Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density

Abstract

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.

More Bone with Less Minerals? The Effects of Dietary Phosphorus on the Post-Cranial Skeleton in Zebrafish

“…Bird and Mabee [ 40 ] serve as reference for normal skeletal development in zebrafish. In addition several publications define malformations of the zebrafish axial skeleton [ 41 , 42 , 43 ]. The absence of any of the described malformation is regarded as normal in this study.…”

“…This could explain why spines are twisted but vertebral centra are not affected in LP zebrafish. In laboratory zebrafish strains, undulated spines also occur linked to conditions other than P deficiency such as increased rearing density [ 43 ] or disturbed somite formation [ 66 ]. In addition, it has been suggested that spine deformities relate to musculature impairment [ 67 , 68 ].…”

“…These findings strengthen the idea that P deficiency alone is not a primary cause of vertebral column abnormalities in zebrafish, and that other or additional factors trigger the development of malformations. Other factors that are currently discussed to cause vertebral column malformation in zebrafish and other teleost species are rearing temperature, excess swimming, increased rearing density or dietary vitamin A supply [ 42 , 43 , 82 , 83 ].…”

Effects of Holding Density on the Welfare of Zebrafish: A Systematic Review

“…For example, higher holding densities can cause increased mortality during the early juvenile phase, 18 decreased growth during the first 3 weeks to 3 months, 51,53 and increased occurrence of skeletal deformities during the first 3 months. 54 To optimize the growth, fish larvae should be held at densities between 12 and 22 fish/L according to Delomas and Dabrowski, 53 and at 9…”

Monsters with a shortened vertebral column: A population phenomenon in radiating fish Labeobarbus (Cyprinidae)

“…In zebrafish, Danio rerio (Hamilton 1822), vertebral column shortening and vertebral fusions are exhibited by type I collagen mutants, as well as by individuals with knockout alleles in two genes involved in type I collagen processing [107]. At the same time, the increased frequency of vertebral deformities in wild-type zebrafish is induced by high rearing densities [108]. The stumpbody phenotypes with the shortened vertebral column in channel catfish Ictalurus punctatus (Rafinesque 1818) [109] and blue tilapia Oreochromis aureus (Steindachner 1864) [12,110] are described as not heritable.…”