Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

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The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37°C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-μL drops at 37°C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1x10 6/mL capacitated and motile spermatozoa. A significant difference (P<0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 ± 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% Cl) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had <37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 ± 24.29, while between 2 fertile dogs it was 22.38 ± 10.02 (P<0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.