Changes in the proportion of X- and Y-chromosome bearing sperm attached to oviductal epithelial cells over time

Recent data indicate that increasing the time from insemination to ovulation inversely affects the subsequent sex ratio of calves (Pursley, et al., 1998, Macfarlane, 2003). Differences in X and Y sperm that allow x sperm to survive longer is one possible explanation for this phenomenon. The objectives of the current study were (1) to develop and validate a real-time PCR based assay for the quantification of Y-chromosome bearing sperm and (2) to use this assay to determine if the proportion of Y sperm that remain attached to cultured oviductal cells deviates over time. To develop a quantitative real-time PCR assay, primers were designed to amplify a portion of the SRY gene and a section of autosomal DNA. Standard curves were developed from plasmids containing the cloned fragments of the SRY gene and the 1.715 satellite region. Sperm were co-incubated with in vitro cultured oviductal cells for 2h, 12h, 24h, or 36h. Sperm that remained attached to the oviductal cells after each time-point were removed and percent Y sperm for each sample was determined using the assay. Sperm attached to oviductal cells consisted of 41.01 +/- 1.75% Y sperm at 2h, 48.37 +/- 1.89% Y sperm at 12h, 33.59 +/- 1.49% Y sperm at 24h, and 41.30 +/- 1.59% Y sperm at 36h (mean +/- SEM). Differences between time points were only significant for 12h versus 24h (P < 0.05), indicating more Y sperm became detached during this time.