The aim of this work will be to evaluate how supplementing freezing media with reduced glutathione (GSH) or BHT affects in-vitro sperm fertility and its impact when a single insemination is performed at different intervals before ovulation. Experiment 1- In total 5 healthy boars from the Landrace x Largewhite, 2-3 years age will be used in this study. Ejaculates will be collected and pooled. Thus, there will be, at least, five replicates per treatment. The treatments will be: 1) cryopreserved semen without antioxidants (Control), 2) cryopreserved semen with 1 mM BHT and 3) cryopreserved semen with 2mM GSH. All semen evaluation will be performed at 30, and 240 min after thawing. The analysis will consist of CASA, sperm membrane integrity, membrane fluidity, lipid peroxidation and superoxide anion production. In Experiment 2- Gilts will be induced into estrus with PG600 and then estrus synchronized by feeding 15 mg×gilt×d of Matrix for 14 d as a top-dress on a standard sow gestation diet. Gilts will be treated with 200µg Tripitorelin (OvuGel) 130 hours of the last Matrix feeding to synchronize ovulation. Gilts (n=20/treatment) will be inseminated at 20 or 30 hours after OvuGel with a single fixed time artificial insemination with 2 x 109 cryopreserved sperm without antioxidants (Control), cryopreserved semen with 1 mM BHT and cryopreserved semen with 2mM GSH. Gilts will be slaughtered between days 28 and 34 of gestation and reproductive tracts collected. The tracts will be identified and processed for pregnancy status, number of normal fetuses, number of degenerative fetuses, number of corpora lutea (CL). Continuous response variables will be analyzed using MIXED and binary response variables analyzed using the GENMOD procedures of SAS (SAS Institute Inc., Cary, NC).
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