381 Production of a transgenic piglet by a new sperm injection technique.

The technique for intracytoplasmic sperm injection (ICSI) has, until now, focused on scoring the tail of the sperm prior to catching and aspiration into the injection pipette. This is in spite of the fact that damage to the head would more closely simulate what occurs during normal fertilization. In addition, to aid in visualizing the injection process so that a reduced volume can be injected, the oocyte is generally centrifuged to clear a portion of the cytoplasm. Thus, with conventional ICSI, the sperm are immobilized with polyvinylpyrrolidone, repeatedly frozen and thawed, treated with DTT or Triton X-100, and severed between the head and tail; the oocyte is centrifuged or activated. All of the above treatments are designed to compensate for the intrinsic defects in conventional ICSI. Our objective was to use a modified ICSI procedure whereby aggressively motile sperm were captured onto the broken tip of an injection pipette and then injected into noncentrifuged oocytes. Damage to the head of the sperm occurred on the pipette or while pushed through the zona pellucida. These procedures are based on the work of Yong et al. 2003 Hum. Reprod. 18, 2390, where they achieved an improvement in development in vitro as compared to conventional methods. Ovaries were collected from prepubertal gilts, and oocytes were aspirated and matured in vitro. Sperm were collected from a transgenic boar carrying the green fluorescent protein (GFP) and frozen. After thawing, aggressively motile sperm were captured and injected through the zona pellucida and into the cytoplasm of the in vitro-matured oocytes. A total of 452 injected oocytes (43-171 oocytes per recipient) were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. GFP expression was observed in the nose and hooves by direct epifluorescent examination of the newborn piglet. This pattern of GFP expression is identical to that in non-ICSI-derived GFP pigs in this line. This result showed for the first time that this new sperm injection technique could be used for production of a viable transgenic piglet using in vitro-matured oocytes and frozen-thawed sperm.