Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
Shopping Cart: ( empty )
You are here: Home > Journals > RD > RD08238
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.


References

Ashworth, P. J. C. , Harrison, R. A. P. , Miller, N. G. A. , Plummer, J. M. , and Watson, P. F. (1994). Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod. Fertil. Dev. 6, 173–180.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS | Brogliatti G., Barreiro G., Larraburu G., and Laborde A. (2004). CASA evaluation of sexed and non-sexed frozen bull semen. Reprod. Fertil. Dev. 16, 127–128. [Abstract] doi:10.1071/RDV16N1AB10

Burtis C. A., Ashwood E. R., and Bruns D. E. (2006). ‘Tietz Textbook of Clinical Chemistry and Molecular Diagnostics.’ (Elsevier Saunders: St. Louis.)

Caballero, I. , Vazquez, J. M. , Centurion, F. , Rodriguez-Martinez, H. , Parrilla, I. , Roca, J. , Cuello, C. , and Martinez, E. A. (2004). Comparative effects of autologous and homologous seminal plasma on the viability of largely extended boar spermatozoa. Reprod. Domest. Anim. 39, 370–375.
| Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | Cattaneo L., Aguilar J., Caballero J., Perez S., Cerrate H., and Brogliatti G. (2002). Relationship of the initial sperm concentration and motility of bovine ejaculates to the efficiency of sperm sexing using flow sorting. Theriogenology 57, 745. [Abstract]

Cattaneo L., Martin P., Caballero J. O., Calvi M., Vietri B., Puyo E., and Medina M. (2004). Pregnancy rate in ewes inseminated with sexed semen in Argentina. Reprod. Fertil. Dev. 16, 284. [Abstract] doi:10.1071/RDV16N1AB329

Cran, D. G. , Johnson, L. A. , Miller, N. G. , Cochrane, D. , and Polge, C. (1993). Production of bovine calves following separation of X- and Y-chromosome bearing sperm and in vitro fertilisation. Vet Rec. 132, 40–41.
| PubMed |  CAS | Evans G., and Maxwell W. M. C. (1987). ‘Salamon’s Artificial Insemination Of Sheep and Goats.’ (Butterworths: Sydney.)

Garcia, E. M. , Vazquez, J. M. , Parrilla, I. , Calvete, J. J. , Sanz, L. , Caballero, I. , Roca, J. , Vazquez, J. L. , and Martinez, E. A. (2007). Improving the fertilizing ability of sex-sorted boar spermatozoa. Theriogenology 68, 771–778.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS | Lindsey A. C., Muckle L. K., and Squires E. L. (2003). Effects of caffeine stimulation on stallion sperm motion characteristics following 18-h storage, flow sorting, and cryopreservation. Theriogenology 59, 510. [Abstract]

Lu, K. H. , Cran, D. G. , and Seidel, G. E. (1999). In vitro fertilization with flow-cytometrically sorted bovine sperm. Theriogenology 52, 1393–1405.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS | Suh T. K., and Schenk J. L. (2003). Pressure during flow sorting of bull sperm affects post-thaw motility characteristics. Theriogenology 59, 516. [Abstract]

Vazquez, J. M. , Martinez, E. A. , Parrilla, I. , Roca, J. , Gil, M. A. , and Vazquez, J. L. (2003). Birth of piglets after deep intrauterine insemination with flow-cytometrically sorted boar spermatozoa. Theriogenology 59, 1605–1614.
| Crossref | GoogleScholarGoogle Scholar | PubMed |

Wilson, R. D. , Fricke, P. M. , Leibfried-Rutledge, M. L. , Rutledge, J. J. , Penfield, C. M. S. , and Weigel, K. A. (2006). In vitro production of bovine embryos using sex-sorted sperm. Theriogenology 65, 1007–1015.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |

Zhang, M. , Lu, K. H. , and Seidel, G. E. (2003). Development of bovine embryos after in vitro fertilization of oocytes with flow-cytometrically sorted, stained and unsorted sperm from different bulls. Theriogenology 60, 1657–1663.
| Crossref | GoogleScholarGoogle Scholar | PubMed | CAS |