Altered B cell phenotype and CD27+ memory B cells are associated with clinical features and environmental exposure in Colombian systemic lupus erythematosus patients.

Background: B lymphocytes are dysregulated in Systemic Lupus Erythematosus (SLE) including the expansion of extrafollicular B cells in patients with SLE of African American ancestry, which is associated with disease activity and nephritis. The population of Colombia has a mixture of European, Native American, and African ancestry. It is not known if Colombian patients have the same B cell distributions described previously and if they are associated with disease activity, clinical manifestations, and environmental exposures. Objective: To characterize B cell phenotype in a group of Colombian Systemic Lupus Erythematosus patients with mixed ancestry and determine possible associations with disease activity, clinical manifestations, the DNA methylation status of the IFI44L gene and environmental exposures. Materials and methods: Forty SLE patients and 17 healthy controls were recruited. Cryopreserved peripheral B lymphocytes were analyzed by multiparameter flow cytometry, and the DNA methylation status of the gene IFI44L was evaluated in resting Naive B cells (rNAV). Results: Extrafollicular active Naive (aNAV) and Double Negative type 2, DN2 (CD27- IgD- CD21- CD11c+) B cells were expanded in severe active patients and were associated with nephritis. Patients had hypomethylation of the IFI44L gene in rNAV cells. Regarding environmental exposure, patients occupationally exposed to organic solvents had increased memory CD27+ cells (SWM). Conclusion: aNAV and DN2 extrafollicular cells showed significant clinical associations in Colombian SLE patients, suggesting a relevant role in the disease's pathophysiology. Hypomethylation of the IFI44L gene in resting Naive B cells suggests that epigenetic changes are established at exceedingly early stages of B cell ontogeny. Also, an alteration in SWM memory cells was observed for the first time in patients exposed to organic solvents. This opens different clinical and basic research possibilities to corroborate these findings and deepen the knowledge of the relationship between environmental exposure and SLE.

Construction of a questionnaire to characterise environmental exposures in patients with systemic lupus erythematosus / Construcción de un cuestionario para caracterizar exposiciones ambientales en pacientes con lupus eritematoso sistémico

ABSTRACT Introduction: As the knowledge of systemic lupus erythematosus (SLE) advances, the need to characterise the burden of environmental exposure in patients becomes increasingly relevant. However, there is currently no tool validated to measure such exposure. For this reason, the objective of this work was the construction of a questionnaire-type tool, in order to detail the exposure to multiple environmental factors previously associated with SLE. Methods: The literature was reviewed to identify relevant environmental factors associated with SLE, and the first version of the questionnaire was constructed. After expert review and feedback, the questionnaire was consolidated and applied to 40 patients and 20 healthy controls. Finally, exploratory Rasch analysis was performed to determine the performance of the tool's response function. Results: The tool showed a favourable performance in the exploratory analysis of its psychometric properties. Additionally, it allowed the characterisation of 10 environmental factors and the differences in the frequencies of exposure between patients with SLE and healthy controls. A high co-occurrence of exposures was identified, as most of the patients had positive exposures to three or more factors simultaneously. Conclusion: A practical and easy-to-apply tool has been constructed, with a favourable performance in its psychometric properties. To our knowledge, this is the first tool in the Spanish language for the characterisation of multiple environmental exposures, and constructed explicitly for patients with SLE with a validation analysis. It enabled us to identify that most of the lupus patients were exposed to the sum of three or more environmental factors.

RESUMEN Introducción: Con el avance en el conocimiento del lupus eritematoso sistémico (LES), se hace relevante la necesidad de caracterizar la carga de la exposición ambiental en los pacientes. Sin embargo, en la actualidad no se dispone de un instrumento validado para medirlo. Por consiguiente, el objetivo de este trabajo fue la construcción de un instrumento tipo cuestionario, a fin de detallar la exposición a múltiples factores ambientales previamente asociados a LES. Métodos: Se llevó a cabo una revisión de la literatura para identificar los factores ambientales asociados a LES y se construyó la primera versión del cuestionario. El instrumento se consolidó luego de la revisión y retroalimentación por expertos y se aplicó a 40 pacientes y 20 participantes sanos. Finalmente, se hizo análisis de Rasch exploratorio para determinar el desempeño en la función de respuesta. Resultados: El instrumento mostró un desempeño favorable en sus propiedades psicométricas. Adicionalmente, permitió la caracterización de 10 factores ambientales y sus diferencias en las frecuencias de exposición entre pacientes con LES y participantes sanos. Se identificó una alta co-ocurrencia de exposiciones reflejada en que la mayoría de los pacientes mostró exposición a tres o más factores simultáneamente. Conclusión: Se construyó un instrumento práctico y de fácil aplicación, con un desempeño favorable en sus propiedades psicométricas. Este instrumento, que es el primero para la caracterización de múltiples exposiciones ambientales específicamente diseñado para pacientes con LES, validado y en idioma español, permitió identificar que la mayoría de los pacientes con lupus estuvieron expuestos a la sumatoria de tres o más factores ambientales.

DNA methylation changes on immune cells in Systemic Lupus Erythematosus.

DNA methylation as a process that regulates gene expression is crucial in immune cells biology. Global and gene specific methylation changes have been described in autoimmunity, especially in Systemic Lupus Erythematosus. These changes not only contribute to the understanding of the disease, but also some have been proposed as diagnostic or disease activity biomarkers. The present review compiles the most recent discoveries on this field on each type of immune cells, including specific changes in signalling pathways, genes of interest and its possible applications on diagnosis or treatment.

Embryo development after vitrification of immature and in vitro-matured equine oocytes.

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO2 (20.3 ± 4.6%). Cleavage for MAT CR- CO2 and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO2), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality.

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 µM Resveratrol (CR, C- respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C-VR, CRVR) or without (C-V-, CRV-) 0.5 µM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in CRVR group compared with CRV- group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but CRV- and CRVR groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C-VR) restored GSH content (P < 0.05) to the level found in the CR group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 µM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.

Ascorbic acid-cyclodextrin complex alters the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl-ß-cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC-CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC-CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap-frozen for gene expression analysis by RT-qPCR. Besides, in vitro-produced zygotes were cultured with 100 µM of AC-CD and blastocysts were as well snap-frozen for gene expression analysis. A group without AC-CD (control- ) and other with CD (control+ ) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC-CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC-CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC-CD, while in blastocysts derived from zygote cultured with AC-CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC-CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC-CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.

In vitro embryo production from cumulus-oocyte complexes type II in Bos indicus cattle / Producción in vitro de embriones a partir de complejos cúmulos oocitos tipo II en bovinos Bos indicus / Produção in vitro de embriões de cumulus oócito complexo de tipo II de bovinos Bos indicus

Abstract The morphological selection of cumulus-oocyte complexes (COCs) is an important step for in vitro embryo production. It has been suggested that COC showing signs of atresia have the ability to generate embryos. The objective of this work was to evaluate the effect of COC morphology from Bos indicus animals with signs of early atresia versus no signs of atresia on in vitro embryo production. COC were classified in: Type I (TI): homogeneous ooplasm with ≥ 4 layers of compact cumulus cells (CC) and Type II (TII): granular ooplasm and ≥ 4 layers of CC slightly expanded. The COC were matured in vitro for 24 hours in TCM199 medium and subsequently fertilized in vitro for 18 h. The suspected zygotes were cultured in vitro for seven days in modified SOFaa medium. Embryonic quality was determined by blastomeric count following staining with Hoechst 33342. Student test was used to determine statistical differences for cleavage, blastocyst rate and blastomeric counts between types of COC. The cleavage rate for TI (n = 220) and TII (n = 161) was 88 ± 4% and 89 ± 8% respectively (p> 0.05); embryo development rate was 36 ± 7% and 33 ± 8% (p>0.05) respectively. The blastomeric count for both groups was 101 and 104 cells for TI and TII respectively (n = 10), (p>0.05). These results demonstrate that there is no difference in the quantity and quality of embryos produced in vitro using COC type I or type II, suggesting that both types could be used for bovine in vitro embryo production in Bos indicus cows.

Resumen La selección morfológica de los complejos cúmulo-oocito (COC) es crucial para la producción de embriones in vitro. Se ha sugerido que COC que muestran signos de atresia poseen capacidad de generar embriones. El objetivo de este trabajo fue evaluar el efecto de la morfología de los COC provenientes de animales Bos indicus con signos de atresia temprana y sin signos de atresia sobre la producción de embriones in vitro. Se clasificaron COC obtenidos de ovarios de faenado en dos grupos: Tipo I (TI): ooplasma homogéneo con ≥ 4 capas de células del cumulo (CC) compactas y Tipo II (TII): ooplasma granular y ≥ 4 capas de CC ligeramente expandidas. Los COC fueron madurados por 24 horas en medio TCM199 y posteriormente fueron fertilizados in vitro durante 18 h. Los presuntos cigotos se cultivaron in vitro por siete días en medio SOF modificado. La calidad embrionaria se determinó por conteo de blastómeras posterior a tinción con Hoechst 33342. Se usó la prueba t para determinar diferencias estadísticas. La tasa de clivaje para los COC, TI (n=220) y TII (n=161), fue 88 ± 4% y 89 ± 8% respectivamente (p>0,05); la tasa de desarrollo embrionario fue 36 ± 7% y 33 ± 8% (p>0,05) respectivamente. El conteo de blastómeras para ambos grupos fue de (TI:101,TII:104) (n=10), (p>0,05). Los resultados de este trabajo permiten concluir que no hay diferencia en la cantidad y calidad de embriones producidos in vitro utilizando COC tipo I o tipo II, sugiriendo que ambas calidades podrían ser usadas en la producción de embriones in vitro a partir de animales Bos indicus.

Resumo A seleção morfológica dos complexos cumulus-oócito (COC) é crucial para a produção in vitro de embriões. Relata-se que COC que apresentam sinais de atresia têm a capacidade de gerar embriões. O objetivo deste estudo foi avaliar o efeito morfológico dos COC de animais Bos indicus com sinais de atresia precoce ou sim sinais de atresia sobre a produção in vitro de embriões. COC obtidos de ovários de animais abatidos foram classificados em dois grupos: tipo I (TI): ooplasma homogéneo com ≥ 4 camadas de células do cúmulos (CC) compactos e Tipo II (TII): ooplasma granular e ≥ 4 camadas CC levemente expandidas. Os COC foram submetidos à maturação por 24 horas em meio TCM199 e depois fertilizados in vitro por 18 h. Os prováveis zigotos foram cultivados in vitro por sete dias em meio SOFaa modificado. A qualidade embrionária foi determinada pela contagem dos blastômeros após coloração com Hoechst 33342. O teste t foi utilizado para determinar diferenças estatísticas significativas. A taxa de clivagem para os COC TI (n = 220) e TII (n = 161) foi de 88 ± 4% e 89 ± 8%, respectivamente (p>0,05); a taxa de desenvolvimento embrionário foi de 36 ±7% e 33 ±8% (p>0,05), respectivamente. E a contagem de blastômeros para ambos os grupos foi (TI: 101, TII: 104) (n = 10) (p>0,05). Com base nos resultados deste trabalho conclui-se que não existe diferença na quantidade e qualidade dos embriões produzidos in vitro, utilizando COC tipo I ou tipo II, sugerindo que ambas as qualidades podem ser utilizadas na produção in vitro de embriões em animaes Bos indicus.

Exposure to cigarette smoke causes DNA damage in oropharyngeal tissue in dogs.

More than 40 mutagenic and carcinogenic agents present in cigarette smoke have been identified as causative factors of human cancer, but no relation has been clearly documented in companion animals. In dogs, in addition to smoke inhalation and transdermic absorption, exposure to smoke includes oral ingestion of particles adhered to the animal's fur. This study evaluates the presence and type of histological alterations and DNA integrity in oropharyngeal tissue in dogs exposed and non-exposed to household cigarette smoke by means of histopathology and comet assay studies on biopsy and swab samples. A non-probabilistic convenience sample of 12 dogs were selected and classified in two groups: exposed and non-exposed to cigarette smoke. Non-parametric Kruskal-Wallis test was carried out on biopsy and swab data and a Chi(2) test was performed on the information obtained by histopathology. A significance level was set at P<0.05. Statistically significant differences were found between groups in comet assays carried out on biopsy samples. No differences (P>0.05) were found between groups based on comet assays swab samples and histopathology assessment. In conclusion, exposure to cigarette smoke causes DNA damage in dog oropharyngeal tissue. The use of dogs as sentinels for early DNA damage caused by exposure to environmental genotoxic agents like cigarette smoke is reported for the first time.

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle.

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.

Perfil hormonal del envejecimiento muscular / Hormonal profile of muscle aging

El concepto de sarcopenia implica la pérdida de la masa y la fuerza musculares asociadas con el envejecimiento. Se produce por múltiples factores, como el daño oxidativo, pérdida de unidades motoras alfa de la médula espinal, disminución de la calidad y masa musculares, descenso de hormonas anabólicas como dehidroepiandrosterona, testosterona, estrógenos, hormona del crecimiento, factor de crecimiento ligado a la insulina tipo IGF-1, hormona paratiroidea e insulina, así como aumento de diferentes interleuquinas y del cortisol, inactividad física voluntaria y dieta hipocalórica. En esta revisión analizamos el papel de algunas hormonas que afectan el balance dinámico entre estímulos anabólicos y catabólicos en el músculo y la relación entre estos cambios hormonales y el desarrollo de sarcopenia.

The concept, sarcopenia, implies the loss of muscle mass and strength with aging. It is produced by multiple factors, such as oxidative damage, loss of alpha motor units of the spinal medulla, reduction of muscle quality and mass, reduction of anabolic hormones such as dehydroepiandrosterone, testosterone, estrogens, human growth hormone, insulin like growth factor IGF-1, parathyroid hormone and insulin, as well as increase in various interleukins and cortisol, voluntary physical inactivity and a hypocaloric diet. In this review we discuss the role of some hormones which affect the dynamic balance between the anabolic and catabolic stimuli in the muscle and the relation of these hormonal changes with the development of sarcopenia.

Simplificación de la fertilización de ovocitos durante la producción in vitro de embriones bovinos / Simplificação da fecundação de oócitos durante produção in vitro de embriões de bovinos / Simplification of oocytes fertilization during in vitro production of bovine embryos

El objetivo de este estudio fue evaluar un nuevo procedimiento de fertilización in vitro (FIV) de ovocitos bovinos, en el cual se omitió la centrifugación de los espermatozoides para los procesos de lavado y selección espermática. Dentro de cada uno de los pozos de los platos convencionales de cuatro pozos (pozos externos) se introdujo un nuevo pozo más pequeño (pozos internos) de aproximadamente 50 μl hecho en material de vidrio (0.3 mm de alto por 0.8 mm de diámetro). El procedimiento consistió en llenar los pozos interno y externo con 700 μl de medio de fertilización, hasta que el pozo interno quedara cubierto de medio, luego se depositaron 10 CCOs por pozo interno; luego 30 μl de semen previamente descongelado se depositaron en el fondo del pozo externo. Después de una hora se removió todo el contenido del pozo externo y se dejó el contenido del pozo interno con el medio y las células germinales por un periodo de incubación de 16 h. Los presuntos cigotos fueron cultivados en medio CR1aa por un periodo de siete días con un cambio de medio a las 72h. Como control, se utilizó un gradiente de Percoll 45-90% para la selección espermática. La tasa de de ovocitos divididos fue de (70.8 vs. 73.1) y la proporción de ovocitos que llegaron al estadio de morúla y blastocisto fue de (18.8 vs. 20.3), respectivamente. Los resultados obtenidos con la nueva propuesta metodológica son similares a los obtenidos con la metodología convencional (p<0.05). Estos resultados indican que ambos métodos producen resultados similares pero la nueva propuesta metodológica permite ahorrar tiempo, es menos laboriosa, consume menos reactivos y permite reducir la manipulación de las células espermáticas con buenos efectos eventuales en el mejoramiento de los procedimientos de reproducción in vitro.

This study was conducted to evaluate an in vitro fertilization (IVF) procedure that avoids the washing-selection-centrifugation of spermatozoa prior to coincubation with oocytes. A special dish was constructed as follows: in the middle of each of the 4-wells of a conventional plastic dish (larger wells), another little (≈50 μl) glass dish (inner well) was placed (0.3 mm-tall x 0.8 mm in diameter), in such a way that when the larger plate was filled with 700 μl of IVF media (Fert–TALP), the inner well was also filled up to overflowing. Ten in vitro matured oocytes were set into the inner well, and 30 μl of thawed semen were placed on the bottom of the outer well. After one hour of incubation, the media from the outer well was removed, and the well with the remaining media and the germ cells, were incubated for 16 hours. Presumptive zygotes were cultured in a CR1aa media for seven days with a culture media replace at 72 h. As a control, a conventional IVF, using the classical 45%-90% Percoll gradient selection, was carried out. The cleavage rates (70.8 vs. 73.1) and the proportion of oocytes reaching morula and blastocyst stages (18.8 vs. 20.3) by the “easy-IVF” were similar to the results obtained by the classical method (p<0.05). These results indicate that the two methods yield similar results but the “easy-IVF” definitely saves time, human effort, reagents, and after all reduces the manipulation of the sperm with eventual good effects in the long run toward improving in vitro reproduction procedures.

O objetivo deste estudo foi avaliar um novo procedimento de fertilização in vitro (FIV) de oócitos bovinos, que foi omitida na centrífuga esperma lavar os processos de seleção e de esperma. Dentro de cada um dos poços pratos convencionais quatro poços (poços externa) um novo bem menor (poços internos) cerca de 50 μl fato em vidro (0,3 mm de altura por 0,8 mm de diâmetro) foram introduziu. O procedimento foi de encher poços interna e externamente com 700 μ l meio de fertilização, até que a fossa foi coberto por meios internos e, em seguida, depositadas 10 CCOs em poços internos; então 30 μl previamente descongelados sêmen foi depositado no fundo da cova fora. Depois de uma hora é retirada a todo o conteúdo da cova, e foi deixada fora da doméstica no conteúdo do bem com o ambiente e células germinativas por um período de incubação de 16 h. A alegada zygotes foram cultivados em meio CR1aa por um período de 7 dias com uma mudança no meio-72h. Como um controle, utilizado uma gradiente de Percoll 45-90% de selecção de espermatozóides. A taxa de oócitos foi dividida (70.8 vs 73.1) e da proporção de oócitos que atingiu o estádio e foi Mórula blastocisto (18.8 vs 20.3), respectivamente. Os resultados obtidos com a nova metodologia proposta são semelhantes aos obtidos com a metodologia convencional (p<0.05). Estes resultados indicam que ambos os métodos produzem resultados semelhantes, mas a nova proposta metodológica economiza tempo, é menos trabalhoso, consome menos reativo e reduzem a manipulação de células espermáticas com bons potenciais efeitos na melhoria dos procedimentos reprodução in vitro.

Efecto de la centrifugación sobre la membrana plasmática y el ADN de espermatozoides bovinos / Efeito da centrifugação sobre a membrana plasmática e o DNA de espermatozóides bovinos / Effect of centrifugation on plasma membrane and DNA of bovine spermatozoa

El objetivo de este estudio fue determinar si la integridad de la membrana plasmática y el ADN de los espermatozoides pueden ser afectados por la centrifugación realizada en el proceso de lavado y selección. Los espermatozoides fueron sometidos a diferentes tiempos de centrifugación (10, 30 y 45 min); se utilizó un control negativo con espermatozoides no centrifugados y un control positivo con espermatozoides sometidos a estrés oxidativo con peroxido de hidrógeno (H2O2) (200 μM). Para evaluar la integridad de la membrana se utilizó la prueba hipoosmótica (HOST) y para evaluar la fragmentación del ADN se utilizó el ensayo cometa: El análisis estadístico se realizó mediante la prueba de Fisher. La centrifugación durante 10, 30 y 45 min, presentó un efecto estadísticamente significativo sobre el daño en el ADN, respecto de los espermatozoides no centrifugados (p<0.05). Además, se observó diferencia estadística significa (p<0.05) entre los espermatozoides centrifugados a diferentes tiempos con respecto al control positivo realizado con H2O2 (200 μM). La comparación de medias no indicó diferencia estadística significativa entre los espermatozoides no centrifugados y los centrifugados por un periodo de 10 y 30 min (p>0.05) en la reacción positiva a la prueba HOST, lo cual sí sucedió (p<0.05) al comparar los no centrifugados y los centrifugados por 45 min. El control positivo realizado con H2O2 presentó diferencia significativa (p<0.05) con el resto de los tratamientos. En conclusión, los resultados del presente trabajo sugieren que la centrifugación de los espermatozoides durante 10, 30 ó 45 min a 700 x g para la realización del gradiente diferencial de Percoll, tiene un efecto deletéreo sobre el ADN de los espermatozoides bovinos y que la centrifugación por 45 min además de causar daño en el ADN produce pérdida de la integridad de la membrana plasmática.

O objetivo de este estudo foi determinar se a integridade da membrana plasmática e o DNA dos espermatozóides podem ser afetados pela centrifugação realizada no processo de lavado e seleção. Os espermatozóides foram submetidos a diferentes tempos de centrifugação (10, 30 e 45 min); foi utilizado um controle negativo com espermatozóides não centrifugados e um controle positivo com espermatozóides submetidos a estresse oxidativo com peróxido de hidrogênio (H2O2) (200 μM). Para avaliar a integridade da membrana foi utilizado o teste hipoosmótica (HOST) e para avaliar a fragmentação do DNA Fo utilizado o ensaio cometa: A análise estatística se realizou mediante o teste de Fisher. A centrifugação durante 10, 30 e 45 min, apresentou um efeito estatisticamente significativo sobre o dano no DNA, em relação aos espermatozóides não centrifugados (p<0.05). Além disto, observou-se diferença estatisticamente significativa (p<0.05) entre os espermatozóides centrifugados a diferentes tempos em relação ao controle positivo realizado com H2O2 (200 μM). A comparação de medias não detectou diferença estatisticamente significativa entre os espermatozóides não centrifugados e os centrifugados por um período de 10 e 30 min (p>0.05) na reação positiva à prova HOST, o qual ocorreu ao comparar os não centrifugados e os centrifugados por 45 min (p<0,05). O controle positivo realizado com H2O2 apresentou diferença significativa (p<0.05) quando comparado contra os outros tratamentos. Em conclusão, os resultados do presente trabalho sugerem que a centrifugação dos espermatozóides durante 10, 30 ou 45 min a 700 x g para realização do gradiente diferencial de percoll, têm um efeito deletério sobre o DNA dos espermatozóides bovinos e que a centrifugação por 45 min além de causar dano no DNA produz perda da integridade da membrana plasmática.

The aim of this study was to evaluate the effects of different times of centrifugation on plasma membrane integrity and DNA of bovine sperm cells, by means of the hyposmotic test (HOST) and the comet assay, respectively. The sperm cells were centrifuged at 700 x g for 10, 30 or 45 min. No-centrifuged thawed semen served as negative control whereas hydrogen peroxide (H2O2) (200 mM) treated sperm cells were used as a positive control. The results indicate that while the integrity of the plasma membrane was not affected by centrifugation, bovine sperm DNA was damaged independently of centrifugation times. Significant differences between negative control and treatments were found (p<0.05) and in the same way, between treatments and positive control, where the damage for oxidative stress was greater. These results indicate that centrifugation could be detrimental for in vitro bovine embryo production. Additionally, some grade of DNA fragmentation in not centrifuged sperm cells (negative control) was registered, suggesting that DNA of bovine sperm cells could be affected by other factors, probably freezing procedure.

El Ensayo Cometa: una técnica para evaluar genotoxicidad en el ADN de oocitos bovinos / The Comet Assay: a technic to evaluate genotoxicity in DNA bovine oocytes

La electroforesis en gel de células individuales o Ensayo Cometa

Actividad antioxidativa de algunos extractos vegetales / Antioxidant activity of some vegetable extracts

La información acerca de extractos vegetales con actividad antioxidativa tiene gran interés para la medicina, las industrias farmacéutica y de alimentos. En este trabajo es evaluada la capacidad antioxidativa de varios extractos vegetales utilizando dos métodos: la decoloración del radical libre estable 1,1-difenil-2-picril hidracilo (DPPH) y la inhibición de la lipoperoxidación de las biomembranas de cerebro de rata. Se encuentra que los extractos activos en la decoloración del DPPH inhiben también la lipoperoxidación de biomembranas inducida por Fe2+-ascorbato, pero no se observa una correlación alta entre la decoloración del DPPH en el medio metanólico y la inhibición de la lipoperoxidación de biomembranas en el medio acuoso. Además, se muestra que los extractos difieren no solamente por la cantidad del material antioxidativo, sino también por las características cinéticas de la reacción de los antioxidantes presentes con radicales libres. Para dos extractos de mayor actividad antioxidativa, se evalúa la cito- y genotóxicidad, mostrando que no poseen efectos tóxicos considerables.