Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male.

Intracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14-15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.