A simplified, one step technique for disinfection of non-hardened rainbow trout eggs with tosylchloramide (Chloramine T) and peroxide (Wofasteril) compounds and the effects on bacterial load and microbiome composition in comparison to iodophore disinfection.

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.