Introduction
Integrating selective breeding with microbiome management is widely proposed as a way to make aquaculture more sustainable by improving disease resistance, stress tolerance, and reducing chemical inputs (Lorgen-Ritchie et al., 2023). Despite this potential, the role of the host-associated microbiome in influencing gamete quality and fertilization success in fish remains insufficiently explored. Recent advances in species-specific probiotic feeding strategies suggest that microbial communities in the gut—as well as in the immediate environment of gametes—can contribute not only to enhanced growth performance, as widely documented (Amoah et al., 2023), but also to improved reproductive outcomes in fish (Enzeline et al., 2024). We aimed to develop a probiotic formulation based on a bacterial strain isolated from the carp gut with previously identified beneficial effects by our research group. This probiotic was subsequently evaluated in a recirculating aquaculture system in feeding trials using multiple concentrations.
Materials and Methods
Three experimental groups were established. The control group (C) received a standard compound feed without probiotic supplementation, while the two treatment groups were fed diets supplemented with bacteria at concentrations of 106 CFU g-1 (T6) and 108 CFU g-1 (T8), respectively. Sperm quality was assessed based on the following parameters: osmolality, pH, motility and kinetic parameters, concentration, and relative sperm count (spermatozoa per kilogram of body weight; spz bwkg-1). Five males were examined per group. During propagation, pooled egg batches from females of the control group were used for fertilization. To evaluate the effect of males, fertilization and hatching rates were determined. Microbiological analyses included the determination of colony-forming units (CFUs) from sperm samples as well as gut samples of both males and females, using non-selective TSA and selective MRSA media. In addition, the composition of the gut microbial community was analyzed. Gut samples were collected from five males and five females from each experimental group, and microbial community structure was characterized using Illumina 16S rRNA gene (V3–V4 region) paired-end sequencing.
Results
No significant differences were found among the three groups (C, T6, T8) in sperm motility, pH, osmolality, or concentration and relative sperm count. Similarly, no significant differences were detected in fertilization rate. However, a significant difference was observed in hatching rates among the groups. Compared to the control group, the hatching rates of both the T6 and T8 groups were significantly higher. In sperm samples, the number of colony-forming units (CFUs) determined on TSA medium was significantly higher in the T8 group compared to the T6 group. No significant differences were observed between treatments on the selective MRS agar medium. The bacterial diversity in the digestive tract was similar between the control group and the 106 CFU g-1 treatment group, whereas lower diversity values were observed in the 108 CFU g-1 group. In most individuals, the gut microbiota was dominated by members of the phyla Fusobacteriota (3.5–92%), Pseudomonadota (1–34%), and Spirochaetota (0–63%). Within the Pseudomonadota phylum, sequences belonging to the genus Aeromonas were predominant in the control and 106 CFU g-1 groups; however, their relative abundance decreased in fish fed the 108 CFU g-1 bacteria-supplemented diet.
Discussion
Probiotic supplementation did not affect key sperm quality parameters or fertilization rate, indicating that basic reproductive functions remained unchanged. However, the significantly higher hatching rates in the treated groups suggest a beneficial effect at later developmental stages, possibly through improved microbial conditions or enhanced embryo viability. The increased bacterial counts in sperm samples and shifts in gut microbiota composition, particularly the reduced abundance of Aeromonas at higher probiotic doses, indicate that microbial community modulation may play a role in these outcomes. Although microbial diversity decreased in the highest dose group, this did not negatively affect performance. The decrease in Aeromonas abundance may be considered beneficial due to its potential role as an opportunistic pathogen. Overall, the results highlight the potential of probiotic application to improve reproductive success in aquaculture, mainly by enhancing hatching efficiency rather than altering sperm quality.
Acknowledgment
This work was funded by the project 2022-1.2.6-T��T-IPARI-TR-2022-00002 within the framework of the National Research, Development and Innovation Fund announced by the Ministry of Culture and Innovation.
References
Amoah, K., Tan, B., Zhang, S., Chi, S., Yang, Q., Liu, H., Yang, Y., Zhang, H., & Dong, X. (2023). Host gut-derived Bacillus probiotics supplementation improves growth performance, serum and liver immunity, gut health, and resistive capacity against Vibrio harveyi infection in hybrid grouper (���Epinephelus fuscoguttatus × ���Epinephelus lanceolatus). Animal Nutrition, 14, 163 - 184. https://doi.org/10.1016/j.aninu.2023.05.005.
Enzeline, V., Widanarni, W., Sudrajat, A., Alimuddin, A., & Nasrullah, H. (2024). Improving reproductive performance and larvae survival by dietary administration of probiotic Bacillus cereus NP5 in female African catfish Clarias gariepinus. Aquaculture International, 32, 7629 - 7646. https://doi.org/10.1007/s10499-024-01532-1.
Lorgen-Ritchie, M., Webster, T., McMurtrie, J., Bass, D., Tyler, C., Rowley, A., & Martin, S. (2023). Microbiomes in the context of developing sustainable intensified aquaculture. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1200997.