Time Dependent Impact of Reactive Oxidants on Seminal Attributes, Mitochondrial Membrane Potential, Lipid Peroxidation and Capacitation-Like Changes of Karan-Fries (KF) Bulls During Cryopreservation

Abstract

BACKGROUND: Cryopreservation of semen is a valuable technique; however, it is also known to be detrimental to the structure of spermatozoa and fertility due to cryo-injury and subsequent generation of reactive oxidants. OBJECTIVE:To determine the time-dependent impact of reactive oxidants on seminal attributes, mitochondrial membrane potential (MMP), lipid peroxidation status (LPO) and early capacitation like changes. MATERIALS AND METHODS:Semen samples were collected by artificial vagina technique from six Karan-Fries (KF) bulls and subsequently examined at 0 h (before cryopreservation) and at 24 hours, 15 days and 2-months of storage for various seminal attributes, MMP (Δψm) , and early capacitation-like changes. Simultaneously, LPO (TBARS) was determined in fresh and post-thaw seminal plasma. RESULTS: A sharp decrease (P<0.01) in semen quality was observed only after 24 h of cryopreservation except for viability and acrosomal integrity. Sperm viability and acrosome integrity reduced significantly up to 2 months of cryopreservation. The lipid peroxidation status was found to be lower in fresh seminal plasma (2.63±0.22 vs. 3.51±0.34 units/mL) as compared to post-thaw. Furthermore, the active Δψm of fresh semen showed a significant (P <0.01) decrease after 24 hours (77.92±0.387 vs. 54.52±0.28%) of cryopreservation, while there was a non- significant decrease in active MMP after 15 d and 2-months (53.68±0.138 and 52.76±0.16%). Moreover, significant (P <0.01) early capacitation-like changes were found in post-thaw spermatozoa (25.72±0.12 vs. 9.1±0.19%) as compared to fresh ejaculate. CONCLUSION: Spermatozoa incur the majority of damages during the early phase of cryopreservation, however the damage associated by different stressors cannot be neglected.