Flow cytometry evaluation of DNA integrity of alpaca sperm after cryopreservation with analogues of superoxide dismutase

Se evaluó la estabilidad del ADN en espermatozoides de alpaca mediante citometría de flujo en muestras congeladas con antioxidantes análogos de superóxido dismutasa (Tempo y Tempol). Doce muestras de semen de alpaca fueron congeladas utilizando un dilutor en base a leche descremada, fructosa, yema de huevo y etilenglicol. Cada muestra fue dividida en tres porciones: grupo control, grupo Tempo (1 mM) y grupo Tempol (1 mM). Los antioxidantes fueron agregados al dilutor durante la curva de enfriamiento (10 °C). Las muestras fueron descongeladas y fijadas en una solución de formaldehido al 2% y permeabilizadas utilizando una solución de Triton X-100 al 0.8%. La integridad del ADN espermático se evaluó con la técnica de TUNEL y una sonda fluorescente para determinar la viabilidad celular (Ioduro de propidio [PI]). Los espermatozoides marcados con la sonda del TUNEL y PI fueron considerados células permeabilizadas con el ADN fragmentado. Las muestras fueron analizadas mediante citometría de flujo utilizando un láser de 488 nm, contando un mínimo de 10 000 células por muestra y utilizando microscopía de fluorescencia. La frecuencia de espermatozoides con ADN fragmentado en el grupo control (38.8 ± 16.2%) fue significativamente mayor (p<0.05) al grupo Tempol (16.7 ± 8.4%), mientras que los resultados del grupo Tempo (25.4 ± 5.8%) fueron estadísticamente similares a los grupos Control y Tempol. Se concluye que la adición del análogo de superóxido dismutasa, Tempol (1 mM), durante el proceso de criopreservación de espermatozoides de alpaca puede prevenir parcialmente el daño al ADN espermático.DNA integrity in alpaca spermatozoa was evaluated by flow cytometric analysis in sperm cryopreserved using antioxidants analogues of superoxide dismutase (Tempo and Tempol). Twelve alpaca semen samples were frozen using an extender based on skim milk, fructose, egg yolk, and ethylene glicol. Each sample was divided into three aliquots: Control group, Tempo group (1 mM), and Tempol group (1 mM). Antioxidants were added during cooling at 10 °C. After thawing, samples were fixed using 2% formaldehyde solution and permeabilizated using 0.8% Triton X-100 solution. TUNEL assay and Iodure propidium (PI) were used for evaluation of DNA integrity and cell permeability. Spermatozoa labeled with TUNEL and PI was classified as cells with DNA damaged. Samples were analyzed by flow cytometric using a 488 nm laser, counting at least 10 000 cells per sample, and by epifluorescene microscopy. Frequency of DNA damaged sperm in control group (38.8 ± 16.2%) was significantly higher (p<0.05) than in the Tempol group (16.7 ± 8.4%), whereas the results in the Tempo group (25.4 ± 5.8) were similar to the control and Tempol groups. It was concluded that the addition of superoxide dismutase analogue Tempol (1 mM) during cooling (10 °C) in alpaca sperm cryopreservation partially prevents sperm DNA damage

Melatonin Scavenger Properties against Oxidative and Nitrosative Stress: Impact on Gamete Handling and In Vitro Embryo Production in Humans and Other Mammals

Oxidative and nitrosative stress are common problems when handling gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because their absence or overproduction causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during in vitro fertilization (IVF) has deleterious or positive effects, depending on the concentration used in the culture medium, demonstrating the delicate balance between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in humans and other mammals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology

Sperm membrane functionality in the dog assessed by flow cytometry

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity staining; phosphatidylserine (PS) translocation by Annexin-V-FITC and PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin ⁄ PI labelling; and mitochondrial membrane potential (DYm) by staining with. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high DYm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translo-cation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = 0.5816;>p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.Fil: Cheuquemán, Carolina. Universidad de La Frontera; ChileFil: Bravo, P.. Universidad de La Frontera; ChileFil: Treulén, Favian. Universidad de La Frontera; ChileFil: Giojalas, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Villegas, Juana. Universidad de La Frontera; Chile. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Sánchez, Raúl. Universidad de La Frontera; Chile. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Risopatrón, Jennie. Universidad de La Frontera; Chile. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; Argentin

Revealing the associated microflora hosted by the globally significant parasite Trichostrongylus colubriformis

Abstract Trichostrongylus colubriformis is a parasitic helminth that primarily infects small ruminants, causing substantial economic losses in the livestock industry. Exploring the microbiome of this helminth might provide insights into the potential influence of its microbial community on the parasite’s survival. We characterised the intestinal microbiome of T. colubriformis that had been collected from the duodenum of sheep, and compared the helminth microbiome with the duodenal microbiome of its host, aiming to identify contributions from the helminth’s environment. At the same time, we explored the isolation of fastidious organisms from the harvested helminth. Primary alpha and beta diversity analyses of bacterial species revealed statistically significant differences between the parasite and the host, in terms of species richness and ecological composition. 16S rRNA differential abundance analysis showed that Mycoplasmoides and Stenotrophomonas were significantly present in T. colubriformis but not in the duodenal microbiome of the sheep. Furthermore, two bacteria, Aeromonas caviae and Aeromonas hydrophila, were isolated from T. colubriformis. Examinations of the genome highlight differences in genome size and profiles of antimicrobial resistance genes. Our results suggest that T. colubriformis carries a specific bacterial community that could be supporting the helminth’s long-term survival in the host’s digestive system