yo-pro-1 and ethidium-homodimer

SummaryThe identification of early changes in the sperm plasmalemma is currently a factor in the improvement of freezing protocols. We analysed the presence of active caspases in freeze-thawed (FT) dog spermatozoa, and evaluated straws from eight dogs using flow cytometry and fluorescence microscopy with fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) combined with ethidum homodimers. Apoptotic-like changes were evaluated using the YO-PRO-1/ethidium homodimer combination, and changes in mitochondrial membrane potential were monitored with JC-1. Sperm motility post-thaw was evaluated using a CASA system. FITC-VAD-fmk stained sperm cells in situ and the subcellular labelling pattern was consistent with known localization of caspases. On average, a high proportion of FT canine sperm showed caspase activity, ranging from 30.2 to 70.7% of the live sperm compared with 7.3 to 24.0% in dead spermatozoa. This observed differentiation between caspase activity in dead and live spermatozoa may be a simple method to disclose subtle differences in sperm quality, since this staining allowed us to find statistically significant differences among dogs. Notably, the sperm sample with overall better results in all sperm parameters studied after thawing had a lower percentage of active caspases in both dead and live spermatozoa.

Topics: Animals; Apoptosis; Benzoxazoles; Caspases; Cryopreservation; Dogs; Ethidium; Flow Cytometry; Male; Mitochondria; Quinolinium Compounds; Semen Preservation; Sperm Motility; Spermatozoa

Sperm morphometric indexes obtained after principal component analysis were used to investigate its value as diagnostic tests for freezability. Semen from six dogs was frozen-thawed following a standard protocol. Before freezing, computer-assisted analysis of sperm morphometry (CASMA) was performed. The principal component analysis (a statistical technique for simplifying a dataset, by reducing multidimensional datasets to lower dimensions for analysis) performed in the data obtained after the CASMA analysis gave four morphometric indexes. After thawing, ejaculates were evaluated for early changes in sperm membranes using the combination of two fluorescent probes, YO-PRO-1 and ethidium homodimer and flow cytometry. Significant differences in the percentages of intact membranes post-thaw were observed among dogs (p < 0.01). Significant non-parametric correlations were found between index 3 and the percentage of intact membranes after thawing (R = 0.432 p < 0.05). Receiving operating system curves demonstrate a good diagnostic value for indexes 2 and 3 in the prediction of freezability, with areas under the curve of 0.798 and 0.786, respectively.

Topics: Animals; Benzoxazoles; Cell Membrane; Cryopreservation; Dogs; Ethidium; Flow Cytometry; Fluorescent Dyes; Image Processing, Computer-Assisted; Male; Predictive Value of Tests; Principal Component Analysis; Quinolinium Compounds; ROC Curve; Spermatozoa

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin-V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF-1, YO-PRO-1 and ethidium homodimer. Four ejaculates from five different boars were frozen-thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin-V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with stable membranes (SNARF-1 positive cells), and three with compromised membranes, one of YO-PRO-1+/Eth- cells, one ethidium homodimer+ spermatozoa and, finally spermatozoa stained both with YO-PRO-1 and ethidium homodimer (YO-PRO-1+/Eth+). The latter three categories corresponded to dead spermatozoa, but with different degree of membrane damage, being YO-PRO+/Eth- an earlier stage of membrane destabilization, (manifested by an increase in membrane permeability, while still maintaining membrane integrity) than YO-PRO+/Eth+. A method agreement analysis between both methods was performed revealing good agreement, although the percentage of live cells was 9.44% larger for the triple stain than the annexin-V assay. The new assay stained all sperm sub-populations present in the sample, making it especially suitable for both fluorescence microscopy and flow cytometry, facilitating the exclusion of debris and egg-yolk particles when using FC.

Topics: Animals; Annexin A5; Benzopyrans; Benzoxazoles; Cell Membrane; Cell Membrane Permeability; Cryopreservation; Ethidium; Flow Cytometry; Fluorescent Dyes; Male; Microscopy, Fluorescence; Naphthols; Quinolinium Compounds; Rhodamines; Semen Preservation; Spermatozoa; Staining and Labeling; Sus scrofa

Using a highly sensitive membrane permeability assay, a viral infection was discovered in the lungs of virus antibody free (VAF) Swiss-Webster mice purchased for respiratory toxicology studies. The assay is based on the uptake of a charged fluorescent compound by cells lacking an intact plasma membrane. Lungs from 74% of the untreated animals from a single vendor tested positive for injury in this assay. High-resolution histopathologic analysis of 1-microm epoxy resin sections from affected animals identified increased peribronchiolar lymphocytic infiltration and markers of epithelial cell injury. Viral particles were directly observed to be budding from the membranes of infiltrating lymphocytic cells by transmission electron microscopy. Standard histological analysis of paraffin-embedded tissues from lungs of the same mice failed to detect obvious pathology. Serological analyses failed to detect the presence of a virus in the affected mice. Therefore, we conclude that (1) a pathogenic condition was present in the respiratory systems of mice judged pathogen free by standard methodologies, (2) the observed condition produced a pattern of injury comparable to those caused by pulmonary toxicants, (3) high-resolution histopathology and advanced imaging techniques can increase the potential for detection of pathological conditions, and (4) apparently healthy animals can have unrecognized infections with the potential for confounding respiratory toxicology studies.

Topics: Animals; Benzoxazoles; Bronchi; Cell Membrane; Cell Membrane Permeability; Ethidium; Fluorescent Dyes; Male; Mice; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Quinolinium Compounds; Respiratory Mucosa; Respiratory Tract Infections; Retroviridae; Retroviridae Infections; Toxicity Tests