| Abstract |
Skeletal deformities are a significant issue for aquaculture, with negative impacts on
fish biological performance (swimming, respiration etc.), phenotypic quality and
welfare. Their appearance is attributed to both environmental (temperature, pH etc) and
genetic factors. Identifying accurate diagnostic markers linked to deviations of
skeletogenesis and development of skeletal deformities, could offer powerful tools to
the research on the causative factors of abnormalities as well to the commercial
hatcheries. In the present study we used zebrafish as a model species to test whether
particular skeletal defects can be associated with the expression of genes involved in
bone formation and resorption. Primarily we focused on kyphosis, since it can be easily
distinguished macroscopically, it is frequent among rearing species and known to
induce high mortality rates. Zebrafish was chosen because of its similar ontogenetic
pattern to that of the most fish species, its well-characterized skeletal development, easy
maintenance, short life cycle and well-studied genome. Also, it is known how to at-will
cause skeletal deformities, allowing the examination of their development.
Two groups of fish were reared in triplicate, under two different feeding regimes;
one standard (control, with combined use of Artemia nauplii and dry feed), and another
one (D, with dry feed only) which has been associated with elevated rates of skeletal
defects. Skeletal analysis verified the initial hypothesis on the effect of the tested
regimes on the development of skeletal abnormalities, with D group presenting
significantly elevated abnormality rates (p<0.05, G-test). The significance effect of
feeding regimes was observed in the case of the abnormal branchiostegal rays (10±9%
in the control group vs 42 ±12% in the D group, mean ±SD), bended neural processes
(10±8% at the Control group vs 47±8% at the D group), abnormal gill cover (4±3 % in
the control vs 25±4 % in the D group) and caudal-peduncle scoliosis (6±4 % in the
control vs 23±3 % in the D group). In addition to the bibliographically expected
abnormalities, D group presented high rates of abnormal vertebral ossification pattern,
in the form of resorption lacunae and irregular bone formation (4±2 % in the control vs
34±32 % in the D group). Calcein staining of the specimens that were collected during
an additional fourth experimental replicate showed that vertebral abnormalities can be
easily detected, on alive larvae, at the beginning of their formation; well before they are
expressed on the external phenotype. In the case of resorption lacunae, they were first
detected on specimens of ca 6.0 mm SL.
QPCR analysis did not reveal significant difference in transcript levels between
the two experimental groups for the most of the studied genes (bglap, col1a1a, acp5a,
runx2b, mmp13a). In the case of Sp7 gene a significantly different expression level
(p<0.05, Kruskal-Wallis) was found between the control (0.45±0.09SE) and D group
(0.84±0.11SE).
Our results on gene expression differentiation between the two experimental
groups are discussed in association to the potential causative factors and critical
ontogenetic windows of the observed skeletal abnormalities.
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