The theoretical rate of Y chromosome-bearing sperm (Y-sperm) and X chromosome-bearing (X-sperm) in ejaculate is 50:50 in mammals, therefore the sex ratio of the embryos following fertilization is expected to be 50:50. The sex ratio of embryos produced in vitro, however, is skewed towards either males or females. The primary aim of the present study was to investigate the factors of in vitro fertilization (IVF) that affect hte attachment or binding rate of bovine Y chromosome-bearing sperm (Y sperm rate) to the zona pellucidae (ZPe). Oocytes collected from bovine ovaries were cultured for 21 h in vitro under various conditions. ZPe were collected from the oocytes. After frozen-thawed bovine semen and ZPe were co-incubated, the ZPe were mounted on glass slides. Then, sperm attached or bound to the ZPe were subjected to in situ hybridization with a Y chromosome-specific probe to determine the Y-sperm rate. In a preliminary experiment, the Y-sperm rate of frozen-thawed sperm was 50.2% without deviation from the theoretical rate (50%), and the Y sperm rate in Y sperm sorted semen was 93.4% as expected. In Experiment 1, the effect of the sperm-ZP coincubation period on the Y-sperm rate was investigated. Short coincubation periods (5 min and 8 min) deviated the Y-sperm rate (55.1% and 54.9%) from the theoretical rate, but long coincubation periods (300 min) did not affect the Y-sperm rate (49.2%). In Experiment 2, a combination of preincubation of sperm for 3 h prior to sperm-ZP coincubation and extension of the maturation period to 36 h did not change the skewed Y-sperm rate of coincubation of untreated sperm with ZPe derived from 21-h-matured COCs (53.0%, 53.9%, and 54.5%, respectively). In conclusion, the ability of frozen-thawed sperm to attach to the ZPe during the first 5 min or 8 min of coincubation was slightly higher in Y sperm than X sperm although neither sperm preincubation nor extension of the maturation period affected the Y-sperm rate.