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Published on: February 6, 2018
Microscopic analysis of MTT stained boar sperm cells
1Barex Biochemical Products, Seb. Centenweg 45, 1602ML Enkhuizen, The Netherlands.
Abstract:
The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few min in a diffuse staining of the midpiece of the sperm cells. Upon further incubation the staining of the midpiece became more intense, and gradually the formation of packed formazan granules became more visible. At the same time, a small formazan stained granule appeared medially on the sperm head, which increased in size during further incubation. After incubation for about 1 h the midpiece granules were intensely stained and more clearly distinct as granules, while aggregation of sperm cells occurred. Around 90% of the sperm cells showed these staining events. At the end of the staining the formazan granules have disappeared from both the sperm cells and medium, whereas formazan crystals appeared as thin crystal threads, that became heavily aggregated in the incubation medium. It was concluded that formazan is taken up by lipid droplets in the cytoplasm. Further, the use of the MTT assay to test for sperm viability should be regarded as a qualitative assay, whereas its practical use at artificial insemination (AI) Stations is limited.
Insights
The MTT assay visualizes formazan in boar sperm, revealing its uptake by lipid droplets. This microscopic study suggests the MTT assay is qualitative and has limited practical use for sperm viability testing at artificial insemination stations.
Area of Science:
- Reproductive Biology
- Cell Biology
- Biochemistry
Background:
- The MTT assay, based on formazan production by sperm cells, has been used for mammalian sperm viability.
- Microscopic visualization of formazan localization in boar sperm was previously lacking.
Purpose of the Study:
- To microscopically visualize the precise location of formazan within boar sperm cells during the MTT assay.
- To understand the morphological events of MTT staining in boar sperm.
Main Methods:
- Boar sperm cells were incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
- Microscopic observation documented the morphological changes and formazan granule formation over time.
- Sperm cells were analyzed for formazan localization and crystal formation.
Main Results:
- MTT staining initially showed diffuse midpiece staining, progressing to intense granules.
- A distinct formazan granule appeared on the sperm head, increasing in size.
- Formazan granules disappeared, forming aggregated crystal threads in the medium, indicating uptake by lipid droplets.
Conclusions:
- Formazan is localized within lipid droplets in the cytoplasm of boar sperm.
- The MTT assay for sperm viability should be considered qualitative.
- The practical application of the MTT assay at artificial insemination stations is limited due to its qualitative nature.
