394 Young Scholar Award Talk: Evaluation of new Markers to Predict Sperm Longevity and Bull Fertility
Authors: Zoca Saulo Menegatti , Walker Julie , Dalton Joe , Perry George A 
Published: September 2022

Abstract
Bull fertility is currently evaluated through a breeding soundness exam (BSE) that can detect infertile/sterile bulls with high accuracy; however, subfertile bulls are often classified as satisfactory breeders. In the first study, Angus bulls (n=5) were used to evaluate the effect of different insemination doses (10, 20 and 40 million sperm/dose) on pregnancy/fixed-time AI (P/FTAI; cows n=4,866). The interaction between insemination dose and bull was not significant (P=0.53), and there was no effect of insemination dose on P/FTAI (P=0.31); however, there was an effect of bull on P/FTAI (P &lt;0.01). Two bulls had high, one bull had average, and two bulls had low fertility. Post-thaw sperm analysis was performed by computer assisted sperm analysis (CASA) and flow cytometry; however, analysis performed could not explain field fertility differences. In a second study, Angus and Angus crosses bulls were used to investigate differences in sperm longevity between epididymal and ejaculated sperm. Epididymal and ejaculated semen was incubated at pH 5.8, 6.8, or 7.3. Proteins in epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm were also investigated. It was identified that epididymal sperm maintained viability longer compared to ejaculated sperm regardless of media pH (P &lt;0.05). Ejaculated sperm had increased longevity (total motility &gt;20%) at pH 6.8 compared to pH 5.8 or 7.3. There were 458 unique proteins between all samples; 311 and 178 in epididymal fluid and seminal plasma, respectively, and 334 and 298 on epididymal and ejaculated sperm, respectively. Based on comparative proteomics, epididymal sperm energy metabolism is more glycolytic compared to ejaculated sperm. In addition, there were greater numbers of antioxidants available for epididymal sperm likely to maintain reactive oxygen species (ROS) at low concentrations to inhibit premature sperm activation. From this dataset, three proteins that are associated with cell-to-cell interaction were studied [plasma serine protease inhibitor (SERPINA5), dystroglycan (DAG1), and CD9]. Among dairy bulls, SERPINA5 and DAG1 were not associated with field fertility; thus, they are not potential fertility markers. The protein CD9 was evaluated to determine whether induction of sperm capacitation (in vitro) could be used to estimate/predict bulls fertility differences. Semen samples from first study were evaluated for total motility (CASA), and functional sperm analysis (flow cytometry) at pre-wash, post-wash, h 0, 3, 6, and 24 of incubation. Sperm analysis were not able to estimate fertility differences between high and average fertility bulls; however, low fertility bulls had decreased (P &lt;0.05) viability, zinc signature 2, and zinc signature1 + 2, and had increased (P &lt;0.05) dead CD9+ sperm. In summary, the use of an insemination dose of 10-40 million sperm can generate acceptable P/FTAI, and evaluation of CD9, viability and zinc signatures may increase the industry’s ability to estimate fertility of bulls that pass a BSE.