P-95: Flow Cytometry Analysis of Bovine Semen:A Qualitative Study

Background: Although AI practices have been introduced little over 60 years, the success rate remains relatively low. This might be due to the exclusive selection of semen based on motility analysis. Recent advancement in sperm sexing using flow cytometry with an increased throughput from next generation cell sorters, made use of this technology in studding sperm qualitative aspects other than motility become highly feasible. This study was carried out to assess the viability of bovine sperm diluted in Bioxcell® and Andromed using fluorescent assisted cell sorting (FACS) in conjunction with motility assessment using computer assisted sperm analysis (CASA). Materials and Methods: Semen was collected via electro ejaculation twice a week from local breed bulls (n= 7, 2-4 years old) during the period from February to April. Ejaculates were divided in two aliquots and extended (1: 2) in Bioxcell® and Andromed at 37°C. Diluted semen was evaluated for motility using CASA (Sperm Vision®, Minitube). Sperm was liquefied (37°C/10 minutes), washed with 0.9% NaCl, centrifuged (2000 rpm for 3 minutes) and filtered using 40 μm nylon mesh (BD, USA). Semen viability was assessed using LIVE/ DEAD® sperm viability kit (Invitrogen, USA). Samples were analyzed using FACSAria™III (FACSDiva software, BD) with an average flow of 20000 sperm/events for each sample. Results: Our data shows different correlation between sperm motility by CASA and viability via FACS analysis on selected bulls. Interestingly, while some semen samples extended with Bioxcell® exhibited high motility, the viability was low; (71.9, 80.8 and 68.8, 18.9% respectively). This observation was not restricted to Bioxcell ® as similar result was found using Andromed (91.3 vs. 49.9%). Conversely however, using both extenders Bioxcell® and Andromed; low semen motility with high viability (69.4, 76.8% and 50, 57.7; 66.6, 71.2% respectively) was observed. Conclusion: These results demonstrate that this phenomenon can occur regardless of the extender being used. Moreover, our results suggest that motility analysis using CASA alone is insufficient in determining semen suitability for ART; thereby suggesting that FACS analysis should be integrated in AI centers to maximize the selection of elite bulls.