Enhancement of IVF in the mouse by zona-drilling.

Cryopreservation of mouse sperm has become an essential method for the long-term storage of novel, genetically modified mouse lines. Cryopreserved sperm from most hybrid lines can be effectively used for in vitro fertilization (IVF) of mouse oocytes. Unfortunately, IVF recovery with cryopreserved sperm from inbred lines is very inefficient. This is especially troublesome since many transgenic lines are created on the popular C57Bl/6 inbred strain. Cryopreserved sperm from C57Bl/6 inbred and genetically modified lines is generally very inefficient when used in standard IVF recovery experiments, with fertilization rates that can be lower than 10%. Assisted reproductive techniques have been developed to improve the IVF efficiencies of cryopreserved inbred sperm. These techniques include zona-drilling, which introduces a hole into the zona pellucida (ZP) surrounding mouse oocytes, using a chemical solution (acid Tyrode's), mechanical disruption (partial zona dissection or piezo-driven micropipette drilling), or laser photoablation. By allowing direct access of the sperm to the cytoplasmic membrane, zona-drilling can improve the efficiency of IVF fertilization rates with inbred sperm to greater than 90%, thus improving the chances of recovering mouse lines on inbred backgrounds that are maintained with cryopreserved sperm. The technique described in this chapter makes use of a piezo controller to mechanically disrupt the ZP, resulting in dramatic increases in the fertilization efficiency of cryopreserved sperm.