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Article History

Received: 2 July 2024

Accepted: 29 January 2025

First Online: 28 February 2025

Declarations

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: All animals and experimental protocols adhered to the guidelines for the care and use of fish in research, as approved by the Ethics Committee of the Iran Veterinary Organization, Department of Aquatic Animal Health and Disease (Approval Code: 96–40-75133). In the practical part of the experiment, to minimize animal distress and ensure their welfare during the experimental challenge, virus inoculations were performed under light anesthesia with the chosen substances and dosages. Fish in different groups, including those injected with VHSV, the control group (without injection), and the group injected with PHSA, were anesthetized and handled in an immersion bath containing 100 mg/L of a 10% eugenol solution[, ]. Fish exhibiting signs of clinical disease (mortality, exophthalmia, hemorrhaging, anemia, and abnormal behavior involving spiral swimming) underwent intensive monitoring, with checks occurring at least three times a day. Regular water quality checks ensured a clean environment for the fish during the infectious challenge. Dead fish and those displaying signs of distress (swimming abnormally) were promptly removed. After a two-week injection challenge, fish from different groups were observed for clinical symptoms. Disease diagnosis was confirmed via RT-PCR. Subsequently, all fish were humanely euthanized using a lethal dosage of 600 mg/L of a 10% eugenol solution in an immersion bath, adhering to animal welfare guidelines [–].

: Not applicable.

: The authors declare no competing interests.