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Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

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Abstract

Sperm cryopreservation protocols for brown bear (Ursus arctos) require the centrifugation of semen samples to increase sperm concentration and to clean urine in contaminated samples. We evaluated the effect of centrifugation regimes (time and relative centrifugal force—RCF) on the quantity of sperm recovered and the quality of post-thawed sperm. Thirteen brown bears were electroejaculated. The ejaculates were diluted 1:1 in Tris–citric acid–glucose (TCG) extender and centrifuged with different RCF/time combinations: 600×g, 1,200×g and 2,400×g, for 3, 6 or 12 min. After centrifugation, spermatozoa were diluted in TES–Tris–fructose extender with egg yolk and glycerol (final glycerol concentration of 8%) and frozen in 0.25-mL straws. In the post-thawed semen, motility was assessed by CASA, and acrosomal status (PNA-FITC), viability (SYBR-14 with propidium iodide) and chromatin status (SCSA) were determined by flow cytometry. The longest centrifugation time (12 min) significantly decreased some motility parameters. Sperm recovery significantly decreased in brown bear at 600×g. Our results suggest that brown bear spermatozoa are more sensitive to long centrifugation times than to high RCF. Centrifugation regimes showed no effects on the post-thawing chromatin status. We recommend preparing the brown bear semen for freezing by centrifugation 1,200×g or 2,400×g for 6 min, after electroejaculation and dilution 1:1 in TCG extender, since these procedures increase the spermatozoa recovery without harmful effects on the post-thawed quality of brown bear spermatozoa.

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Acknowledgements

This work was supported in part by CICYT (CGL 2007-63748/BOS) and CANTUR S.A. Felipe Martínez-Pastor was supported by the Ramón y Cajal program (Spanish Ministry of Science and Innovation). The authors thank Miguel Angel Marañon and the game keepers of the Cabarceno Nature Park, Susana Gomes Alves, María Mata Campuzano, Manuel Alvarez Rodriguez, Julio Tamayo Canul and Elena Lopez Urueña.

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Authors and Affiliations

  1. ITRA-ULE, INDEGSAL, University of León, 24071, León, Spain

    María Nicolas, Mercedes Alvarez, Enrique Anel, Félix Martínez, Felipe Martínez-Pastor, Paulino de Paz & Luis Anel

  2. Animal Reproduction and Obstetrics, University of León, 24071, León, Spain

    María Nicolas, Mercedes Alvarez, Enrique Anel, Félix Martínez & Luis Anel

  3. Cabárceno Park, Cantabria, Spain

    Santiago Borragán

  4. Cell Biology, University of León, 24071, León, Spain

    Felipe Martínez-Pastor & Paulino de Paz

Authors
  1. María Nicolas
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  2. Mercedes Alvarez
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  3. Enrique Anel
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  4. Félix Martínez
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  5. Santiago Borragán
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  6. Felipe Martínez-Pastor
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  7. Paulino de Paz
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  8. Luis Anel
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Corresponding author

Correspondence to Paulino de Paz.

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Communicated by C. Gortázar

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Nicolas, M., Alvarez, M., Anel, E. et al. Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes. Eur J Wildl Res 58, 77–84 (2012). https://doi.org/10.1007/s10344-011-0544-8

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  • DOI: https://doi.org/10.1007/s10344-011-0544-8

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