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Doctoral thesis1999Open access

In vitro characterisation of cryopreserved canine spermatozoa : with special reference to post-thaw survival time and zona pellucida binding capacity

Ström Holst, Bodil


Methods for cryopreservation of canine spermatozoa have mostly been developed without proper assessment of the cell functional status using in vitro methods. This has complicated the adequate development of new procedures and the refinement o f old ones. In the present study, two methods known to give high pregnancy rates after artificial insemination in the field (the Andersen and CLONE methods), were evaluated in vitro. Sperm survival time post-thaw, assessed as motility during incubation for 3 hrs at 37°C, was short using both procedures. Additionally, a high percentage of the acrosomes were damaged, both when evaluated using light and transmission electron microscopy, indicating that acrosomal damage might be one reason for the reduced post-thaw survival time. Further, the detergent Equex STM paste was added to a Tris-based extender to evaluate its effect on sperm survival time post-thaw. Plasma membrane (PM) integrity immediately post thaw was significantly higher using Equex STM paste, and both motility and PM integrity were prolonged during incubation at 38°C when Equex STM paste was added. Moreover, ffozen-thawed dog spermatozoa were evaluated for their ability to bind to homologous zona pellucida (ZP) in a ZP binding assay (ZBA). The capacity o f canine spermatozoa to bind to the ZP was found to be a feature of the living sperm cell. Chilling spermatozoa for 4 days at 4°C tended to decrease ZP binding ability compared with one day’s storage. The addition o f Equex STM paste to the cryopreservation extender had a significantly beneficial effect on sperm ZP binding capacity. To facilitate the implementation of a ZBA in the canine, it is necessary to find a method for storage of retrieved oocytes. Deep freezing of canine ovaries and salt-storage of oocytes was seen to result in morphological changes of the ZP as detected by scanning electron microscopy, mainly manifested as a wider meshwork o f the outer surface of the ZP. These changes were thought to contribute to the observed reduced sperm-binding capacity compared to fresh oocytes, but did not exclude the use o f stored oocytes in a ZBA. Taken together, the addition of a ZBA to other in vitro tests can be expected to contribute to better assessments o f the damage caused to dog spermatozoa when developing and refining techniques for semen chilling and cryopreservation.


dog; sperm; cryopreservation; in vitro; evaluation; post-thaw; survival time; zona pellucida

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria
1999, number: 62ISBN: 91-576-5445-X
Publisher: Swedish University of Agricultural Sciences

      SLU Authors

    • Ström Holst, Bodil

      • Department of Obstetrics and Gynaecology, Swedish University of Agricultural Sciences

    UKÄ Subject classification

    Clinical Science

    Permanent link to this page (URI)