Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

O1-conotoxin Tx6. 7 cloned from the genomic DNA of Conus textile that inhibits calcium currents

Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDW CPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.(AU)

'Beer yeast' (liquid brewery waste) for sheep and pig feeding: epidemiological and clinical aspects of the poisoning, safety margin and prevention / "Levedo de cerveja" (resíduo líquido de cervejaria) para alimentação de ovinos e suínos: aspectos epidemiológicos e clínicos da intoxicação, margem de segurança e prevenção

Considering the practice of using 'brewer's yeast' (BY) - liquid brewery yeast waste from the brewing industry - in animal feed in some establishments in the south of the State of Rio de Janeiro in Brazil, this study was conducted to establish safety margins and to determine epidemiological and clinical aspects. In addition, this study suggested prophylactic measures that can prevent or minimise alcohol intoxication due to BY in sheep and swine. The main characteristics of the clinical features in both natural poisoning and controlled spontaneous ingestion in swine as well as intoxication by controlled ingestion of BY using a ruminal probe in sheep were found to be staggering gait, tripping and falls. Overall, 3.8 and 8.875mL/kg of ethanol content in the BY caused mild-moderate and severe clinical conditions, respectively, in swine and sheep. The following prophylactic measures are suggested: 1. BY must be adequately diluted in water, whey, or with BY that has been previously stored in the property (old BY); 2. It must be administered in proportion to the animals' weight and size. 3. It must be administered continuously, without interruptions, and with the provision of other food in the trough (such as soybean meal or cornmeal and water ad libitum). In conclusion, although BY is increasingly used in sheep and swine breeding properties - in the south of the State of Rio de Janeiro - many owners use one or more of the suggested prophylactic measures. Hence, ethanol intoxications because of BY are infrequent and rarely cause deaths; therefore, BY can be used provided adequate prophylactic measures are followed.

Considerando-se à prática de utilizar "levedo de cerveja" (LC) - resíduo líquido da indústria cervejeira - na alimentação de animais, em alguns estabelecimentos no sul do Estado do Rio de Janeiro, esse estudo foi realizado para estabelecer as margens de segurança e os quadros epidemiológico e clínico, bem como sugerir medidas profiláticas que impeçam ou minimizem esse tipo de intoxicação alcoólica para ovinos e suínos. Verificou-se que o quadro clínico na intoxicação por ingestão controlada de LC por sonda em ovinos e na intoxicação natural e ingestão espontânea controlada em suínos, caracterizou-se principalmente por andar cambaleante, tropeços e quedas. Concentrações de 3,8 e 8,875mL/kg de etanol contido no LC, causaram quadros clínicos leve a moderado e grave em suínos e ovinos, respectivamente. Como medidas profiláticas sugere-se: diluição adequada do LC com água, soro de leite ou com o LC já estocado na propriedade (LC antigo); administração do LC proporcional ao peso/tamanho dos animais, administração contínua, sem interrupções, disponibilizar outro alimento no cocho, como farelo de soja ou fubá de milho e água à vontade. Conclui-se que apesar de o LC ser cada vez mais utilizado nas propriedades criadoras de ovinos e suínos no sul do Estado do Rio de Janeiro já que, muitos proprietários aplicam uma ou mais medidas profiláticas aqui sugeridas, as intoxicações pelo etanol, contido no LC são pouco frequentes e raramente ocorrem mortes, de forma que esse produto deve ser utilizado, desde que as medidas profiláticas sejam observadas.

Circular RNA protein tyrosine kinase 2 aggravates pyroptosis and inflammation in septic lung tissue by promoting microRNA-766/eukaryotic initiation factor 5A axis-mediated ATP efflux

Purpose: Sepsis is characterized by an acute inflammatory response to infection, often with multiple organ failures, especially severe lung injury. This study was implemented to probe circular RNA (circRNA) protein tyrosine kinase 2 (circPTK2)-associated regulatory mechanisms in septic acute lung injury (ALI). Methods: A cecal ligation and puncture-based mouse model and an lipopolysaccharides (LPS)-based alveolar type II cell (RLE-6TN) model were generated to mimic sepsis. In the two models, inflammation- and pyroptosisrelated genes were measured. Results: The degree of lung injury in mice was analyzed by hematoxylin and eosin (H&E) staining and the apoptosis was by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. In addition, pyroptosis and toxicity were detected in cells. Finally, the binding relationship between circPTK2, miR-766, and eukaryotic initiation factor 5A (eIF5A) was detected. Data indicated that circPTK2 and eIF5A were up-regulated and miR-766 was down-regulated in LPS-treated RLE-6TN cells and lung tissue of septic mice. Lung injury in septic mice was ameliorated after inhibition of circPTK2. Conclusion: It was confirmed in the cell model that knockdown of circPTK2 effectively ameliorated LPS-induced ATP efflux, pyroptosis, and inflammation. Mechanistically, circPTK2 mediated eIF5A expression by competitively adsorbing miR-766. Taken together, circPTK2/ miR-766/eIF5A axis ameliorates septic ALI, developing a novel therapeutic target for the disease.

Gestational follow-up in ewes - placentome size and fecal progesterone metabolite levels

Background: Transabdominal ultrasonography in sheep allows real-time diagnosis and monitoring of pregnancy as well as measurement of the size of placentomes, which are structures that connect the fetal and maternal portions, that increase as pregnancy progresses. Progesterone is involved in the maintenance of gestation, with high levels noted during pregnancy. In this context, it is hypothesized that measurements of placentome and progesterone could help in monitoring pregnancy in sheep. Therefore, the main objectives of this study included evaluating whether placentome size and fecal progesterone levels are associated with gestational age in ewes and whether measurement of the placentome and progesterone could be used as an estimate of gestational age. Materials, Methods & Results: A total of 63 pregnant adult (between 2 and 5 years of age) Santa Inês ewes were monitored in the city of Boa Vista, Roraima. All ewes were kept in the paddock with Panicum maximun cv. tanzania, mineral salt, and water ad libitum, supplemented with 200 g/animal/day of soybean residue. Ewes were subjected to ultrasonography every 21 days from day 42 of pregancy until term to monitor pregnancy. Ultrasonography was performed using a linear probe to measure the placentome, including length × width in centimeters. Also every 21 days, fecal samples were collected for measurement of progesterone level. Feces were collected directly from the rectal ampulla, stored individually in plastic bags, and frozen at -20ºC until processing. In the laboratory, these feces samples were freeze-dried and subjected to hormonal extraction with 80% methanol. Then, the levels of fecal progesterone metabolites were measured by enzyme immunoassay. Data was tabulated and submitted to statistical analyses, including descriptive stastistic, correlation, KruskalWallis test and Tukey's test. The levels of fecal progesterone metabolites increased significantly between the 3rd and 4th months of pregnancy, with a decrease close to delivery. Placentome size increased significantly between the 2nd and 3rd months of pregnancy. Placentome measurements did not correlate with progesterone levels and showed a weak correlation with gestational age. Therefore, we concluded that the measurements of placentome size and a single evaluation of fecal progesterone metabolites were not considered efficient methods for estimating gestational age. Discussion: Measurement of progesterone level in fecal samples is a non-invasive hormone monitoring method that provides values like that of blood levels and is less stressful for the animals at the time of sample collection. The wide physiological variation makes it difficult or even useless to use fecal progesterone, when is measured only once per animal, as an indicator of the presence of pregnancy. Despite this limitation, serial sampling is useful for monitoring of hormones during the pregnancy period and is an important tool for research purposes. Placentome size measurements correlated weakly with gestational age and did not correlate with progesterone level. Thus, the presence of placentomes is indicative of the presence of pregnancy but was considered inefficient for determining gestational age in sheep.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.(AU)

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].(AU)

Alternative use of endocavitary probe to guide minimally invasive partial nephrectomy: is it reasonable?

Purpose: To describe the use of endocavitary ultrasound probe as an auxiliary tool when performing partial nephrectomy in cases of endophytic renal tumors, to standardize the method, and to report the preliminary results achieved with this technique. Methods: Fifteen patients diagnosed with completely endophytic underwent partial nephrectomy with the use of an endocavitary ultrasound probe. This article describes the technique involved in partial nephrectomy and details the preparation of the endocavitary ultrasound probe to ensure its safe use. Results: All the patients had a RENAL score between 8 and 11. The median time of warm ischemia was 26 and 18 minutes for laparoscopic or robot-assisted surgery, respectively. The median duration of surgery was 150 minutes, and the median console time was 145 minutes for the laparoscopic and robot-assisted surgery groups, respectively. The median estimate of blood loss was 200 mL. Only three patients in the laparoscopic group had focal positive surgical margins. There were no cases of infection at the site of probe entry. Conclusions: Intraoperative use of an endocavitary ultrasound probe for partial nephrectomy is possible and a safe alternative to the excision of endophytic tumors when neither robotic probes nor laparoscopic probes are available.

Técnica de dilatación con balón mediante procedimiento de endoscopia en un perro con estenosis esofágica: reporte de caso / Balloon dilatation technique via endoscopy procedure in a dog with esophageal stenosis: case report / Técnica de dilatação por balão via procedimento endoscópico em cão com estenose esofágica: relato de caso

La estenosis esofágica es una alteración morfofuncional que provoca una lesión inflamatoria en las capas submucosas y musculares del esófago, induciéndolas a la fibrosis y alterando el diámetro esofágico. El presente informe aborda el uso de un dilatador de balón como vía auxiliar para corregir la estenosis esofágica en un paciente canino, hembra, Pug, con antecedentes de vómitos recurrentes como principal síntoma. Mediante endoscopia se observó que el esófago torácico estaba inflamado, con mucosa engrosada y fibrótica, además de anillos fibrosos de color blanquecino, lo que dificultaba el paso de la sonda, lo que permitía determinar el diagnóstico de estenosis esofágica. En este informe se optó por el uso de balón dilatador, realizándose tres procedimientos con una semana de diferencia, para mejorar la condición sintomática. Tras el procedimiento dilatador, fue posible el desarrollo favorable del cuadro clínico presentado por el paciente.

Esophageal stenosis is a morphofunctional alteration that causes inflammatory lesion in the submucosal and muscular layers of the esophagus, inducing them to fibrosis and altering the esophageal diameter. The present report addresses the use of a balloon dilator as an auxiliary way to correct esophageal stenosis in a canine, female, Pug patient, with a history of recurrent vomiting as the main complaint. Through endoscopy, it was observed that the thoracic esophagus was inflamed, with thickened and fibrotic mucosa, in addition to whitish colored fibrous rings, which hindered the passage of the probe, enabling the determination of the diagnosis of esophageal stenosis. In this report, we opted for the use of a dilator balloon, with three procedures being performed one week apart, to improve the symptomatic condition. After the dilator procedure, the favorable development of the clinical condition presented by the patient was possible.

A estenose esofágica é uma alteração morfofuncional que ocasiona em lesão inflamatória das camadas submucosa e muscular do esôfago, induzindo-as a fibrose e que altera o diâmetro esofágico. O presente relato aborda a utilização de balão dilatador, como forma auxiliar de correção de estenose esofágica em paciente canino, fêmea, da raça Pug, apresentando histórico de vômitos recorrentes como queixa principal. Por meio da endoscopia, observou-se que o esôfago torácico estava inflamado, com a mucosa espessada e fibrótica, além de anéis fibrosos de coloração esbranquiçada, o que dificultava a passagem da sonda, possibilitando a determinação do diagnóstico de estenose esofágica. Nesse relato, optou-se pelo uso do balão dilatador, sendo feitos três procedimentos intervalados de uma semana entre eles, para a melhora da condição sintomática. Após o procedimento dilatador, foi possível o desenvolvimento favorável do quadro clínico apresentado pela paciente.

Nonlinear models for soil moisture sensor calibration in tropical mountainous soils

Electromagnetic sensors are widely used to monitor soil water content (θ); however, site-specific calibrations are necessary for accurate measurements. This study compares regression models used for calibration of soil moisture sensors and investigates the relation between soil attributes and the adjusted parameters of the specific calibration equations. Undisturbed soil samples were collected in the A and B horizons of two Ultisols and two Inceptisols from the Mantiqueira Range in Southeastern Brazil. After saturation, the Theta Probe ML2X was used to obtain the soil dielectric constant (ε). Several readings were made, ranging from saturation to oven-dry. After each reading, the samples were weighted to calculate θ (m³ m-³). Fourteen regression models (linear, linearized, and nonlinear) were adjusted to the calibration data and checked for their residue distribution. Only the exponential model with three parameters met the regression assumptions regarding residue distribution. The stepwise regression was used to obtain multiple linear equations to estimate the adjusted parameters of the calibration model from soil attributes, with silt and clay contents providing the best relations. Both the specific and the general calibrations performed well, with RMSE values of 0.02 and 0.03 m³ m-³, respectively. Manufacturer calibration and equations from the literature were much less accurate, reinforcing the need to develop specific calibrations.

Calibration of the Pilodyn instrument for the indirect in situ determination of the basic wood density of balsa / Calibração do instrumento Pilodyn para determinação indireta in situ da densidade básica da madeira de pau-de-balsa

Balsa wood is used widely in the nautical, aeronautical and wind-energy industries because of its low-density and high-resistance. In the Brazilian state of Mato Grosso, speculative producers with little knowledge of the culture have established balsa plantations using non-standardized and non-selected seedlings. In this sense, research is needed to improve balsa cultivation under Brazilian conditions to better explore the resource. The present study evaluated the efficiency of the Pilodyn instrument for the indirect determination of the basic wood density (BWD) of balsa. Twenty 12-year old balsa trees cultivated in Alta Floresta, Mato Grosso, were probed with the Pilodyn instrument on both north and south faces of the trunk at the base and at 1.30 and 3.10 m from the ground. Wood discs were subsequently collected from the probed sites and BWD determined according to the water-immersion method. Data were submitted to the Shapiro-Wilk normality test followed by analysis of variance and Pearson's correlation analysis with α=0.05. The BWD of balsa decreased in the direction from the base to the top of the tree. The penetration depth of the Pilodyn probe was inversely proportional to BWD with a correlation that was strong (R2=0.72) at heights of 1.30 and 3.10 m but considered weak (R2=0.46) at the base of the trunk. The reliability of the Pilodyn method was sufficient to allow the development of a linear equation that could be applied for the indirect determination of the BWD of balsa based on the penetration depth of the Pilodyn probe assessed at 1.30 m from the ground.

O pau-de-balsa é amplamente utilizado nas indústrias náutica, aeronáutica e de energia eólica devido à sua baixa densidade. No estado de Mato Grosso, produtores especulativos de balsa têm estabelecido plantios sem conhecimento sólido da cultura usando mudas não padronizadas e não selecionadas. Pesquisas são necessárias para melhorar o cultivo de balsa sob condições brasileiras para melhor explorar este recurso. O presente estudo teve como objetivo avaliar a eficiência do instrumento Pilodyn para a determinação indireta da densidade básica da madeira (DBM) de balsa. Vinte árvores de 12 anos de idade cultivadas em Alta Floresta, Mato Grosso, foram avaliadas com o instrumento Pilodyn nas faces norte e sul do tronco, na base e a 1,30 e 3,10 m do solo. Discos de madeira foram posteriormente coletados dos locais sondados e o DBM determinado de acordo com o método de imersão em água. Os dados foram submetidos ao teste de normalidade Shapiro-Wilk seguido de análise de variância e análise de correlação de Pearson com α=0,05. Os valores de DBM da balsa diminuiu da base em direção ao topo da árvore, enquanto a profundidade de penetração do Pilodyn foi inversamente proporcional. A correlação entre a profundidade de penetração do Pilodyn e o DBM foi fraca na base do tronco (R2=0,46), mas forte (R2=0,72) nos pontos de 1,30 e 3,10 m. O método Pilodyn foi suficientemente confiável que permitiu o desenvolvimento de uma equação para a determinação indireta do DBM do pau-de-balsa considerando o valor da profundidade de penetração a 1,30 m como uma das variáveis.

Padronização de uma PCR multiplex para a detecção de fraude em leite bubalino e caprino por adição de leite bovino / Standardization of a multiplex PCR for detection of fraud in buffalo and goat milk by addition of bovine milk

O consumo de leite de espécies como bubalino e caprino tem se popularizado por representarem uma alternativa para indivíduos que possuem restrições alimentares relacionadas ao leite bovino e em virtude das propriedades nutricionais desses alimentos. No entanto, fatores como a baixa produção e a sazonalidade predispõem a adulterações destes alimentos, principalmente pela adição de leite bovino, visando maior rendimento e lucratividade. Assim, o objetivo do estudo foi padronizar um método de PCR multiplex para autenticação de leites bubalino e caprino. Para isso, amostras de leite exclusivamente de cada espécie foram utilizados para a padronização da técnica. Em seguida, foi realizada a fraude pela adição de leite bovino ao caprino e ao bubalino, em proporções de 0,1% até 100%. A técnica foi eficaz, precisa, rápida e prática para a detecção do DNA de bovino, bubalino e caprino, separadamente e em conjunto. Na fraude experimental, o limite de detecção da técnica ocorreu a partir do menor percentual testado (0,1%) tanto no leite caprino quanto no bubalino. Dessa forma, a PCR multiplex testada mostrou ser uma importante ferramenta para a autenticação de leite, pendendo ser utilizada para fins de fiscalização por órgãos competentes.

Milk consumption of species such as buffalo and goat has become popular due to the nutritional properties of these foods and because they represent an alternative for individuals who have dietary restrictions related to bovine milk. However, factors such as low production and seasonality predispose to adulteration, mainly by the addition of bovine milk, aiming at higher yield and profitability. Thus, the aim of the present study was to standard a multiplex PCR method for buffalo and goat milks authentication. For this, the milks exclusively of each species were used to standardize the technique. Subsequently, fraud was performed by the addition of bovine milk to goat and buffalo in proportions from 0.1% to 100%. The technique was effective and accurate for detecting bovine, buffalo and goat DNA separately and together quickly and practically. In experimental fraud, the detection limit of the technique occurred from the lowest percentage tested (0.1%) in both goat and buffalo milk. Thus, the multiplex PCR tested proved to be an important tool for milk authentication, pending to be used for supervision by competent agencies.

Isolation and molecular identification of Mycobacterium bovis in tissue lesions of cattle slaughtered in slaughterhouses located in the State of Ceará / [Isolamento e identificação molecular de Mycobacterium bovis em lesões teciduais de bovinos abatidos em matadouros-frigoríficos localizados no estado do Ceará]

The objective of this research was to identify Mycobacterium bovis in lesions suggestive of tuberculosis in bovine carcasses in the State of Ceará, by means of bacteriological and molecular diagnostic tests. Between August 2017 and January 2019, the State inspection service (SIE) inspected 59,512 cattle, of which 7.4% (44 / 59,512) presented suggestive lesions. Of these animals, 68 samples were sent, of which 4.5% (31/68) located in the lung, 2.9% (20/68) in lymph nodes, 2.0% (14/68) in the liver, and 0.4% in the carcass (3/68). When performing bacteriological isolation, 15.9% (7/44) of bovines showed colony growth in the samples. The smears of the isolates were submitted to Zielh-Neelsen staining and all confirmed acid-fast bacilli. The polymerase chain reaction identified all isolates 100% (7/7) as M. bovis. The association of diagnostic techniques allowed to identify the presence of the agent in the State and the molecular analysis proved to be a beneficial technique in the monitoring of bovine tuberculosis and can be used as an auxiliary method in the bovine tuberculosis control and eradication program in the State of Ceará.

O objetivo do trabalho foi pesquisar Mycobacterium bovis em lesões sugestivas de tuberculose nas carcaças de bovinos no estado do Ceará, por meio dos testes de diagnóstico bacteriológico e molecular. Entre agosto de 2017 e janeiro de 2019, o Serviço de Inspeção Estadual (SIE) inspecionou 59.512 bovinos; destes, 7,4% (44/59.512) apesentaram lesões sugestivas. Desses animais foram enviadas 68 amostras, das quais 4,5% (31/68) estavam localizadas no pulmão, 2,9% (20/68) nos linfonodos, 2,0% (14/68) no fígado e 0,4% (3/68) na carcaça. Ao realizar o isolamento bacteriológico, 15,9% (7/44) dos bovinos evidenciaram crescimento de colônias nas amostras. Os esfregaços dos isolados foram submetidos à coloração de Zielh-Neelsen e todos eles confirmaram bacilo álcool-ácido resistente. A reação em cadeia da polimerase identificou todos os isolados, 100% (7/7), como M. bovis. A associação das técnicas de diagnóstico permitiu identificar a presença do agente no estado, e a análise molecular demonstrou ser uma técnica benéfica no monitoramento da tuberculose bovina, podendo ser utilizada como um método auxiliar no programa de controle e erradicação da tuberculose bovina no estado do Ceará.

Comparação de métodos de sexagem em filhotes de serpentes da espécie Python brongersmai / Comparing methods of sex identification in Python brongersmai snakelets

Com a ascensão de animais exóticos no mercado pet, observou-se a necessidade de maiores estudos nesta área. Este estudo teve como objetivo comparar diferentes métodos de sexagem em filhotes de serpentes da espécie Python brongersmai (Bloody Python), sendo utilizados 14 filhotes com aproximadamente 1 ano e 9 meses de vida, oriundos do Criadouro Comercial de Fauna Silvestre e Exótica ­ CCF, autorização de manejo N° 414064, localizado no município de Cornélio Procópio ­ PR. Foi utilizado a introdução da probe metálica de sexagem para contagem de escamas, mensurações do Comprimento Rostro-Cloacal (CRC), Comprimento Pós-Cloacal (CPC), Comprimento Total (CT); avaliação ultrassonográfica dos órgãos reprodutivos e perfil genético (DNA), indicando a fidedignidade de cada um. Observou-se uma eficácia de 100% na determinação do sexo através das amostras de DNA, enquanto a sexagem a partir da contagem de escamas via probe metálica e o dimorfismo sexual por mensurações corporais permitiram apenas uma classificação sexual prévia, sofrendo variações quando comparadas, mostrando-se como métodos inconclusivos. Já a ultrassonografia não foi eficaz para o processo de sexagem em serpentes filhotes de até aproximadamente 1 ano e 9 meses de idade da espécie Python brongersmai.(AU)

With the increment of exotic animal's trade in the pet business, the necessity of more studies in this area was observed. This study aimed at comparing different methods of sex identification in Python brongersmai snakelets (Bloody Python), using 14 baby snakes aged 1 year and 9 months approximately, from commercial breeding of wild and exotic fauna, situated in Cornélio Procópio ­ PR. Methods used were the probing technique introduction, measurement of snout-vent length, post-vent length, total length; ultrasound evaluation of reproductive organs and genetic profile exam (DNA samples), denoting the veracity of each one. A 100% efficacy was observed in the study of sexual determination through DNA samples, while the other methods were shown to be inconclusive, whilst the sexing scale count via the probing technique introduction and sexual dimorphism by bodily measurements permitted only a previous sexual classification, suffering variations when compared, showing to be inconclusive methods. The ultrasound evaluation was not efficient for the sexing process in snakelets aged approximately 1 year and 9 months in Python brongersmai.(AU)

Infecção pelo vírus da Febre do Nilo Ocidental em equinos no Estado de São Paulo / West Nile Fever virus infection in horses in São Paulo state, Brazil

Background: The West Nile virus (WNV) antibodies were reported in Brazil in the serum samples taken from horses andbirds in the Midwest region and Paraíba state in 2008 and 2013, respectively. In 2014, the first human case was confirmedin a rural worker in the state of Piauí and, in 2018, the virus was isolated from the central nervous system of a horse withnervous symptoms in the state of Espírito Santo. The virus is a member of the Flaviviridae family of the genus Flavivirus(neurotropic), infecting several mammalian species, with humans and horses being the most susceptible. Approximately35% of horses develop clinical signs, thus they are considered the best sentinels for this disease. The aim of this case reportis to describe the first confirmed cases of West Nile Fever (WNF) in two horses in the state of São Paulo.Cases: Two horses with neurological symptoms were treated at the Veterinary Hospital of Cruzeiro do Sul University (SãoPaulo, SP), in 2019. Both horses came from neighboring regions that have a large Atlantic Forest preservation area and arealso routes for migratory birds, known to be competent hosts for transmitting the West Nile Fever virus, such as the swallow,the falcon and the hawk. The first one had symptoms, such as weakness and sporadic seizures; however, after recovering,it was hospitalized a few days later due to kidney failure and laminitis. The second one showed incoordination, pelviclimb weakness, and was walking in circles, evolving to seizures. Both animals were euthanized, and their central nervoussystem samples and total blood samples were tested for rabies, herpes virus, and WNV; the first 2 tests showed negativeresults. Ribonucleic acids (RNA) were extracted from erythrocytes using the polymerase...

Infecção pelo vírus da Febre do Nilo Ocidental em equinos no Estado de São Paulo / West Nile Fever virus infection in horses in São Paulo state, Brazil

Background: The West Nile virus (WNV) antibodies were reported in Brazil in the serum samples taken from horses andbirds in the Midwest region and Paraíba state in 2008 and 2013, respectively. In 2014, the first human case was confirmedin a rural worker in the state of Piauí and, in 2018, the virus was isolated from the central nervous system of a horse withnervous symptoms in the state of Espírito Santo. The virus is a member of the Flaviviridae family of the genus Flavivirus(neurotropic), infecting several mammalian species, with humans and horses being the most susceptible. Approximately35% of horses develop clinical signs, thus they are considered the best sentinels for this disease. The aim of this case reportis to describe the first confirmed cases of West Nile Fever (WNF) in two horses in the state of São Paulo.Cases: Two horses with neurological symptoms were treated at the Veterinary Hospital of Cruzeiro do Sul University (SãoPaulo, SP), in 2019. Both horses came from neighboring regions that have a large Atlantic Forest preservation area and arealso routes for migratory birds, known to be competent hosts for transmitting the West Nile Fever virus, such as the swallow,the falcon and the hawk. The first one had symptoms, such as weakness and sporadic seizures; however, after recovering,it was hospitalized a few days later due to kidney failure and laminitis. The second one showed incoordination, pelviclimb weakness, and was walking in circles, evolving to seizures. Both animals were euthanized, and their central nervoussystem samples and total blood samples were tested for rabies, herpes virus, and WNV; the first 2 tests showed negativeresults. Ribonucleic acids (RNA) were extracted from erythrocytes using the polymerase...(AU)

Effects of heat-stress on oocyte number and quality and in vitro embryo production in Holstein heifers

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryonic losses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates. Materials, Methods & Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as < 10°C (group 1), 10-25°C (group 2), and > 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P < 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P < 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P < 0.05). Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction and convection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (>25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concluded that performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates.

Use of balloon dilatation technique via endoscopy procedure in a canine patient with esophageal stenosis: case report / Uso da técnica de dilatação por balão via procedimento endoscópico em um paciente canino com estenose esofágica: relato de caso / Uso de la técnica de dilatación con balón mediante procedimiento de endoscopia en un paciente canino con estenosis esofágica: reporte de caso

Esophageal stenosis is a morphofunctional alteration that causes inflammatory lesion in the submucosal and muscular layers of the esophagus, inducing them to fibrosis and altering the esophageal diameter. The present report addresses the use of a balloon dilator as an auxiliary way to correct esophageal stenosis in a canine, female, Pug patient, with a history of recurrent vomiting as the main complaint. Through endoscopy, it was observed that the thoracic esophagus was inflamed, with thickened and fibrotic mucosa, in addition to whitish colored fibrous rings, which hindered the passage of the probe, enabling the determination of the diagnosis of esophageal stenosis. In this report, we opted for the use of a dilator balloon, with three procedures being performed one week apart, to improve the symptomatic condition. After the dilator procedure, the favorable development of the clinical condition presented by the patient was possible.

A estenose esofágica é uma alteração morfofuncional que ocasiona em lesão inflamatória das camadas submucosa e muscular do esôfago, induzindo-as a fibrose e que altera o diâmetro esofágico. O presente relato aborda a utilização de balão dilatador, como forma auxiliar de correção de estenose esofágica em paciente canino, fêmea, da raça Pug, apresentando histórico de vômitos recorrentes como queixa principal. Por meio da endoscopia, observou-se que o esôfago torácico estava inflamado, com a mucosa espessada e fibrótica, além de anéis fibrosos de coloração esbranquiçada, o que dificultava a passagem da sonda, possibilitando a determinação do diagnóstico de estenose esofágica. Nesse relato, optou-se pelo uso do balão dilatador, sendo feitos três procedimentos intervalados de uma semana entre eles, para a melhora da condição sintomática. Após o procedimento dilatador, foi possível o desenvolvimento favorável do quadro clínico apresentado pela paciente.

La estenosis esofágica es una alteración morfofuncional que provoca una lesión inflamatoria en las capas submucosas y musculares del esófago, induciéndolas a la fibrosis y alterando el diámetro esofágico. El presente informe aborda el uso de un dilatador de balón como vía auxiliar para corregir la estenosis esofágica en un paciente canino, hembra, Pug, con antecedentes de vómitos recurrentes como principal síntoma. Mediante endoscopia se observó que el esófago torácico estaba inflamado, con mucosa engrosada y fibrótica, además de anillos fibrosos de color blanquecino, lo que dificultaba el paso de la sonda, lo que permitía determinar el diagnóstico de estenosis esofágica. En este informe se optó por el uso de balón dilatador, realizándose tres procedimientos con una semana de diferencia, para mejorar la condición sintomática. Tras el procedimiento dilatador, fue posible el desarrollo favorable del cuadro clínico presentado por el paciente.

High rDNA polymorphisms in Astyanax lacustris (Characiformes: Characidae): new insights about the cryptic diversity in A. bimaculatus species complex with emphasis on the Paraná River basin

This study aimed to identify species of Astyanax bimaculatus group from four Itaipu Reservoir tributaries (Paraná River Basin) by cytogenetics and molecular markers (COI) to investigate the possible occurrence of cryptic diversity in part of this basin. The four populations showed only one karyotype formula and simple AgNORs. FISH with 18S rDNA probe showed a high variation, and 5S rDNA probes evidenced simple sites in most of the specimens, although multiple sites are present in two specimens. The variations of 5S and 18S cistrons generated 13 cytotypes. The molecular data did not reveal cryptic diversity in the populations; however, its grouping with 82 sequences from other stretches of the Paraná River Basin originated three haplogroups (distances of 3.12% and 8.82%) and 33 haplotypes were identified. DNA Barcode suggests that cytogenetic variations represent a high polymorphism degree, and it identified the analyzed specimens as Astyanax lacustris, which confirms the morphological identification. Our data suggest that the cryptic diversity of this group in the tributaries of the Paraná River Basin is different than the proposed by the synonymizations of A. altiparanae and A. asuncionensis to A. lacustris. This study reinforces the importance of integrative cytogenetics and molecular methods for taxonomy.(AU)

Este trabalho teve como objetivo identificar espécies do complexo Astyanax bimaculatus de quatro afluentes do reservatório de Itaipu (bacia do Rio Paraná) por métodos citogenéticos e moleculares (COI), investigando a possibilidade de ocorrência de diversidade críptica em parte desta bacia. As quatro populações apresentaram apenas uma fórmula cariotípica e AgNORs simples. A FISH com rDNA 18S apresentou alto grau de variação e as sondas de rDNA 5S evidenciaram sítios simples na maioria dos exemplares, embora sítios múltiplos tenham sido evidenciados em dois espécimes. As variações dos cistrons 5S e 18S geraram 13 citótipos. Os dados moleculares não revelaram diversidade críptica nas populações, entretanto, seu agrupamento com 82 sequências de outros trechos da mesma bacia formou três haplogrupos (distâncias de 3,12% e 8,82%) e gerou 33 haplótipos. O DNA Barcode sugere que as variações citogenéticas representam um alto grau de polimorfismo e identificou os espécimes analisados como Astyanax lacustris, confirmando a identificação por caracteres morfológicos. Nossos dados sugerem que a diversidade críptica do grupo nos afluentes da bacia do Rio Paraná é diferente do proposto pelas sinonimizações de A. altiparanae e A. asuncionensis para A. lacustris, reforçando a importância da integração de métodos citogenéticos e moleculares para a taxonomia.(AU)