Calving sex ratio as related to the predicted Y-chromosome-bearing spermatozoa ratio in bull ejaculates

Abstract The first objective was to correlate calving sex-ratio data from semen lots with the semen sex ratio obtained by two duplex polymerase chain reaction (PCR)/gel electrophoresis techniques. The two techniques involved different starting DNA amounts, PCR conditions, agarose gel concentrations, sample placement on the gels, lane size, number of lanes per gel, and duration of electrophoresis. The second objective was to sequence the duplex PCR products to verify their match to genes and chromosomes for which they were designed. Thirty-six ejaculates (lots) from eight Holstein sires were collected. Semen straws were distributed among dairies in three states. Ten straws per lot were used for the different PCR techniques. Sperm DNA was extracted and PCR analysis was done using one primer set to amplify a single copy section of the factor IX precursor (X-chromosome only) and another primer set to amplify a single copy section the sex determining region (Y-chromosome only). The glyceraldehyde phosphate dehydrogenase gene was amplified as an internal control. Standard curves were designed using PCR products in known ratios. Gel electrophoresis and image analysis were used to determine predicted %Y-chromosome-bearing spermatozoa (PredPtY). Sex (male = 1, female = 0) was reported on 526 calves and the ratio of the number of male to total calves (proportion of male calves (PMC)) was determined between sire and lot within sire. The PredPtY and PMC were significantly correlated ( r  = 0.82, P