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RESEARCH ARTICLE
Contents Vol 21(4)

Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze–thaw damage

T. Leahy A C , J. I. Marti B , G. Evans A and W. M. C. Maxwell A
+ Author Affiliations
- Author Affiliations

A Faculty of Veterinary Science, The University of Sydney, Sydney, NSW 2006, Australia.

B Unidad de Tecnologia en Produccion Animal, Centro de Investigacion y Tecnologia, Agroalimentaria de Aragon, 50059 Zaragoza, Spain.

C Corresponding author. Email: tamaral@vetsci.usyd.edu.au

Reproduction, Fertility and Development 21(4) 571-578 https://doi.org/10.1071/RD08238
Submitted: 23 October 2008  Accepted: 26 January 2009   Published: 7 April 2009

Abstract

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

Additional keywords: cryopreservation, flow cytometry, sex preselection, sheep.


Acknowledgements

The authors are grateful to XY, Inc. (USA), and the Spanish Grant PR2006–0186 for supporting this research, and Australian Wool Innovation for scholarship support of T. Leahy. We thank M. Ruckholdt for operation of the SX MoFlo cell sorter and Ms W. Smith, Ms K. Heasman and Mr A. Souter for technical assistance.


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