Marking Juvenile Zebrafish With Fluorescent Substances And Its Effects On Antioxidases

Fish marking techniques have developed in response to the requirements of aquaculture breeding and fish stock conservation. Many of these methods are problematic in terms of marking juvenile fish, and/or are costly or very time-consuming. Each of these methods has advantages and disadvantages that depend on the reason for marking and the required ease of both marking and mark identification. There are some important factors that affect the usefulness of the technique, including fish stress and mortality, labor intensity and cost, and mark retention and detection. Immersing fish in fluorescent dyes reduces handling, stress and mortality rates, and allows large groups of fish to be marked simultaneously. Now domestic studies on immersing fish with fluorescent dyes are focusing on the choice of fluorescent pigments and immersion concentrations, and immersion time is always set at24h. However, there is little known about the effect of marking otolith with different immersion time and the impact degree caused by the stress when immersing fish in fluorescent dyes. In present study, aquatic model organism, juvenile zebrafish (Danio rerio) were immersed in tetracycline hydrochloride (TCH) and calcein with four different concentration and two treatment time, to find out the suitable marking substance, concentration and immersion time for olotith marking of juvenile zebrafish; then the influence of TCH concentration and immersion time on antioxidant enzymes SOD, CAT, and GSH-Px were studied to test its security. These results could provide theoretical basis and reference for marking techniques in living aquatic resources enhancement. The main results were as follows:1. TCH is a suitable substance for otolith marking of juvenile zebrafish, the ratio of TCH marking is100%, and the marking effect is significant. In the three pairs of otoliths, the marks in lapillus and sagitta are very obvious. The I3(BP450-490) and N2.1(BP515-560) excitation lights give the best results for detection of TCH in otoliths.2. Calcein is not suitable for otolith marking of juvenile zebrafish, the ratio of marking is low, yet the marking effect is worse. The marks in lapillus and sagitta are not obvious but always better than asteriscus. The I3(BP450-490) and N2.1(BP515-560) excitation lights give better results for detection of Calcein in otoliths.3. Juvenile stage is suitable for olotith marking by fluorescent dyes. Some embryonic developmental stages may be also suitable, but more studies should be made to ensure the security of embryonic or larvae development.4. In a certain rang, the fluorescent reaction intensity increased with the increasing of the solution concentrations and immersion time; In a certain period after immersion, the fluorescent reaction intensity increased with the juveniles growth.5. By studing the change of SOD、CAT、GSH-Px activity of juvenile zebrafish immersed in TCH solutions, we find that TCH had a induction to the main antioxidant enzymes, and at same solution concentration the activity of antioxidases increased as the immersion treatment time changed from18h to24h;250mg/L for24h may have an impact degree to the juveniles, but the toxic effect can recover after a short time; when the concentration was at100mg/L or150mg/L, the antioxidases could basically protect juvenile from the toxic effects.6. Considering factors as above mentioned,250mg/L for18h was found to be the most appropriate for juvenile zebrafish with100%marking rate and100%survival rate;150mg/L for24h was also more appropriate for the juvenile; in addition,250mg/L for24h had the best mark effect and could also be taken into consideration as the damage which made can recover after a short time.