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.
In Experiment 1, we performed intracytoplasmic sperm injection (ICSI) of frozen-thawed epididymal and fresh ejaculated in vitro-capacitated spermatozoa into in vivo and in vitro-matured porcine oocytes. Within each group, oocytes were sperm-injected, sham-injected or served as handling controls. After subsequent in vitro-culture for 48 h the number of unchanged, fragmented und cleaved oocytes was recorded. The best result (14% cleaved after ICSI vs 2 and 0% with the sham injection and handling controls; P < 0.01) was achieved 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-capacitated spermatozoa, followed by a 5 min-exposure to 0 (control), 50 or 100 microM calcium-ionophore. Comparable groups were sham injected or served as handling controls. It became apparent that Ca-ionophore treatment after injection of spermatozoa was ineffective at 100 microM, where at 50 microM a significant reduction in cleavage rate was observed (6 vs 26% with untreated controls, P < 0.01). Fluorescence staining with Hoechst 33342 revealed that in most cases of sperm-injected oocytes that remained unchanged after 48 h of in vitro-culture, sperm heads had not decondensed. Only few oocytes had continued to the pronucleus stage. In this context no favorable effect of Ca-ionophore was to be observed.
Kolbe
T
T
Institute of Animal Husbandry and Genetics, Goettingen, Germany.
Holtz
W
W
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D015925:Cryopreservation / Q000662:veterinary*; D004542:Ejaculation; D004822:Epididymis; D005260:Female; D005854:Germ Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D012662:Semen Preservation / Q000662:veterinary*; D013075:Sperm Capacitation; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D013552:Swine
D000001:Calcimycin