title: Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen.
abstract: The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.
authors.affiliation: Department of Veterinary Medicine, Federal University of Alagoas, Viçosa-AL. Brazil.
authors.forename: D R
authors.initials: DR
authors.lastname: Câmara
authors.forename: S V
authors.initials: SV
authors.lastname: Silva
authors.forename: F C
authors.initials: FC
authors.lastname: Almeida
authors.forename: J F
authors.initials: JF
authors.lastname: Nunes
authors.forename: M M P
authors.initials: MM
authors.lastname: Guerra
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage*; D002374:Catalase / Q000008:administration & dosage; D002462:Cell Membrane / Q000502:physiology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D005978:Glutathione / Q000008:administration & dosage; D006358:Hot Temperature; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012756:Sheep; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology / Q000648:ultrastructure; D013482:Superoxide Dismutase / Q000008:administration & dosage; D013997:Time Factors
chemical_list: D000975:Antioxidants; D002374:Catalase; D013482:Superoxide Dismutase; D005978:Glutathione