Suitability of the hemi-zona assay for the evaluation of the effect of the length of the equilibration period before cryopreservation.

The aim of this study was to test the suitability of the interspecific hemizona assay (HZA) to predict the fertilizing capacity of bovine sperm after modifying the length of the equilibration period before freezing and thawing. Ejaculates from 10 proven fertile bulls were split after dilution, equilibrated at 4 °C for either 24 h (control sperm = CS) or 6, 48, 72 or 96 h (test sperm = TS) and cryopreserved. Hemizona (HZ) pairs from in vitro matured pig oocytes were used for the heterologous HZA: After thawing and swim-up (1 h) CS and TS were co-incubated with matching HZ (125,000 S/HZ in 25 µL Fert-TALP) for 4 h. Spermatological analyses (progressive motile sperm (PMS), plasma membrane- and acrosome-intact sperm (PMAI), sperm showing a high degree of DNA fragmentation (%DFI)) were performed after 0 and 3 h of incubation after thawing. After an equilibration time of 48 h and 72 h values for PMAI0h were higher (P < 0.05) compared to PMAI0h values of sperm equilibrated for 6 h, and %DFI3h values were higher after 96 h (P < 0.05) compared to 6 h equilibration. Between 12 and 90 TS and 13-97 CS were tightly bound to each HZ, respectively. The mean Hemizona Index (HZI) after a sperm equilibration for 48 h (HZI = 92.3 ± 12.7) or 72 h (HZI = 98.9 ± 16.23) was higher (P < 0.01) than after an equilibration for 6 h (HZI = 73.3 ± 13.93) or 96 h (HZI = 81.3 ± 11.41). The HZI for 96 h equilibration was moderately negatively related to PMS0h and PMS3h (r < -0.35, P < 0.05). Furthermore the HZI for 6 h equilibration was highly negatively correlated with DFI0h (r = -0,46, P < 0.01). On the basis of these results it can be concluded that the hemi-zona assay is a suitable test to detect alterations in the fertilizing capacity of bovine sperm after modifying the equilibration period.

Histone H1 and MAP kinase activities in bovine oocytes following protein synthesis inhibition.

In vitro nuclear maturation is associated with known activity profiles of the M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinases, which are two key regulators of mitotic and meiotic cell cycles. Initiation of meiotic resumption in vitro can be prevented by cycloheximide treatment and after removal of the inhibitor germinal vesicle breakdown takes place nearly twice as fast as in untreated controls. In this study experiments were conducted in order to examine the chromosome condensation status and the dynamics of MPF and MAP kinase activities after cycloheximide treatment (10 microg/ml) of cumulus-enclosed oocytes for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium for various times. Bovine oocytes displayed variations in the degree of chromosome condensation at the end of the inhibitor treatment phase. Following removal of the inhibitor germinal vesicle breakdown occurred after 4-5 h of subsequent culture in inhibitor-free medium. MPF and MAP kinase exhibited low activities during the first 1-3 h following cycloheximide treatment. Increasing levels of enzyme activities were detected 4-7 h following cycloheximide treatment for 17 and 24 h, respectively, and subsequent culture in inhibitor-free medium. The patterns of enzyme activities corresponded with the accelerated nuclear maturation process. It can be concluded that cycloheximide treatment does not lead to a more synchronous course of nuclear maturation and that the activities of both, MPF and MAP kinase are initiated at least 2-5 h earlier in comparison with untreated oocytes.