Effect of Citrus sinensis , Punica granatum , and Citrus limon Peels Oil on the Quality of Frozen–Thawed Ram Sperm

Essential oils are well‐known for their strong antioxidant properties and are valuable supplements for enhancing the quality of thawed sperm. Among these, citrus fruits such as Citrus sinensis , Citrus limon , and Punica granatum are particularly rich in antioxidants, especially in their peels, which exhibit the highest antioxidant activity. In this study, essential oils were extracted from the peels of Citrus sinensis , Citrus limon , and Punica granatum and added to diluted Sanjabi ram semen at different concentrations (0.25%, 0.5%, 1%, and 2% v/v) during the freezing process. The effects of these three essential oils were evaluated using various qualitative parameters after thawing, including sperm total motility, viability, membrane functionality, DNA integrity (DI), total abnormality rates, and malondialdehyde (MDA) levels. The analysis was conducted with seven replicates, and the resulting data were assessed using SPSS software. Duncan’s test was applied to compare the means at a significance level of 5%. Incorporating 0.25% v/v Punica granatum peel oil into the freezing medium for ram sperm significantly enhanced viability (70.19 ± 2.03; p < 0.01), total motility (66.38 ± 1.12; p < 0.01), and membrane integrity (67.53 ± 2.29; p < 0.01) after the freeze–thaw process compared to other treatment groups. Furthermore, both 0.25% v/v Punica granatum (16.02 ± 0.39) and Citrus sinensis peel (16.40 ± 0.51) essential oils in the freezing medium resulted in lower MDA levels ( p < 0.03) and improved DI (89.57 ± 0.1 and 91.44 ± 1.03, respectively; p < 0.0065) after freeze–thaw when compared to other treatments. Additionally, the inclusion of 0.25% v/v Citrus limon essential oil led to a reduction in total sperm abnormalities following freeze–thaw. In contrast, higher concentrations of 1% and 2% v/v of both Citrus limon and Punica granatum peel essential oils significantly decreased viability ( p < 0.01), total motility ( p < 0.01), and both membrane ( p < 0.01) and DI ( p < 0.01) parameters after freeze–thaw compared to the control group. Moreover, the 2% v/v concentrations of Citrus limon (28.63 ± 0.6) and Punica granatum peel (28.41 ± 0.58) essential oils resulted in increased MDA levels ( p < 0.05) compared to the control. Overall, adding appropriate concentrations of essential oils from Citrus sinensis , Citrus limon , and Punica granatum peels, known for their antioxidant properties, significantly improved sperm quality parameters after thawing.