Semen preservation is an important process to ensure semen quality is sufficient for assisted reproductive technology. Studies have showed that suppression of free radicals, crystal formation and regulation of extracellular osmotic pressure is essential to ensure survivability. Past studies have indicated that utilizing protein already existent in the body could be used as a free radical scavenger and osmotic regulator -a potential preservation agent. Thus, the objective of this study was to determine the effect of different albumin-V concentrations on pooled bovine spermatozoa. Pooled cryopreserved semen straws were thawed and incubated in different concentrations of albumin-V (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg albumin-V/ml Bio-excel ® extender) at 4°C. Sperm motility and viability were analyzed at 0, 2, 24 and 48 hours using Makler Chamber. Results indicated that motility and viability were noticeably reduced against time except for sperm incubated in 0.4 mg/ml. Membrane elasticity study using atomic force microscope (AFM) was then conducted on 0, 0.4, and 1.0 mg/ml 48 hours spermatozoa. We had observed that sperm membrane in the 0.4 mg/ml had a lower elasticity value (1.7 ± 0.6 kPA) compared to fresh semen (5.5 ± 1.0 kPA), 0 mg/ml (5.5 ± 0.1 kPA) and 1.0 mg/ml (5.3 ± 0.9 mg/ml). It is very likely that the motility and viability reduction was partly due to the changes in spermatozoa membrane elasticity. We conclude that albumin-V is a potential molecule for 4°C semen preservation; varying albumin concentration will have an influence on the spermatozoa membrane and affect the overall outcome of semen quality. Further study is required to elucidate the function and mechanism of albumin-V in preservation and its direct effect on ART outcome.