Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (5)

Single-cell Motility Analysis of Tethered Human Spermatozoa


Single-cell Motility Analysis of Tethered Human Spermatozoa

William M Skinner et al. Bio Protoc.


Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA).

Keywords: Amyloid fibrils; Flagellar beat frequency; High-speed imaging; Human spermatozoa; Seminal plasma; Single-cell motility analysis; Sperm Swim-up; Sperm flagellum; Sperm motility.

Conflict of interest statement

Competing interests We declare there are no competing interests.


Figure 1.
Figure 1.
Fully assembled high-speed video microscopy rig with perfusion setup, on a vibration-isolation table
Figure 2.
Figure 2.. Assembled perfusion chamber for recording.
A. The recording chamber is placed inside the stage adapter and connected to perfusion lines via a perfusion manifold. The suction line is attached to a metal suction tube, which is positioned on the stage adapter with a magnetic clamp. B. Top view of the same setup.
Figure 3.
Figure 3.. Homemade perfusion line.
The large syringe is used to hold perfusion fluid, and the small syringe is used to purge air bubbles from the system. The Perfusion Manifold allows for up to 8 perfusion lines to be connected simultaneously. Extra manifold ports are blocked when not in use.
Figure 4.
Figure 4.. Semen sample and tubes of HTF fluid.
The semen sample is in the collection container at right. The donor’s anonymized identity number and time of collection is labeled on the collection container (but redacted for publication). One ml of semen will be underlaid into each of the tubes of HTF on the left. In this case, only 3 tubes of HTF were used, as the ejaculate volume was roughly 3 ml.
Figure 5.
Figure 5.. Semen sample before and after swim-up.
A. HTF fluid in falcon tube before addition of semen. B. One ml of semen carefully underlaid at the bottom of the tube. C. The same tube after 1 h of incubation at 37 °C. Note the supernatant is cloudier, and the boundary between the semen and the HTF fluid less defined. This shows that sperm has swum up into the supernatant. D. The same tube after removing several milliliters of supernatant.
Figure 6.
Figure 6.. Sperm settling and concentration.
A. Three ml of HTF supernatant was removed from each swim-up tube and pooled into one 15 ml conical tube. B. The cloud of sperm cells that have settled to the bottom of the tube after 30–60 min standing at RT. C. Several milliliters of supernatant removed to concentrate the sperm cells. D. Sperm cloud resuspended by gentle pipetting.
Figure 7.
Figure 7.. Example frames illustrating the measurement of one full beat cycle of a human spermatozoa.
The numbers above each frame indicate specific frame position out of 2,310 frames collected. There is a 12-frame gap between each image displayed here, and the scale bars represent approximately 5 μm. The image was contrast enhanced in ImageJ for visual clarity. This spermatozoon has a period of 60 ms, and therefore a frequency of approximately 17 Hz. Video (.avi) and TIFF files are included as supplemental materials.

Similar articles

See all similar articles


    1. Austin CR (1951). Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B 4(4): 581–596. - PubMed
    1. Burnett LA, Blais EM, Unadkat JD, Hille B, Tilley SL and Babcock DF (2010). Testicular expression of Adora3i2 in Adora3-knockout mice reveals a Role of mouse A3Ri2 and human A3Ri3 adenosine receptors in sperm. J Biol Chem 285: 33662–33670. - PMC - PubMed
    1. Chang MC (1951). Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168(4277): 697–698. - PubMed
    1. Cooper TG and Yeung CH (2006). Computer-aided evaluation of assessment of “grade a” spermatozoa by experienced technicians. Fertil Steril 85(1): 220–224. - PubMed
    1. Fredricsson B, Möller L, Pousette A and Westerholm R (1993). Human sperm motility is affected by plasticizers and diesel particle extracts. Pharmacol Toxicol 72(2): 128–133. - PubMed

LinkOut - more resources