Centrifugation on puresperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen

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Theriogenology xx (2011) xxx

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Centrifugation on PureSperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen
J. Doradoa,*, L. Alcaráza, N. Duartea, J.M. Porteroa, D.Achaa, S. Demydab, A. Muñoz-Serranob, M. Hidalgoa
a

Animal Reproduction Group, Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Cordoba, 14071 Córdoba, Spain b Department of Genetics, Faculty of Veterinary Medicine, University of Cordoba, 14071 Córdoba, Spain Received 21 October 2010; received in revised form 3 January 2011; accepted 27 February 2011Abstract The main objective of this study was to investigate if centrifugation through PureSperm® density-gradient can improve the post-thaw semen quality of dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm®. Assessments of sperm motility (assessed by computerized-assisted semenanalysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of Propidium iodine/ isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, unselected samples and selected preparations. Cryopreservation had a significant (P 0.001) effect on all studied semen parameters. PureSperm® centrifugation yielded sperm suspensionswith improved motility and viability (P 0.001). The washing step significantly reduced (P 0.001) all of the kinematics parameters evaluated as well as reduced the proportion of viable spermatozoa with intact acrosomes (P 0.05). We concluded that PureSperm® centrifugation is a successful method for improving the quality of frozen-thawed dog spermatozoa. However, washing after density-gradientcentrifugation dramatically reduces the post-thaw semen quality, indicating that the inclusion of such a washing step is unnecessary. © 2011 Published by Elsevier Inc.
Keywords: Sperm separation; CASA; Sperm viability; Cryopreservation; Dog spermatozoa

1. Introduction Cryopreservation induces a series of osmotic, chemical and mechanical stresses to sperm, causing death of some sperm and severepost-thaw damage in surviving cells, reducing fertilizing ability [1]. Therefore, current pregnancy and live birth success rates of assisted reproduction techniques (ART) are not completely satis-

* Corresponding author. Tel: 0034 957 212136; fax: 0034 957 211093. E-mail address: jdorado@uco.es (Jesús M. Dorado Martín). 0093-691X/$ – see front matter © 2011 Published by Elsevier Inc.doi:10.1016/j.theriogenology.2011.02.026

factory with frozen-thawed dog semen [2]. The selection of the most viable spermatozoa (potentially fertile) from a population where the majority has been damaged or is dead might be one of the prerequisites for achieving optimal conception rates after ART with frozen-thawed dog semen. Centrifugation through layers of colloid has been satisfactorily used to separatemotile, chromatin-intact and morphologically normal spermatozoa from the extended semen [3]. Recently, some speciesspecific formulations have been developed for use in human ART and also in animal ART [3], including

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dogs [4]. PureSperm®, one of those commercial products, has also been used inter-species with acceptable results [5,6]. However, to our knowledge, studies on the effect of PureSperm® centrifugation on frozen-thawed dog semen have not been...