A RAPID MICRORADIOGRAPHIC METHOD TO DETECT SKELETAL ANOMALIES IN EARLY JUVENILE FISH

Introduction Skeletal anomalies affect 昀椀sh growth, welfare and health, increase mortality and decrease product marketability (Boglione et al., 2013). Skeletal anomalies of early juveniles can be identi昀椀ed through an in toto double staining procedure, whose execution takes several days thus delaying diagnoses. Recently, a rapid microradiographic method to identify skeletal anomalies in early sea bream Sparus aurata juveniles (total length ≥ 25 mm) was proposed (Pousis et al., 2022). The aim of the present study was to assess the use of microradiography to identify skeletal anomalies in gilthead sea bream of younger age than previously reported and to extend the use of this method to the European sea bass Dicentrarchus labrax, the common carp Cyprinus carpio and the silver carp Hypophthalmichthys molitrix. Material and Methods Gilthead sea bream (N = 92; total length, TL, 18-31 mm), European sea bass (N = 145; TL 16-21 mm), common carp (N= 51; TL 18-31mm) and silver carp (N= 87; TL 17-23 mm) were sampled in 昀椀sh farms operating in Italy and in Albania, anaesthetized, 昀椀xed in 10% buffered formalin and then stored in 50% ethanol. The 昀椀sh were then micro-radiographed in latero-lateral projection. Since the high-resolution 昀椀lm is sensitive to water, a transparent polyethylene 昀椀lm was placed between the photographic 昀椀lm and the specimens. X-ray exposure was set up at 12 kV and 18 mA. The specimens <20 mm TL were subjected to 6500 X-ray shots, while the specimens ≥20 mm TL underwent 7500 shots. Films were then developed with Kodak HC-110, 昀椀xed in Kodak UNIFIX, and dried at room temperature. After microradiography, all the samples were stained with a routine in toto double staining procedure for bone and cartilage (Boglione et al., 2013). Results and Discussion In gilthead sea bream, the majority of the analyzed individuals (82.6%) showed at least one skeletal anomaly and most of them showed more than one anomaly (average: 3.3 ± 2.4). A total of 248 anomalies were recorded of which 21.4% were several anomalies such as haemal lordosis, vertebral body shape anomalies, vertebral body fusion and cephalic deformities. In European sea bass specimens, a total of 533 anomalies were recorded of which 10.3% were several anomalies such as cephalic deformities, kyphosis, lordosis and vertebral body fusion. The frequency of individuals with at least one (severe or slight) anomaly was found to be 93.1% while the average of anomalies per individual were 3.9 ± 3.1. In silver and common carp, a total of 1119 and 131 anomalies were respectively recorded. In silver carp 93.1% of the analyzed specimens showed almost one anomaly (average: 13.8 ± 15.4) while in common carp the frequency of deformed specimens was 78.4% (average: 3.5 ± 3.0). The most common severe anomalies in both species were vertebral body shape anomalies and vertebral body fusion, while no cephalic deformities were found. The frequency of specimens with almost one severe anomaly was 57.5% and 41.2%, respectively. In all the analyzed specimens the most common slight anomalies were deformed neural and haemal arches. All the skeletal anomalies observed after double staining method were also identi昀椀ed in the microradiographic plates (Fig. 1). The present study suggests microradiography as a useful tool for the rapid identi昀椀cation of skeletal anomalies in early juveniles of different species, provided that X-ray exposure conditions appropriate to the size of the examined samples are set up. Financial grants provided by the EU Programme H2020 (GA 862658, NewTechAqua), and by the Apulian Region to C.P. (POR PUGLIA FESR-FSE 2014/2020 – Asse X-Azione 10.4—Research for Innovation – REFIN).