Tween-20 transiently changes the surface morphology of PK-15 cells and improves PCV2 infection.

BACKGROUND: Low concentrations of nonionic surfactants can change the physical properties of cell membranes, and thus and in turn increase drug permeability. Porcine circovirus 2 (PCV2) is an extremely slow-growing virus, and PCV2 infection of PK-15 cells yields very low viral titers. The present study investigates the effect of various nonionic surfactants, namely, Tween-20, Tween-28, Tween-40, Tween-80, Brij-30, Brij-35, NP-40, and Triton X-100 on PCV2 infection and yield in PK-15 cells. RESULT: Significantly increased PCV2 infection was observed in cells treated with Tween-20 compared to those treated with Tween-28, Tween-40, Brij-30, Brij-35, NP-40, and Triton X-100 (p < 0.01). Furthermore, 24 h incubation with 0.03% Tween-20 has shown to induce significant cellular morphologic changes (cell membrane underwent slight intumescence and bulged into a balloon, and the number of microvilli decreased), as well as to increase caspase-3 activity and to decrease cell viability in PCV2-infected PK-15 cells cmpared to control group; all these changes were restored to normal after Tween-20 has been washed out from the plate. CONCLUSION: Our data demonstrate that Tween-20 transiently changes the surface morphology of PK-15 cells and improves PCV2 infection. The findings of the present study may be utilized in the development of a PCV2 vaccine.

A scanning electron microscopy study of frozen/thawed dog sperm during in vitro gamete interaction.

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.