Studies on in vitro fertilization and embryo production by intracytoplasimc sperm injection (ICSI) in cattle

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The research was aimed to establish an intracytoplasmic sperm injection (ICSI) in cattle by studying various conditions for spermatozoa and oocytes. For spermatozoa, (1) sperm pre-treatment, (2) sperm immobilization (3) concentrations of polyvinylpyrolidone (PVP), and for oocytes, (1) oocyte maturation period, (2) centrifugation to polarize the ooplasmic lipid, (3) oocyte activation methods, were investigated to prove a hypothesis that oocyte activation after ICSI is not necessary in cattle, and the blastocysts were transferred to recipient heifers to obtain normal calves. The results obtained were; (1) Bull difference varied the outcomes by conventional in vitro fertilization (IVF) and by ICSI. Namely, male pronuclear formation rate was improved by ICSI using bull semen with low IVF rates. (2) There was no significant difference in the male pronuclear formation rate among the sperm types (motile, immotile or dead sperm). Ca ionophore and sperm immobilization enhanced the fertilizability. (3) Oocyte survival and normal fertilization rate were improved by centrifugation, 4% PVP concentration and sperm-tail cutting. The subsequent cleavage and blastocyst development were 72% and 23%, respectively. (4) After transfer 8 blastocysts derived by ICSI without oocyte activation, 4 out of 7 heifers were pregnant and gave 5 normal calves. These results indicate that a bovine ICSI method was been established, and showed that oocyte activation after ICSI is not necessary in cattle.