The objective of this study was to identify the effect of application Dithiothreitol within Intracytoplasmic Sperm Injection (ICSI) process. Semen sample from commercial bovine frozen sperm producer were used in this study. For the treatments, we used 2 mM, 5 mM and 20 mM concentration of DTT to treat the sperm and 3 incubation lengths,30, 60 and 120 min.Our study was corroborating to previous study that DTT affect to sperm decondensation is depending on its concentration and the length of incubation (P<0.05). The results showed that the biggest sperm swelling in the 20 mM for 120 min (49.19±3.63)μm2, and gradually smaller in the lower concentration and quicker incubation time. Sperm DNA fragmentation in the sperm treatment was detected by Acridine Orange method. Although accuracy of this method is still questionable, however reliable method for measuring DNA fragmentation in sperm using Acridine Orange (AO) test was successfully developed. In this study we measure the red fluorescence intensity of each sperm then classified them into 6 scores in 3 categories. We found that the DTT treatment has significant correlation to sperm DNA fragmentation, higher concentration excessively damaging sperm DNA (P<0.01, r= 0.962). However, the quantity of the high fragmented sperm DNA is not significantly affect to the cleavage rate of bovine embryo produced by ICSI (P>0.05, R=-0.158).To find out the causes of embryo fragmentation in bovine 2-cell embryo, we revealed that sperm DNA fragmentation level caused by DTT is significantly correlated (P<0.01, R= 0.591) to the incidence of 2-cell stage embryo fragmentation in the first day culture. In the last experiment, to get the differences between treatments to the morula stage of bovine embryo, we found that the highest rate (55.88%) achieved by the third treatment which is sperm treated with 5 mM of DTT in a 37.5% fragmented sperm. In this case, sperm DNA Fragmentation level seems likely has a maximum limit of quantity which is still in the acceptable level to the developmental competence of bovine embryo.Our results suggest that using Dithiothreitol (DTT) as reducing agents to induce decondensation in the bovine sperm head within ICSI process need to be consider, in order getting the normal sperm DNA, which affect to successful development of ICSI embryo.