The current study evaluated the physiochemical quality and gene expression profile of post‐thawed buck semen after supplementation with antioxidants [melatonin (M), L‐carnitine (LC), cysteine (Cys), LC + M, M + Cys, LC + Cys, LC + Cys + M] in comparison with the non‐treated control group. Physical and biochemical characteristics of semen were evaluated following freezing and thawing. Transcript abundance of six selected candidate genes was profile using quantitative real‐time PCR. The data demonstrated significant enhancement of post‐freezing total motility, progressive motility, percentage of live sperm, CASA parameters, plasma membrane and acrosome integrity in all groups supplemented with Cys, LC, M + Cys and LC + Cys compared with the control group. The biochemical analysis of semen indicated that semen groups supplemented with LC and LC + Cys recorded increased levels of GPX and SOD that were coupled with up‐regulation of antioxidant genes (SOD1, GPX1 and NRF2) and mitochondrial transcripts (CPT2 and ATP5F1A). Moreover, H2O2 level and DNA fragmentation percentage were reduced compared with other groups. In conclusion, supplementation of Cys alone or in combination with LC positively improved the post‐thaw physiochemical properties of rabbit semen through activation of bioenergetics‐related mitochondrial genes and cellular antioxidant defence mechanism.

Background: Sperm cryopreservation is necessary for genetic sharing on a national and international scale.Objective: This study aimed to examine, after treatment with various antioxidants, the link between buck age and sperm quality using the computer-assisted sperm analysis (CASA) method. Material and methods: Collected semen from 16 younger and 14 older New Zealand rabbit bucks. The obtained semen was assessed by the CASA system using ejaculates with at least 70% progressive motility. Separated semen samples into eight liquors and diluted (1:1 v/v) with rabbit semen extender supplemented with all antioxidant combinations (melatonin (M), L-carnitine (LC), cysteine (Cys), LC+M, M+Cys, LC+Cys, and LC+Cys+M) and untreated group. Results: The casa's characteristics were higher in young than older bucks. In comparison to controls, which recorded the lowest values, the addition of antioxidants increases total and progressive sperm motility in all experimental groups significantly. All experimental group's antioxidant supplementation levels and the control group were significantly lower than fresh pooled semen. Additionally, all CASA parameters increased compared to the control group when buck semen was supplemented with different antioxidants. Conclusion:In contrast to younger bucks, administering several antioxidants (LM, LS, MS, and LMS) has a more substantial positive effect on sperm motility and CASA parameters in older bucks. Thus, age has a substantial role in defining semen's quality. Additionally, treatment with either a single (L, M, and S) or a combination of antioxidants enhances buck semen's motility and CASA properties (LM, LS, MS, and LMS).