HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES
Fig. 7. Oocyte just prior to sperm injection. The polar body can be seen at the twelve o’clock position of the oocyte.
4. Sperm Processing
Equine embryos have successfully been produced using spermatozoa from semen sources of varying degrees of quality and processing styles, even in- cluding dead sperm and lyophilized sperm.4 Most commonly, spermatozoa derived from cryopreserved semen are used for the ICSI procedure. Since only a small number of spermatozoa are needed, a small portion of the frozen straws is cut while under liquid nitrogen. The cut portion of the straw is placed directly into a thawing medium at 38°C. The
thawed semen is further processed by either wash- ing and centrifugation or a swim-up, allowing sper- matozoa to swim up from the bottom of the tube for 15 minutes. Two microliters of sperm suspension, collected either from the centrifuged sample or from immediately above the straw in the swim-up tube, placed in a 5-L drop of a polyvinylpyrrolidone (PVP) solution under mineral oil on the ICSI manip- ulation plate. The water soluble polymer PVP’s high viscosity slows the sperm cells down, aiding in their evaluation and manipulation and eases the sperm injection process. Prior to sperm cell selec- tion, the sharp injection pipette is rinsed in PVP solution to decrease adhesion of sperm to the pi- pette. The injection pipette is lowered into the PVP solution containing the sperm sample, and after an evaluation of the sperm population, a single sperm cell is chosen subjectively based on its vigor and morphology. The selected sperm cell is immobi- lized by dragging the injection pipette over the tail of the sperm, essentially trapping it between the pi- pette and the bottom of the dish. After immobili- zation of the sperm, the bevel of the injection pipette is used to fracture, but not completely sever, the tail in mid-shaft. Fracturing of the sperm tail breaks the cell membrane of the sperm, aiding in release of cytosolic sperm factors which are important in oocyte activation.5 The sperm cell is loaded broken tail first into the injection pipette and the pipette is then moved to the droplet containing the oocytes (Fig. 6).
Fig. 8. The sperm cells can be visualized in this oocyte after injection. AAEP PROCEEDINGS Vol. 65 2019 367
