Intracytoplasmic injection (ICSI) of in vivo or in vitro matured oocytes with fresh ejaculated or frozen-thawed epididymal spermatozoa and additional calcium-ionophore activation in the pig

摘要

In experiment 1, frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa were injected into in vivo and in vitro-matured pig oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as controls. After subsequent in vitro-culture for 48 h, the number of intact, fragmented and cleaved oocytes was recorded. The best result (14% of oocytes cleaved after intracytoplasmic sperm injection vs. 2 and 0% with sham injection and controls respectively; P<0.01) wasachieved with fresh in vitro-capacitated spermatozoa injected into in vivo-matured oocytes. In vitro-matured oocytes displayed high fragmentation rates. In experiment 2, in vitro-matured oocytes were injected with freshly ejaculated in vitro-capacitatedspermatozoa, followed by a 5-min exposure to 0 (control), 50 or 100μM calcium-ionophore. Comparable groups were sham injected or served as controls. Ca-ionophore treatment after injection of spermatozoa was ineffective at 100μM. The cleavage rate waslower (P<0.01) at 50μM calcium-ionophore (6%) than in controls (26%). Fluorescence staining with Hoechst 33342 indicated that in most sperm-injected oocytes that remained intact after 48 h of in vitro-culture, sperm heads did not decondense. Only a fewoocytes developed to the pronucleus stage.