Abstract
Disinfection of the interior of non-hardened eggs with iodophors (Buffodine®) is an established hygienic practice in salmonid aquaculture to prevent pathogen transmission from the broodstock fish to their offspring. As iodophors inhibit sperm motility, fertilization is first performed in a 0.75 % NaCl solution, followed by egg disinfection in a second step after fertilization is complete. Although this two-step egg disinfection procedure is simple to perform under laboratory conditions, it presents challenges for fish farms using mass stripping. The process involves two highly time-sensitive steps, requiring precise execution, as any errors can lead to fertilization failure or ineffective disinfection. A more practical approach would be to simplify disinfection into a single-step procedure. The present study demonstrates that non-hardened rainbow trout eggs can be fertilized and disinfected simultaneously using a one-step method with tosylchloramide (100 mg/l Chloramine T®) or peroxide (100 µl Wofasteril®) compounds in 0.75 % NaCl for 40 min. This procedure is feasible as the applied concentrations of Chloramine T and Wofasteril have only low impact on sperm motility. The one-step methods do also not negatively impact embryo and early larval development. Non-hardened rainbow trout egg disinfection methods with Chloramine T and Wofasteril were as effective as the conventional Buffodine method in reducing total bacterial load of eggs at 3 h post-fertilization (hpf), the point at which water hardening is complete. Reanalysis of total bacterial load after 22 days of development (embryo stage at the onset of eye pigmentation) proved Chloramine T more effective than Wofasteril and Buffodine. Microbiome composition differed significantly across developmental stages and disinfection treatments. Notable variations were observed between non-disinfected controls and eggs treated with Buffodine, Chloramine T, or Wofasteril, in persistent bacterial communities, stage-specific bacteria, and bacteria colonizing the chorion during embryogenesis. Buffodine treatment increased bacterial diversity, while Chloramine T and Wofasteril led to reduced diversity compared to the control. These findings are discussed in the context of microbiome stability and resilience, key factors for long-term fish health. In summary, the simplified one-step disinfection protocol offers practical advantages in reducing time-sensitive handling steps and improving efficiency in large-scale aquaculture operations. Moreover, it is environmentally preferable as both Wofasteril and Chloramine T are used at lower concentrations than Buffodine and degrade rapidly in water, leaving no harmful residues.