ABSTRACT Growing evidence indicate that male reproduction is in a decline path. Cytometry is a powerful tool in assessing sperm viability with remarkably high precision, short period of time and provide simultaneous evaluation of multiple parameters. The objective of this study was to assess the viability of semen in a variety of species: bovine, human and two endangered species; the Arabian tahr/ibex and the Arabian leopard using fluorescent assisted cell sorting (FACS) in conjunction with the conventional method of motility evaluation by computer assisted sperm analysis (CASA). Semen was collected during the breeding seasons via electro ejaculation from breeding domestic Omani breed bulls (n= 10, 3-4 years old), Arabian tahr/ibex (n = 12, 2- 14 years old) in addition to an 18hr postmortem epididymis biopsy from an Arabian tahr/ibex(-12 years old) and Arabian leopard (n=2, 16-18 years old). Human semen (n = 5, 37-64 years old) was prepared in a local fertility clinic. Animal ejaculates were diluted (1:2) in three commercial extenders. Diluted semen was first evaluated for motility using CASA (Sperm Vision®, Minitube), then followed by washing with 0.9% NaCl centrifuged (2000 rpm for 3min) and filtered using a 40um nylon mesh (BD, USA) to eliminate debris and clumps that interfere with the FACS apparatus. Semen viability was assessed using the LIVE/DEAD® sperm viability kit (Invitrogen, USA) that segregates cells according to SYBR14 (live) vs. PI (dead) florescence uptake. While chromatin integrity test (Acridine Orange test) was evaluated for Arabian tahr and human semen, acrosome integrity (PSA-FITC/ PI) was carried out in the Arabian leopard spermatozoa. Samples were analyzed using FACSAriaTMIII (FACSDiva software, BD) with an average flow of 20,000 sperm/events for each sample. to Bioxcelleters, hower and Bioxca Our data shows different correlation between sperm motility by CASA and viability via FACS analysis on selected animals regardless of the extender being used. In bovine semen samples, extenders AndroMed® and Bioxcell® had similar effect on maintaining several motion parameters, however, AndroMed® improve the viability of semen compared to Bioxcell®. The correlation between sperm motility and viable sperm was low (r = -0.233- 0.307). Similar trend were witnessed in the Arabian tahr/ibex, a low correlation was observed (r= 0.1202 – 0.3339). While semen extended in AndroMed® showed great motility, Triladyl® maintained higher viability. Season and age have a drastic effect on the quality of spermatozoa. It's worth noting that the deceased Arabian tahr semen had high viability (42%) which can be effectively used in ART programs. Interestingly, Arabian leopard spermatozoa exhibited considerably high motility (88.30%), viability (40- 46%) and acrosomal integrity (20%) that can be successfully used in ART. Unlike sperm analysis from animals, human cases were evaluated individually and proper recommendations were given based on FACS data. Our results indicate that the commonly used motility analysis method CASA alone is insufficient in determining semen suitability for ART, thereby suggesting that FACS analysis should be integrated in Al centers to maximize the selection of elite animals. The proposed standard will greatly enhance the outcome of other advanced biotechnological techniques such as AI, IVF, IUI and ICSI.