| 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. |
