The objective of the study was to determine the effect of cholesterol-loaded cyclodextrin (CLC) on the quality parameters of semen from Aksaray Malakli Shepherd dogs of different age groups. Forty-eight male dogs were divided into 3 groupings according to their ages (young age (Y): ≤3years, n: 20; middle age (M): 4–6 years, n: 20; old age (O): ≥7 years; n: 8). The sperm-rich portion of the ejaculate from each dog was divided into four aliquots and extended with either tris as a control (C) or tris loaded with 0.5, 1.0, and 1.5mg/120×106 CLC as low (L), intermediate (I), and high (H) doses, respectively. Following equilibration for at least half an hour, the straws were frozen in nitrogen vapor and then stored in liquid nitrogen at least for 48h. Later, the frozen straws were thawed in a water bath for spermatological evaluation. Significant differences were observed between different age groups in terms of the spermatological parameters (p < 0.05). The evidence suggests that increasing age is associated with poor in-vitro spermatological parameters and CLC was able to protect the acrosome integrity from cryo-damage during the freeze-thawing process. Better semen freezability characteristics were obtained at young ages, considering the overall parameters.