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Oviductal sperm counting

WW Wipawee Winuthayanon
CW Carmen J Williams

Last updated date: Apr 2, 2021 Views: 788 Forks: 0

original An abbreviated version of this protocol was published in eLife in Dec, 2015
Oviductal estrogen receptor α signaling prevents protease-mediated embryo death
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  1. Dissect the oviducts from the uteri as close to the uterine horn as possible
  2. Place the oviduct (one at a time) on a glass slide under a dissecting microscope
  3. Make a small cut (just a nick to insert the needle) in the ampulla below the cumulus-oocyte complexes (COCs) using butterfly scissors
  4. Use a blunt-ended 30 Gauge needle on a tuberculin syringe filled with ~30 µL diH2O for flushing
  5. Insert the needle into the ampulla through the cut
  6. Hold the needle in place using forceps
  7. Push diH2O from the ampulla toward the isthmus, beginning distal to the cumulus mass, using ∼30 µL water via the 30 G needle. (Note that the water will immobilize the sperm)
  8. Remove the oviduct from the slide after flushing
  9. Cover the slide with a coverslip
  10. Count the number of sperm flushed from each oviduct on the entire slide using a 40X objective following the motion below.
Diagram, text Description automatically generated

11. Repeat the procedure with the other side oviduct and combine the number for the total sperm number in both oviducts

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Winuthayanon, W and Williams, C(2021). Oviductal sperm counting. Bio-protocol Preprint. bio-protocol.org/prep993.
  2. Winuthayanon, W., Bernhardt, M. L., Padilla-Banks, E., Myers, P. H., Edin, M. L., Lih, F. B., Hewitt, S. C., Korach, K. S. and Williams, C. J.(2015). Oviductal estrogen receptor α signaling prevents protease-mediated embryo death. eLife. DOI: 10.7554/eLife.10453

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