Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Science

First Advisor

Becky L. Sartini

Abstract

The goal of this dissertation research was to characterize the spermatozoal transcript profile of bovine cryopreserved spermatozoa. The main goal was to sequence the complete transcript profile from a population of sires across a wide fertility range utilizing RNA-Sequencing (RNA-Seq). Sequencing was then conducted to compare the spermatozoal transcript profiles of higher and lower fertility sires in an effort to determine the presence or absence of spermatozoal transcripts that may aid in improving fertility testing in sires. In addition to RNA-Seq analysis, other semen parameters such as morphology, DNA fragmentation, and RNA amount were analyzed for relationships with fertility. The first chapter of this dissertation is a literature review detailing the artificial insemination industry, male fertility, spermatogenesis, spermatozoal RNAs, and RNASeq high-throughput sequencing. The review details what is currently known about the spermatozoal RNA population as well as the potential application of RNA-Seq to advance the knowledge of this spermatozoal RNA population. The second chapter is a manuscript that was published in the journal Biology of Reproduction in January 2013 and was co-first-authored by Elizabeth Anderson. This manuscript was the first study to sequence the spermatozoal transcript profile using RNA-Seq for any species. RNA-Seq analysis of pooled semen from multiple sires sequenced a bovine spermatozoal transcriptome consisting of 6,166 transcripts, including several previously identified and novel candidate transcripts for further functional study. The third chapter of this dissertation is a manuscript being prepared for submission to the Journal of Dairy Science. In this manuscript, the spermatozoal transcript profiles of higher and lower fertile sires was sequenced. Target fertility candidate transcripts were selected on the basis of expression differences between the two populations and then correlation of these spermatozoal transcripts with sire fertility was examined. Appendix 1 consists of unpublished data examining the relationship of sperm RNA amount with semen parameters such as morphology, DNA fragmentation index, motility, and sperm RNA quantity. Sperm morphology analysis was performed by the Parrish lab while DNA fragmentation index analysis was performed by the Evenson lab. This sperm RNA isolation method is the same procedure as described in Appendix One and therefore I was unable to validate this transcript population. Appendix 2 reports an unpublished initial RNA-Seq study done with spermatozoal RNA isolated with a column based method and mRNA amplification that differs from these protocols reported in Chapter 2. I was unable to validate the spermatozoal transcript profile generated with these RNA isolation and amplification methods leading to the development of a second RNA isolation procedure that is reported in Chapter 2. Through this work it is evident that there was much we did not know about the bovine spermatozoal transcript profile but the use of RNA-Seq in this study has helped us understand the population more. This new technology has allowed us to sequence the entire transcript profile while determining what transcripts are full-length. Not only were we able to sequence a general population but we compared lower and higher fertility populations and found many differences between the two. Utilizing all of our data we were able to identify four individual transcripts that have correlations with sire fertility that may prove useful as alternatives for in vitro fertility tests.

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