Cryopreservation induces sub lethal damage to the spermatozoa, thereby reduce their fertile life. There are some biochemical additives that may enhance buffalo semen freezability; Amphora coffeaeformis is one of these biochemical semen additives. Till now, the exact effects of Amphora coffeaeformis on buffalo semen processing outcomes haven't been
discovered. The current study aimed to clarify Amphora coffeaeformis roles during buffalo semen cryopreservation. Cryopreserved semen was assessed for post- thawing motility, viability, acrosomal integrity, antioxidant enzymes concentration, lipid peroxidation and ultrastructure changes. Current results indicated that addition of 100 µl/ml Amphora
coffeaeformis to semen extender significantly (P<0.05) improved post-thawing motility, viability and acrosomal integrity (63.33±9.28 %, 133.33±9.40 and 12.33± 2.02 %, respectively) compared with control (43.33±6.01 %, 74.16± 10.93 and 24.67±2.03 %, respectively). Moreover, 100 µl/ml Amphora coffeaeformis significantly increased (P<0.05) total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx) and ascorbic acid concentrations (0.58 ± 0.05 mµ/ml, 95.00 ± 4.16 U/ml, 87.61 ± 5.20 U/L and 58.33 ± 2.16, respectively) with respect to the control (0.15 ± 0.06 mµ/ml, 31.25 ± 1.65 U/ml, 51.73 ± 2.32 U/L and 22.14 ± 7.84, respectively). Furthermore, 100 µl/ml Amphora coffeaeformis significantly decreased (P<0.05) lipid peroxidation of the cryopreserved spermatozoa compared with the control semen (9.04 ± 0.69 vs 24.58 ± 1.60 nmol/ml). Likewise, at this concentration. Ultra-structure examination illustrated that addition of
100 µl/ml Amphora coffeaeformis to semen protected the plasma membrane, acrosomal region and mitochondria and maintained the ultrastructure integrity of the cryopreserved spermatozoa compared with the control spermatozoa .In conclusion the addition of 100 µl/ml Amphora coffeaeformis to the freezing extender improves fine structure integrity through enhancing the antioxidant defense of buffalo sperm cells and decreasing the rate of lipid peroxidation.Therefore, amphora coffeaeformis as a natural antioxidant may reduce cryodamage and improve sperm cryopreservation quality.