Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette

Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so-called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos.