Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献   

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and rhodamine 123; protocol 2: PI, FITC‐PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC‐PSA and CMXRos; and protocol 4: PI, H342, FITC‐PSA and JC‐1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility ≥80% and abnormal morphology ≤10%. The semen was diluted in Modified Tyrode’s medium (TALP) (25 × 10⁶ spermatozoa/ml) and split into two aliquots, one sample was flash‐frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash‐frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R² = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R² = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R² = 0.84, 0.93 and R² = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC‐1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.     

Multiple-step melting in two-dimensional hexatic liquid-crystal films

An unexpected three-stage melting transition has been observed in two-dimensional (2D) free-standing liquid-crystal films by in situ electron-diffraction and optical-reflectivity measurements. These data suggest the existence of two phases between the 2D solid and liquid: a hexatic phase and, at a higher temperature, an intermediate liquid phase with hexatic-like positional correlations ( approximately 40 angstroms) but no long-range orientational order. Previous high-resolution heat-capacity measurements have revealed a divergent-like anomaly at the hexatic-liquid transition that sharply contradicts the predictions of 2D melting theories. The observation of an intermediate isotropic phase may alter our understanding of 2D melting and lead to reconciliation between current experiments and theories.     

Effects of Prolonged in vitro Culture and Cryopreservation on Viability,DNA Fragmentation,Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long‐term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.     

Aleutian disease in the ferret

Ferrets inoculated with Aleutian disease (AD)-infective material of mink origin harbored the infective agent for at least 136 days after inoculation. In contrast, hamsters given a similar inoculum of infective material no longer harbored the infective agent at 21 days postinoculation. During the infective period, neither ferrets nor hamsters had any detectable illness. However, in certain ferrets, massive periportal lymphocytic infiltrates were observed. A survey of ferrets from different ranches revealed similar lymphocytic infiltrates only in ferrets raised on a ranch which had AD-infected mink.