veterinary medicine in recent times. Among the others, indications for their application include the use as coccidiostats to boost immunity and improve the performance of poultry, to prevent diarrhea in piglets (due to their antimicrobial properties), as fish diet supplementation, to use against Varroa destructor in beekeeping or against Malassezia pachydermatis and ticks in pets, etc. (28–31). In ruminants, the possible benefits of EOs among the others include the improvement of ruminal fermentation and digestion and the reduction of methanogenesis and nitrogen excretion (29). In addition, various studies have demonstrated the efficacy of EOs against GINs in sheep. The list is wide and includes oregano, thyme, coriander, tea tree, and lavender as well as various species of lemongrass, eucalyptus, lippia, mint, etc. (32, 33).

Peppermint (Mentha x piperita L.) represents a well-known aromatic and medicinal herb derived from crossing between spearmint (Mentha spicata L.) and water mint (Mentha aquatica L.). It belongs to the Lamiaceae family and is native to the Mediterranean region but is now cultivated in various parts of the world (34). Peppermint is one of the most important plant species in the pharmaceutical and cosmetic industries, whose EO is widely produced and used. Many pharmacological properties of peppermint have been previously demonstrated and include analgetic, antiviral, antibacterial, antifungal, and antiparasitic effects (35, 36). Moreover, the effect of M. piperita EO against sheep GINs was also demonstrated in various studies, whereby it showed promising results against different parasite stages (37–39). However, only the efficacy under laboratory conditions has been proven so far, which requires the confirmation of these results in field trials. Therefore, along with in vitro, the aim of the present study was to examine the in vivo effect of M. piperita EO against GINs of sheep, as well as to evaluate the safety of its use in the hosts.

The urgent problem associated with the development and spread of AR in sheep GINs requires actively searching for alternative strategies, whereby botanical anthelmintics are considered one of the most promising options (50). However, finding the most suitable plant formulation among the wide number of species, both from an efficacy and safety point of view, as well as from the aspect of price, is a challenging task. Thus, EOs and extracts from a wide number of plants have been tested so far on the efficacy against sheep GINs, as discussed earlier (32, 33). As can be noticed, most of these studies refer to in vitro testing. Although these tests are appropriate for the initial evaluation of anthelmintic potential due to their speed, reproducibility, easy application, absence of animals, and price (48), in vivo tests and evaluation of safety are also crucial parts of the process of developing a new anthelmintic drug and are needed before the formulation is introduced into practice (51). The choice of assays is also important, with the EHT and FECRT being recommended due to their accuracy and reliability. In fact, these tests are most commonly used for such studies (23, 52).

Considering the number of studies conducted so far and their results, the EO of M. piperita can be considered one of the potential candidates for the development of a new anthelmintic agent. Thus, in a study by Katiki et al. (37), it showed significant activity against Haemonchus contortus on the EHT with an obtained IC₅₀ value of 0.26 mg/mL. Along with the ovicidal activity, EO also exhibited an effect on larval development and feeding with IC₅₀ values of 0.26 and 0.07 mg/mL, respectively. Next, a study by Chagas et al. (38) has demonstrated moderate ovicidal (IC₅₀ = 1.44 mg/mL) but high larvicidal (IC₅₀ = 0.10 mg/mL) activity of the aforementioned EO, also against H. contortus. In our last study (39), M. piperita EO showed a strong, dose-dependent (R² = 0.98) activity against a mixture of sheep GIN genera (Hemonchus, Trichostrongylus, Teladorsagia, and Chabertia) with an inhibition of egg hatchability varying from 72.5–99.8% (IC₅₀ = 0.28 mg/mL) in the concentration range of 0.049–50 mg/mL. From that perspective, the results of the EHT in the present study showed a lower ovicidal effect of M. piperita EO, which may be attributed to the use of different EO samples in comparison with that study. In addition, in the present study, we used a different method to obtain concentrations (dilution method in comparison with micropipettes in the last study).

In the FECRT, the tested formulation (EO diluted in sunflower oil) exhibited an anthelmintic activity of 46.04% in total on Day 14 at the tested dose of 150 mg/kg (Table 3). As in our study performed with oregano EO (41), the efficacy was better in Farm 2 compared with Farm 1 (63.35 and 28.73%, respectively, on D14) (Graph 3), which can be explained by the differences in animal husbandry. That is, on the second farm, sheep were kept in boxes during the treatment, which facilitated the manipulation of animals and the application of formulations. In contrast, in the first farm, sheep were completely kept free-range. Other factors such as differences in feed (due to differences in animal husbandry), and consequently composition and volume of rumen content, may also affect the results. On the other hand, albendazole showed a high effect (>95%) on both farms on Days 7 and 14, indicating that anthelmintic resistance did not develop in the tested farms. The lower efficacy of the tested sample compared with the positive control can be explained by interactions of phytochemicals along the gastrointestinal tract and thus their partial inactivation and consecutive limited bioavailability (29). Next, the high standard deviation values of EPGs in both farms in all treatment groups suggest a large variation in the degree of parasite burden of individual animals. This is usually a characteristic of GINs, where the largest number of parasites accumulates in a smaller number of animals in certain herd (3). On the other hand, this suggests the importance of TST strategies during the treatments (19–21).

According to the GC–MS analyzes, menthol and menthone were most responsible for the anthelmintic effect shown, as their percentage representations were 32.6 and 22.0%, respectively. Indeed, these two compounds are considered the most important for various pharmacological properties of M. piperita EO and its wide use (35). However, pure menthol failed to reduce the EPG of H. contortus during in vivo testing. In contrast, the whole EO of M. arvensis, whose main component was menthol (86.7%), showed an efficacy of approximately 50% on Days 1, 14, and 21 (38). In other similar studies, whole EOs were also usually more effective than their isolated major components, as shown for Croton Zehtneri - anethole (53) and Thymus vulgaris–thymol (54). This suggests the importance of all EO compounds for their activity, potentially due to their synergistic effect. The absence of an effect of pure menthol in mentioned research was explained by its limited bioavailability and by the fact that menthol is excreted in the form of glucuronides via urine (38).

In comparison with the composition of M. piperita EO in our previous study (39), differences in the present compounds and their percentages may be observed. That is, the main compounds of that sample were piperitone (25.4%) and trans-dihidrocarvone (14.6%). In other mentioned studies, the chemical composition of M. piperita EO corresponds more to the sample of the present study. Thus, menthol (42.5%) followed by menthone (27.4%) were the main compounds of the sample in a study by Katiki et al. (37), and menthol (30.5%) was also the main compound of the sample in a study by Chagas et al. (38). This suggests that the composition of EO may vary considerably, even among those obtained from the same plant species. From that aspect, various factors such as the age and part of the plant, geographical origin, precipitation, light, and soil properties that refer to pH, structure, and salinity, as well as the presence of various organisms and microorganisms in a plant environment may be involved. In addition, the methods used for the extraction of oils, the producers from whom the oils are sourced, and the way they were stored prior to their use may also affect chemical composition (55, 56). Consequently, these factors may also lead to smaller or larger differences in efficacies, as can be noticed if the results of the mentioned studies, including the present one, are compared. The question that arises is to what extent these variations may hinder the development of commercial preparation, and whether ways to standardize the composition of certain EOs should be a topic of further studies.

Despite their increasing popularity, one of the main problems associated with the wider use of EOs in practice appears to be the lack of toxicity studies (33). Potential toxic and side effects of EOs and their constituents are usually tested in experimental animals such as rodents, whereas acute toxicity tests conducted so far in rats showed that most EOs have an LD₅₀ of 1–20 g/kg, indicating their low toxicity (57). However, studies evaluating the effects on the host are rarely conducted. In the present study, the results of the preliminary toxicity tests showed the absence of side and toxic effects of M. piperita EO on sheep, i.e., their behavior, hematological parameters, as well as kidney and liver functions. This suggests the safety of the use of the applied formulation in sheep, at least when it comes to short-term effects. The same results were obtained for EO of lemongrass (Cymbopogon schoenanthus L.) applied to sheep (180 and 360 mg/kg, p.o.), and for the encapsulated combination of anethole and carvone applied to lambs (20 and 50 mg/kg, p.o.), with the absence of toxic effects on animal behavior, as well as liver and kidney functions, suggesting their safety (58, 59).

In a study by Chagas et al. (38), no toxicity symptoms were observed in sheep treated with M. arvensis EO. This and the results of the present study may be considered to be expected since the main compound of both of these EOs, menthol, has very low acute oral toxicity (LD₅₀ > 2000 mg/kg b.w.) and is therefore generally recognized as safe by the FDA (U.S. Food and Drug Administration). Moreover, the FDA has approved the use of menthol in food (38, 60). According to the Assessment Report on Mentha x piperita L., folium and aetheroleum (61) can be safely used in humans at the recommended doses (oral, cutaneous, and inhalation) for the treatment of various gastrointestinal disorders or the symptomatic relief of mild tension-type headaches. However, although Mentha species are considered safe, special attention should be paid to the content of pulegone and menthofuran in the formulation due to their reported hepato-toxicity (62, 63). Thus, according to the Public Statement on the use of herbal medicinal products containing pulegone and menthofuran (64), the intake of their combination in 37.5 mg/person/day in humans is considered acceptable for herbal-medicinal products, which is set up as a limit for a life-long exposure. Such studies should be provided for animals including sheep.

The results of the coproculture examination showed the presence of four GIN genera before the treatment on the tested farms: Trichostrongylus (44%), Teladorsagia (41.33%), Haemonchus (10.5%), and Chabertia (4.17%). Since their percentage representation did not change significantly (p > 0.05) after the treatment, it can be concluded that the treatment with M. piperita EO is not specific for only a single genus. However, the results of some other studies showed higher activity of EOs against H. contortus in comparison with Trichostrongylus spp. after per-oral administration of the formulations (65–67), suggesting that there may be differences in sensitivity on EOs within the GIN genera (e.g., due to their position in the gastrointestinal tract). Anyhow, the mechanism of nematicidal activity of M. piperita EO compounds is still not fully elucidated. In a study by Choudhary et al. (68), menthol was shown to potentiate acetylcholine and levamisole responses in the receptor sensitive to levamisol but not to nicotine. The same study also showed that menthol can significantly potentiate the contraction of Ascaris suum somatic muscle strips at each concentration of acetylcholine. A study by Khan et al. (69) showed that methanol and the extract of M. arvensis cause high apoptotic effect in the muscles, gonads, and uterus (eggs) of the free-living nematode Caenorhabditis elegans, which was used as a model organism. The results also showed that stress genes (gst-4 and hsp-16.2) were highly expressed in the affected nematodes compared to normal C. elegans.

Although the applied oil significantly reduced the number of EPG after the treatment, with the achieved in vivo efficacy being one of the highest obtained for EOs against GINs (32, 33), it is still insufficient for its independent use. However, studies conducted so far showed that none of the aforementioned possible control methods appears to be sustainable when applied alone (6). Thus, as discussed earlier, it is generally agreed that a combination of different strategies in the form of integrated parasite management is the only sustainable solution for long-term parasite control (6, 16–19, 70). An example is the results obtained in a mentioned study by Choudhary et al. (68), where the positive allosteric modulatory properties of menthol were proven, which could be used in combination therapy with cholinergic anthelmintics.

From all the above, results obtained in the present study suggest that M. piperita EO can play a valuable role in an integrated approach to control parasites. In general, the use of botanical anthelmintics offers many advantages, such as their rich chemical composition with a lot of compounds belonging to different chemical groups (lower susceptibility to the development of resistance), their natural origin (lower amount of residues in animal products and the environment), suitable price, etc. (13, 14, 48, 50). Moreover, in vivo efficacy of M. piperita EO could be further improved with the use of an encapsulation technique that can protect its active ingredients from degradation, and thus enable their higher bioavailability (71, 72). On the other hand, better results can also be achieved by increasing the dose or using multiple applications over several consecutive days instead of single administration although the use of a higher dose or multiple administration should also be evaluated from the toxic point of view. Efficacy can also be improved, as well as control release enabled by other ways of applications such as lick blocks that contain plant-based compounds, and may provide long-term use (73).

The development and spread of anthelmintic resistance in nematodes require an urgent search for alternatives. The results of the present study suggest that M. piperita EO is suitable for use against GINs in sheep based on the efficacy and preliminary toxicity tests performed. Therefore, in combination with other strategies (rational use of anthelmintics or other alternatives), the tested formulation could play a valuable role in an integrated, environmentally friendly control of these parasites. Further studies should be performed to confirm the safety after long-term use of peppermint oil in practice to achieve even higher efficacy in field trials, as well as to test its efficacy against GINs resistant to commercial drugs.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University of Naples (PG/2021/0130480, 16 December 2021). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

FŠ: conceptualization, methodology, formal analysis, investigation, data curation, and writing original draft preparation. SK: conceptualization, resources, and writing review and editing. DS: conceptualization, validation, and supervision. RR: conceptualization, methodology, and resources. NS: investigation, resources, and writing review and editing. DO: investigation and resources. LR: methodology, validation, writing review and editing, and supervision. EC: methodology and investigation. MM: methodology and validation. GC: validation and supervision. AB: conceptualization, methodology, investigation, data curation, and writing review and editing. All authors contributed to the article and approved the submitted version.

This research was cofounded by the Ministry of Education, Science, and Technological Development of the Republic of Serbia, rescript No. 451–03-1183/2021–14.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.