This study combined in silico analyses of albumin-cholesterol (CHOL) interactions with in vitro assessment of the effects of species-specific (ovine serum albumin, OSA) and non-species-specific albumins (bovine: BSA; human: HSA; caprine: CSA) in cooled ram sperm. Docking, molecular dynamics, and quantum biochemical calculations characterized albumin-CHOL binding. Experiments used 10 ejaculates from 10 rams cooled for 3 h (15 °C) or 24 h (5 °C) and incubated in TALP with BSA, HSA, OSA or CSA. OSA combined energetically favorable poses with stable complexes, at FA1-FA9 and FA1, and was the most consistent CHOL binder across in silico methods. In 3-h samples, OSA decreased total motility (44.0 ± 2.4%; P < 0.05) versus the other groups, reduced viability (28.8 ± 5.3%, P < 0.05) versus control (50.1 ± 4.2%), and increased acrosomal damage (40.2 ± 3.5%; P < 0.05) compared with control and BSA (24.4 ± 1.6%, 23.5 ± 1.4%). In 24-h samples, OSA reduced motility and viability (48.8 ± 3.8%, 21.9 ± 1.6%; P < 0.05) compared with BSA (72.2 ± 4.5%, 44.4 ± 4.3%), increased apoptosis (93.9 ± 0.6%) versus all the other albumins, and decreased mitochondrial ROS levels (6.1 ± 0.4%; P < 0.05) versus BSA and HSA (22.4 ± 3.9% and 16.4 ± 2.8%). Both HSA and OSA increased acrosomal damage (35.1 ± 2.3%, 30.8 ± 1.4%; control: 19.2 ± 4.2%). OSA thus induces pronounced membrane and acrosome alterations under capacitating conditions, likely linked to favorable OSA-CHOL binding, but concomitantly compromises motility, viability and mitochondrial ROS in cooled samples. These results indicate that the species-specific albumin is detrimental for cooled ram spermatozoa and cannot be recommended as a substitute for BSA under the present conditions. Nevertheless, the species-related effects of albumins indicate that strategies tailored for each species will help refine in vitro sperm handling protocols.