The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

Abstract 

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or −196°C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4°C (11%) and +25°C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (≥10spikes/h) was higher in the non-dried control group (79%; P<0.05) than in the FD groups (58, 55, and 54% for storage at −196, +4, and +25°C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P<0.05) resumed meiosis than those injected with +25, +4 and −196°C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P<0.05) than those in all the FD groups (34, 27, and 28% for storage at −196, +4, and +25°C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or −196°C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.

Keywords: Bovine spermatozoa, Calcium oscillations, Freeze-drying, Homogenous ICSI, Interspecies ICSI