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Research article - Peer-reviewed, 2012

Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96

Balao da Silva, C.; Macias-Garcia, B.; Morillo Rodriguez, A.; Gallardo Bolanos, J. M.; Tapia, J. A.; Aparicio, I. M.; Morrell, J. M.; Rodriguez-Martinez, H.; Ortega-Ferrusola, C.; Pena, F. J.


The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 mu M of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 mu M or greater (P < 0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 mu M (P < 0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 mu M the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 mu M. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT. (C) 2012 Elsevier B.V. All rights reserved.


Stallion; Sperm; CASA; Hoechst 33342; Sex sorting

Published in

Animal Reproduction Science
2012, volume: 131, number: 3-4, pages: 165-171

Authors' information

Balao da Silva, C. M.
University of Extremadura
Macías García, B.
University of Extremadura
Morillo Rodriguez, A.
University of Extremadura
Gallardo Bolaños, J.M.
University of Extremadura
Tapia, J.A.
University of Extremadura
Aparicio, I.M.
University of Extremadura
Swedish University of Agricultural Sciences, Department of Clinical Sciences
Rodriguez-Martinez, H.
Linköping University
Ortega-Ferrusola, C.
University of Extremadura
Peña, F.J.
University of Extremadura

UKÄ Subject classification

Veterinary Science
Animal and Dairy Science

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