Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96.
Abstract: 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 μM 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 μM 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 μM (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 μM 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 μM. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-01-24 PubMed ID: 22325925DOI: 10.1016/j.anireprosci.2012.01.003Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research paper investigates the impact of Hoechst 33342, a stain used in sperm sexing technology, on the motility and membrane integrity of stallion sperm when used in varying concentrations. The study found that the negative effects of the stain vary based on the medium used for incubation, with stallion sperm showing more tolerance when incubated in an INRA-Tyrodes modified medium compared to a KMT medium.
Introduction and Research Objective
- The research seeks to evaluate the effects of the fluorescent dye Hoechst 33342 on the motility and membrane integrity of stallion sperm.
- The dye is used in the Beltsville sexing technology, which separates X and Y chromosomal sperm based on their DNA content.
Research Methodology
- Various concentrations of Hoechst 33342 dye were prepared and applied to the ejaculate samples.
- They were then incubated for up to 90 minutes in two different media – KMT and INRA-Tyrodes modified medium.
- After 40 minutes and 90 minutes of incubation, the motility of the sperm was measured using Computer-assisted Sperm Analysis (CASA), and membrane integrity was analyzed using flow cytometry after YoPro-1/Eth staining.
Findings and Conclusions
- In the KMT extender medium, sperm motility significantly decreased after 45 minutes when sperm were incubated with concentrations of Hoechst of 45 μM or greater.
- In contrast, when incubated in a modified INRA96 medium, stallion sperm tolerated higher concentrations of Hoechst, showing decrease in motility only when incubated in presence of 90 μM.
- Additionally, incubation in the INRA96 medium did not affect membrane integrity, unlike in the KMT medium.
- The findings suggest that the modified INRA96 medium could be an effective alternative to KMT for incubating and analysing stallion sperm.
- The study also emphasizes that the deleterious effects of Hoechst 33342 on stallion sperm varies based on the incubation medium used.
Cite This Article
APA
Balao da Silva C, Macías-García B, Morillo Rodriguez A, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Morrell JM, Rodriguez-Martínez H, Ortega-Ferrusola C, Peña FJ.
(2012).
Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96.
Anim Reprod Sci, 131(3-4), 165-171.
https://doi.org/10.1016/j.anireprosci.2012.01.003 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction, Veterinary Teaching Hospital, Department of Medicine, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
MeSH Terms
- Animals
- Benzimidazoles / pharmacology
- Cytoprotection / drug effects
- Fluorescent Dyes / pharmacology
- Horses / physiology
- Male
- Sex Preselection / veterinary
- Sperm Motility / drug effects
- Spermatozoa / drug effects
- Staining and Labeling
- Time Factors
Citations
This article has been cited 3 times.- Marin DFD, de Souza EB, de Brito VC, Nascimento CV, Ramos AS, Filho STR, da Costa NN, Cordeiro MDS, Santos SDSD, Ohashi OM. In vitro embryo production in buffaloes: from the laboratory to the farm. Anim Reprod 2019 Oct 23;16(2):260-266.
- Feng L, Gan H, Zhao W, Liu Y. Effect of transplantation of olfactory ensheathing cell conditioned medium induced bone marrow stromal cells on rats with spinal cord injury. Mol Med Rep 2017 Aug;16(2):1661-1668.
- Egyptien S, Deleuze S, Ledeck J, Ponthier J. Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions. Animals (Basel) 2023 Oct 6;13(19).
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