Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Animal Science

First Advisor

Julie Walker

Second Advisor

George Perry


Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. After insemination, sperm must traverse the female barriers and undergo capacitation to complete fertilization; however, there are differences in fertility even among bulls that successfully pass a breeding soundness exam. For any potential marker of fertility, there must be variability expressed among animals. The series of studies in this dissertation had the objective 1) to evaluate differences between epididymal and ejaculated sperm and respective fluids proteins to understand increased longevity of epididymal sperm; 2) to evaluate the potential of dystroglycan (DAG1) and plasma serine protease inhibitor (SERPINA5) as fertility markers; and 3) to evaluate whether post in vitro capacitation changes in sperm could be used to estimate fertility differences between bulls. In summary, it was observed that 1) epididymal sperm was able to maintain viability longer than ejaculated sperm when cultured under the same conditions, also, sperm energy metabolism appears to be more glycolytic compared to sperm in the ejaculate, based on the greater number of proteins related to this pathway only present in epididymal samples. Sperm also has a greater number of antioxidants available in the epididymis that is likely to be maintaining reactive oxygen species (ROS) at low concentrations to inhibit premature sperm activation. This is supported by a greater mitochondrial membrane potential of epididymal sperm compared to ejaculated sperm and the fact that epididymal sperm was able to maintain viability longer than ejaculated when cultured under the same conditions. Then, it was observed that 2) DAG1 and SERPINA5 proteins, that are associated with cell to cell interactions, were localized on the bovine sperm head, also, SERPINA5 was localized on the sperm tail, and concentrations of DAG1 and SERPINA5 on the sperm head were correlated with each other (P = 0.01, r2 = 0.54), also, SERPINA5 was correlated with embryo cleavage rate (P = 0.04, r2 = 0.48), and the percentage of tail labeled for SERPINA5 was correlated with sperm viability (P = 0.05, r2 = 0.44); however, neither protein was associated with sire conception rate (SCR; i.e., field fertility). Thus, SERPINA5 may be related with sperm protection and/or oocyte fertilization while DAG1 may be related to sperm transport or formation of the sperm reservoir in the oviduct. Lastly, it was observed that 3) multiple analyses over time in capacitation media of viability, zinc signature 2, zinc signature 1 + 2, and dead CD9+ were able to estimate differences between low fertility bulls to high and intermediary fertility bulls. The inclusion of a viability, a zinc signature, or CD9 protein assay in artificial insemination (AI) studs’ quality control measurements may have the potential to predict bull fertility; however, a larger number of bulls with known fertility need to be evaluated to validate these results. In conclusion, more research is necessary to understand: 1) the role of the proteins only identified in epididymis and their role in increased sperm longevity; 2) the role of SERPINA5 and DAG1 in vivo; and 3) the potential of viability, zinc signature and CD9 protein analyses post sperm capacitation as predictors of bull fertility.

Number of Pages



South Dakota State University


Supplemental files:

Raw files from LCMS / MS - Size: 1.4 GB

Six Table files:
Table 1: Spectra count fluid protein
Table 2: Spectra count sperm protein
Table 3: Gene ontology fluid protein
Table 4: Gene ontology sperm protein
Table 5: KEGG fluid protein
Table 6: KEGG sperm protein (1144244 kB)
Raw files from LCMS / MS

Table 1 - Spectra count Fluid protein.xlsx (35 kB)
Spectra count fluid protein

Table 2 - Spectra count Sperm protein.xlsx (36 kB)
Spectra count sperm protein

Table 3 - Gene Ontology Fluid protein.xlsx (19 kB)
Gene ontology fluid protein

Table 4 - Gene Ontology Sperm protein.xlsx (20 kB)
Gene ontology sperm protein

Table 5 - KEGG Fluid protein.xlsx (16 kB)
KEGG fluid protein

Table 6 - KEGG Sperm protein.xlsx (15 kB)
KEGG sperm protein

Available for download on Friday, December 15, 2023



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