373 OOCYTE-ACTIVATING CAPACITY OF BOVINE SPERMATOGENIC CELLS AND ACTIVATION PROTOCOLS FOR INTRACYTOPLASMIC INJECTION OF BOVINE ROUND SPERMATIDS

In vitro spermatogenesis can be applied to generate spermatids or spermatozoa and produce a genetically modified male germ line. Intracytoplasmic injection of the spermatids or spermatozoa is an important technique for effective production of offspring. The objective of this study is to evaluate oocyte-activation capacity of bovine spermatids or spermatozoa and to determine the effective activation treatment for in vitro development of bovine oocytes injected with round spermatids. Cryopreserved testicular spermatogenic cells and cauda epididymal spermatozoa obtained from a 1-year-old Japanese bull were used. In the first experiment, we injected bovine round (ROS) and elongated (ELS) spermatids, or testicular (TES) and cauda epididymal (CES) spermatozoa into mouse oocytes to examine their oocyte-activating capacity. The presence of pronuclei within whole-mounted oocytes was observed 4 h after injection. In the second experiment, we injected similar spermatids and spermatozoa into bovine oocytes without additional activation, and examined cleavage and blastocyst development. In the third experiment, bovine oocytes injected with ROS were activated with 7% ethanol or 5 µM ionomycin for 5 min (1 × Et or 1 × Iono) immediately after injection; some were further activated repeatedly at 3 h after injection (2 × Et or 2 × Iono), and some of these were subjected to 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h after the second activation (2 × Et + DMAP or 2 × Iono + DMAP). Data were analyzed by the chi-square test in all experiments. The vast majority of bovine ROS failed to activate mouse oocytes (activation rate 10%). Activation rates of mouse oocytes injected with bovine ELS, TES, and CES were 61%, 75%, and 91%, respectively. The results suggest that oocyte-activation capacity is acquired during transformation from ROS to ELS. Cleavage and blastocyst rates of bovine oocytes injected with CES (59% and 19%, respectively) were significantly higher ( P < 0.05) than the rates obtained with injections of TES (37% and 9%) and ROS (5% and 0%) without additional activation. However, cleavage and blastocyst rates of bovine oocytes injected with ROS in the groups of 2 × Et + DMAP (80% and 19%) and 2 × Iono + DMAP (76% and 19%) were significantly higher ( P < 0.05) than those in the groups of 1 × and 2 × Et (37% and 2%, 59% and 4%), 1 × and 2 × Iono (10% and 7%, 22% and 4%), or those receiving a sham injection and activated with 2 × Iono + DMAP (43% and 4%). These results demonstrate that intracytoplasmic injection of ROS with repeated Et or Iono activation followed by DMAP treatment is more efficient than single or double Et or Iono activation.