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.