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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Livestock Bio-Systems » Research » Publications at this Location » Publication #355328

Research Project: Improving Livestock Production by Developing Reproductive and Precision Management Technologies

Location: Livestock Bio-Systems

Title: Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity

item MENEGATTI ZOCA, SAULO - South Dakota State University
item NORTHROP, EMMALEE - South Dakota State University
item WALKER, JULIE - South Dakota State University
item Cushman, Robert - Bob
item PERRY, GEORGE - South Dakota State University

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/22/2022
Publication Date: 3/2/2022
Citation: Menegatti Zoca, S., Northrop-Albrecht, E.J., Walker, J.A., Cushman, R.A., Perry, G.A. 2022. Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity. Theriogenology. 184:51-60.

Interpretive Summary: The objective of the current study was to identify methods to improve long-term storage of bull sperm without cryopreservation. In the epididymis, sperm are stored for a long period of time, but upon ejaculation motility is increased and lifespan is decreased. Epididymal and ejaculated sperm were collected from bulls and biochemical and proteomic analyses were performed to identify mechanisms contributing to the increased longevity of epididymal sperm. Mitochondrial function was superior in epididymal sperm compared to ejaculated sperm. Proteins associated with sperm motility and mitochondrial function were identified from the proteomic analyses. These proteins may be useful in storage buffers to slow the depletion of mitochondrial function, allowing the long-term storage of bull sperm without cryopreservation.

Technical Abstract: Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 ºC. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 ºC. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P <= 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR < 0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm’s increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH.