Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.

We studied the effects of water temperature, density as measured by density index (DI), and food deprivation (alternating cycles of daily feeding and fasting) on dorsal fin erosion of steelhead Oncorhynchus mykiss through a factorial experiment lasting 22 weeks. Dorsal fin index (DFI) correlated inversely with age, length, and weight of fish and was significantly affected by crowding (i.e., DI). Increasing DI (from 0.0 to 0.5) also improved feed conversions and slightly depressed survival, but DI had no significant effects on weight gain, condition (K ), or carcass composition. Water temperature (10ЊC or 15ЊC) had a strong and significant effect on DFI; colder water improved DFI while slowing body growth. Low temperature treatments improved survival slightly and altered carcass composition by increasing moisture at the expense of fat and protein.The effects of density and temperature were also strongly interactive. Food deprivation slightly improved final DFI at low DIs, but greatly reduced the average size of fish affected by severe fin erosion. Food-deprived fish exhibited carcass composition trends similar to fish reared in 10ЊC water. Feed conversion improved significantly with alternating fasting and feeding cycles. Histological evaluation proved inconclusive for physical fin nipping evidence or for the presence of detrimental microorganisms.

An equation was developed for comparing the degree of fin erosion among populations of the same fish species, regardless of fish size, or against a reference population with uneroded fins: fin factor (%) = (fin length x 100)/(total length). This replaces more subjective approaches used in the past for estimating the degree of fin missing.

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