The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved ones (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.(AU)

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.(AU)

Efficiency of chemical preservatives used in raw milk samples for bacterial counts by flow cytometry

The objective of this study was to evaluate and compare two chemical preservatives in terms of their sample preservation capabilities, considering the individual bacterial count (IBC) and time and temperature variables. Samples were collected in expansion tanks in three commercial dairy farms located in the northwest of Rio Grande do Sul, characterized as G1: low IBC values, G2: average IBC values, and G3: high IBC values. The tanks were stored at three different temperatures (4, 10, and 25 °C) for 14 d. Samples supplemented with the preservative Azilat in G1 (lower IBC group) exhibited the best results at a temperature of 4 °C, whereas for G2 and G3, the results showed no statistically significant difference between temperatures 4 and 10 °C. The temperature 25 °C exhibited the worst results. For samples preserved with Azidiol, regardless of the studied group (G1, G2, and G3), the temperatures of 4 and 10 °C did not present a significant difference regarding the preservation of the samples, with the temperature of 25 °C exhibiting the worst results. Azilat was effective in keeping the samples conserved when they presented low IBC, being able to fluctuate with the increase in IBC and temperature variation. Azidiol was effective regardless of the initial IBC level.

Association of Palm gene mutations with structure and function of paralemmin proteins in Lori-Bakhtiari and Zel sheep Breeds

The aim of current study was to survey genetic variability of PALM gene'sexon 3 and 4 by PCR-SSCP and DNA sequencing in Zel and Lori Bakhtiari sheep breeds. TheSIFT(Sorting Intolerant from Tolerant) and PHyre2 program were used to predict the possible impact of amino acid substitutions on performance and structure of the paralemmin protein. A total of 140 animal's from 2 Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Zel sheep breeds were considered. The results showed that there are two polymorphic sites including a nonsynonymous substitution and an insertion mutation (49bp). Non-synonymous mutation deduced Thr20Ala amino acid exchange and ensuing two different structures for paralemmin protein that could be potentially affect protein structure and function during the interaction with glutamate in the cytosolic surface of plasma membrane. PALMgene, according to evolutionary path, is classified into two separate categories. In first covey, Gallus gallusand in second one, other species in several branches, so that the sequence of cow and sheep is placed in a sub-branch which forms a clade beside goat. Comparison of illustrated coding region sequences, PALMgene among different species, is of orthologous which are derived from a common ancestor.(AU)

Impacto da qualidade seminal e dos fatores relativos ao macho na produção in vitro de embriões bovinos / Impact of semen quality and other male-related factors on in vitro embryo production in cattle

A importância da qualidade do sêmen no processo comercial de produção in vitro de embriões (PIVE) é bem conhecida, ainda que não devidamente relatada na literatura. Existe não apenas uma significativa diferença entre touros nas taxas de clivagem e de blastocistos, mas também nas taxas de prenhezes subsequentes. Adicionalmente, há evidências de interação entre touro e tecnologia de processamento do sêmen (particularmente na separação de espermatozoides por sexo), entre touro e protocolo de preparação do sêmen para fertilização in vitro, e ainda entre touro e doadora. Controlar estes efeitos em uma rotina comercial tem sido um desafio crescente para os laboratórios, particularmente com a alta oferta de novos touros decorrente da recente adoção da seleção genômica. O presente trabalho aborda algumas destas questões, com base na experiência da Bio Biotecnologia da Reprodução Animal nesta área.(AU)

The importance of semen quality in a commercial in vitro embryo production (IVEP) routine is well-known, although underreported in the literature. There is not only a significant difference among sires on cleavage and blastocyst rates, but also on subsequent pregnancy rates. Moreover, there are evidences of interaction between sire and sperm processing technology (particularly in the case of sex-sorted semen), between sire and the protocol for sperm preparation for in vitro fertilization, and between sire and donor. Controlling such effects in a commercial routine has been a growing challenge for the laboratories, especially due to the high turnover of sires caused by the recent adoption of genomic selection in most breeds. The current study discusses some of these aspects, from the perspective of the experience of Bio Biotecnologia da Reprodução Animal in this field.(AU)

CD4+ and CD8+ T lymphocyte counts and ratio in cats with chronic gingivostomatitis and naturally infected with feline immunodeficiency virus: a preliminary study / Razão e contagem de linfócitos T CD4+ e linfócitos T CD8+ em gatos com gengivoestomatite crônica e infectados naturalmente pelo vírus da imunodeficiência felina: estudo preliminar

This study aimed to evaluate the CD4+ and CD8+ T lymphocytes counts and CD4+: CD8+ ratio in a colony of cats with chronic gingivostomatitis (CGS). We used forty domestic short-haired cats inhabiting the same colony. Ten cats with CGS were immunodeficiency virus-positive (group IV), and ten with CGS were immunodeficiency virus-negative (group III). As a control, twenty cats without CGS were used: ten cats were immunodeficiency virus-positive (group II) and ten cats were immunodeficiency virus-negative (group I). We employed flow cytometry to count CD4+ and CD8+ T lymphocytes. In cats infected with the immunodeficiency virus, the presence of CD4+ lymphocytes were lower both for animals with and without CGS. Conversely, not immunodeficiency virus-infected cats with CGS had a higher amount of CD4+ when compared to seronegative animals without CGS. The counts of CD8+ T lymphocytes showed no significant difference among cats with CGS, whether infected with immunodeficiency virus or not. The CD4+: CD8+ ratio was only different for group III, which was higher than any other group. No difference was observed for total lymphocyte number and CD8+ among groups. By contrast, mean CD4+ levels were different, with cats from groups III and IV showing higher levels than those from groups I and II. The flow cytometry could be a useful tool for the diagnosis and prognosis of cats with CGS infected by the immunodeficiency virus.

Este estudo teve como objetivo avaliar a contagem e a razão de linfócitos T CD4+ e CD8+ em uma colônia de gatos com gengivoestomatite crônica (CGS). Foram analisados quarenta gatos domésticos que habitavam a mesma colônia. Dez gatos com CGS foram positivos para o vírus da imunodeficiência (grupo IV), e dez com CGS foram negativos para o vírus da imunodeficiência (grupo III). Como controle, vinte gatos sem CGS foram usados: dez gatos foram positivos para o vírus da imunodeficiência (grupo II) e dez gatos foram negativos para o vírus da imunodeficiência (grupo I). Empregou-se a citometria de fluxo para contagem de linfócitos T CD4+ e CD8+. Nos gatos infectados pelo vírus da imunodeficiência, a presença de linfócitos CD4+ foi menor tanto para os animais com e sem CGS. Por outro lado, gatos não infectados e com CGS apresentaram maior quantidade de linfócitos CD4+ quando comparados a animais soronegativos sem CGS. A contagem de linfócito T CD8+ não mostrou diferença significativa entre gatos com CGS, infectados ou não com o vírus da imunodeficiência. A razão CD4+:CD8+ foi diferente apenas para o grupo III, que foi maior do que qualquer outro grupo. Não foi observada diferença para o número total de linfócitos e CD8+ entre os grupos. Em contraste, os níveis médios de CD4+ foram diferentes, com os gatos dos grupos III e IV apresentando níveis mais elevados do que os dos grupos I e II. A citometria de fluxo pode ser uma ferramenta útil para o diagnóstico e prognóstico de gatos com CGS infectados pelo vírus da imunodeficiência.

Evaluation of novel SexedULTRA-4M technology for in vitro bovine embryo production

SexedULTRA-4M™ is made using an improved method of sex-sorting sperm in a less damaging environment for better retaining sperm integrity throughout the sorting process. The objective of this research was to compare conventional (CONV) and SexedULTRA-4M™ (ULTRA-4M) semen for bovine IVP using four Angus bulls. Matured slaughterhouse oocytes (n = 4000) were divided into the CONV group and the ULTRA-4M group (2000 COCs for each semen type). The IVF process was implemented with CONV and ULTRA-4M semen from the same bull. The cleavage rates, eight cell embryos and blastocysts on day 7 of culture were evaluated for each semen type and each bull. The statistical analysis was carried out with the ANOVA procedure SAS software. The results were 54.45% ± 1.03 and 58.10% ± 1.07; 35% ± 1.57 and 39.15% ± 1.62; 22.8% ± 1.09 and 27.15% ± 1.12 for CONV and ULTRA-4M, respectively, for cleavage rate, eight cell embryos and blastocysts on day 7 for the average of all bulls, comparing only the semen type. Concerning only the semen type, ULTRA-4M was significantly superior to CONV for cleavage rates (P = 0.01) and blastocysts on day 7 (P = 0.009). There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analyzed for Bull 1 and Bull 4, however, for Bull 2 ULTRA-4M was significantly superior to CONV for cleavage rates and blastocysts on day 7 (P< 0.05). In Bull 3, ULTRA-4M was significantly higher (P< 0.05) for blastocysts on day 7 compared to CONV. In conclusion, under the conditions of this research the ULTRA-4M and CONV semen produced similar bovine IVP results overall.(AU)

Interactive effects of digestible protein levels on thermal and physical stress responses in Nile tilapia

The effects of dietary digestible protein (DP) levels (22, 26, 29, 32, and 34%) and different stressors (cold-induced stress, CIS; heat/dissolved oxygen-induced stress, HDOIS; transport-induced stress, TIS; and size-sorting-induced stress, SSIS) on hemato-biochemical parameters were evaluated. Four hundred and forty Nile tilapia fingerlings were distributed into 40-250 L aquaria and fed experimental diets for 110 days, and fed each of the five experimental diets, that were randomly distributed to eight replicates per treatment. Then, different groups of fish were subjected to one type of stress. Groups of 40 fish were used on CIS (17 °C), HDOIS (32 °C), and TIS (4 h), and a group of 140 fish on SSIS (15 min air exposure and 60 s handling). There was no effect on hemato-biochemical profile when DP levels were compared, neither before nor after stress; however, there was a significant stress effect. Digestible protein did not mitigate stress response under SSIS and CIS; lymphopenia and neutrophilia were the main cell-mediated immune response; dietary 22 and 26% DP impaired oxygenation on SSIS and TIS; fish under HDOIS and SSIS demanded more energy using triglycerides as an energy source; the diet formulated to contain 22% DP was not adequate to keep homeostasis under temperature stress. Cluster analysis showed that, for DP levels below the requirement for growth, SSIS and CIS were considered the most stressful conditions. At 34% DP level, HDOIS response was comparable to that of non-stressing conditions.(AU)

Monitoring and baseline of glyphosate-resistant sourgrass in the main soybean growing regions of Brazil / Monitoramento e baseline da resistência de capim-amargoso ao herbicida glifosato nas principais regiões produtoras de soja no Brasil

Frequent application of glyphosate for consecutive years has enhanced the selection pressure on sourgrass (Digitaria insularis) populations, which resulted in the development of glyphosate-resistant biotypes. Therefore, this work was developed with the objective of monitoring sourgrass resistance to glyphosate, develop a baseline of sourgrass susceptibility to this molecule and, consequently, identify the discriminatory dose between resistant and susceptible populations. This work was divided into three steps. The first step consisted of identifying and sorting sourgrass resistant and susceptible biotypes among 30 samples. In the second step, glyphosate baseline was elaborated considering exclusively the glyphosate-susceptible biotypes, which allowed the definition of a discriminatory dose. At the end, the third step, monitoring of glyphosate-resistant biotypes was achieved, considering five growing seasons (2016 ­2020) and 809 samples of sourgrass populations, collected throughout 12 states of Brazil. Glyphosate baseline was elaborated to sourgrass and ideal discriminatory rate was identified as 960 g ha-1.Glyphosate-resistant populations of sourgrass were found in all soybean growing regions sampled. Among 809 populations, 25.96% were considered resistant to glyphosate. The states with the highest frequency of glyphosate-resistant populations were Rio Grande do Sul, Mato Grosso do Sul, Bahia, Mato Grosso and Paraná.

Este trabalho foi desenvolvido com o objetivo de monitorar a resistência de capim-amargoso ao herbicida glyphosate desenvolver uma baseline de suscetibilidade da espécie e, por consequência, identificar a dose discriminatória de glifosato entre populações resistentes e suscetíveis de capim-amargoso. Todo o trabalho foi dividido em três fases. A primeira fase consistiu da análise de 30 amostras de capim-amargoso, identificando-as e classificando-as em resistentes ou suscetíveis. Na segunda fase, foi elaborada uma baseline para suscetibilidade do capim-amargoso ao herbicida glyphosate, o que permitiu a definição de uma dose discriminatória. Ao final, na terceira fase, obteve-se o monitoramento dos biótipos de capim-amargoso quanto à resistência, considerando-se cinco safras (2016 ­2020) e 809 amostras de capim-amargoso, oriundas de 12 estados brasileiros. Assim sendo, a baseline de suscetibilidade de capim-amargoso ao glyphosate foi estimada, cuja dose discriminatória ideal foi de 960 g ha-1. Populações de capim-amargoso resistentes ao glyphosate foram encontradas em todas as regiões produtoras de soja amostradas. Dentre 809populações, 25,96% foram consideradas resistentes ao glyphosate. Os estados com maior frequência de populações resistentes foram: Rio Grande do Sul, Mato Grosso do Sul, Bahia, Mato Grosso e Paraná.

Validação prática de modelos de infravermelho próximo para tomate: sólidos solúveis e acidez / Practical validation of near infrared models for tomato: soluble solids and acidity

O tomate é a hortaliça mais produzida e consumida, tendo aceitabilidade tanto para o consumo in natura quanto para a industrialização. Apesar da ampla aceitação dos tomates, o consumidor apresenta como exigência para aquisição do fruto a qualidade,mensurada através de parâmetros como sólidos solúveis (SS) e acidez titulável (AT). Uma técnica de análise química, não destrutiva e não invasiva e de resposta rápida, é a de espectroscopia de absorção na região do infravermelho próximo que tem sido bastante utilizada em várias indústrias, desde agrícola a petroquímica.Considerando a produção em alta escala, a qualidade do tomate exigida pelos consumidores e por se tratar de uma técnica não destrutiva e não invasiva da espectroscopia no infravermelho próximo (Near Infrared Spectroscopy ­NIR),faz-se necessário modelos testados em condições comerciais garantindo um modelo de infravermelho próximo para tomate facilitando a classificação. Realizou-sea validação de forma externa e prática dos modelos de infravermelho próximo para tomate in natura, comparando com os métodosdestrutivos e conferindo a acurácia dos modelos na qualificação do fruto quanto aos teores de sólidos solúveis e acidez. O presente trabalho apresenta modelos do projeto executado de 2018 a 2019, construídos atravésdos aplicativos Model Builder e The Unscrambler e selecionados a partir de parâmetros como coeficiente de calibração, coeficiente da validação cruzada, erro médio do conjunto de calibração, e da validação cruzada e cálculo do desvio do resíduo de calibração. Os resultados foram obtidos através da previsão do modelo para os atributos sólidos solúveis (SS) e acidez titulável (AT), do tomate de mesa com maior variabilidade de produção. O modelo para SS apresenta potencialidade para uso comercial, seja na determinação de ponto de colheita, seja na quantificação de qualidade do vegetal. Já para AT, o NIR portátil não produziu um modelo aplicável pela limitação do comprimento de onda.(AU)

Tomatoes are the most commonly used and consumed vegetables, being acceptable for fresh consumption as well as for industrialization. Despite the wide acceptance of tomatoes, the consumer presents quality as a requirement for the acquisition of the fruit, which is measured through parameters such as soluble solids (SS) and titratable acidity (TA). A chemical analysis technique, non-destructive and non-invasive and with fast response, is an absorption spectroscopy in the near infrared region that has been widely used in several industries, from agricultural to petrochemicals. Demanding large-scale production, the quality of tomatoes demanded by consumers and as it is a non-destructive and non-invasive technique of near infrared spectroscopy (NIR), it is still necessary to test models under commercial conditions, ensuring a near infrared model for tomatoes making sorting easy. An external and practical validation of the near infrared models for fresh tomatoes was carried out, comparing them with the destructive methods and checking the accuracyof the models in qualifying the fruit in terms of soluble solids and acidity. This paper presents project models obtained from 2018 to 2019, built from the Model Builder and The Unscrambler programs and selected from parameters such as calibration coefficient, cross validation coefficient, average error of the calibration set, and cross validation and calculation of the residual calibration deviation. The results obtained through the prediction of the model for the soluble solid attributes and titratable acidity, of the table tomato with greater production variability. The model for SS of table tomato has potential for commercial use, either in determining the harvest point, or quantifying the vegetable's quality. For AT or portable NIR, it did not produce an applicable model due to the wave length limitation(AU)

Uncertainties and risks in delimiting species of Cambeva (Siluriformes: Trichomycteridae) with single-locus methods and geographically restricted data

Cambeva contains species with complex taxonomy or poorly delimitated in terms of morphology and geopraphic distribution. We conducted an extensive review of Cambeva populations from coastal drainages of Southern to Southeastern Brazil to evaluate species geographic limits with an integrative analysis including morphological and molecular data (COI). We test if two single-locus methods, Bayesian Poisson Tree Processes (bPTP) and Generalized Mixed Yule Coalescent (GMYC), are efficient to delimit species boundaries in Cambeva by the comparison with the diagnosable morphological units. Using GMYC, we also evaluated the combination of tree and molecular clock priors to reconstruct the input phylogeny and assessed how well the implemented model fitted our empirical data. Eleven species were identified using a morphological diagnosability criterion: Cambeva balios, C. barbosae, C. botuvera, C. cubataonis, C. davisi, C. guaraquessaba, C. iheringi, C. tupinamba, and C. zonata and two treated as undescribed species. In contrast with previous knowledge, many of them have wider distribution and high intraspecific variation. Species delimitation based on single-locus demonstrated incongruences between the methods and strongly differed from the morphological delimitation. These disagreements and the violation of the GMYC model suggest that a single-locus data is insufficient to delimit Cambeva species and the failure may be attributable to events of mitochondrial introgression and incomplete lineage sorting.(AU)

Cambeva contém espécies com taxonomia complexa ou mal delimitadas em termos morfológicos e de distribuição geográfica. Realizamos uma extensa revisão de populações de Cambeva das drenagens costeiras do Sul ao Sudeste do Brasil para avaliar os limites das espécies com uma análise integrativa incluindo dados morfológicos e moleculares (COI). Testamos se dois métodos de locus único, Implementação Bayesiana dos Processos da Árvore de Poisson (bPTP) e Coalescente de Yule Misto Generalizado (GMYC), são eficientes para delimitar os limites das espécies em Cambeva pela comparação com as unidades morfológicas diagnosticáveis. Usando o GMYC, também avaliamos a combinação de árvores e relógios moleculares para reconstruir a filogenia e avaliamos o quão bem o modelo implementado se ajustava aos nossos dados empíricos. Foram identificadas 11 espécies usando o critério morfológico: Cambeva balios, C. barbosae, C. botuvera, C. cubataonis, C. davisi, C. guaraquessaba, C. iheringi, C. tupinamba e C. zonata e duas tratadas como espécies não-descritas. Em contraste com o conhecimento prévio, muitas delas têm distribuição mais ampla e alta variação intraespecífica. A delimitação das espécies baseada em locus único demonstrou incongruências entre os métodos e diferiu fortemente da delimitação morfológica. Essas discordâncias e a violação do modelo GMYC sugerem que os dados de locus único são insuficientes para delimitar as espécies de Cambeva e a falha pode ser atribuída a eventos de introgressão mitocondrial e sorteio incompleto da linhagem.(AU)

Citometria de Fluxo em andrologia: da pesquisa básica para a rotina clínica / Flow citometry in andrology: from basic research to clinical routine

Os primeiros estudos de espermatozoides com citometria de fluxo com espermatozoides iniciaram no final da década de 1970. Com os avanços tecnológicos, hoje contamos com equipamentos com alta sensibilidade e eficiência que, em conjunto com amplo catálogo de sondas fluorescentes, podemos mensurar com alta precisão características celulares. Como exemplo, destacam-se dano em membrana plasmática, atividade mitocondrial, produção de espécies reativas de oxigênio, dano ao DNA espermático e muito mais. Na presente revisão, as potencialidades e limitação para a implementação da citometria de fluxo na análise seminal de espécies domésticas são exploradas e comentadas.

The first flow cytometry studies with spermatozoa were published in the late 1970s. With the technological advances in the following years, today we have equipments with high sensitivity and efficiency that, together with a large number of commercially available fluorescent probes, allow us to measure different cell characteristics with high accuracy. Some of the most evaluated characteristics are plasma membrane damage, mitochondrial activity, production of reactive oxygen species, sperm DNA damage, and much more. In this review, the potentials and limitations for the implementation of flow cytometry in the seminal analysis of domestic species are explored and commented.

A comparative evaluation of methods for estimating the colostrum quality in sows / Avaliação comparativa de métodos para estimar a qualidade do colostro em porcas

Colostrum is the main source of immunoglobulins (Ig) for neonate piglets and plays a crucial role within the health and growth of the piglet. Currently in pig farming, there are still no widespread practical methods for measuring the Ig concentration in colostrum at herd level. We evaluated sows' colostrum IgG concentration using an optical and a digital Brix refractometer and their performance was correlated to an IgG ELISA test, and flow cytometry. Colostrum concentrations of IgG and IgA averaged 74.05 ± 21.37mg/mL and 20.2 ± 5.32mg/mL respectively. The mean value of the Brix percentages for optical refractometer was 26.32%, and for digital was 28.32%. The Brix refractometer measurements of colostrum samples presented high correlation for IgG content analyzed by ELISA (Optical = 0.74, Digital = 0.87; P <0.001). Considering the immunophenotyping, the values for IgG and IgA lymphoblasts indicated a highly significant relationship to ELISA (IgG=0.77, IgA=0.84; P<0.001). The Brix refractometer can be considered a useful tool to be included in a colostrum monitoring program to improve potentially neonatal health. In addition, we demonstrated that flow cytometry can be an important tool to analyze and characterize the immunological potential of sow colostrum.(AU)

O colostro é a principal fonte de imunoglobulinas (Ig) para leitões recém-nascidos e desempenha um papel crucial na saúde e no crescimento dos leitões. Atualmente, na suinocultura, ainda não existem métodos amplamente utilizados na prática de produção para medir a concentração de imunoglobulinas no colostro suíno. Avaliou-se a concentração de IgG no colostro de porcas usando refratômetros Brix óptico e digital, e o desempenho foi comparado com ELISA e citometria de fluxo. As concentrações de IgG e IgA no colostro foram 74,05 ± 21,37mg/mL e 20,2 ± 5,32mg/mL, respectivamente. A percentagem de Brix média das amostras de colostro para o refratômetro óptico foi 26,32%, e para o digital foi 28,32%. As medições dos refratômetros de Brix apresentaram elevada correlação com a concentrações de IgG medidas por ELISA (óptico=0,74, digital=0,87; P<0,001). Considerando a imunofenotipagem, os valores dos linfoblastos IgG e IgA apresentaram alta correlação com o ELISA (IgG=0,77, IgA=0,84; P<0,001). O refratômetro Brix pode ser considerado uma ferramenta útil para ser incluída em um programa de monitoramento de colostro para melhorar a saúde neonatal. Além disso, foi demonstrado que a citometria de fluxo pode ser uma ferramenta importante para analisar e caracterizar o potencial imunológico do colostro de porcas.(AU)

Cytometric blood phenotyping in free-living white-eyed parakeet (Psittacara leucophthalmus) / Fenotipagem citométrica do sangue de maritacas jovens de vida-livre (Psittacara leucophthalmus)

This study aimed at performing cytometric phenotyping of the blood samples from free-living, young white-eyed parakeets (Psittacara leucophthalmus), stained with 3,3-dihexyloxacarbocyanine [DiOC6(3)]. DiOC6(3)-stained whole blood samples from 19 free-living, young white-eyed parakeets were analyzed by flow cytometry and cell types were distinguished by their typical fluorescence in blue laser channel (FL-1) and SSC (side scatter). It was possible to differentiate erythrocytes (58.3±13.6) from leukocytes (32.4±13.1) and some of the leucocyte subpopulations: lymphocytes/thrombocytes (29.7±7.7), monocytes (30.6±8.5), and granulocytes (5.9-26). However, lymphocytes and thrombocytes could not be sorted in the plots. Our study determined that the predominant population in white-eyed parakeet (P. leucophthalmus) was lymphocytes, thrombocytes, and monocytes in the leucocytes gates in comparison to the granulocyte population. The cytometry method and use of DiOC6(3) stain was available for parakeets blood samples and can be studied and applied to other species of parrots.(AU)

Este estudo teve como objetivo realizar a fenotipagem citométrica com 3,3-di-hexiloxacarbocianina [DiOC6 (3)] de amostras de sangue de maritacas jovens de vida-livre (Psittacara leucophthalmus). As amostras de sangue total, coradas com DiOC6(3) de 19 maritacas de vida livre, foram analisadas por citometria de fluxo e os tipos de células foram distinguidos por sua fluorescência típica no canal laser azul (FL-1) e SSC (dispersão lateral). Foi possível diferenciar eritrócitos (58,3±13,6) de leucócitos (32,4±13,1) e algumas subpopulações de leucócitos: linfócitos/trombócitos (29,7±7,7), monócitos (30,6±8,5) e granulócitos (5,9-26), entretanto, linfócitos e trombócitos não puderam ser diferenciados em duas populações distintas. Nosso estudo determinou que a população predominante P. leucophthalmus foi mononuclear agranulocítica em comparação com a taxa de aquisição da população granulocítica. A metodologia de citometria de fluxo com uso da coloração de DiOC6(3) foi aplicável a amostras sanguíneas das maritacas e pode ser estudado e aplicado para outras espécies de psitacídeos.(AU)

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-olds lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.(AU)

Cytometric blood phenotyping in free-living white-eyed parakeet (Psittacara leucophthalmus) / Fenotipagem citométrica do sangue de maritacas jovens de vida-livre (Psittacara leucophthalmus)

This study aimed at performing cytometric phenotyping of the blood samples from free-living, young white-eyed parakeets (Psittacara leucophthalmus), stained with 3,3-dihexyloxacarbocyanine [DiOC6(3)]. DiOC6(3)-stained whole blood samples from 19 free-living, young white-eyed parakeets were analyzed by flow cytometry and cell types were distinguished by their typical fluorescence in blue laser channel (FL-1) and SSC (side scatter). It was possible to differentiate erythrocytes (58.3±13.6) from leukocytes (32.4±13.1) and some of the leucocyte subpopulations: lymphocytes/thrombocytes (29.7±7.7), monocytes (30.6±8.5), and granulocytes (5.9-26). However, lymphocytes and thrombocytes could not be sorted in the plots. Our study determined that the predominant population in white-eyed parakeet (P. leucophthalmus) was lymphocytes, thrombocytes, and monocytes in the leucocytes gates in comparison to the granulocyte population. The cytometry method and use of DiOC6(3) stain was available for parakeets blood samples and can be studied and applied to other species of parrots.(AU)

Este estudo teve como objetivo realizar a fenotipagem citométrica com 3,3-di-hexiloxacarbocianina [DiOC6 (3)] de amostras de sangue de maritacas jovens de vida-livre (Psittacara leucophthalmus). As amostras de sangue total, coradas com DiOC6(3) de 19 maritacas de vida livre, foram analisadas por citometria de fluxo e os tipos de células foram distinguidos por sua fluorescência típica no canal laser azul (FL-1) e SSC (dispersão lateral). Foi possível diferenciar eritrócitos (58,3±13,6) de leucócitos (32,4±13,1) e algumas subpopulações de leucócitos: linfócitos/trombócitos (29,7±7,7), monócitos (30,6±8,5) e granulócitos (5,9-26), entretanto, linfócitos e trombócitos não puderam ser diferenciados em duas populações distintas. Nosso estudo determinou que a população predominante P. leucophthalmus foi mononuclear agranulocítica em comparação com a taxa de aquisição da população granulocítica. A metodologia de citometria de fluxo com uso da coloração de DiOC6(3) foi aplicável a amostras sanguíneas das maritacas e pode ser estudado e aplicado para outras espécies de psitacídeos.(AU)

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line / [Mudanças metabólicas induzidas por melitina em células da linhagem Madin-Darby Bovine Kidney]

In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10µg/mL). Flow cytometry analysis revealed that melittin at 2µg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2µg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5µg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.(AU)

Neste estudo, os efeitos tóxicos da melitina em células Madin-Darby Bovine Kidney (MDBK) foram analisados quanto à funcionalidade mitocondrial, por redução de MTT e citometria de fluxo, potencial de apoptose, necrose, produção de espécies reativas de oxigênio (ROS), peroxidação lipídica e fragmentação de DNA, utilizando-se citometria de fluxo e desestabilização da membrana celular, por microscopia confocal. A toxicidade apresentou características dose-dependentes e a atividade mitocondrial foi inibida até 78,24±3,59% (P<0,01, n = 6) em células MDBK expostas à melitina (10µg/mL). Análises por citometria de fluxo revelaram que a melitina a 2µg/mL apresentou o maior índice necrótico celular (P<0,05). A maior lipoperoxidação de membranas também foi na concentração de 2µg/mL de melitina (P<0,05), o que foi posteriormente confirmado por microscopia confocal. A maior produção de ROS aconteceu quando as células foram expostas a 2,5µg/mL de melitina (P<0,05), e essa concentração também aumentou a fragmentação de DNA (P<0,05). Houve uma significativa correlação positiva entre a lipoperoxidação de membranas e a produção de ROS (R=0,4158), funcionalidade mitocondrial (R=0,4149) e apoptose (R=0,4978). Portanto, o estresse oxidativo gerado pela melitina culminou na elevação de ROS intracelular, que inicia uma cascata de eventos tóxicos nas células MDBK.(AU)

Accuracy and physical characteristics of total mixed rations and feeding sorting behavior in dairy herds of Castro, Parana

The objective of this study was to assess the accuracy of the content of total mixed rations (TMR) offered to high-producing cows from 20 dairy herds of Castro, Paraná State, Brazil. The average milk yield during the sample collection period was 38.0±6.8 kg/day, with 3.47±0.25% milk fat and 3.05±0.18% milk protein. The particle size distribution of samples of fresh diet and leftovers was evaluated using the Penn State Particle Separator (PSPS) and chemical analysis was also conducted. The TMR homogeneity was estimated by the coefficient of variation (CV) of particles retained on the two intermediary sieves (1.18 mm), in which a CV higher than 5% indicated a poorly mixed TMR. The TMR offered exceeded the formulated TMR only for NDF (+7.3%) and was lower than the formulated TMR for CP (−6.5%), ADF (−10.1%), and ash (−18.6%) contents. These differences are due to daily variations in feed quality, mixing equipment, and ingredient mixing order, mostly for forage. Differences between leftovers and TMR offered were substantial: −9.4% for CP, +25.1% for NDF, +31.6% for ADF, and +13.1% for ash, suggesting a feed sorting effect against long forage particles and in favor of small concentrate particles. The proportion of the offered TMR retained in the top PSPS sieve showed positive correlations with NDF (r = 0.58) and ADF (r = 0.54) contents of the refusals, which indicates that cows sort against long particles. Positive correlations were also found between the CV in the second sieve and NDF (r = 0.56) and ADF (r = 0.47) contents of refusals, suggesting that poorly mixed diets are more easily sorted by cows. Therefore, evaluating the chemical and physical properties of TMR, especially for forage, is extremely important for providing a consistent diet.

Taxonomic and phylogenetic beta diversity in headwater stream fish communities of the Paraná and Paraguai River basins

Patterns of species replacement and richness differences along environmental gradients or ecoregions shed light on different ecological and evolutionary mechanisms acting on community structure. Communities of aquatic ecosystems of different watersheds are supposed to host distinct species and lineages. Quantifying and understanding the degree to which these differences are affected by environmental and biogeographical factors remains an open question for these environments, particularly in the Neotropical region. We investigated patterns of taxonomic and phylogenetic composition of headwater streams of the Paraná and Paraguai River basins to understand how local and biogeographical factors affect the assembly of fish communities. We also quantified taxonomic and phylogenetic beta diversity by decomposing them into nestedness and turnover components. We found that local environmental factors are the main factors influencing the composition of stream fish communities. Whereas pH affected both taxonomic and phylogenetic turnover, water velocity was responsible for phylogenetic turnover and pH was the main driver of phylogenetic nestedness. Our results indicate an effect of local environmental factors in determining the structure of headwater stream fish communities through a combination of a species sorting mechanism (water velocity and pH) and phylogenetic habitat filtering (pH).(AU)

Padrões de substituição de espécies ou diferenças de riqueza ao longo de gradientes ambientais ou ecoregiões lançam luz sobre diferentes processos e mecanismos ecológicos atuando na estruturação das comunidades. Supõe-se que comunidades aquáticas pertencentes a diferentes bacias pertençam a linhagens evolutivas distintas. Quantificar e entender o grau em que tais diferenças são resultado de fatores ambientais locais e/ou processos biogeográficos ainda é uma questão pouco explorada. Neste estudo nós investigamos os padrões de composição taxonômica e filogenética em riachos de cabeceira das bacias dos Rios Paraná e Paraguai, para entender como fatores locais e biogeográficos afetam a estruturação das comunidades de peixes. Nós quantificamos a diversidade beta taxonômica e filogenética decompondo estas em aninhamento e substituição. Encontramos que os fatores ambientais locais são os principais determinantes da composição das comunidades de peixes destes riachos. Enquanto o pH afetou tanto a substituição de linhagens e de espécies, a velocidade da água foi responsável por uma substituição de linhagens, enquanto o pH foi o principal responsável pelo aninhamento de linhagens. Nossos resultados indicam a importância dos fatores locais através da combinação entre mecanismos de preferência de nicho (velocidade da água e pH) e filtragem ambiental de linhagens (pH).(AU)

Taxonomic and phylogenetic beta diversity in headwater stream fish communities of the Paraná and Paraguai River basins

Patterns of species replacement and richness differences along environmental gradients or ecoregions shed light on different ecological and evolutionary mechanisms acting on community structure. Communities of aquatic ecosystems of different watersheds are supposed to host distinct species and lineages. Quantifying and understanding the degree to which these differences are affected by environmental and biogeographical factors remains an open question for these environments, particularly in the Neotropical region. We investigated patterns of taxonomic and phylogenetic composition of headwater streams of the Paraná and Paraguai River basins to understand how local and biogeographical factors affect the assembly of fish communities. We also quantified taxonomic and phylogenetic beta diversity by decomposing them into nestedness and turnover components. We found that local environmental factors are the main factors influencing the composition of stream fish communities. Whereas pH affected both taxonomic and phylogenetic turnover, water velocity was responsible for phylogenetic turnover and pH was the main driver of phylogenetic nestedness. Our results indicate an effect of local environmental factors in determining the structure of headwater stream fish communities through a combination of a species sorting mechanism (water velocity and pH) and phylogenetic habitat filtering (pH).(AU)

Padrões de substituição de espécies ou diferenças de riqueza ao longo de gradientes ambientais ou ecoregiões lançam luz sobre diferentes processos e mecanismos ecológicos atuando na estruturação das comunidades. Supõe-se que comunidades aquáticas pertencentes a diferentes bacias pertençam a linhagens evolutivas distintas. Quantificar e entender o grau em que tais diferenças são resultado de fatores ambientais locais e/ou processos biogeográficos ainda é uma questão pouco explorada. Neste estudo nós investigamos os padrões de composição taxonômica e filogenética em riachos de cabeceira das bacias dos Rios Paraná e Paraguai, para entender como fatores locais e biogeográficos afetam a estruturação das comunidades de peixes. Nós quantificamos a diversidade beta taxonômica e filogenética decompondo estas em aninhamento e substituição. Encontramos que os fatores ambientais locais são os principais determinantes da composição das comunidades de peixes destes riachos. Enquanto o pH afetou tanto a substituição de linhagens e de espécies, a velocidade da água foi responsável por uma substituição de linhagens, enquanto o pH foi o principal responsável pelo aninhamento de linhagens. Nossos resultados indicam a importância dos fatores locais através da combinação entre mecanismos de preferência de nicho (velocidade da água e pH) e filtragem ambiental de linhagens (pH).(AU)