The study aimed to evaluate the influence of semen collection frequency at different protocols on deleterious the interaction between the enzymes of the bulbourethral gland and egg yolk during the dilution and of buck semen. Five bucks aged 2.5-3 years were included in this study. Four different protocols of semen collection frequency were applied; once a week, twice a week at an interval of two days, thrice a week at an interval of one day, and twice daily at an interval of one hour. Buck semen ejaculates were evaluated using computer assisted sperm analysis that includes the percentage of motile sperm (MS), percentage of progressive motile sperm (PMS), velocity curvilinear (VCL), velocity average path (VAP), velocity straight line (VSL), linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head movement (ALH), and beat cross frequency (BCF). Results showed that sperm motility parameters had decreased significantly after dilution. Sperm motility parameters (VCL, VSL, VAP and ALH) of the second ejaculate were higher significantly (P<0.05) after dilution, when ejaculates collected twice and thrice a week and twice a day. Conversely, motility parameters of third ejaculates were less than those of first and second ejaculates when ejaculates collected thrice a week. In conclusion, there are effects of semen collection frequency on the ability of buck semen dilution, and the best results were when ejaculates had collected twice a day at an interval of one hour, accordingly we recommended this protocol for preparation of buck semen for artificial insemination.