Introduction

Invertebrates constitute an important component of the global diet, and the culinary value of molluscan species is no exception [1]. Edible gastropods are known for their nutritional value, mainly for their high protein content and essential minerals [2, 3]. In addition to extensive studies on the histology, physiology, and breeding of helicine species [4–8], their meat quality has also been investigated due to their widespread consumption throughout Europe [9, 10]. Moreover, edible snail tissue is considered a premium food product, with its high protein content and low fat highlighted as key aspects of its nutritional value [9].

Apostolou et al. [11] recorded a total of 61 operational snail farms in Greece sharing a common gene pool for broodstock [12]. Notably, the highest values in snail growth were observed in open field combined with low density, while the heaviest snail fillet was achieved through a combination of net-covered greenhouses and low density [13]. It is important to highlight that nearly all snail farmers utilize a compound diet as a feed for their snails. The nutritional requirements of terrestrial snails, and in particular their macronutrient requirements, are poorly known. In nature, terrestrial snails feed on a variety of organic materials, such as plants or decaying plant material [14]. Moreover, farmed snail diets have been either empirically formulated or adapted from those designed for other farmed animals, such as poultry, partly due to the limited scientific understanding of snail nutrition [15–17].

Essential oils are complex mixtures of volatile compounds produced by different plant species as secondary metabolites [18]. Currently, more than 3,000 types of essential oils are known, but only about a tenth of them are suitable for use [19]. Phytochemicals are widely recognized as safe for animals, consumers, and the environment. Regarding usage in aquaculture, these products have great potential due to the beneficial biological effects reported in aquatic species, such as growth enhancement, appetite stimulation, immunomodulatory, and antioxidant activity [20]. They contribute to the maintenance of gastrointestinal tract health and functionality through regulation of the native microbiota, which plays a crucial role in host physiology, including protection against pathogenic microorganisms, efficient nutrient absorption, and immune regulation [21].

These properties are attributed to the ability of essential oils to act as prebiotics within the intestinal environment, regulating the composition of the gut microbiota. In addition, essential oils may affect both the quantity and the composition of secretions produced by the intestinal mucosa and alter the physical and chemical characteristics of the intestinal environment, resulting in enhanced disease resistance. Stimulation of intestinal secretions facilitates improved regulation of digestion and nutrient absorption by the gut microbiome. This process may increase the availability of amino acids for protein synthesis and, consequently, lead to increased body protein content in reared animals [22].

When essential oils are included in poultry diets, feed intake has been reported to remain unchanged or slightly decreased. In contrast, improvements in weight gain and feed conversion ratio are consistently observed. These positive effects are attributed to two commonly accepted mechanisms: stimulation of digestive enzyme secretion and stabilization of the gut microbial community [23]. In swine, however, the effects of essential oils on feed intake remain inconsistent. Reported increases in intake are probably related to improved growth rate, a response commonly observed with growth-promoting feed additives. Enhanced feed digestibility following diet enrichment with essential oils has been recorded. The improvement may be attributed to increased bile and enzyme secretion and to a lesser extent, to reduced endogenous nutrient losses [23].

Several studies have investigated the use of essential oils in livestock nutrition, including poultry [24], rabbits [25], pigs [26], and fish [27]. In addition, several studies have examined the proximate composition of poultry, bovine, and pork meat following enrichment of the diet with essential oils such as oregano and rosemary [28–30]. Despite the potential benefits, relatively few studies have examined the effects of vegetable oil supplementation on the lipid composition of farmed snails (Helix aspersa maxima) fillet [15].

Studies on the use of essential oils in snails have mainly focused on their application for snail control [31–34], while only a limited number have evaluated their inclusion in snail diets and the associated effects on growth performance [35, 36]. Linhart and Thompson [35] used 6 chemotypes of Thymus vulgaris in a feeding experiment with adult and juvenile Cornu aspersum aspersum (C. a. aspersum), whereas Lemjallad et al. [37] enriched snail diets with rosemary powder. The only study conducted in Greece by Vokou et al. [36] used three terrestrial snail species [C. a. aspersum, Eobania vermiculata (E. vermiculata), Helix lucorum] fed with diets enriched with oregano leaves or oregano essential oil. To date, no studies have evaluated the effects of essential oil-enriched diets on snail fillet composition. This study aimed to investigate the effects of nanoemulsion containing various essential oils on feed acceptability, growth performance, chemical composition of snail fillet, and digestive glands’ tissue morphology in Cornu aspersum maximum (C. a. maximum). Digestive gland morphology was used as a rapid and accessible indicator of animal welfare. By evaluating these quality factors, this study seeks to establish acceptable inclusion rates of essential oils in the diets of farmed C. a. maximum.

Materials and methods

Results

All groups of snails promptly accepted the respective diets, and feed consumption was similar (P > 0.05) among all groups (Table 2). While weight values remained similar throughout most of the experiment, a statistically significantly lower value was observed in the T3 group compared to the control group on day 42. At the end of the experiment, the mean final body weight of snails from all treatments ranged from 14.70–15.14 g (Table 2), and there were not found significant differences among them. The same result also applies to weight gain and growth rate.

Shell diameter at the end of the experiment was smaller in snails fed with the control diet compared to the other treatments, but this was not statistically significant (Table 3). Overall, the diameter values did not show any statistically significant difference at any point in the feeding experiment.

Mortality was observed in all experimental groups during the trial. When the mortality rate was calculated as mean values over the entire experimental period, the control group showed the highest overall mortality (6.2%), while the lowest was recorded in T2 (3.69%). Mortality rate during the experiment in T1 and T3 was similar, at approximately 5%.

Table 4 shows the snail production characteristics, such as shell weight, soft body weight, and fillet mass, at the end of the experimental period. All snail groups exhibited similar (P > 0.05) shell weights and did not exhibit significant differences among treatments (Table 4). There was a trend of higher soft body weights in snails fed the nanoemulsion-treated diets compared to the control group, but this was not significant (P > 0.05).

Finally, fillet mass despite being highest in the experimental treatment T3 and lowest in the control group, did not exhibit significant differences among treatments. According to Table 4, the fillet mass of the snails remained unchanged despite the increasing nanoemulsion concentration.

Fillet moisture and ash contents did not exhibit significant differences among the groups (Table 5). Fillets of experimental treatment T1, T2, and T3 exhibited significantly higher protein content (60.12%, 60.20%, and 59.53%, respectively) compared to the control diet (55.20%). Fillet fat of snails fed with the enriched diets T1 and T2 presented significantly (P < 0.05) higher content (2.79% and 2.42%) than that of the control (1.18%) and the T3 group (1.93%). According to Table 5, diet enrichment affected carbohydrate content of snail fillet while gross energy remained stable.

The digestive tubules of snails fed with the control diet are shown in Figure 1A. The digestive cells appeared morphologically normative. Embedded in the epithelium of the digestive tubules, calcium cells that possess spherules were observed. The spherules’ appearance was moderate. The presence of excretory cells could be identified. The haemolymphatic sinus was surrounded by connective tissue mainly consisting of supporting adipose cells. Due to intracellular activity of digestive cells of snails fed the T1 diet (Figure 1B), the hepatic duct appeared slightly dilated, while there were few calcium cells. In Figure 1C (T2 diet), digestive cell apoptosis was apparent, with living cells pushing dying neighboring cells towards the lumen. The rearrangement of the remaining digestive cells and the generation of new ones filled the gaps left by the dead ones, thereby maintaining the integrity of the epithelium of the digestive tubule. Αpoptotic bodies could be seen within the dilated hepatic ducts. There appeared to be very few calcium cells present, which did not contain well-formed spherules. There was a vacuole containing an undigested residue granule. There was necrosis of digestive and calcium cells of snails fed the T3 diet (Figure 1D). Hepatic duct borders could not be differentiated easily from the digestive tubule’s matrix since the digestive cells were fragmented within the lumens. Furthermore, calcium cells appeared to have dissolved cytoplasm, while a significantly thinned epithelium lining was observed.

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Figure 1. Digestive gland micrographs of snails fed with a) control diet, b) T1 diet, c) T2 diet, d) T3 diet. Transverse sections were used for Ηematoxylin and Εosin staining. AB: apoptotic bodies; AC: adipose cell; CC: calcium cell; DC: digestive cell; EC: excretory cell; ECG: excretory cell with granule; HS: haemolymphatic sinus; L: lumen; *: thinned epithelium lining; black arrow: necrotic cells; red arrow: multinucleated digestive cells. Scale bar: 50 μm.

Discussion

The presence of nanoemulsion in the snail diet led to increased weight without reducing feed consumption. In contrast, Linhart and Thompson [35] reported that, among six chemotypes of Thymus vulgaris tested in adult and juvenile C. a. aspersum snails, only those fed exclusively with linalool exhibited an increase in weight. Furthermore, Vokou et al. [36] in a feeding experiment on three species of terrestrial snails (C. a. aspersum, E. vermiculata, Helix lucorum) observed that Origanum vulgare hirtum oil reduced feed consumption in all three species, while C. a. aspersum and E. vermiculata snails actively avoided areas with high essential oil concentrations. The different results in the present study may be attributed to the concentration of essential oil or to the combination of aromatic plants included in the nanoemulsion.

According to the literature and by comparing the results of our feeding experiment with previous studies, we observed a similar snail growth rate (0.22 g/snail/day). In a feeding experiment in which C. a. aspersum snails were fed with an enriched diet containing rosemary powder, Lemjallad et al. [37] reported a maximum growth rate of 0.2 g/snail/day, although the average rate was considerably lower. Furthermore, Apostolou et al. [13] recorded a mean growth rate of 0.20 and 0.18 g/snail/day for C. a. maximum.

Mean weight of snail’s fillet in our dietary experiment ranged from 2.6 to 2.8 g, surpassing the value (2.1 g) reported by Kougiagka et al. [9] for the fillet of farmed C. a. maximum snails. This notable difference can be attributed to the reduced quantity of feed supplied to the snails by farmers [11]. No statistically significant differences in mean snail fillet mass were observed among treatments of our experiment, although enriched diets showed a tendency toward higher values. In contrast, Apostolou et al. [13] recorded snail fillet mass inside a net-covered greenhouse of 4.51 g, due to higher final (17.74 g) weight of snails.

Mortality rate remained low throughout the experiment, especially in the experimental treatments fed with an enriched diet. These findings demonstrated an improvement in snail in snails’ viability, reducing the mortality rate through the incorporation of nanoemulsion. Similar results were obtained by Lemjallad et al. [37] and El Khayari et al. [40], where the mortality rate was lower in groups fed with an enriched diet. The significantly increased mortality observed in the T3 group during the final two weeks of the experiment (approximately twice that of the other groups) may be associated with the higher nanoemulsion dose incorporated into the diet. A similar effect has already been demonstrated by Ali et al. [41] involving snails of E. vermiculata and Monacha obstructa species.

Diet enrichment did not affect moisture and ash content. Regarding fat content, fillet from snails fed the T1 and T3 diets exhibited increased levels, whereas Ranucci et al. [29] reported that dietary enrichment reduced fat content in pork meat.

Snail fillets from T1 and T3 groups exhibited higher protein content, a finding that may be explained by enhanced protein digestibility in enriched diets, as suggested by previous studies. Dias Junior et al. [42] reported increased crude protein digestibility in lambs fed with diets containing increasing levels of essential oils. Similarly, Cheng et al. [43] observed that supplementation with oregano essential oil in chicken diet may enhance muscle protein deposition by improving crude protein absorption. In contrast, no changes in protein content were reported in chicken and beef meat following dietary enrichment with a mixture of essential oils, including oregano and rosemary, incorporated in a nanoemulsion [28, 30].

Mc Donnell et al. [33] investigated the adverse effects of pesticides on snail welfare, noting that snails are frequently regarded as pests. The authors examined 11 essential oils to determine the optimal oil concentration for controlling C. a. aspersum. Essential oils are known to have molluscicidal properties. Similarly, Ferreira et al. [31, 32] reported that thymol negatively affects the growth, reproduction, and survival of Subulina octona and Bradybaena similaris.

Although comparisons with studies conducted in vertebrates such as poultry and pigs may provide useful indications regarding potential mechanisms, these findings should be interpreted with caution due to the physiological differences between vertebrates and gastropods.

As regards the histomorphology findings, enrichment of snail feed with the T1 diet did not result in notable alterations in digestive or calcium cells. At the end of the feeding trial, a limited number of calcium cells were observed, without a significant presence of calcium spherules. Calcium spherules are considered part of a complex intracellular detoxification mechanism [44]. According to Delakorda et al. [45], calcium spherules in digestive gland cells are intracellular membrane-bound structures, and their structure and chemical composition may naturally vary depending on environmental conditions and the snail’s life cycle. The lumen of the digestive tubules appeared slightly enlarged, consistent with observations reported by Ziegler et al. [46] in a study investigating the effects of the antidepressants citalopram and venlafaxine on the aquatic snail Planorbarius corneus.

In snails fed the T2 diet, apoptotic phenomena were observed in epithelial digestive cells, accompanied by a reduction in calcium cells and their content. Abdel-Azeem et al. [47], who exposed Helix aspersa snails to titanium dioxide nanoparticles at two concentrations via spraying, reported luminal dilation and the presence of cellular blebs at the lower concentration, indicative of impending cell death. Rearrangement of remaining cells and the generation of new cells to fill the gaps left by dead cells contribute to the maintenance of epithelial structural integrity [48]. In the present study, a few calcium cells were observed in the T2 group, and these did not contain well-formed spherules.

These findings are consistent with those reported by Hamed et al. [49], who observed in land snail E. vermiculata a marked reduction in both the number of calcium cells and the size of calcium spherules following administration of carbamate-treated baits, although this was accompanied by complete degeneration of the tubular epithelium. In contrast, in our study, the digestive tubules maintained their overall structural integrity.

In snails fed the T3 diet, necrosis of both digestive and calcium cells was observed. Cellular swelling and necrotic changes were evident across all cell types, and excretory cells were scarcely identifiable within the digestive tubules. Hamed et al. [49] reported that, following fourteen days of exposure to methiocarb-treated baits, the cytoplasm of calcium cells appeared dissolved, while the cytoplasm of digestive cells was completely degenerated, resulting in a severely compromised epithelial structure. Similar observations were reported by Farfar et al. [8], who noted a pronounced reduction in epithelial lining height and extensive necrosis in Cornu aspersum snails exposed to cadmium and copper. Additionally, Mohammadein et al. [50] observed that the boundaries between digestive and calcium cells in the digestive glands of E. vermiculata became indistinguishable between polluted and unpolluted sites, reflecting severe histological disruption.