Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Resveratrol-Mediated Reversal of Doxorubicin-Induced Osteoclast Differentiation.

Secondary osteoporosis has been associated with cancer patients undertaking Doxorubicin (DOX) chemotherapy. However, the molecular mechanisms behind DOX-induced bone loss have not been elucidated. Molecules that can protect against the adverse effects of DOX are still a challenge in chemotherapeutic treatments. We investigated the effect and mechanism of DOX in osteoclast differentiation and used the Sirt 1 activator resveratrol (RES) to counteract DOX-induced effects. RAW 264.7 cells were differentiated into osteoclasts under cotreatment with DOX and RES, alone or combined. RES treatment inhibited DOX-induced osteoclast differentiation, reduced the expression of osteoclast fusion marker Oc-stamp and osteoclast differentiation markers Rank, Trap, Ctsk and Nfatc1. Conversely, RES induced the upregulation of antioxidant genes Sod 1 and Nrf 2 while DOX significantly reduced the FoxM1 expression, resulting in oxidative stress. Treatment with the antioxidant MitoTEMPO did not influence DOX-induced osteoclast differentiation. DOX-induced osteoclastogenesis was studied using the cathepsin-K zebrafish reporter line (Tg[ctsk:DsRed]). DOX significantly increased ctsk signal, while RES cotreatment resulted in a significant reduction in ctsk positive cells. RES significantly rescued DOX-induced mucositis in this model. Additionally, DOX-exposed zebrafish displayed altered locomotor behavior and locomotory patterns, while RES significantly reversed these effects. Our research shows that RES prevents DOX-induced osteoclast fusion and activation in vitro and in vivo and reduces DOX-induced mucositis, while improving locomotion parameters.

New insights into benzo[⍺]pyrene osteotoxicity in zebrafish.

Persistent and ubiquitous organic pollutants, such as the polycyclic aromatic hydrocarbon benzo[⍺]pyrene (BaP), represent a major threat to aquatic organisms and human health. Beside some well-documented adverse effects on the development and reproduction of aquatic organisms, BaP was recently shown to affect fish bone formation and skeletal development through mechanisms that remain poorly understood. In this work, zebrafish bone-related in vivo assays were used to evaluate the osteotoxic effects of BaP during bone development and regeneration. Acute exposure of zebrafish larvae to BaP from 3 to 6 days post-fertilization (dpf) induced a dose-dependent reduction of the opercular bone size and a depletion of osteocalcin-positive cells, indicating an effect on osteoblast maturation. Chronic exposure of zebrafish larvae to BaP from 3 to 30 dpf affected the development of the axial skeleton and increased the incidence and severity of skeletal deformities. In young adults, BaP affected the mineralization of newly formed fin rays and scales, and impaired fin ray patterning and scale shape, through mechanisms that involve an imbalanced bone remodeling. Gene expression analyses indicated that BaP induced the activation of xenobiotic and metabolic pathways, while negatively impacting extracellular matrix formation and organization. Interestingly, BaP exposure positively regulated inflammation markers in larvae and increased the recruitment of neutrophils. A direct interaction between neutrophils and bone extracellular matrix or bone forming cells was observed in vivo, suggesting a role for neutrophils in the mechanisms underlying BaP osteotoxicity. Our work provides novel data on the cellular and molecular players involved in BaP osteotoxicity and brings new insights into a possible role for neutrophils in inflammatory bone reduction.

New Insights on Vitamin K Metabolism in Senegalese sole (Solea senegalensis) Based on Ontogenetic and Tissue-Specific Vitamin K Epoxide Reductase Molecular Data.

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 (vkorc1) and vkorc1 like 1 (vkorc1l1) were determined during the larval ontogeny of Senegalese sole (Solea senegalensis), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1, and ovary and brain for ssvkorc1l1. Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis.

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (-150 °C) performing cooling rate (-66 °C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with -66 °C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.

Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Osteotoxicity of 3-methylcholanthrene in fish.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

Electric ultrafreezer (- 150 °C) as an alternative for zebrafish sperm cryopreservation and storage.

Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (- 150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (- 20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (- 66 °C/min) could improve post-thaw sperm quality. Freezing at - 20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (- 66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The - 66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.

Improved regeneration and de novo bone formation in a diabetic zebrafish model treated with paricalcitol and cinacalcet.

Bone changes related to diabetes have been well stablished, but few strategies have been developed to prevent this growing health problem. In our work, we propose to investigate the effects of calcitriol as well as of a vitamin D analog (paricalcitol) and a calcimimetic (cinacalcet), in fin regeneration and de novo mineralization in a zebrafish model of diabetes. Following exposure of diabetic transgenic Tg(ins:nfsb-mCherry) zebrafish to calcitriol, paricalcitol and cinacalcet, caudal fins were amputated to assess their effects on tissue regeneration. Caudal fin mineralized and regenerated areas were quantified by in vivo alizarin red staining. Quantitative real-time PCR was performed using RNA from the vertebral column. Diabetic fish treated with cinacalcet and paricalcitol presented increased regenerated and mineralized areas when compared with non-treated diabetic group, while no significant increase was observed in non-diabetic fish treated with both drugs. Gene expression analysis showed an up-regulation for runt-related transcription factor 2b (runx2b), bone gamma-carboxyglutamic acid-containing protein (bglap), insulin a (insa) and insulin b (insb) and a trend of increase for sp7 transcription factor (sp7) in diabetic groups treated with cinacalcet and paricalcitol. Expression of insra and vdra was up-regulated in both diabetic and nondiabetic fish treated with cinacalcet. In nondiabetic fish treated with paricalcitol and cinacalcet a similar increase in gene expression could be observed but not so pronounced. The increased mineralization and regeneration in diabetic zebrafish treated with cinacalcet and paricalcitol can be explained by increased osteoblastic differentiation and increased insulin expression indicating pro-osteogenic potential of both drugs.

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

ESSA1 embryonic stem like cells from gilthead seabream: a new tool to study mesenchymal cell lineage differentiation in fish.

Embryonic stem (ES) cells are a promising tool for generation of transgenic animals and an ideal experimental model for in vitro studies of embryonic cell development, differentiation and gene manipulation. Here we report the development and initial characterization of a pluripotent embryonic stem like cell line, designated as ESSA1, derived from blastula stage embryos of the gilthead seabream (Sparus aurata, L). ESSA1 cells are cultured in Leibovitz's L-15 medium supplemented with 5% fetal bovine serum and, unlike other ES cells, without a feeder layer. They have a round or polygonal morphology, grow exponentially in culture and form dense colonies. ESSA1 cells also exhibit intense alkaline phosphatase activity, normal karyotype and are positive for stage-specific embryonic antigen-1 (SSEA1) and octamer-binding transcription factor 4 (Oct4) markers for up to 30 passages. Upon treatment with all-trans retinoic acid, ESSA1 cells differentiate into neuron-like, oligodendritic, myocyte and melanocyte cells; they can also form embryoid bodies when seeded in bacteriological plates, a characteristic usually associated with pluripotency. The capacity of ESSA1 cells to differentiate into osteoblastic, chondroblastic or osteoclastic cell lineages and to produce a mineralized extracellular matrix in vitro was demonstrated through histochemical techniques and further confirmed by immunocytochemistry using lineage-specific markers. Furthermore, ESSA1 cells can be used to produce chimera, where they contribute to the development of a variety of tissues including the trunk and gut of zebrafish embryos and fry. Thus, ESSA1 cells represent a promising model for investigating bone-lineage cell differentiation in fish and also highlight the potential of piscine stem cell research.

mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

Distinguishing the Effects of Water Volumes versus Stocking Densities on the Skeletal Quality during the Pre-Ongrowing Phase of Gilthead Seabream (Sparus aurata).

Gilthead seabream (Sparus aurata) production is a highly valued aquaculture industry in Europe. The presence of skeletal deformities in farmed gilthead seabream represents a major bottleneck for the industry leading to economic losses, negative impacts on the consumers' perception of aquaculture, and animal welfare issues for the fish. Although past work has primarily focused on the hatchery phase to reduce the incidence of skeletal anomalies, this work targets the successive pre-ongrowing phase in which more severe anomalies affecting the external shape often arise. This work aimed to test the effects of: (i) larger and smaller tank volumes, stocked at the same density; and (ii) higher and lower stocking densities maintained in the same water volume, on the skeleton of gilthead seabream fingerlings reared for ~63 days at a pilot scale. Experimental rearing was conducted with gilthead seabream juveniles (~6.7 ± 2.5 g), which were selected as 'non-deformed' based on external inspection, stocked at three different densities (Low Density (LD): 5 kg/m3; Medium Density (MD): 10 kg/m3; High Density (HD): 20 kg/m3) in both 500 L and 1000 L tanks. Gilthead seabream were sampled for growth performance and radiographed to assess the skeletal elements at the beginning and end of the experimental trial. Results revealed that (i) LD fish were significantly longer than HD fish, although there were no differences in final weights, regardless of the water volume; (ii) an increase in the prevalence of seabream exhibiting cranial and vertebral axis anomalies was found to be associated with increased density. These results suggest that farmers can significantly reduce the presence of some cranial and axis anomalies affecting pre-ongrown gilthead seabream by reducing the stocking density.

Exploring Omega-3's Impact on the Expression of Bone-Related Genes in Meagre (Argyrosomus regius).

Dietary supplementation with Omega-3 fatty acids seems to promote skeletal health. Therefore, their consumption at imbalanced or excessive levels has offered less beneficial or even prejudicial effects. Fish produced in aquaculture regimes are prone to develop abnormal skeletons. Although larval cultures are usually fed with diets supplemented with Omega-3 Long Chain Polyunsaturated fatty acids (LC-PUFAs), the lack of knowledge about the optimal requirements for fatty acids or about their impact on mechanisms that regulate skeletal development has impeded the design of diets that could improve bone formation during larval stages when the majority of skeletal anomalies appear. In this study, Argyrosomus regius larvae were fed different levels of Omega-3s (2.6% and 3.6% DW on diet) compared to a commercial diet. At 28 days after hatching (DAH), their transcriptomes were analyzed to study the modulation exerted in gene expression dynamics during larval development and identify impacted genes that can contribute to skeletal formation. Mainly, both levels of supplementation modulated bone-cell proliferation, the synthesis of bone components such as the extracellular matrix, and molecules involved in the interaction and signaling between bone components or in important cellular processes. The 2.6% level impacted several genes related to cartilage development, denoting a special impact on endochondral ossification, delaying this process. However, the 3.6% level seemed to accelerate this process by enhancing skeletal development. These results offered important insights into the impact of dietary Omega-3 LC-PUFAs on genes involved in the main molecular mechanism and cellular processes involved in skeletal development.

Reversal of Doxorubicin-Induced Bone Loss and Mineralization by Supplementation of Resveratrol and MitoTEMPO in the Early Development of Sparus aurata.

Doxorubicin is a widely used chemotherapeutic drug known to induce bone loss. The mechanism behind doxorubicin-mediated bone loss is unclear, but oxidative stress has been suggested as a potential cause. Antioxidants that can counteract the toxic effect of doxorubicin on the bone would be helpful for the prevention of secondary osteoporosis. We used resveratrol, a natural antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, to counteract doxorubicin-induced bone loss and mineralization on Sparus aurata larvae. Doxorubicin supplemented Microdiets increased bone deformities, decreased mineralization, and lipid peroxidation, whereas Resveratrol and MitoTEMPO supplemented microdiets improved mineralization, decreased bone deformities, and reversed the effects of doxorubicin in vivo and in vitro, using osteoblastic VSa13 cells. Partial Least-Squares Discriminant Analysis highlighted differences between groups on the distribution of skeletal anomalies and mineralization of skeleton elements. Calcium and Phosphorus content was negatively affected in the doxorubicin supplemented group. Doxorubicin reduced the mRNA expression of antioxidant genes, including catalase, glutathione peroxidase 1, superoxide dismutase 1, and hsp90 suggesting that ROS are central for Doxorubicin-induced bone loss. The mRNA expression of antioxidant genes was significantly increased on resveratrol alone or combined treatment. The length of intestinal villi was increased in response to antioxidants and reduced on doxorubicin. Antioxidant supplements effectively prevent bone deformities and mineralization defects, increase antioxidant response and reverse doxorubicin-induced effects on bone anomalies, mineralization, and oxidative stress. A combined treatment of doxorubicin and antioxidants was beneficial in fish larvae and showed the potential for use in preventing Doxorubicin-induced bone impairment.

Regular Supplementation with Antioxidants Rescues Doxorubicin-Induced Bone Deformities and Mineralization Delay in Zebrafish.

Osteoporosis is characterized by an abnormal bone structure with low bone mass and degradation of microarchitecture. Oxidative stress induces imbalances in osteoblast and osteoclast activity, leading to bone degradation, a primary cause of secondary osteoporosis. Doxorubicin (DOX) is a widely used chemotherapy drug for treating cancer, known to induce secondary osteoporosis. The mechanism underlying DOX-induced bone loss is still not fully understood, but one of the relevant mechanisms is through a massive accumulation of reactive oxygen and nitrogen species (i.e., ROS and NOS) leading to oxidative stress. We investigated the effects of antioxidants Resveratrol and MitoTEMPO on DOX-induced bone impairment using the zebrafish model. DOX was shown to increase mortality, promote skeletal deformities, induce alterations on intestinal villi, impair growth and mineralization and significantly downregulate osteoblast differentiation markers osteocalcin 2 and osterix/sp7. Lipid peroxidation was significantly increased in DOX-supplemented groups as compared to control and antioxidants, suggesting ROS formation as one of the key factors for DOX-induced bone loss. Furthermore, DOX affected mineral contents, suggesting an altered mineral metabolism. However, upon supplementation with antioxidants, DOX-induced effects on mineral content were rescued. Our data show that supplementation with antioxidants effectively improves the overall growth and mineralization in zebrafish and counteracts DOX-induced bone anomalies.