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Morphofunctional Characterization of Cooled Sperm With Different Extenders to Use in Equine-Assisted Reproduction
Authors:Shirley Andrea Florez-Rodriguez  Rubens Paes de Arruda  Maíra Bianchi Rodrigues Alves  Fernanda Jordão Affonso  Henrique Fulaneti Carvalho  Kleber Menegon Lemes  Renata Lançoni  André Furugen Cesar de Andrade  Eneiva Carla Carvalho Celeghini
Institution:1. Laboratory of Semen Biotechnology and Andrology, Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, College of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), Pirassununga, São Paulo, Brazil;2. Laboratory of Andrology and Embryo Technology of Swine, Department of Animal Reproduction, University of São Paulo, Pirassununga, São Paulo, Brazil
Abstract:The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen.
Keywords:Stallion  Sperm quality  Cooling  BSA  Skim milk
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