Abstract
In Japan, low availability of equine oocytes makes it very difficult to improve the in vitro fertilization (IVF) system of horses. In this situation, an assay for sperm penetrability into zona-free hamster oocytes could be useful as a simulator of the IVF system. The aim of this study was to clarify the optimum conditions of the pretreatment of frozen-thawed stallion spermatozoa with caffeine and ionophore A23187 (IA) in the penetration assay. Frozen-thawed stallion spermatozoa were washed and then pretreated with IA in modified BO solution (mBO) supplemented with caffeine. After the pretreatment, the spermatozoa were used for the penetration assay with zona-free hamster oocytes. At first, effect of increasing concentrations of caffeine (0-10 mM) were examined on the penetration of spermatozoa pretreated with 1 μM IA for 60 sec. The highest penetration rate after 10 h co-culture with oocytes was obtained for spermatozoa penetrated in the presence of 1 mM caffeine (49.0%). Next, when spermatozoa were pretreated with 0-1.0 μM IA for 0-180 sec in the presence of 1 mM caffeine, significantly higher penetration rates after 10 h co-culture with oocytes were obtained under the condition of 0.1 μM IA for 120 sec (52.2%) and 1.0 μM IA for 60 sec (40.9%). When oocytes were fixed 2, 4, 6, 8 and 10 h after IVF using spermatozoa treated with 0.1 μM IA for 120 sec in mBO containing 1 mM caffeine, the penetration rates increased significantly from 2 h (10.4%) to 4 h (21.7%) and from 6 h (27.5%) to 8 h (49.0%). These results indicate that pretreatment with 0.1 μM IA for 120 sec in the presence of 1 mM caffeine effectively promotes the penetration of frozen-thawed stallion spermatozoa into zona-free hamster oocytes, and that under this condition, spermatozoa might complete the acrosome reaction during 6 to 8 h of incubation after IA treatment.
