Resumen Antecedentes: En la mayoría de los análisis morfométricos y alométricos se ignora el grado de variación atribuible a causas no biológicas, los efectos de esta variación en los análisis estadísticos posteriores se desconocen. No está claro si la conservación de la muestra induce una variación sustancial en la forma y si dicha variación afecta la inferencia estadística posterior. En los estudios osteológicos, los peces se preparan rutinariamente utilizando un procedimiento conocido comúnmente como transparentación-tinción. Sin embargo, las muestras se miden con frecuencia después de este procedimiento. En diversos estudios se ha determinado que la fijación de peces produce deformación, con disminución del tamaño, pero no se ha evaluado el efecto del proceso de transparentación-tinción en el análisis morfométrico y alométrico. Objetivo: Determinar el efecto del proceso de transparentación-tinción en la cuantificación de la variación morfológica a partir de análisis morfométricos. Métodos: Se utilizaron 33 especímenes de la especie Chloroscombrus chrysurus (Linnaeus, 1766), previamente fijados en formol al 10%, se midieron antes y después de ser transparentados y teñidos con rojo de alizarina “S”. Se llevaron a cabo tres análisis: análisis morfométrico, análisis de deformación y análisis alométrico. Para el reporte solo se tomaron en cuenta los parámetros estadísticamente significativos. Resultados: Se encontró que el procedimiento afectó el 90% de los índices morfométricos, así como el 90.9% de las medidas morfométricas, donde el 54.5% disminuyó y el 36.4% aumentó. Con respecto al análisis alométrico se obtuvo que el 80% de las relaciones longitud-longitud son iguales. Conclusiones: El proceso de transparentación-tinción influye en la variación morfológica de C. chrysurus y está determinada a partir del análisis de las variables morfométricas usando métodos morfométricos y alométricos.

Abstract Background: In morphometric and allometric analysis, the degree of variation attributable to non-biological causes is ignored. The effects of this variation on subsequent statistical analyzes are unknown. It is unclear whether sample conservation induces substantial variation in shape and whether such variation affects subsequent statistical inference and interpretation. Therefore, in fish skeletal studies, fish are routinely prepared for osteological studies using a common procedure known as clearing and staining, but clearing samples are frequently measured after this process. In various studies it has been determined that the fixation of fishes produces deformation, with a decrease in the size, but the effect has not been evaluated process of clearing-staining on the morphometric and allometric analysis. Objective: Determine the effect of the clearing and staining process on the morphometric and allometric analyses. Methods: Thirty-three specimens of the species Chloroscombrus chrysurus (Linnaeus, 1766) previously fixed within 10% formalin. These were measured before and after the clearing process and alizarin red S staining. Three shape analyzes were applied: morphometric analysis, deformation analysis and allometric analysis. Only the statistically significant results were used. Results: It was found that the procedure affected 90% of the morphometric indices, as well as 90.9% of the morphometric measurements, where 54.5% decreased and 36.4% increased. With respect to the allometric analysis, 80% of the length-length relationships remain the same. Conclusions: The clearing and staining process affects C. chrysurus’s morphological variation, determined by the morphometric analysis variables using morphometric and allometric methods.

Abstract Human periodontal ligament stem cells (hPDLSCs) are promising cells for dental and periodontal regeneration. Objective This study aimed to develop novel alginate-fibrin fibers that encapsulates hPDLSCs and metformin, to investigate the effect of metformin on the osteogenic differentiation of hPDLSCs, and to determine the regulatory role of the Shh/Gli1 signaling pathway in the metformin-induced osteogenic differentiation of hPDLSCs for the first time. Methodology CCK8 assay was used to evaluate hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red S staining, and the expression of osteogenic genes were evaluated. Metformin and hPDLSCs were encapsulated in alginate-fibrinogen solutions, which were injected to form alginate-fibrin fibers. The activation of Shh/Gli1 signaling pathway was examined using qRT-PCR and western blot. A mechanistic study was conducted by inhibiting the Shh/Gli1 pathway using GANT61. Results The administration of 50 μM metformin resulted in a significant upregulation of osteogenic gene expression in hPDLSCs by 1.4-fold compared to the osteogenic induction group (P < 0.01), including ALP and runt-related transcription factor-2 (RUNX2). Furthermore, metformin increased ALP activity by 1.7-fold and bone mineral nodule formation by 2.6-fold (P<0.001). We observed that hPDLSCs proliferated with the degradation of alginate-fibrin fibers, and metformin induced their differentiation into the osteogenic lineage. Metformin also promoted the osteogenic differentiation of hPDLSCs by upregulating the Shh/Gli1 signaling pathway by 3- to 6- fold compared to the osteogenic induction group (P<0.001). The osteogenic differentiation ability of hPDLSCs were decreased 1.3- to 1.6-fold when the Shh/Gli1 pathway was inhibited, according to ALP staining and alizarin red S staining (P<0.01). Conclusions Metformin enhanced the osteogenic differentiation of hPDLSCs via the Shh/Gli1 signaling pathway. Degradable alginate-fibrin hydrogel fibers encapsulating hPDLSCs and metformin have significant potential for use in dental and periodontal tissue engineering applications. Clinical Significance Alginate-fibrin fibers encapsulating hPDLSCs and metformin have a great potential for use in the treatment of maxillofacial bone defects caused by trauma, tumors, and tooth extraction. Additionally, they may facilitate the regeneration of periodontal tissue in patients with periodontitis. (hPDLSCs alginatefibrin alginate fibrin ShhGli1 ShhGli Shh Gli1 Gli Shh/Gli metformininduced time CCK (ALP evaluated alginatefibrinogen fibrinogen solutions qRTPCR qRT PCR blot GANT61 GANT 5 1.4fold 14fold 1.4 1 4 P 0.01, 001 0.01 , 0 01 0.01) runtrelated runt related factor2 factor 2 factor- RUNX2. RUNX2 RUNX . (RUNX2) Furthermore 1.7fold 17fold 1.7 7 2.6fold 26fold 2.6 6 P<0.001. P0001 P<0.001 (P<0.001) lineage 3 1.3 13 1.6fold 16fold 1.6 inhibited P<0.01. P001 P<0.01 (P<0.01) applications Alginatefibrin Alginate trauma tumors extraction Additionally periodontitis GANT6 4fold 14 1. 00 0.0 (RUNX2 7fold 17 6fold 26 2. P000 P<0.00 (P<0.001 16 P00 P<0.0 (P<0.01 0. (RUNX (P<0.00 P0 P<0. (P<0.0 P<0 (P<0. P< (P<0 (P<

Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures. Proteaseactivated Protease activated receptor2 2 receptor- PAR (PAR2 characteristics periodontitis (PDLSCs vitro 7 14 0.1 01 0 1 (0. U/mL, UmL U/mL , U mL U/mL) SLIGRLNH2 SLIGRLNH SLIGRL NH2 NH (SLIGRL-NH2 100 (10 nM, nM nM) FSLLRYNH2 FSLLRYNH FSLLRY (FSLLRY-NH2 nM. . RTqPCR RT qPCR (RT-qPCR ELISA (ELISA factors metabolism cytokines (ALP staining assessed 5 performed p 0.05, 005 0.05 05 0.05. Additionally kappaΒ kappa Β RANKL (RANKL <0 05. 05) findings resorption procedures (PAR 0. (0 (SLIGRL-NH 10 (1 (FSLLRY-NH 00 0.0 (

Resumo O Neem tem sido utilizado como medicamento devido às suas propriedades benéficas, tais como os efeitos antimicrobianos. Os produtos Neem para aplicação oral estão a aumentar. Antes da recomendação para uso terapêutico no ser humano, os seus efeitos nas atividades celulares precisam ser examinados. Por conseguinte, o objectivo deste estudo era testar os efeitos do extracto bruto de neem etanólico nas células de polpa dentária e osteoblastos em termos de viabilidade celular, mineralização e expressões genéticas. O extracto de neem etanólico derivado de folhas secas de neem foi sujeito a identificação química utilizando GC-MS. As células estaminais de polpa dentária humana (hDPSCs) e os pré-osteoblastos (MC3T3) foram tratados com várias concentrações do extrato bruto de neem. A viabilidade celular, mineralização, e expressões genéticas foram investigadas pelo ensaio MTT, PCR em tempo real, e o ensaio S vermelho de alizarina, respectivamente. A análise estatística foi realizada por ANOVA unidirecional seguida pelo teste Dunnett. O GC-MS detectou vários grupos de substâncias como o esquisterpeno. Doses baixas a moderadas do extracto bruto de neem (4 - 16 µg/ml) não afetaram a viabilidade do hDPSC e MC3T3, enquanto 62,5 µg/ml do extracto de neem diminuiu a viabilidade do MC3T3. Doses elevadas do extrato bruto de neem (250 - 1.000 µg/ml) reduziram significativamente a viabilidade de ambas as células. O extrato bruto de neem a 1.000 µg/ml também diminuiu a viabilidade de hDPSC e MC3T3 diferenciados e a sua mineralização. Além disso, 4 µg/ml de neem inibiu a viabilidade do hDPSC diferenciado. Não há diferença estatística nas expressões genéticas relacionadas com a diferenciação celular. Em conclusão, o extrato bruto do neem afetou a viabilidade celular e a mineralização. A viabilidade celular alterou-se diferentemente dependendo das doses, tipos de células, e fases celulares. O extrato bruto do neem não afetou a diferenciação celular. O rastreio do seu efeito em vários aspectos deve ser examinado antes da aplicação para uso humano.

Abstract Neem has been used as a medicine due to its beneficial properties such as anti-microbial effects. Neem products for oral application are on the rise. Before recommendation for therapeutic use in human, its effects on cellular activities need to be examined. Therefore, the aim of this study was to test the effects of the ethanolic neem crude extract on dental pulp cells and osteoblasts in terms of cell viability, mineralization, and gene expressions. The ethanolic neem extract derived from dry neem leaves was subjected to chemical identification using GC-MS. Human dental pulp stem cells (hDPSCs) and pre-osteoblasts (MC3T3) were treated with various concentrations of the neem crude extract. Cell viability, mineralization, and gene expressions were investigated by MTT assay, real-time PCR, and alizarin red S assay, respectively. Statistical analysis was performed by one-way ANOVA followed by Dunnett test. GC-MS detected several substance groups such as sesquiterpene. Low to moderate doses of the neem crude extract (4 - 16 µg/ml) did not affect hDPSC and MC3T3 viability, while 62.5 µg/ml of the neem extract decreased MC3T3 viability. High doses of the neem crude extract (250 - 1,000 µg/ml) significantly reduced viability of both cells. The neem crude extract at 1,000 µg/ml also decreased viability of differentiated hDPSC and MC3T3 and their mineralization. Furthermore, 4 µg/ml of neem inhibited viability of differentiated hDPSC. There is no statistical difference in gene expressions related to cell differentiation. In conclusion, the neem crude extract affected cell viability and mineralization. Cell viability altered differently depending on the doses, cell types, and cell stages. The neem crude extract did not affect cell differentiation. Screening of its effect in various aspects should be examined before the application for human use.

Our study attempted to compare the efficacies of bone morphogenetic protein (BMP) 2, 6, and 9 in inducing osteogenic differentiation of preodontoblasts (PDBs). We immortalized PDBs by introducing a reversible SV40 T antigen-based immortalization system. Cell proliferation capability was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The effects of BMP2, 6, and 9 on the osteogenic differentiation of immortalized preodontoblasts (iPDBs) were measured by alkaline phosphatase (ALP) activity assays and alizarin red S staining. The expression of osteogenic markers was evaluated by semiquantitative real-time polymerase chain reaction analysis. To assess ectopic bone formation, rat-derived iPDBs were transfected in culture with adenoviral vectors designated Ad-BMP2, 6, and 9 and subcutaneously or intramuscularly injected into mice. Several BMPs retained endogenous expression in PDBs and regulated the mRNA expression of mineralized tissue-associated proteins. ALP activity and mineralized nodule formation were significantly increased in the Ad-BMP9-transfected group relative to the control group. In addition, the most significant hard tissue formation was in this group. The results indicated that BMP signaling was involved in the osteogenic differentiation of iPDBs. BMP9 could be an efficacious accelerant of the osteogenic differentiation of iPDBs.

Abstract Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP – ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Fluoride, which is often added to toothpaste or mouthwash in order to protect teeth from decay, may be a novel therapeutic approach for acceleration of periodontal regeneration. Therefore, we investigated the effects of fluoride on proliferation and mineralization in human periodontal ligament cells in vitro. The periodontal ligament cells were stimulated with various concentrations of NaF added into osteogenic inductive medium. Immunohistochemistry of cell identification, cell proliferation, alkaline phosphatase (ALP) activity assay, Alizarin red S staining and quantitative real-time-polymerase chain reaction (RT-PCR) were performed. Moderate concentrations of NaF (50-500 μmol/L) had pro-proliferation effects, while 500 μmol/L had the best effects. ALP activity and calcium content were significantly enhanced by 10 μmol/L NaF with osteogenic inductive medium. Quantitative RT-PCR data varied in genes as a result of different NaF concentrations and treatment periods. We conclude that moderate concentrations of NaF can stimulate proliferation and mineralization in periodontal ligament cells. These in vitro findings may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of suitable concentrations of NaF into the medication for periodontitis treatment, i.e., into periodontal packs and tissue patches.

A spectrophotometric method for the determination of AlIII in hemodialysis water employing alizarin red S complexing agent in the presence of polyvinylpyrrolidone 40 surfactant is described. The complex, formed in pH 4.5 buffered media, is detected at 510 nm. Optimal concentrations of all reagents were investigated as well as the appropriate detection wavelength. A linear analytical curve in the range of 5.0-320 µg L-1 was obtained, providing a limit of detection (3s, n = 7) of 2 µg L-1 and quantification limit of 5 µg L-1. Results were compared with those generated using inductively coupled plasma mass spectrometry (ICP-MS) as an independent technique and method validation was also demonstrated by analysis of AccuStandard certified reference material QCS‑02-R1-5, appropriately diluted to 10 µg L-1. Equivalent results (t-test at a 95% confidence level) with a relative standard deviation of 4% were obtained. Real samples, spiked at a level of 10 µg L-1 to conforme to Brazilian legislated limits, showed recoveries between 90-110%. The procedure is simple and fit-for-purpose with sufficient detection power to screen for Al at trace levels in hemodialysis water without pre-concentration of the samples.

Homogeneous liquid-liquid microextraction via flotation assistance (HLLME-FA) was investigated for the extraction of molybdenum from the water samples. Alizarin Red S and cetyl trimethylammonium bromide (CTAB) were used as a complexing ligand and ion-pairing reagent, respectively. The enriched analyte in the floated organic phase was determined by electrothermal atomic absorption spectrometry (ETAAS). In this work, low density organic solvent was used and no centrifugation was required in this procedure. A special extraction cell was designed to facilitate the collection of the low-density extraction solvent. By using air flotation, the organic solvent was collected at the conical part of the designed cell. Under the optimum conditions, the method performance was studied in terms of linear dynamic range (0.5-200 µg L-1), linearity (r2 > 0.991), precision (repeatability < 10.0 %). Also, the limit of detection (LOD) of 0.1 µg L-1 was obtained for molybdenum. The proposed method has been successfully applied for the determination of the molybdenum in water samples.

The degradation of Alizarin Red S by electro-generated species using Pt and BDD electrodes was performed. The results were explained by the generation of OH•; radical, S2O8(2-) at BDD electrode and active chlorine species at Pt electrode. The slow degradation is affected by the current density, initial pH, temperature, initial dye concentration and the nature of the supporting electrolyte. However, the ionic strength showed a negligible effect on both electrodes. In the presence of KCl, the intermediates produced during the degradation are similar at both electrodes. In the presence of sulfate (at BDD electrode), the rate and the mechanism of the degradation are different from those in the presence of KCl. TOC analysis showed total mineralization of AR S.

Daily deposition of otolith increments was validated for juveniles of 8 intertidal fish species in central Chile, Helcogrammoides chilensis, Helcogrammoides cunninghami (Tripterygiidae), Auchenionchus microcirrhis, Auchenionchus crinitus (Labrisomidae), Hypsoblennius sordidus (Blenniidae), Gobiesox marmoratus, Sicyaces sanguineus (Gobiesocidae) and Myxodes viridis (Clinidae). Validation was performed by Alizarin Red-S labelling followed by 6 days of growth.

Infusions of Orthosiphon stamineus Benth., Lamiaceae, leaves are widely used in Southeastern Asia to treat different illnesses. Nonetheless, no data is available on the safety of O. stamineus for pregnant women and their babies. This study was undertaken to evaluate the developmental toxicity of O. stamineus standardized aqueous extract in female Sprague Dawley rats (n=21) at 0, 250, 500, 1000 and 2000 mg/kg/day, by gavage on gestation days 6-20. Clinical signs of maternal toxicity, body weight gain, and food and water consumption were recorded. Caesarean sections were performed on gestation day 21; resorptions and living and dead fetuses were counted. Fetuses were weighed and examined for external abnormalities. Half of the fetuses from each litter were cleared and stained with Alizarin red S for skeleton evaluation. O. stamineus standardized aqueous extract did not alter pregnancy body weight gain and food and water consumption and caused no other sign of maternal toxicity. Embryolethality and prenatal growth retardation were not observed either. O. stamineus standardized aqueous extract increased a few skeleton variations and a skull bone malformation (hyoid bone absent) in a non-dose dependent manner. Anogenital distance was increased in male and female fetuses exposed to the highest O. stamineus standardized aqueous extract dose, an indication that the extract could possibly contain androgenic compounds.

Juvenis de pejerrey, Odontesthes bonariensis, foram expostos a Vermelho de Alizarina S (ARS) 0,1% de duas formas, sozinho ou com uma imersão anterior em 2,2% de solução salina (Indução osmótica, IO) para melhorar o método de marcação ARS. Os procedimentos foram realizados no campo e os peixes foram liberados em gaiolas (tanques-rede) de 1 m3 na lagoa "La Salada de Monasterio", Chascomús, Buenos Aires, Argentina. Após 73 dias, marcas claras foram observadas nos otólitos, raios da nadadeira caudal e escamas em ambos os tratamentos, sendo que a intensidade do sinal nas escalas de IO + ARS de peixe tratado foi superior. Por outro lado, não foram observados marcas no grupo controle sobre as mesmas estruturas. Aproximadamente um ano pós-tratamento (385 dias), apenas marcas nos raios da nadadeira caudal foram encontrados claramente nos peixes tratados com IO+ARS. Entre os peixes observados, após este período, não houve diferenças significativas no comprimento total ou peso entre o grupo controle e marcados, ademais, a mortalidade variou entre 30-40% em todas as gaiolas. Estes resultados fornecem fortes evidências e um grande potencial para aplicação desta técnica rentável de marcação que diferencia o pejerrey selvagem e o produzido em cativeiro. O sucesso na marcação dos raios da nadadeira caudal também é de grande importância, pois sua verificação é fácil e não requer o sacrifício de peixes

Juveniles of pejerrey, Odontesthes bonariensis, were exposed to 0.1% Alizarin Red S (ARS) alone or with a previous immersion in 2.2% saline solution (Osmotic Induction, OI) to enhance the ARS marking method. Fish were marked in the field and immediately released in 1 m3 cages in "La Salada de Monasterio" lagoon, Chascomús, Buenos Aires , Argentina. After 73 days, clear marks were observed in the otoliths, caudal fin rays and scales with both treatments, being the intensity of the signal in the scales of OI+ARS treated fish higher. On the other hand, no marks were observed in the control group on the same structures. Approximately one year post-treatment (385 days), only marks in caudal fin rays were found clearly in OI+ARS treated fish. After this period, no significant differences in total length or weight between marked or control fish were observed and the mortality ranged between 30-40 % in all cages. These results provide strong evidence for the potential applicability of this cost-effective marking technique in differentiation of wild and hatchery-produced pejerrey. The success in the caudal fin rays marking is also important because it is easy to do and does not require the sacrifice of fish.