title: Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage.
abstract: A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer-assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro-1) and mitochondrial membrane potential (JC-1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL-2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL-2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL-2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro-1 negative) sperm post-thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Cáceres, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Morillo Rodriguez
authors.forename: C
authors.initials: C
authors.lastname: Balao da Silva
authors.forename: B
authors.initials: B
authors.lastname: Macías-García
authors.forename: J M
authors.initials: JM
authors.lastname: Gallardo Bolaños
authors.forename: J A
authors.initials: JA
authors.lastname: Tapia
authors.forename: I M
authors.initials: IM
authors.lastname: Aparicio
authors.forename: C
authors.initials: C
authors.lastname: Ortega-Ferrusola
authors.forename: F J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000818:Animals; D002462:Cell Membrane; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004126:Dimethylformamide / Q000494:pharmacology*; D006736:Horses / Q000502:physiology*; D008297:Male; D012662:Semen Preservation / Q000379:methods; D013081:Sperm Motility; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents; D004126:Dimethylformamide