Image Analysis Quantification of Decondensation of Bovine Spermatozoa and PCR Amplification of a Y Chromosome Specific Sequence.
Date of Award
Doctor of Philosophy (PhD)
Dairy Science (Animal, Dairy, and Poultry Sciences)
John E. Chandler
The first objective of this research was to develop a decondensation treatment for bovine spermatozoal DNA that worked consistently across bulls and breeds. The second objective was to use the decondensed DNA for polymerase chain reaction analysis of levels of Y-bearing spermatozoa in different ejaculates and in sex sorted spermatozoa. Decondensation of spermatozoa with dithiothreitol and potassium hydroxide was compatible with in vitro amplification and did not inhibit amplification. The polymerase chain reaction and image analysis were used to differentiate between proportions of Y spermatozoa by measuring fluorescent intensities of electrophoresed, polymerase chain reaction-amplified DNA. There were differences in intensities of amplified DNA between bulls and between ejaculates within bulls. Using the mean intensity measure from the analysis, percent Y-bearing spermatozoa was found to range from 26.5% to 95.5% with an average across all ejaculates of bulls of 50.8%. Intensities of sex chromosome sorted spermatozoa from 5 bulls were measured and differences in fluorescent intensity of DNA of Y-enriched sperm samples were found in 1 of 5 bulls.
Steinholt, Hilde Christina, "Image Analysis Quantification of Decondensation of Bovine Spermatozoa and PCR Amplification of a Y Chromosome Specific Sequence." (1996). LSU Historical Dissertations and Theses. 6218.