Oocytes can either be recovered just prior to ovulation from the large dominant follicle each cycle or from the smaller subordinate follicles at any time.   Most commonly we collect from both the dominant and subordinate follicles at the same time each cycle.  A typical mare will have one dominant and 7-8 subordinate follicles each cycle.  The average oocyte collection rate is about 75% per follicle for the dominants and 60-70% for the subordinates giving an average yield of .7 oocytes from the dominant follicle and 5 from the subordinates per cycle.  This is highly variable from mare to mare, as some mares have many subordinate follicles and some quite few; older mares tend to have somewhat fewer subordinate follicles.  The oocyte from the dominant follicle is usually of higher quality but since the oocytes from the subordinate follicles are generally more numerous approximately 2/3 of the pregnancies produced come from these oocytes from the small follicles.

Oocytes are generally recovered as a cumulus-oocyte complex, an oocyte surrounded by the cumulus cells that nourish it, dislodged from the follicle wall.  Initially as a follicle grows, the cumulus cells are tightly adhered to the follicle wall and the mass of cells itself is very dense; the cells are very close together.  As the follicle grows and becomes a dominant follicle the cumulus mass starts to expand as the cells secrete an extracellular matrix that spreads them apart.  Nearing ovulation this expansion makes the attachment to the follicle wall weaker and weaker so that during ovulation the oocyte can be swept from the follicle into the oviduct.  The loosening of the attachment makes the oocyte somewhat easier to recover nearing ovulation, although as the follicle enlarges it is more difficult to create the turbulence necessary to dislodge the oocyte.  For these reasons, aspiration of the immature follicles with their firmly attached cumulus-oocyte complex, tend to have the highest recovery rate when they are between 10 and 20 mm in size, while the dominant follicles are best aspirated less than 14 or 15 hours before an expected ovulation.

Individual sperma are selected under the microscope and immobilized by crushing the tail with the injection pipette.  Once in the oocyte, this damage to the cell membrane covering the tail will facilitate the passage of “sperm factor” into the oocyte.  Sperm factor is a chemical that is critical in activating the oocyte to start the process of cell division.

For the ICSI procedure itself suction on the holding pipette is used to position and stabilize the oocyte.  The oocyte is positioned so that the injection will take place away from the polar body (extruded excess genetic material but near the genetic material still in the cell) and into an area of good visualization if possible.

Once the pipette enters the ooplasm (cytoplasm of the oocyte) some ooplasm is aspirated into the pipette to assure that the oolemma (oocyte cell membrane) has been completely penetrated and to help with oocyte activation.  The oolemma is very elastic and can be difficult to puncture, even with the pipette inserted well into the oocyte.  In this video there are two oocytes injected.  In the first, the oolemma was pierced by the pipette itself and can be seen sliding back into its original position before the sperm was injected.  The oolemma did not rupture spontaneously in the second oocyte so aspiration was used to pierce it.  The oolemma can be seen being aspirated into the pipette before it ruptures and allows aspiration of ooplasm and the ejection of the sperm.