Spontaneous skeletal deformities in the Zebra Danio (Brachydanio rerio) bred for fish toxicity tests

Zebra Danio ( Brachydanio rerio , Hamilton-Buchanan) tropical fish are easily maintained and bred under laboratory conditions; procedures for maintenance and breeding are described. Intensive inbreeding of this fish gave rise to a high incidence of spontaneous skeletal deformities which appear to reflect a genetic defect rather than an environmental stimulus. This finding casts doubt on the suitability of the Zebra Danio for fish toxicity testing.     

Breeding the convict cichlid (Cichlasoma nigrofasciatum) for use in laboratory fish toxicity tests

A procedure is described for breeding Convict Cichlid ( Cichlasoma nigrofasciatum ) tropical fish under laboratory conditions. Three generations of Convict Cichlids were bred successfully using this procedure and there was no significant incidence of deformities from in–breeding to the F₃ , which contrasts with a high incidence of skeletal deformities observed when Zebra Danio tropical fish were inbred to the F ₃ generation. The Convict Cichlid is a suitable species for laboratory toxicity testing, particularly studies to assess effects on breeding performance.     

Effects of phosphorus and vitamin C deficiency,vitamin A toxicity,and lipid peroxidation on skeletal abnormalities in Atlantic halibut (Hippoglossus hippoglossus)

Dietary nutrients play an important role in skeletal tissue metabolism of fish. Deficiency and toxicity of certain nutrients have been linked to bone deformities in larval and juvenile fish. The pathogenesis of skeletal disorders in larval and juvenile fish from the same genetic stock, cultured under similar environment conditions is often difficult to distinguish when marginal deficiencies of multiple nutrients are involved. A study was conducted to characterize the skeletal deformities linked to the deficiency of phosphorus and ascorbic acid, vitamin A toxicity and lipid peroxidation in juvenile halibut. Five experimental diets containing a low level of phosphorus (0.5% dry matter basis), no vitamin C supplement, high level of vitamin A (80 000 IU kg^?1) and oxidized marine fish oil (peroxide value, 7.53 meq kg^?1) and a control diet based on cod fillet and vitamin free casein were fed to juvenile Atlantic halibut for 14 weeks in an attempt to characterize the skeletal deformities. Phosphorus, ascorbic acid, retinol, and α‐tocopherol concentrations of liver and kidney were measured at 0 and 14 weeks. Reduced vertebral ash and phosphorus content were observed in fish fed the low phosphorus diet. Skeletal abnormalities included abnormal hemal and neural spines in the hemal region and scoliosis in the cephalic and hemal regions of the vertebral column. Hepatic and kidney ascorbic acid concentrations were significantly lower in the group fed no ascorbic acid supplement. Skeletal abnormalities were scoliosis and lordosis primarily in the hemal region of the vertebral column. High levels of vitamin A in the diet caused increased hepatic retinol content and scoliosis spanning the cephalic/prehemal and anterior hemal regions of the vertebral column. Fish fed the oxidized oil diet showed increased thiobarbituric acid (TBA) value in the liver and muscle tissue with no significant decrease in hepatic vitamin E concentration. The most frequent skeletal deformity observed was scoliosis, spanning the cephalic/prehemal regions as well as the anterior hemal region of the vertebral column. The pattern and type of abnormalities observed in fish fed these experimental diets were similar to those observed in a commercial halibut hatchery.     

Incidence of spinal deformities in natural populations of Aphanius fasciatus Nardo, 1827 from the Gulf of Gabes, Tunisia

Spinal deformities in Aphanius fasciatus were observed in fish collected from polluted (S1) and non-polluted (S2) areas in the Gulf of Gabes in Tunisia. Kyphosis, scoliosis and lordosis were detected and these basic deformities frequently co-occur in varying degrees of severity. A total of 612 specimens of A. fasciatus were caught and 54 were found to be deformed, which represents 8.82%. Deformed fish were eight times more frequent in S1 than in S2, possibly indicating a relationship between pollution levels and skeletal malformation. In both sexes, variation of deformities incidence with length was very similar. The highest occurrence of deformities was observed in fish with total length <25 mm and decreasing thereafter with fish length. The possible causes for such deformities as well as the suitability of this kind of study for environmental monitoring are discussed.     

Vitamin K deficiency inhibits mineralization and enhances deformity in vertebrae of haddock (Melanogrammus aeglefinus L.)

Vitamin K has been known to regulate bone formation through osteocalcin synthesis by osteoblasts, which is important for mineralization and bone structure. The mechanism underlying the relationship of vitamin K with the changes of microanatomy is not fully understood, and our goal is to test whether bone deformities develop in association with vitamin K deficiency. Fish were fed a semi-purified diet containing either devoid (0.00 mg/kg diet) or adequate (40.0 mg/kg diet supplemented but 20.8 mg/kg analyzed) levels of vitamin K (menadione sodium bisulphite) for 20 weeks. At the end of 8 and 20 weeks, fish were subjected to gross examination and X-ray, and mineral content of the vertebrae was measured. The vertebrae were also subjected to histological, histomorphometric and enzyme histochemical examinations to determine the bone formation and resorption. Vitamin K deficiency primarily decreased bone mineralization and subsequently a decrease in bone mass thus resulted in an increased susceptibility to bone deformity. The occurrence of bone deformities coincided with an increased amount of osteoid tissue and decreased bone mineral content. Number of osteoblasts and osteoclasts were not affected by dietary vitamin K. In conclusion, vitamin K deficiency can impair bone mineralization and enhances bone deformities.     

Studies on the appearance of skeletal anomalies in red porgy: effect of culture intensiveness,feeding habits and nutritional quality of live preys

Despite the great interest of red porgy as a new species for Mediterranean aquaculture, its commercial production is constrained by the high incidence of skeletal deformities occurring in this species under culture conditions. Several studies have been conducted to better understand the origin of these anomalies in this species, using different system intensiveness, rotifers enrichment products or rotifers docosahexaenoic acid content. The first study showed that culture intensification increased the number of fish with an extra vertebrae, what was probably related to the different nutritional quality of live preys employed in each treatment, since water temperature, salinity and genetic background were identical for the different batches of fish studied. Total incidence of skeletal abnormalities was higher in the intensive system, particularly cranial abnormalities and kyphosis in the cephalic vertebrae. In both rearing systems the most common skeletal anomalies were vertebral column disorders, lordosis and fused vertebrae, their localization along the column being affected by the culture intensiveness. Rotifer enrichment, predominantly its docosahexaenoic acid content significantly affected deformities occurrence. A marked positive effect of rotifer docosahexaenoic acid content was found on larval survival. X‐ray studies denoted elevated levels of bone abnormalities associated, in both trials, to low docosahexaenoic acid content in live preys. Among different anomalies, the presence of fused vertebrae was the most frequent deformity for both rearing trials. A 50% reduction in the number of deformed fish for each type of deformity was obtained when the larvae were fed higher docosahexaenoic acid levels, denoting the important role of this fatty acid in bone development. Further studies are needed to elucidate the importance of essential fatty acids on the development of bone deformities in fish, since the functions of these fatty acids differ among them and can lead to very different effects in fish metabolism, including bone formation.     

Zinc deficiency and the activities of lipoprotein lipase in plasma and tissues of rats force-fed diets with coconut oil or fish oil

The present study was performed to investigate the effect of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and tissues of rats fed diets containing either coconut oil or fish oil as dietary fat, using a bifactorial experimental design. To ensure an adequate food intake, all the rats were force-fed by gastric tube. Experimental diets contained either 0.8 mg zinc/kg (zinc-deficient diets) or 40 mg zinc/kg (zinc-adequate diets). The effects of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and postprandial triglyceride concentrations and distribution of apolipoproteins in serum lipoproteins depended on the type of dietary fat. Zinc-deficient rats fed the coconut oil diet exhibited a reduced activity of lipoprotein lipase in postheparin serum and adipose tissue, markedly increased concentrations of triglycerides in serum, and a markedly reduced content of apolipoprotein C in triglyceride-rich lipoproteins and high density lipoproteins compared with zinc-adequate rats fed coconut oil. By contrast, zinc-deficient rats fed the fish oil diet did not exhibit reduced activities of lipoprotein lipase in postheparin serum and adipose tissue and increased concentrations of serum lipids compared with zinc-adequate rats fed the fish oil diet. This study suggests that a reduced activity of lipoprotein lipase might contribute to increased postprandial concentrations of serum triglycerides observed in zinc-deficient animals. However, it also demonstrates that the effects of zinc deficiency on lipoprotein metabolism are influenced by dietary fatty acids.     

Vitamin C deficiency in Channa punctatus Bloch

The results of a series of experiments on Channa punctatus previously conditioned to a complete synthetic diet and later fed vitamin C deficient diet for 210 days followed by 30 days of recovery with fortified vitamin C ration are reported. The survival, growth, morphological abnormalities and biochemical changes in tissue ascorbic acid and cholesterol levels were studied.
Deficiency of vitamin C resulted in a mortality of up to 25% and retarded growth accompanied by anorexia and loss of weight. Marked skeletal abnormalities, viz. , lordosis, scoliosis and degenerative changes in the thoracic vertebrae were revealed by X-ray studies. Biochemical investigations showed a significant decrease in blood and kidney ascorbic acid levels within a month of feeding the deficient diet. A prolonged deficiency beyond 150 days resulted in a rapid rise in the cholesterol content of the liver. These biochemical changes were reversed when vitamin C was added to the ration.     

Effect of rearing techniques on skeletal deformities and osteological development in red porgy Pagrus pagrus (Linnaeus, 1758) larvae

Red porgy is a candidate species for marine aquaculture diversification. The objective of the present study was to describe the osteological development in this species and the occurrence of skeletal deformities in relation to the intensification of the rearing system. Fish samples were periodically collected along the development from hatching to juveniles (95 days after hatching). Osteological development and the presence of skeleton abnormalities were evaluated. Larvae reared under S‐IS showed a better growth in terms of total length in comparison with IS reared ones. Regarding to osteological development for red porgy, this was similar between fish from both culture systems, but differing in timing of apparition and ossification of skeletal elements. X‐ray studies revealed a high number of fish with skeletal deformities (Semi‐intensive: 38.8%; Intensive: 46.5%), but no significant effect of the rearing technique on the incidence of deformities such as lordosis or fused vertebrae was found. However, cranial abnormalities and kyphosis incidences were significantly higher in intensive system cultured red porgy. These results, suggest a relationship among rearing technique, osteological development and the apparition of certain deformities.     

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

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 analyses the response of the skeleton to different rearing densities and describes 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^–1; medium density (MD), 8 fish l^–1 and low density (LD), 2 fish l^–1. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens’ average standard length, which decreased with the increase in the rearing density. Differences in 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 specimen, 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 the rearing density on the development of different skeletal phenotypes.     

Weak compensatory growth makes short-term starvation an unsuitable technique to mitigate body deformities of <Emphasis Type="Italic">Tinca tinca</Emphasis> juveniles in intensive culture

Juvenile tench, Tinca tinca (L.) (initial mean weight 0.67 g) were continuously fed at high (5.0% of fish biomass) or low (2.5% of fish biomass) daily doses of a commercial formulated diet, or starved for 6 days, then fed these doses. The experiment lasted 40 days. Visible skeletal deformities occurred in fish fed the high doses, and the 6-day food deprivation mitigated the percentage of deformed fish from 37.3 to 12.1%. Deformities were associated with higher condition coefficient value. Faster growing individuals were more susceptible to body malformations within the feeding groups. No compensatory growth in body weight was observed in juveniles fed high or low doses. Lack of compensation was supported by lower carbon/nitrogen ratio in starved-re-alimented fish. Morphometric indices (condition coefficient and height/length ratio) suggested only partial compensation observed mostly during the first few days after the end of starvation. The possible mechanisms underlying this weak compensatory response in T. tinca juveniles may be associated with their slow growth rate and low oxygen consumption. Short starvation mitigates body deformities in intensively fed tench juveniles, however, this technique is not recommended in aquaculture due to their weak compensatory growth response.     

Historical overview of spinal deformities in ancient Greece

Little is known about the history of spinal deformities in ancient Greece. The present study summarizes what we know today for diagnosis and management of spinal deformities in ancient Greece, mainly from the medical treatises of Hippocrates and Galen. Hippocrates, through accurate observation and logical reasoning was led to accurate conclusions firstly for the structure of the spine and secondly for its diseases. He introduced the terms kyphosis and scoliosis and wrote in depth about diagnosis and treatment of kyphosis and less about scoliosis. The innovation of the board, the application of axial traction and even the principle of trans-abdominal correction for correction of spinal deformities have their origin in Hippocrates. Galen, who lived nearly five centuries later impressively described scoliosis, lordosis and kyphosis, provided aetiologic implications and used the same principles with Hippocrates for their management, while his studies influenced medical practice on spinal deformities for more than 1500 years.     

Benign coxsackievirus damages heart muscle in iron-loaded vitamin E-deficient mice

Several oxidative stressors (dietary selenium deficiency, dietary vitamin E deficiency coupled with fish oil feeding, genetic reduction of glutathione peroxidase activity) allow a normally benign coxsackievirus B3 (CVB3/0) to damage heart muscle in host mice. This study investigated whether dietary iron overload, another oxidant stress, would also permit CVB3/0 to exert a cardiopathologic effect in vitamin E-deficient (-VE) mice. Four groups of mice were fed either a -VE or a +VE diet containing either an adequate or an excessive (30x) amount of iron. After 4 weeks of feeding, the mice were inoculated with CVB3/0 and heart damage was assessed at various times postinfection. Mice fed a diet sufficient in VE with excess iron developed heart damage equivalent to mice fed a diet deficient in vitamin E without excess iron. However, severe heart damage occurred in the group fed a diet deficient in VE with excess iron, which was the most pro-oxidative diet. The highest heart viral titers were found in mice fed the -VE/excessive iron diet. However, the extent of heart damage did not always correlate with the formation of TBARS in liver homogenates. Further research is needed to clarify the role of oxidative stress and iron overload in determining the course of viral infection.     

Senegalese sole bone tissue originated from chondral ossification is more sensitive than dermal bone to high vitamin A content in enriched Artemia

Several studies have evaluated the effects of dietary vitamin A (VA) on the incidence of skeletal deformities during early ontogeny of fish, but little is known about its effects on bones depending on their process of ossification (dermal or chondral). We examined the incidence of skeletal deformities along development (30 and 48 dph) by double staining technique, in dermal (haemal and caudal vertebral bodies) and chondral (neural and haemal spines, epural, parahypural and hypurals) bones in Senegal sole post metamorphosed larvae fed with different dietary VA levels (37 000, 44 666, 82 666 and 203 000 UI total VA kg^?1 DW) during Artemia feeding phase (6–37 dph, at 18°C). Results obtained in this study showed that dietary VA disrupted the skeletogenesis in Senegalese sole post metamorphosed larvae by increasing the incidence of skeletal deformities in the axial skeleton and caudal fin complex, which were dependent on both bone morphogenesis and ossification processes. Fish fed with the highest dietary VA content showed the highest incidence of skeletal deformities and its value increased along ontogeny. However, when we compared the incidence of deformities in skeletal structures considering their ossification process, most skeletal structures derived from chondral ossification showed a significant higher increase in deformity incidences in fish fed an excess of VA (44 666, 82 666 and 203 000 UI kg^?1 DW), however within chondral bones, hypurals deformity incidence only increased in sole larvae fed Artemia highest VA content. In contrast, this dietary dose‐response effect was only noted in dermal bones from fish fed the highest dose of VA (203 000 UI kg^?1 DW). In addition, the incidence of deformities in chondral bones increased even when the dietary imbalance of VA was corrected, whereas dermal bones were not affected at later ages. These results indicated that depending on the ossification process from which different skeletal structures are derived, bones might be differentially affected by high dietary VA content. Those directly originated from the connective tissue with a preliminary cartilage stage were more sensitive to dietary VA excess than those formed by intramembranous ossification.     

Variance in prey abundance influences time budgets of breeding seabirds: evidence from pigeon guillemots Cepphus columba

We use data on pigeon guillemots Cepphus columba to test the hypothesis that discretionary time in breeding seabirds is correlated with variance in prey abundance. We measured the amount of time that guillemots spent at the colony before delivering fish to chicks ("resting time") in relation to fish abundance as measured by beach seines and bottom trawls. Radio telemetry showed that resting time was inversely correlated with time spent diving for fish during foraging trips (r=−0.95). Pigeon guillemots fed their chicks either Pacific sand lance Ammodytes hexapterus , a schooling midwater fish, which exhibited high interannual variance in abundance (CV=181%), or a variety of non-schooling demersal fishes, which were less variable in abundance (average CV=111%). Average resting times were 46% higher at colonies where schooling prey dominated the diet. Individuals at these colonies reduced resting times 32% during years of low food abundance, but did not reduce meal delivery rates. In contrast, individuals feeding on non-schooling fishes did not reduce resting times during low food years, but did reduce meal delivery rates by 27%. Interannual variance in resting times was greater for the schooling group than for the non-schooling group. We conclude from these differences that time allocation in pigeon guillemots is more flexible when variable schooling prey dominate diets. Resting times were also 27% lower for individuals feeding two-chick rather than one-chick broods. The combined effects of diet and brood size on adult time budgets may help to explain higher rates of brood reduction for pigeon guillemot chicks fed non-schooling fishes.     

Food Provisioning in Red-Necked Grebes (Podiceps Grisegena) at Common Carp (Cyprinus Carpio) Ponds

Parental feeding patterns were studied in red-necked grebe (Podiceps grisegena) broods throughout the entire period of parental care in a common carp (Cyprinus carpio) fish-pond area in SE Poland in 1993–2002. Fish formed a substantial part of prey provided to the flightless young from their second week of life. Although the numbers of large invertebrates and tadpoles, the alternative prey to fish, did not decrease during the chick rearing period, grebe parents gradually shifted from delivering predominately invertebrates to delivering fish, and the average size of fish fed to chicks increased with brood age. Broods with relatively high fledging success (at least two chicks fledged) had a larger proportion of fish in their diet than broods seriously reduced because of undernourishment. The dive duration of foraging grebe parents did not differ between carp, wild fish and non-fish prey, but carp prey required significantly more time for handling. The percentage of prey rejected by chicks increased over the prefledging period from 2 to 24%. Of the prey rejected, 82% were fish apparently too large for the young to swallow. Fish prevalence in the diet of red-necked grebe chicks at carp ponds contradicts the results of other studies on the feeding habits of the nominative subspecies during breeding season. However, the red-necked grebe is a gape-limited predator and the piscivory of the chicks is limited to small-bodied fish.     

Gestational changes in concentrations of selenium and zinc in the porcine fetus and the effects of maternal intake of selenium

The effect of maternal dietary selenium (Se) and gestation on the concentrations of Se and zinc (Zn) in the porcine fetus were determined. Mature gilts were randomly assigned to treatments of either adequate (0.39 ppm Se) or low (0.05 ppm Se) dietary Se. Gilts were bred and fetuses were collected throughout gestation. Concentrations of Se in maternal whole blood and liver decreased during gestation in sows fed the low-Se diet compared to sows fed the Se-supplemented diet. Maternal intake of Se did not affect the concentration of Se in the whole fetus; however, the concentration of Se in fetal liver was decreased in fetuses of sows fed the low-Se diet. Although fetal liver Se decreased in both treatments as gestation progressed, the decrease was greater in liver of fetuses from sows fed the low-Se diet. Dietary Se did not affect concentrations of Zn in maternal whole blood or liver or in the whole fetus and fetal liver. The concentration of Se in fetal liver was lower but the concentration of Zn was greater than in maternal liver when sows were fed the adequate Se diet. These results indicate that maternal intake of Se affects fetal liver Se and newborn piglets have lower liver Se concentrations compared to their dams, regardless of the Se intake of sows during gestation. Thus, the piglet is more susceptible Se deficiency than the sow.     

Occurrence of skeletal deformities and osteological development in red porgy Pagrus pagrus larvae cultured under different rearing techniques

The present study describes the osteological development and the occurrence of skeletal deformities in red porgy Pagrus pagrus larvae in relation to the intensification of the rearing system. Eggs obtained from natural spawning were cultured under two different rearing systems: intensive (100 eggs l^?1) in 2000 l and semi‐intensive (mesocosm) system (5 eggs l^?1) in 40 000 l conico‐cylindrical tanks. Fish samples were periodically collected along the development from hatching to juveniles at 95 days post hatching (dph). Osteological development, meristic counts and the presence of skeletal deformities were evaluated. Despite the external appearance of the juveniles being similar to wild standards, X‐ray studies revealed a high number of fish (semi‐intensive: 37·8%; intensive: 45·5%) with skeletal deformities. Regardless of the rearing system, no significant interaction was found between the per cent of the most common deformities, axial deviations (lordosis and presence of fused vertebrae). Cranial deformities and kyphosis incidences, however, were significantly higher in intensively cultured P. pagrus. Also, the fused vertebrae in these fish were located mainly in the caudal area instead of pre‐haemal area for semi‐intensively reared P. pagrus. Moreover, a significant interaction was found between the total number of vertebrae and the type of rearing system used; fish from the intensive system showing a higher number of fish with an extra vertebrae (10 abdominal + 15 caudal). Present results suggest a relationship among feeding sequence, osteological development and deformity incidence and location in P. pagrus larvae.