The notochord constitutes the main axial support during the embryonic and larval stages, and the arrangement of collagen fibrils within the notochord sheath is assumed to play a decisive role in determining its functional properties as a fibre-wound hydrostatic skeleton. We have found that during early ontogeny in Atlantic salmon stepwise changes occur in the configuration of the collagen fibre-winding of the notochord sheath. The sheath consists of a basal lamina, a layer of type II collagen, and an elastica externa that delimits the notochord; and these constituents are secreted in a specific order. Initially, the collagen fibrils are circumferentially arranged perpendicular to the longitudinal axis, and this specific spatial fibril configuration is maintained until hatching when the collagen becomes reorganized into distinct layers or lamellae. Within each lamella, fibrils are parallel to each other, forming helices around the longitudinal axis of the notochord, with a tangent angle of 75-80 ° to the cranio-caudal axis.The helical geometry shifts between adjacent lamellae, forming enantiomorphous left-and right-handed coils, respectively, thus enforcing the sheath. The observed changes in the fibre-winding configuration may reflect adaptation of the notochord to functional demands related to stage in ontogeny. When the vertebral bodies initially form as chordacentra, the collagen lamellae of the sheath in the vertebral region are fixed by the deposition of minerals; in the intervertebral region, however, they represent a pre-adaptation providing torsional stability to the intervertebral joint. Hence, these modifications of the sheath transform the notochord per se into a functional vertebral column. The elastica externa, encasing the notochord, has serrated surfaces, connected inward to the type II collagen of the sheath, and outward to type I collagen of the mesenchymal connective tissue surrounding the notochord. In a similar manner, the collagen matrix of the neural and haemal arch cartilages is tightly anchored to the outward surface of the elastic membrane. Hence, the elastic membrane may serve as an interface between the notochord and the adjacent structures, with an essential function related to transmission of tensile forces from the musculature. The interconnection between the notochord and the myosepta is discussed in relation to function and to evolution of the arches and the vertebra. Contrary to current understanding, this study also shows that notochord vacuolization does not result in an increased elongation of the embryo, which agrees with the circular arrangement of type II collagen that probably only enables a restricted increase in girth upon vacuolization, not aiding elongation. As the vacuolization occurs during the egg stage, this type of collagen disposition, in combination with an elastica externa, also probably facilitates flexibility and curling of the embryo.

The aim of the present study was to elucidate if increasing levels of added vitamin K in the feed influenced fish growth, health or the incidences of bone deformities in Atlantic salmon (Salmo salar L.) from onset of start feeding to 100 g in freshwater. Duplicate groups of Atlantic salmon fry (0.20 g) were fed eight levels (0, 2.5, 5, 7.5, 10, 15, 20 and 50 mg menadione kg )1 ) of the vitamin K derivate menadione nicotinamide bisulphite (MNB) in a regression design for 28 weeks. All fish maintained high growth rates throughout the experiment, and showed no significant differences in specific growth rate, condition factor, whole body proximate analysis, blood coagulation time, vertebra morphology or mechanical properties of vertebrae. We found a dose-response between whole body vitamin K concentration and the dietary MNB supplementation level. Analysis of liver c-glutamylcarboxylase activity revealed significant dose-dependent differences between groups given the 0, 10 and 50 mg MNB kg )1 diets. In conclusion, Atlantic salmon seems to require low levels of dietary vitamin K, and the amount of vitamin K found naturally in the presently used feed ingredients may be enough to maintain optimal growth, health and bone strength in Atlantic salmon fry from start feeding. KEY WORDS

BackgroundIn teleosts such as Atlantic salmon (Salmo salar L.), segmentation and subsequent mineralisation of the notochord during embryonic stages are essential for normal vertebrae formation. However, the molecular mechanisms leading to segmentation and mineralisation of the notochord are poorly understood. The aim of this study was to identify genes/pathways acting in gradients over time and along the anterior-posterior axis during notochord segmentation and immediately prior to mineralisation of the vertebral bodies in Atlantic salmon.ResultsNotochord samples were collected from unsegmented, pre-segmented and segmented developmental stages. In each stage, the cellular core of the notochord was cut into three pieces along the longitudinal axis (anterior, mid, posterior). RNA was sequenced (22 million pair-end 100 bp/ library) and mapped to the salmon genome. 66569 transcripts were predicted and 55775 were annotated. In order to identify possible gradients leading to segmentation of the notochord, all 71 notochord-expressed hox genes were investigated, most of them displaying a typical anterior-posterior expression pattern along the notochord axis. The clustering of hox genes revealed a pattern that could be related to notochord segmentation. We further investigated how mineralisation is initiated in the notochord, and several factors related to chondrogenic lineage were identified (sox9, sox5, sox6, tgfb3, ihhb and col2a1), suggesting a cartilage-like character of the notochord. KEGG analysis of differentially expressed genes between stages revealed down-regulation of pathways associated with ECM, cell division, metabolism and development at onset of notochord segmentation. This implies that inhibitory signals produce segmentation of the notochord. One such potential inhibitory signal was identified, col11a2, which was detected in segments of non-mineralising notochord.ConclusionsAn incomplete salmon genome was successfully used to analyse RNA-seq data from the cellular core of the Atlantic salmon notochord. In transcriptome we found; hox gene patterns possibly linked to segmentation; down-regulation of pathways in the notochord at onset of segmentation; segmented expression of col11a2 in non-mineralised segments of the notochord; and a chondroblast-like footprint in the notochord.

Vitamin K belongs to the lipid soluble vitamins, and occurs naturally as phylloquinone (vitamin K1) and menaquinone (vitamin K2). In addition, there is a synthetic provitamin, menadione (vitamin K3), primarily used as a vitamin K source in animal feed. Menadione is unstable during feed processing and storage and the dietary content may reach critically low levels. Recent publications also question the availability of menadione in feed for salmonids. Vitamin K plays vital roles in blood coagulation and bone mineralization in fish, but the suggested minimum requirement varies considerably depending on the vitamin K source used. Vitamin K deficiency is characterized by mortality, anaemia, increased blood clotting time and histopathological changes in liver and gills. However, one should assess both inherent and supplemented forms of vitamin K in feeds for exact determinations, as relevant novel feed ingredients of plant origin may be sufficient to meet the requirement for vitamin K. The current review gives an overview of the biochemical role of vitamin K, and discusses vitamin K requirement in fish in light of updated literature, with special emphasis on salmonids.

The tissue-specific gene expression of the vitamin K-dependent proteins bone c-carboxyglutamate-protein (BGP) and matrix c-carboxyglutamate-protein (MGP) in Atlantic salmon (Salmo salar L.) was investigated. In previous studies, BGP, the most abundant non-collagenous protein of bone, was almost exclusively associated with bone, whereas the non-structural protein MGP has a more widespread tissue distribution. In-situ hybridization of juvenile Atlantic salmon (40 g, fresh water) vertebrae demonstrated expression of bgp and mgp mRNA in osteoblasts lining the trabecular bone, whereas no staining was observed in the compact bone. By separating the trabecular and compact bone of both juvenile (40 g, fresh water) and adult (1000 g, sea water) Atlantic salmon, we observed that the two vertebral bone compartments displayed different levels of bgp, whereas no such differences were seen for mgp. Measurements of the mineral content and Ca ⁄ P molar ratio in adult salmon revealed no significant differences between trabecular and compact bone. In conclusion, the osteoblasts covering the salmon vertebrae have unique gene expression patterns and levels of bgp and mgp. Further, the study confirms the presence of mRNA from the vitamin K-dependent proteins BGP and MGP in the vertebrae, fin and gills of Atlantic salmon.