Various tests can be used to evaluate the anthelmintic efficacy of standard anthelmintics and the detection of AR (44). The use of in vitro tests in veterinary parasitology has numerous advantages over in vivo tests. Namely, these tests are rapid, inexpensive, reproducible, usually simple and reduce the number of animals needed for field trials, which can be expensive, laborious and time-consuming (43). The two most commonly used in vitro methods for testing the efficacy of anthelmintics are the EHT and the larval development test (45, 46). These methods are also extremely useful for screening substances of plant origin for their activity against helminths and are therefore commonly used for this purpose. The EHT is used to evaluate the ovicidal activity of an active substance and provides comparable, reliable and precise results for the evaluation of the efficacy of e.g., benzimidazoles and thus forms a basis for other tests (45). However, the results of EHT and in vivo tests such as FECRT do not always correlate with each other, as many factors can influence activity under field conditions (43, 45). Therefore, the efficacy of novel agents is confirmed after laboratory tests are complemented by field studies, with in vitro tests providing a good basis for selecting the most promising anthelmintic agents (47, 48, 49, 50).
In the present study, the tested EOs showed high, dose-dependent (R2 values close to 1) ovicidal activity against GIN eggs, which can be compared in different ways. According to the criteria established by the World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) for evaluating the efficacy of various anthelmintics tested in vitro, compounds with those greater than 90% are considered effective in controlling nematodes, including GINs (51, 52). From this point of view, the three highest concentrations of O. basilicum and M. spicata, two of C. medica and one of H. officinalis EO reached the required efficacy, which was comparable to the positive control (p > 0.05). All tested concentrations (even the lowest) of H. officinalis, C. sativum, L. officinalis and O. basilicum showed significantly higher effects than both negative controls (p < 0.05). However, a commonly used pharmacological parameter for comparison of drug-inhibitory effects is the half-maximal inhibitory concentration (IC50), which is precise as it counts all tested concentrations (53). According to this parameter, the most effective oils were M. spicata, O. basilicum and H. officinalis with IC50 values of 0.07, 0.08 and 0.19 mg/ml, respectively. These results largely correspond to the criteria mentioned above.
Some of the EOs tested in the present study were previously investigated in vitro for their anthelmintic effects against sheep GINs. Thus, Sousa et al. (54) tested the ovicidal activity of EOs from 12 different cultivars of O. basilicum, as well as their individual compounds (linalool, methyl chavicol, citral and eugenol) and their combinations, against the eggs of H. contortus. The effect of the EO samples was variable, with IC50 values ranging from 0.56 to 2.22 mg/ml, with the highest active variety consisting mainly of linalool and methyl chavicol. In fact, the isolated combination of these compounds in a ratio of 64:11 (similar to that of O. basilicum EO in the present study, along with other compounds) was the most potent combination with an IC50 of 0.44 mg/ml, indicating the great potential of these compounds for inclusion in anthelmintic formulations.
L. angustifolia EO was tested in a study by Ferreira et al. (43), where it showed ovicidal activity (IC50 = 0.316 mg/ml) that was higher than that of the other oils tested. Moreover, the concentration of 3.125 mg/ml and all higher concentrations were more than 90% effective, although the concentrations of 0.195 mg/ml and below were ineffective (< 10%). In the same study, Lavandula EO showed also larvicidal activity and had an effect on adult worm motility. The main compound of that sample was linalool acetate (36.0%), along with camphor (5.54%) and eucalyptol (4.87%). Finally, in a study by Gaínza et al. (55), the EO of C. sinensis, with limonene as the main constituent (96%), showed high ovicidal activity with IC50 and IC90 values of 0.27 and 0.99 mg/ml, respectively. To our knowledge, the rest of the EOs from our study were not tested against sheep GINs, at least not with regard to their ovicidal activity.
The anthelmintic potential of EOs derives from their chemical composition, i.e. the bioactive compounds that compose them (56), with the main compound usually being the most important (57). From this perspective, the acyclic monoterpene tertiary alcohol linalool, and the terpenoid ketones carvone and pinocamphone were the most abundant in the most effective EOs. The individual anthelmintic activity of linalool and carvone against sheep GINs is very well known and described (54, 58, 59, 60), while pinocamphone has not yet been studied and is unknown for this property. The anthelmintic activities of isolated EO ingredients can vary greatly (58), and therefore the components that make up the EO composition are critical to their pharmacological properties. Their percentage content is also important, especially that of the main compound. Thus, the EOs of O. basilicum, consisting of linalool at percentage 62.8%, showed a dominant activity compared to L. angustifolia, where linalool was also the main compound but in the percentage of 37.5%. This fact was already demonstrated in our previous study with eleven EOs (38), where the oils with a high percentage of one compound were the most effective (oregano, fennel etc). However, the presence of other compounds is also important due to synergistic effects, as previous studies have shown that whole EOs generally have the higher effect than isolated compounds (52, 61).
The results of these studies indicate that the number of compounds is not a decisive factor. While in the present study the three most effective oils (O. basilicum, M. spicata and H. officinalis) had the highest number of compounds, in the previous study (38) the number of compounds was inversely proportional to their efficacy. In general, the chemical composition of the EOs may vary depending on many factors related to the plant`s environment, such as soil properties (hydrology, pH, salinity), light, precipitation and season, but also age and part of the plant used for EO extraction, its genetic characteristics etc. (33, 62). The presence of certain animals and microorganisms may also be involved, as they stimulate the plants to produce oils in sight of defense mechanism (63). Finally, the method used for extraction and the post-extraction process before use can also play a role (33). All these factors can lead to differences in the chemical composition of EO extracted from different, but also from the same plant species, and consequently, to differences in their biological properties including anthelmintic activity, as shown in previous studies (38, 54, 61).
The problem of standardization of EO composition may hinder the commercialization of these products. Along with these, they possess some undesirable properties that are avoided to a certain extent in the pharmaceutical field: hydrophobicity and insolubility in water, instability due to hydrolysis and oxidation, and high volatility (20, 64). Although some of the EOs such as oregano (65) and peppermint (66) have already shown noticeable field efficacy against GINs with no side effects noted for the sheep, research efforts are currently focused on developing innovative formulations based on encapsulation techniques that can overcome these problems. Indeed, encapsulation can protect active ingredients that are sensitive to oxygen, light and moisture and prevent interactions with other compounds. In this way, the stability and bioavailability of EOs can be further increased, while reducing toxicity and volatility. In addition, it can enable controlled release, which is important for various uses of oils (20, 67). Nevertheless, there are still some unknowns related to the potential application of EOs against GINs, including their mechanism of action. So far, they are known to have significant negative effects on the survival, reproduction, development, behavior and metabolic pathways of nematodes (68). Understanding the molecular mechanism of action of EOs activity is important for the development of the most appropriate formulation (69), but also for accurate risk assessment and remediation (70).
With regard to the aforementioned disadvantages of the exclusive use of commercial anthelmintics in the control of sheep GINs, EOs have many advantages. On the one hand, the possibility of resistance development in nematodes is most likely lower due to the diversity of their ingredients with potentially different mechanisms of action (18, 43). On the other hand, EOs as herbal anthelmintics are often considered safer for the host than synthetic, chemical compounds (71, 72). The environmental and public health aspects also favor herbal anthelmintics, as they are biodegradable (73). Finally, the financial aspect also plays a role, as they tend to be cheaper than conventional treatments, especially in countries with developed biodiversity (43, 71, 72). Considering all these aspects, the use of herbal anthelmintics, including EO, seems to be a promising option that can help reduce the use of synthetic drugs, along with other alternative options (74, 75, 76), as mentioned earlier. Alternatively, they can be combined with the rational use of commercial drugs based on different refugia strategies (77), as some previous studies have shown that they can increase their efficacy (78). In any case, an integrated approach seems to be the backbone for the future management of GINs in small ruminants.