Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation |
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Authors: | Martínez-Pastor F Martínez F Alvarez M Maroto-Morales A García-Alvarez O Soler A J Garde J J de Paz P Anel L |
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Institution: | a Biology of Reproduction Group, National Wildlife Research Institute (IREC), CSIC-UCLM-JCCM, and Institute for Regional Development (IDR), 02071 Albacete, Spain b Animal Reproduction and Obstetrics, University of León, 24071 León, Spain c Regional Center of Animal Selection and Reproduction (CERSYRA), JCCM, Valdepeñas, 13300 Ciudad Real, Spain d Molecular Biology, University of León, 24071 León, Spain |
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Abstract: | We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×106 mL−1 and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl®, Andromed®, Bioxcell®, and UL with 8% LDL (low-density lipoproteins). Triladyl® and Andromed® performed better than Bioxcell® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl® and Andromed® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing. |
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Keywords: | Sperm cryopreservation Electroejaculation Extenders Red deer Semen fractions |
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