LOCALIZATION ON SPERM, QUANTIFICATION AND MOLECULAR FEATURES OF TWO SEMINAL PROTEINS

by Dawson, George Ray.

16 Objective markers to identify higher fertility individuals are needed to maximize livestock breeding success. Two heparin-binding proteins, which are reflective of fertility in bulls, have been biochemically identified as fertility-associated antigen (FAA) and tissue inhibitor of metalloproteinases-2 (TIMP-2). These four studies were designed to examine the importance of those proteins in relation to reproduction in bulls and other livestock species. In the first study, indirect immuno-fluorescent microscopy was performed to localize FAA and TIMP-2 to livestock sperm. FAA was localized on spermatozoal acrosomes of bulls and rams, but no cross-reactivity was observed for stallions. TIMP-2 labeling was observed on acrosomes and posterior heads, which was species dependent. Localization patterns for FAA and TIMP-2 were further investigated during heparininduced capacitation and acrosome reactions of bovine sperm. In study two, an enzyme-linked immunosorbent assay (ELISA) was developed to determine concentrations of FAA in bovine seminal plasma (SP). A commercially available TIMP-2 ELISA was utilized to quantify TIMP-2. Respective mean concentrations of FAA and TIMP-2 in SP were 6.66±1.487 ug/ml and 1.18±0.045 mg/ml. Concentrations of FAA in SP did not correspond to bull fertility potential, however, older bulls with higher concentrations of TIMP-2 in SP sired more calves. The third study evaluated utility of an amplified fragment length polymorphism with bovine TIMP-2 gene specific primers to amplify a 700 bp genomic DNA (gDNA) product from sperm. From 53 bulls screened, 22.6% were negative for the 700 bp 17 amplicon. There was a three-fold likelihood for 700 bp negative bulls to not sire a calf compared to 700 bp positive bulls. The product was cloned and sequenced, but no homology to TIMP-2 was detected. Therefore, the product represented novel bovine gDNA sequence. The fourth study identified an equine homologue to the bovine FAA gene. Immuno-based diagnostics had not detected FAA in stallion semen. The equine DNA homologue was 88.5% identical in nucleotide and 86% in amino acid sequences to bovine FAA. Subtle differences in the amino acid sequence are likely responsible for the inability to detect FAA in stallion semen with FAA antibodies to bovine FAA.