The effects of different rearing protocols and peat substrate types on cocoon and hatchling production parameters in medicinal leech cultivation are presented in Table 2. In this study, four different protocols were evaluated within the hybrid model, while the classical pool model was used as the control group.
In the hybrid model groups using new peat, Protocol A yielded a mean number of 2.28±0.63 cocoons per broodstock and 23.28±7.24 hatchlings per broodstock (Table 2). In contrast, the corresponding values in the pool model were 1.02±0.44 cocoons per broodstock and 11.56 ±6.49 hatchlings per broodstock, respectively.
In groups using used peat, both the hybrid model and the pool model exhibited markedly lower numbers of cocoons and hatchlings per broodstock (Table 2). In some groups with used peat, no cocoon formation or hatchling production was observed.
Cocoon weight values in the hybrid model groups using new peat ranged between 0.63±0.25 g and 0.82± 0.29 g. Lower cocoon weight values were recorded in the pool model and in groups using used peat (Table 2).
As illustrated in, the mean numbers of cocoons and hatchlings per broodstock are comparatively presented according to peat substrate type and applied production protocols.
Studies on the laboratory-based production of medicinal leeches have largely relied on classical rearing approaches centered on individual care under fixed temperature and humidity parameters. However, in these systems, the reproductive process is generally conducted under limited environmental control and it has been reported that environmental fluctuations and inter-individual interactions (particularly in multi-individual pool systems) play a decisive role in reproductive performance. Indeed, reproductive success in
Hirudo species has been shown to be closely associated with environmental factors such as temperature, humidity and overall environmental stability and even minor variations in these parameters can significantly affect cocooning periods and hatchling yield (
Petrauskienë et al., 2011).
Nevertheless, classical production approaches are reported to involve substantial limitations in terms of both productivity and sustainability, mainly due to high stocking density, restricted individual monitoring and stress-related adverse effects (
Ceylan et al., 2015).
The hybrid model developed within the scope of this study was designed by restructuring the classical pool system in order to overcome the aforementioned limitations and it offers a semi-modular production approach that allows broodstock leeches to be monitored in separate compartments. Through this system, cocooning frequency and hatchling output could be recorded in a more regular and systematic manner, while key parameters directly influencing the reproductive process (such as ambient temperature, humidity and substrate moisture) could be managed in a more stable and controlled way (
Petrauskienë et al., 2011;
Ceylan et al., 2015).
Moreover, compared with conventional pool systems, the hybrid model reduced inter-individual interactions and provided a lower-stress rearing environment, thereby contributing to the maintenance of individual reproductive behaviors under conditions closer to their natural course (
Ceylan et al., 2015).
The quantitative findings clearly support these structural advantages. Under Protocol A of the hybrid model, a total of 16 cocoons and 163 hatchlings were obtained from only 7 broodstock leeches, corresponding to a mean of 2.28 cocoons and 23.28 hatchlings per broodstock. In contrast, under Protocol A of the classical pool model, 163 cocoons and 1423 hatchlings were obtained from 126 broodstock individuals, resulting in mean values of 1.02 cocoons and 11.56 hatchlings per broodstock, respectively.
The statistically significant differences detected between the two models with respect to both cocoon and hatchling numbers (p<0.01) indicate that the hybrid model provides approximately two-fold higher reproductive efficiency compared with the conventional pool system (Table 2). Furthermore, the absence of broodstock mortality throughout the experimental period in the hybrid model represents an important finding supporting the system’s capacity to maintain low stress levels and environmental stability.
The biological basis of the enhanced reproductive performance observed in the hybrid model can be explained by the reduction of stress and physical or microbial disturbances during the critical stages of the reproductive process. In hirudiniform leeches, a foamy secretion released around the clitellum during cocoon deposition gradually hardens to form a “spongy outer layer.” It has been reported that inter-individual contact during the formation of this layer may interfere with proper cocoon enclosure, which can be associated with cocoon deformation and defects in embryonic development. Indeed, increased stocking density has been shown to elevate cocoon deformation rates and in some deformed cocoons, dehydration may accompany the process, preventing successful hatchling development (
Ceylan et al., 2023).
In this context, the provision of individual isolation and the limitation of inter-individual interactions in the hybrid model constitute a fundamental biological mechanism that may enhance hatchling yield by reducing both mechanical disturbances and stress responses.
In addition, reproductive success has been shown to be highly sensitive to environmental stability. Under conditions involving controlled temperature (approximately 25-26/27°C), strict regulation of humidity, adequate ventilation and the use of an appropriate peat-soil substrate, increased hatchling yield and reduced rates of defective cocoons and mortality have been reported. Furthermore, a slightly acidic environment (pH 5.0-6.5) and high humidity levels (70-85%) have been demonstrated to be favorable for embryonic development and successful hatchling emergence
(Aminov et al., 2025).
Accordingly, the combination of low inter-individual contact, a stable microclimate and appropriate substrate conditions implemented in the hybrid model contributed to improved cocoon integrity and enhanced embryonic survival, ultimately leading to more successful reproductive outcomes.
These findings do not reflect a phenomenon specific solely to medicinal leech production; rather, they are consistent with the broader aquaculture literature highlighting the effects of environmental stability and stress management on biological productivity in controlled production systems. Studies conducted on various aquatic organisms have reported that production systems ensuring microenvironmental stability are associated with reduced stress levels, maintenance of metabolic balance and enhanced reproductive or growth performance
(Nottanalan et al., 2025; Tanuja et al., 2025). Similarly, modular and controllable production systems have been emphasized for their role in preserving organism health and supporting long-term production sustainability
(Kumara et al., 2023).
In this context, the high hatchling yield achieved in the hybrid model can be attributed to the implementation of isolated incubation management, maintenance of controlled temperature and humidity conditions, regulation of water exchange frequency and optimization of cocoon monitoring intervals (Table 1, Fig 1). Accordingly, the hybrid model represents a biologically rational and generalizable controlled production approach that is not limited to medicinal leech cultivation.
Finally, the absence of a statistically significant difference between the two production models in terms of cocoon morphometric parameters (p>0.05) indicates that these morphological characteristics are more closely associated with species-specific biological factors rather than with the production system itself
(Xiong et al., 2020). In contrast, the marked reduction in production efficiency observed with the use of used peat substrate highlights the critical role of the substrate’s microbiological load and chemical properties in reproductive success. In particular, increased microbial load and waste accumulation in pool systems using used peat have been associated with cocoon deformities and fungal infections (Table 2).
These findings demonstrate that the success of the hybrid model depends not only on structural modifications but also on the quality, hygiene and standardization of the production materials employed
(Donahue et al., 2022). In this context, achieving a sustainable and high-yield reproductive cycle requires moving beyond a purely qualitative classification of substrates and taking into account parameters such as microbiological load, pH balance and oxygen capacity during production processes.
Several limitations of the present study should also be acknowledged. The hybrid model was implemented using a limited number of broodstock individuals and was evaluated within a single population, with the findings therefore constrained to specific environmental conditions and a relatively short production period. Long-term monitoring, assessment across multiple reproductive cycles and testing of the model under different populations and environmental settings would enhance the generalizability of the results.
Nevertheless, the controlled production approach offered by the hybrid model provides important implications for the conservation of medicinal leeches and for sustainable commercial production. The high reproductive efficiency observed may reduce the need for harvesting individuals from natural populations, while individual monitoring and low mortality rates support a more ethical and traceable production process. In this respect, the hybrid model can be considered a promising alternative with the potential to balance conservation biology objectives and commercial production goals in medicinal leech cultivation.
