Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation, to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials, and the potential risk of total loss of the frozen stock. Theoretically it has been supposed that the FD sperm could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator or even ambient temperature. However, since FD spermatozoa after rehydration loose their motility that is essential requirement to complete the physiological fertilization, relatively difficult and specialized technique as ICSI must be applied to the FD sperm. The low efficacy of ICSI technique in cattle constitutes a species-specific obstacle against the successful application of FD bull spermatozoa. The characteristics of FD bull sperm to trigger the early post-fertilization events (induction of calcium oscillations and oocyte activation) and the dynamics of epigenetic reprogramming in the FD sperm-derived pronuclear zygotes (active demethylation of paternal genome) have not been well-understood. Therefore in the present study, the contributions of the FD bull sperm to oocyte activation, paternal genome demethylation and embryonic development were investigated. The earliest function of the sperm cells at fertilization is to induce repetitive increases of calcium ion concentration in the ooplasm, the so-called calcium oscillations. The calcium oscillations are responsible for the initiation and completion of many post-fertilization events. The first indicator for calcium oscillations induced by the sperm is the release of the oocytes from the M-II arrest and the pronuclear formation. In the first series of this study (Chapter 2), bull spermatozoa freeze-dried and then stored for one year at +25, +4 or –196 oC were microinseminated into ovulated mouse oocytes to evaluate their ability for inducing the intracellular calcium oscillations, and into in vitro-matured bovine oocytes to evaluate the resumption of meiosis. The original freezing-drying protocol includes primary dehydration process for 14 h at 0.37 hPa followed by secondary dehydration process for 3 h at 0.001 hPa. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected mouse oocytes, with exception of a few oocytes injected with FD sperm stored at +25 or +4 oC that exhibited a single increase or no response (non-oscillated). The proportion of oocytes oscillated with high frequency (≥10 spikes per hour) was higher in the control oocytes injected with FT sperm than those in the oocytes injected with FD sperm. After homogenous ICSI, the proportion of bovine oocytes resuming meiosis and developing to the pronuclear stage (complete activation) was higher in the control group than those in all the FD groups. 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 oC, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage. Although the routine bovine ICSI technique includes application of exogenous stimuli for oocyte activation, stronger stimuli may be required when the FD bull sperm are used for bovine embryo production by ICSI. In the early stages of embryonic development, the gametic methylation marks are erased (demethylation) and replaced with the embryonic marks that are important for embryonic development and acquirement of toti- or pluripotency. The paternal genome is subjected to replication-independent, genome-wide active demethylation during the first few hours after fertilization while maternal genome maintains its methylation level until the beginning of mitotic division stage when both maternal and paternal genomes undergo replication-dependent, passive demethylation with each mitotic cycle. Although the active demethylation process of the paternal genome is considered to be controlled by a function of an ooplasmic factor(s), this process may be facilitated either by a sperm factor(s) or by male pronuclear chromatin composition. In the next series of this study (Chapter 3), demethylation dynamics of paternal genome in pronuclear-stage bovine zygotes produced by IVF, or intracytoplasmic injection of FT and FD spermatozoa were investigated. The FD sperm were stored for one year at +4 or –196 oC. In conventional IVF-derived zygotes, the overall average of the relative methylation (RM; male/female) significantly decreased from 0.92 at 8 hpi to 0.69 at 10 hpi without any additional decrease at 14 and 18 hpi. This was accompanied with higher proportions of zygotes showing RM<0.6 at 10, 14 and 18 hpi, compared to the proportion at 8 hpi. The overall averages of the RM in the FT-ICSI derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in the corresponding IVF-derived zygotes (8 and 14 hpi), but higher proportion of the 6 hpic zygotes showed RM<0.6 compared to the 8 hpi zygotes. The proportions of the FD-ICSI derived zygotes at 12 hpic showing RM<0.6 were higher than that in the FT-ICSI derived zygotes. Thus, bovine paternal genome rapidly demethylated within 10 h after IVF and 6 h after ICSI, and at least the freeze-drying and/or the storage process have no adverse effect on demethylation of the paternal genome. The demethylation extent in pronuclear-stage bovine zygotes was moderate with 0.4≤RM<0.6. The ICSI technique has been used not only as a research tool for understanding gamete interactions around the fertilization, but also as a practical alternative to conventional IVF in rescuing severe male infertility. In the last series of this study (Chapter 4), in vitro production of bovine blastocysts from FD-ICSI derived zygotes was attempted. The developmental potential of bovine ICSI oocytes into blastocysts in vitro or live calves in vivo is still low, unstable and far from satisfactory regardless of the numerous efforts. Therefore as preliminary parts of this experiment, effects of oocyte centrifugation to ensure sperm injection, sperm pretreatment with DTT to reduce the disulfide bonds, and supplementary regimens for oocyte activation on the blastocyst yields from ICSI oocytes were investigated. Only oocyte activation regimen among these factors contributed to improve the bovine ICSI technique as below. Following ICSI, oocytes were treated with ionomycin, ethanol, ionomycin followed by ethanol, ionomycin followed by CHX, or ionomycin followed by 6-DMAP. Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29–30%) comparing to the non-treated oocytes (12%), but the other regimens did not (9–18%). None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Therefore, the new activation regimen composed from two triggers for single calcium increase (5 μM ionomycin for 5 min immediately after ICSI plus 7% ethanol for 5 min 4 h after ICSI) without using either protein synthesis or protein phosphorylation inhibitors was applied to the oocytes injected with FD sperm. The FD sperm were prepared either by the original 2-setp freezing-drying protocol or the shorter 1-step protocol without the secondary dehydration process, and stored for 1–7 days at +4 oC. The cleavage rates of FD-ICSI oocytes (37–43%) were ~50% lower than that of FT-ICSI oocytes (68%). Regardless the protocol for FD sperm preparation, the blastocyst development rate after FD-ICSI (1–2%) was much lower than that after the control FT-ICSI (21%), thus further improvement of the freeze-drying protocol for bull spermatozoa is required. It remains to be clarified whether the functional damages induced in sperm cells by the suboptimal freezing-drying process occurred at the level of the DNA. In conclusions, freeze-drying protocol employed here slightly reduced the ability of the sperm to induce calcium oscillations and resumption of meiosis, but it had no adverse effect on the active demethylation dynamics of paternal genome during the early stage after fertilization. However, bull sperm freeze-dried by this protocol participated very little into the embryonic development to the blastocyst stage, even after improvement of blastocyst yield from ICSI oocytes by applying exogenous activation stimuli with ionomycin and ethanol. Therefore, the conditions during the freeze-drying process, which were found practical originally in rodents, need to be optimized for bull spermatozoa.