The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study is designed to analyse the response of the skeleton to different rearing densities and provides a description of the skeletal deformities. Wild type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD) 32 fish/L, medium density (MD) 8 fish/L and low density (LD) 2 fish/L. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualise mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that rearing density significantly influenced the specimens' average standard length, which decreased with increasing rearing density. Differences concerning meristic counts among the three groups were not observed. Rearing density-independent malformations affected the ribs, neural arches and the spines of the abdominal region as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimens, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of rearing density on the development of different skeletal phenotypes.
Oryzias latipes is increasingly used as a model in biomedical skeletal research. The standard approach is to generate genetic variants with particular skeletal phenotypes which resemble skeletal diseases in humans. The proper diagnosis of skeletal variation is key for this type of research. However, even laboratory rearing conditions can alter skeletal phenotypes. The subject of this study is the link between skeletal phenotypes and rearing conditions. Thus, wildtype medaka were reared from hatching to an early juvenile stage at low (LD: 5 individuals/L), medium (MD: 15 individuals/L), and high (HD: 45 individuals/L) densities. The objectives of the study are: (I) provide a comprehensive overview of the postcranial skeletal elements in medaka; (II) evaluate the effects of rearing density on specific meristic counts and on the variability in type and incidence of skeletal anomalies; (III) define the best laboratory settings to obtain a skeletal reference for a sound evaluation of future experimental conditions; (IV) contribute to elucidating the structural and cellular changes related to the onset of skeletal anomalies. The results from this study reveal that rearing densities greater than 5 medaka/L reduce the animals’ growth. This reduction is related to decreased mineralization of dermal (fin rays) and perichondral (fin supporting elements) bone. Furthermore, high density increases anomalies affecting the caudal fin endoskeleton and dermal rays, and the preural vertebral centra. A series of static observations on Alizarin red S whole mount-stained preural fusions provide insights into the etiology of centra fusion. The fusion of preural centra involves the ectopic formation of bony bridges over the intact intervertebral ligament. An apparent consequence is the degradation of the intervertebral ligaments and the remodeling and reshaping of the fused vertebral centra into a biconoid-shaped centrum. From this study it can be concluded that it is paramount to take into account the rearing conditions, natural variability, skeletal phenotypic plasticity, and the genetic background along with species-specific peculiarities when screening for skeletal phenotypes of mutant or wildtype medaka.
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