Found this....
The opening can be made anywhere in the shell that effects viable fertilization, but is typically made in an area of the shell that is near the germinal disc. While an egg may be manipulated so as to place the germinal disc at different regions of the egg, the germinal disc in a newly oviposited egg is located at the large end of the shell, which overlies the air cell adjacent to the yolk. Once an opening has been created in the shell, the sperm sample is preferably delivered by introducing the needle, pipette, etc., through the air cell and beneath a membrane lying below the air cell (inner shell membrane). The sperm number can be increased or decreased, depending on where and in what form the sperm are administered. In a further preferred embodiment, the sperm sample is delivered into the egg using a needle. In nature, the sperm cells must penetrate the inner perivitelline membrane and fuse with the oolema for successful fertilization to occur. With IOF, the sperm cells must also penetrate the outer perivitelline membrane before successful fertilization can occur. To increase the fertilization efficiency, one can treat the OPL. Any treatment which rendered the OPL or yolk membrane more permeable to sperm could be utilized, for example, a non-toxic acid, a proteolytic enzyme or physical abrasion.
In one embodiment, the needle, pipette, etc., is advanced through the shell at an angle of approximately 15°, penetrating the membrane lining the shell. In a method of the invention, the needle, pipette, etc., can be advanced through the air cell, until it meets the inner shell membrane. A person practicing the method of the invention will know that the tip of the needle, pipette, etc., has encountered the membrane when slight resistance to further advancement of the tip is felt. As the tip is gently advanced, the resistance from the membrane gives way and the tip is allowed to barely penetrate the membrane. The sperm sample can then be delivered into the egg, adjacent to a region of the membrane and that is adjacent to the germinal disc. Therefore, the sperm can be delivered just under the membrane, a process known as intracytoplasmic sperm injection (ICSI). Typical volumes of the sperm sample are as small as 0.005 ml or as large as 0.10 ml. A typical volume of injected sperm sample is about 0.01 ml.
Preferably, to prevent contamination of the egg and death of an embryo, the opening in the shell is sealed. A non-toxic adhesive can be applied directly to the opening in the shell to seal it. Alternatively, a piece of eggshell can be used as a patch to close the opening and may be attached to the shell with a non-toxic adhesive. In one embodiment, the non-toxic adhesive is Elmer's
glue. In another embodiment, the adhesive is silicone sealant. Moreover, any tissue glue can also be used to seal the shell. A tissue glue is a sterile, non-toxic adhesive used during surgical, operative procedures to bind tissues together.
