title: Post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender.
abstract: The performance of cryopreserved semen in ovine artificial insemination still needs improvement. Some antioxidants have been tested, with variable success. We cryopreserved semen from Churra rams using TES-Tris-fructose with 4% glycerol and 10% egg yolk (EY) or 3.5% soybean lecithin (SL), with 1 mM of reduced glutathione (GSH), Trolox, crocin, or cysteamine. Samples were analyzed after thawing and incubation (6 hours, 38 °C) for motility (computer-assisted sperm analysis [CASA]), viability, acrosomal integrity, apoptosis, mitochondrial activity, chromatin status, and lipoperoxidation (malondialdehyde production). Interactions (antioxidant/extender/incubation) were significant for most variables. Extenders yielded similar results, although SL depressed mitochondrial activity and linearity (P < 0.001), it improved motility (P < 0.05), DNA fragmentation (P < 0.05), and acrosomal damage (P < 0.001). The control, GSH, and Trolox showed greater viability with SL (P < 0.01). Cysteamine depressed motility (0 hours: 51.6 ± 2.0% vs. 32.3 ± 4.3%; 6 hours: no motility vs. 32.5 ± 1.9%; P < 0.001), but improved viability when using EY (P = 0.004). Crocin increased acrosomal damage (P = 0.022) but improved linearity-related parameters after thawing (P = 0.014). Trolox considerably reduced malondialdehyde production in both extenders (8.6 ± 0.4 nmol per 10(8) cells vs. 14.2 ± 0.3 in EY and 20 ± 0.6 in SL; P < 0.001). Interestingly, thiol antioxidants (cysteamine and GSH) increased DNA fragmentation (percentage of DNA fragmentation index), whereas crocin reduced it (P < 0.05). Interactions between extender and antioxidant must be taken into account for improving sperm cryopreservation. Soybean lecithin seems to be a suitable replacement for EY, but its effect on mitochondria must be investigated. Trolox and crocin might be useful for ram semen freezing.
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Mata-Campuzano
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Álvarez-Rodríguez
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Álvarez
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Division for Research and Post-graduate Studies, Technological Institute of Conkal, Yucatán, México.
authors.forename: Julio
authors.initials: J
authors.lastname: Tamayo-Canul
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Anel
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
authors.forename: Paulino
authors.initials: P
authors.lastname: de Paz
authors.affiliation: Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain. Electronic address: felipe.martinez@unileon.es.
authors.forename: Felipe
authors.initials: F
authors.lastname: Martínez-Pastor
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015925:Cryopreservation / Q000662:veterinary*; D004530:Egg Yolk; D005990:Glycerol; D054709:Lecithins; D008297:Male; D012662:Semen Preservation / Q000662:veterinary*; D012756:Sheep / Q000502:physiology*; D013048:Specimen Handling; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D000975:Antioxidants; D054709:Lecithins; D005990:Glycerol
keywords: Antioxidant; Cryopreservation; Extender; Ram; Spermatozoa