Despite many cryopreservation techniques in bovine semen, various stressors' detrimental effects remain a significant issue. The present study targeted to assess the role of semen quality parameters, sperm function tests, lipid peroxidation, reactive oxygen species (ROS), and different antioxidants in the cryopreservation of bovine semen. Further, the kinetics of lipid peroxidation, ROS, and antioxidants on repeated semen collection under short ejaculatory abstinence were studied. We designed a comparative study where bulls were grouped into good and low freezable semen groups (Freeze-groups) based on their post-thaw motility. All the bulls included had similar initial motility and qualified minimum standards for initial semen parameters viz. semen volume and sperm concentration. The present study detected a higher lipid peroxidation and ROS viz. superoxide anions (•O2-) and a lower total antioxidant capacity (TAC) in the low freeze-group compared to the good freeze-group. The ROS and antioxidants showed unique kinetics on repeated semen collection at short intervals, and no significant change was detected in semen volume, sperm motility, and sperm concentration. This study detected higher head abnormalities and poor acrosome integrity in the low freeze-groups. The present study results indicated that the sperm head might be the most vulnerable part of the sperm to cryopreservation stress. The present study finds significantly higher lipid peroxidation and ROS levels and reduced antioxidant capacity as the primary reasons for low cryopreservability. Further, repeated semen collection with a shorter or lack of abstinence does not impose any significant change in the semen volume and sperm concentration; moreover, it could be beneficial for higher antioxidant levels and lower lipid peroxidation levels. As seminal plasma has both inhibitory and stimulatory roles in sperm function and cryopreservation, identifying the critical role players of seminal plasma and identifying sperm related changes in cryopreservation could predict the cryopreservability potential of semen. • For the first time in bovine, influence of ejaculatory abstinence on ROS and antioxidant systems are studied. • We demonstrate Sperm head is more vulnerable to cryopreservation damages. • We substantiate that high levels of ROS and low TAC could be responsible for poor cryopreservability of bovine semen. • The non-enzymatic or small molecular weight antioxidant molecules of seminal plasma are important in cryopreservability. • We demonstrate that semen collection, in bovine, with shorter abstinence period is beneficial for high TAC and low LPO. [ABSTRACT FROM AUTHOR]