348 DEVELOPMENT OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY FOR QUANTIFICATION OF Y-CHROMOSOME-BEARING SPERM IN CATTLE

Abstract

Recent data from our laboratory indicate that increasing the time from AI until ovulation in cattle affects the sex ratio of offspring in favor of females (Pursley et al. 1998 J. Dairy Sci. 81, 2139-2144, and unpublished data). One possible explanation for this phenomenon is that the population of X- and Y-chromosome-bearing sperm (X-sperm, Y-sperm) deviate over time to the detriment of Y-sperm. Thus, at the time of ovulation, fewer Y-sperm would constitute the population available for fertilization. Co-culture of sperm and oviductal cells is one way to test whether a greater percentage of Y-sperm detaches from oviductal cells following prolonged incubation. In order to test this, a reliable high-throughput method to determine numbers of Y-sperm is required. The objective of this study was to develop and validate a quantitative PCR (q-PCR)-based assay to accurately determine numbers of Y-sperm in samples from cattle. Primers were designed to amplify a 57-base pair (bp) portion of the SRY gene (for quantification of Y-sperm) and a 21-bp section of autosomal DNA from the 1.715 satellite (for estimation of total sperm numbers). Standard curves were developed from plasmids containing cloned fragments of the SRY gene and the 1.715 satellite region from which primers were designed. Average R² of 0.9977 and 0.9886 were obtained for the 1.715 satellite and SRY standard curves. Assay of fixed amounts of male genomic DNA revealed between and within assay coefficients of variation of 2.17% and 1.54% for the 1.715 satellite and 8.79% and 9.08% for the SRY assay, respectively. Specificity of the assay for the SRY gene was verified using female and male genomic DNA. Similar copy numbers per unit DNA were obtained for the 1.715 satellite sequence in both male and female DNA samples, but the SRY sequence was undetectable in female DNA samples. Samples of separated semen with known amounts of X- and Y-sperm (donated by XY, Inc., Fort Collins, CO, USA) were used to determine accuracy of the q-PCR assay in quantification of numbers of Y-sperm. DNA was extracted from sorted semen samples containing 16.8%, 32%, 66.5%, and 79% Y-sperm and copies of SRY and 1.715 satellite DNA determined in duplicate samples. Percent recovery of genomic DNA from sorted semen samples was calculated using 1.715 satellite primers and was used to adjust expected number of SRY copies for each sample of sorted semen analyzed. Differences in observed copies of SRY gene versus expected copies of SRY gene were determined by Student's t-test. No differences in observed versus expected copies of the SRY gene were detected for each sample (P > 0.05). In conclusion, an assay that accurately quantifies numbers of Y-sperm was developed and validated. This assay will be utilized in future studies to determine if the proportions of Y-sperm released from cultured oviductal cells deviate over time.