Journal of Biomechanics 48 (2015) 29412947

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Journal of Biomechanics
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Effect of non-Newtonian uid properties on bovine sperm motility

Toru Hyakutake a,n, Hiroki Suzuki b, Satoru Yamamoto b
a
b

Faculty of Engineering, Yokohama National University, 79-5 Hodogaya, Yokohama 240-8501, Japan
Graduate School of Engineering, Yokohama National University, 79-5 Hodogaya, Yokohama 240-8501, Japan

art ic l e i nf o

a b s t r a c t

Article history:
Accepted 3 August 2015

The swimming process by which mammal spermatozoa progress towards an egg within the reproductive
organs is important in achieving successful internal fertilization. The viscosity of oviductal mucus is more
than two orders of magnitude greater than that of water, and oviductal mucus also has non-Newtonian
properties. In this study, we experimentally observed sperm motion in uids with various uid rheological properties and investigated the inuence of varying the viscosity and whether the uid was
Newtonian or non-Newtonian on the sperm motility. We selected polyvinylpyrrolidone and methylcellulose as solutes to create solutions with different rheological properties. We used the semen of Japanese
cattle and investigated the following parameters: the sperm velocity, the straight-line velocity and the
amplitude from the trajectory, and the beat frequency from the fragellar movement. In a Newtonian uid
environment, as the viscosity increased, the motility of the sperm decreased. However, in a non-Newtonian uid, the straight-line velocity and beat frequency were signicantly higher than in a Newtonian
uid with comparable viscosity. As a result, the linearity of the sperm movement increased. Additionally,
increasing the viscosity brought about large changes in the sperm agellar shape. At low viscosities, the
entire agellum moved in a curved apping motion, whereas in the high-viscosity, only the tip of the
agellum apped. These results suggest that the bovine sperm has evolved to swim toward the egg as
quickly as possible in the actual oviduct uid, which is a high-viscosity non-Newtonian uid.
& 2015 Elsevier Ltd. All rights reserved.

Keywords:
Bovine sperm
Viscosity
Non-Newtonian uid
Flagellum
Image analysis

1. Introduction
The movement of a spermatozoon, the male reproductive cell,
toward an egg, the female reproductive cell, enables fertilization. The
swimming process toward an egg within the reproductive organs is
important for the mammal spermatozoa to achieve successful internal fertilization. Each sperm cell has a agellum, and the propulsion of
the sperm is caused by the active motion of the agellum. This active
force arises from the action of inner- and outer-arm dynein motors. It
is presumed that this movement mode evolved in response to the
surrounding environment of the sperm; consequently, there exist
several agellar waveforms that depend on the species. For example,
in the case of the sea urchin, the sperm swims in the sea (external
fertilization). Conversely, in mammals, the sperm moves within the
reproductive organs (internal fertilization). Therefore, understanding
how the mechanism of sperm motility corresponds to the surrounding uid environment is extremely important.
In this study, we consider the sperm motility of mammals. As
mentioned above, mammal sperm participates in internal fertilization. When the sperm moves toward the ovary through the
n

Corresponding author. Tel./fax: 81 45 339 3882.

E-mail address: hyaku@ynu.ac.jp (T. Hyakutake).

http://dx.doi.org/10.1016/j.jbiomech.2015.08.005
0021-9290/& 2015 Elsevier Ltd. All rights reserved.

oviduct, it is inuenced by several circumstances. The oviductal

mucus is composed of various uids, including macromolecules
and gels. Mammalian sperm migrates through the oviduct, where
the viscosity is quite high compared to that of water. Moreover, the
sperm moves against the ow of the oviductal uid because of
tubal peristalsis. In addition, a change in the sperm motion, called
hyperactivation, may occur by a signal transduction mechanism
through a medium of calcium ions on the way to the ovary.
Therefore, we can say that sperm motility is signicantly inuenced by the rheology, shear stress, and chemical composition of
the surrounding uid. In the present study, we focused on the
effect of the uid rheological properties of the oviductal mucus on
sperm motility.
Many researchers have conducted rigorous studies on the
motion characteristics of sperm from theoretical (Gray and
Hancock 1955; Lighthill, 1976; Higdon, 1979; Phan-Thien et al.,
1987), experimental (Brokaw 1965; Phillips 1972; Mortimer et al.,
1997; Crenshaw et al., 2000; Woolley 2003; Denissenko et al.,
2012), and numerical (Gillies et al., 2009; Smith et al., 2010; Elgeti
et al., 2010; Tam and Hosoi 2011; Gurarie et al., 2011; Guerrero
et al., 2011) standpoints. Focusing on the bovine sperm used in the
present experiment, Rikmenspoel and Herpen (1957) indicated
that sperm velocity is proportional to the frequency of the sperm
rotation. Furthermore, they observed the elongation of the

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

agellum and suggested that bovine sperm rotates in three

dimensions. Rikmenspoel (1965) distinguished between nonrotating and rotating sperm heads and concluded that sperm with
nonrotating heads is unhealthy. Focusing on the rotation of the
sperm head, Drake (1974) claried that the sperm head continuously rotates 360. Rikmenspoel (1984) experimentally
observed the relationship between temperature and viscosity.
Ishijima et al. (1992) investigated the rotational movement of a
spermatozoon around its longitudinal axis in sperm from various
species, including bovine sperm. Friedrich et al. (2010) conducted
high-precision measurements of head and agellum motion of
bull spermatozoa as they swam along circular paths near a surface.
In addition, several studies recently successfully tracked the threedimensional trajectory of the human spermatozoon (Sheng et al.,
2007; Corkidi et al., 2008; Su et al., 2012). However, most of these
studies investigated sperm motility in a diluted solution. Wolf
et al. (1977) found that the mucus in the uterine tube is a viscoelastic uid that contains gelatinous materials and macromolecules. Lai et al. (2007) measured the viscosity of fresh human
cervical mucus samples as a function of the shear rate and showed
that the viscosity of the mucus is greater than that of water by two
orders of magnitude. Furthermore, they indicated that the mucus
is a non-Newtonian uid. Several studies have focused on the
effect of the surrounding environment of the sperm, i.e., the uid
characteristics of the oviduct, on the sperm motility, particularly
the viscosity of the surrounding uid (Katz et al., 1978; Rikmenspoel 1984; Smith et al., 2009; Kirkman-Brown and Smith 2011).
Suarez and Dai (1992) experimentally investigated mouse sperm
motility in a viscoelastic uid. Several numerical studies (Fu et al.,
2007; Teran et al., 2010) have been examined on the motion of
sperm in non-Newtonian uids. However, since few experimental
studies have focused on the differences between high-viscosity
Newtonian and non-Newtonian uid environments, the effect of a
non-Newtonian surrounding uid on sperm motility is not clear.
Therefore, it is necessary to observe sperm motility in a highviscosity non-Newtonian uid to understand the essential
mechanism of sperm motility.
Given this background, we experimentally investigated the
effect of a non-Newtonian uid on the motion characteristics of
bovine sperm. In particular, by comparing Newtonian and nonNewtonian uid environments, we analyzed the trajectory of the
sperm motion and investigated the effect of the rheological
properties of the surrounding environment on the sperm velocity
and the amplitude from the sperm trajectory. Additionally, we
investigated the inuence of the uid rheological properties on the
agellar shape of the observed sperm. The obtained experimental
results will be useful in clarifying the mechanics of sperm motility
under their actual environmental conditions. Furthermore, the
results will provide valuable information for the reproduction
industry of the animal husbandry eld. Additionally, this study
may provide useful data that will contribute to understanding
sperm motility in the microuidic sperm sorter that has been
developed for infertility treatment (Cho et al., 2003; Schuster et al.,
2003; Hyakutake et al., 2009; Matsuura et al., 2012).
2. Materials and methods
We observed the sperm motion using an optical microscope (OlympusIX71,
Olympus, Japan) and obtained pictures using a CCD camera (K-II, Kato Koken,
Japan). We used Japanese cattle semen (Suzukane, Animal Genetics Japan Co., Ltd.,
Japan), which was cryopreserved in a liquid nitrogen tank. First, we thawed it and
added a Triscitric acidglucose solution, which has similar components to those of
the diluted solution used when the semen was cryopreserved. This was added to
prevent the loss of fertilization ability from damage to and breakdown of the sperm
acrosome. As a result, we were able to sustain the sperm motility and extend the
observation time. Next, to facilitate observation, we separated the bovine semen
into sperm and seminal plasma using a centrifugal separator for a duration of

PVP-K90 4.0 %
PVP-K90 10 %
PVP-K90 15 %

100

Viscosity [Pa*s]

2942

MC100 0.6 %
MC100 2.0 %
MC4000 1.0 %

10-1

10-2

10-3

10
Shear rate [1/s]

100

Fig. 1. Relation between the shear rate and viscosity of PVP-K90, MC100, and
MC4000. The lled triangle represents PVP-K90 4.0%, the lled diamond represents
PVP-K90 10%, the lled inverted triangle represents PVP-K90 20%, the open triangle
represents MC100 0.6%, the open diamond represents MC100 2.0%, and the open
inverted triangle represents MC4000 1.0%.

10 min. The separated sperm was then diluted with a phosphate buffered saline,
where we fused it with polyvinylpyrrolidone (PVP) and methylcellulose (MC)
solutions to change the rheological properties of the sperm solution. This suspension was warmed in a water bath at 38.5 C, and the temperature of the suspension was maintained using a thermoplate during the observation. The suspension was placed in a glass slide with a pool of depth 0.1 mm, and covered with a
coverslip. Since there was a gap of 0.1 mm between the bottom of the pool and the
coverslip, the motion of the sperm was not restricted in the vertical direction.
We used a rheometer (ARES-G2, TA Instruments) to measure the viscosity of
the PVP and MC solutions at a temperature of 38.5 C. We selected PVP-K90 (Wako
Pure Chemical Industries, Ltd., Japan) for the PVP solutions, and MC100 and
MC4000 (Wako Pure Chemical Industries, Ltd., Japan) for the MC solutions. Three
PVP-K90 concentrations, 4.0%, 10%, and 15%, were selected. Two MC100 concentrations, 0.6% and 2.0%, and one MC4000 concentration, 1.0%, were selected.
Fig. 1 shows the relationship between the shear rate and the viscosity of the six
solutions. The experimental data in this gure were taken from one measurement.
The viscosities of the PVP-K90 solutions were almost constant as a function of the
shear rate, which means that PVP-K90 is a Newtonian uid. An increase in the PVPK90 concentration caused an increase in the viscosity. The MC solution generally
has viscoelastic properties (Amari and Nakamura, 1973). The viscosities of the
MC100 solutions did not greatly change for different shear rates; therefore, we
considered the non-Newtonian properties of MC100 to be weak. However, the
viscosity of the MC4000 solution decreased with an increase in the shear rate, and
its values were between the viscosities of the PVP-K90 solutions with concentrations of 10% and 15%. The viscosity when the shear rate was 1.0 s  1 was approximately three times that when shear rate was 100 s  1. Therefore, we considered the
MC4000 solution to have strong non-Newtonian properties.
We observed the sperm motion under a microscope and obtained images at a
rate of 200 fps using a high-speed camera. For image analysis, we employed a
particle tracking velocimetry (PTV) technique analysis using the DIPP-Motion Pro
uid analysis software (Ditect Co., Ltd., Japan). We obtained the trajectory of the
sperm motion by marking the sperm head in the images. At a low viscosity, the
sperm agellum was assumed to have three-dimensional movement because the
sperm head rotates. Strictly speaking, we needed to observe the sperm motion in
three dimensions because of the rotating sperm head. However, exact threedimensional observation is very difcult. Therefore, we obtained the two-dimensional velocity for the observed plane in the present experiment. On the other
hand, at a high viscosity, the sperm hardly rotates, so we can consider the agellar
wave plane to become parallel to the observed plane as the projected area of the
sperm head becomes larger. Therefore, we extracted sperm when we observed the
projected area of the sperm head to be large. From the obtained trajectory, we
calculated two velocities using the MATLAB digital image analysis software
(MathWorks, USA). The rst is the sperm velocity VSP, which was calculated by
averaging the velocities determined using the change in sperm position in each
pair of successive images. The other is the straight-line velocity of the sperm VST,

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

2943

Fig. 2. Trajectories of sperm for different solutions and trajectory times: (a) PVP-K90 4.0%, 1 s; (b) MC4000 1.0%, 2 s. The red line represents the progress axis of the sperm
trajectory, which was obtained by using the least-squares method from the obtained trajectory point. We dened half the distance between the furthest two points of both
sides of the progress as the amplitude of the sperm trajectory (A). (For interpretation of the references to color in this gure legend, the reader is referred to the web version
of this article.)

3. Results
Fig. 3 shows the relation between the straight-line velocity VST
and the sperm velocity VSP for the seven different solutions. From
this gure, it is clear that VST increases with VSP, demonstrating a
correlation between the straight-line and sperm velocities
depending on each solution. For the diluted solution and the PVPK90 concentrations of 4%, the experimental data were mostly
distributed in the area above the line passing through the origin
with a slope of VSP/VST 3.0. This large slope means that the
straight-line velocity is small compared to the sperm velocity; that
is, the linearity of the sperm trajectory is low. However, for the
PVP-K90 concentrations of 10% and 15%, the decreases in VSP and
VST were signicant. The experimental data for these concentrations were mostly distributed in the area above the line passing
through the origin with a slope of VSP/VST 1.0; this slope corresponds to sperm moving in a straight line. Regarding the effect of
the viscosity on the two velocities, increasing the concentration
caused a large decrease in both velocities. In the case of the diluted
solution, the sperm velocity was in the range of approximately
110220 m/s. Conversely, when the PVP-K90 concentration was
15%, which corresponds to a viscosity of 0.45 Pa s, the sperm
velocity range decreased remarkably to approximately 712 m/s.
The experimental data of the MC solutions showed different tendencies than that of PVP solutions. In the MC100 solutions, both
velocities decreased as the concentration increased. Conversely,

250

200

V SP [m/s]

which was calculated using the distance between the sperm positions in the rst
and last frames. Furthermore, we also calculated the amplitude of the sperm trajectory, A. First, we obtained the progress axis of the sperm trajectory by using the
least-squares method from the obtained trajectory point (red line in Fig. 2(a)). Next,
we obtained the furthest two points on both sides of the progress axis and dened
half the distance between these two points as the amplitude of the sperm trajectory. For illustrative purposes, Fig. 2(a) and (b) shows the trajectories of the sperm
motion and the progress axis for a PVP concentration of 4.0% and an MC4000
concentration of 1.0%, respectively. In the case of PVP-K90, 19, 16, and 15 sperm
samples were taken at 4.0%, 10.0%, and 15.0%, respectively. In the case of MC100, 22
and 18 sperm samples were taken at 0.6% and 2.0%, respectively. In the case of
MC4000, 15 sperm samples were taken. In addition, we experimented on the case
of the diluted solution onlythat is, we did not add the reagent to increase viscosity. In this case, 17 sperm samples were taken.

150

100
Diluted solution
PVP-K90 4.0 %
PVP-K90 10 %
PVP-K90 15 %
MC100 0.6 %
MC100 2.0 %
MC4000 1.0 %

50

20

40
VST [m/s]

60

80

Fig. 3. Straight-line velocity (VST) versus sperm velocity (VSP). The lled square
represents the diluted solution, the lled triangle represents PVP-K90 4.0%, the
lled diamond represents PVP-K90 10%, the lled inverted triangle represents PVPK90 20%, the open triangle represents MC100 0.6%, the open diamond represents
MC100 2.0%, and the open inverted triangle represents MC4000 1.0%.

the straight-line velocity VST in the MC4000 solution did not

decrease, and VST of some sperm increased despite the fact that
the viscosity of MC4000 was larger than that of MC100. As shown
in Fig. 1, the viscosity of MC4000 decreased with increasing shear
rate in the range of 1.0 to 100 s1, and its values were between the
viscosities of the PVP-K90 solutions with concentrations of 10%
and 15%. However, the velocity characteristics of MC4000 were
drastically different from those of the PVP-K90 solutions. The
straight-line velocity in the MC4000 solution increased signicantly compared to that in PVP solutions with comparable
viscosities (PVP-K90 10% and 15%). The maximal straight-line
velocity in the MC4000 solution was approximately 50 m/s,
which is comparable to that of the diluted solution.

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T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

250

3
Diluted solution
PVP-K90 4.0 %
PVP-K90 10 %
PVP-K90 15 %
MC100 0.6 %
MC100 2.0 %
MC4000 1.0 %

Ad [-]

200

m/s

150

100

0.2

0.4

0.6

0.8

50

L [-]
Fig. 4. Linearity (L) versus normalized amplitude (Ad). The lled square represents
the diluted solution, the lled triangle represents PVP-K90 4.0%, the lled diamond
represents PVP-K90 10%, the lled inverted triangle represents PVP-K90 20%, the
open triangle represents MC100 0.6%, the open diamond represents MC100 2.0%,
and the open inverted triangle represents MC4000 1.0%.

0
Diluted
solution

PVP-K90 PVP-K90 PVP-K90

4.0%
10%
15%

V_SP
To investigate these different tendencies in more detail, we
dened the linearity as the reciprocal of the slope in Fig. 3.

(1)

L 1 means that the sperm moves in a straight line, and L 0

means that the sperm does not progress at all. Fig. 4 shows the
relation between the linearity L and the amplitude Ad of the sperm
trajectory normalized by the length of the average sperm head (5
m). This gure shows that increasing concentration in PVP-K90
and MC100 solutions led to decreasing amplitude and increasing
linearity. However, in the non-Newtonian MC4000 solution,
increasing viscosity had almost no effect on the amplitude and
brought about a large increase in linearity. The linearity of most
spermatozoa was over 0.8, and they moved in almost straight lines
(see Fig. 2(b)).
Fig. 5 summarizes the variation in the average values of VSP and
VST for the seven different solutions. The average sperm velocity in
the 0.0% PVP-K90 solution was approximately 200 m/s, whereas
that in the 15% PVP-K90 solution was approximately 9.2 m/s, a
considerable decrease of over 95%. Similarly, the average straightline velocity decreased from 40 to 3.7 m/s as the viscosity
increased. Regarding the MC solutions, the average sperm velocity
in the 0.6% MC100 solution was approximately 122 m/s and that
in the 1.0% MC4000 solution was approximately 37 m/s, a
decrease of approximately 70%. However, the average straight-line
velocities in the MC100 and MC4000 solutions were almost
equivalent.
Next, we calculated the beat frequency of the agellar movement. Fig. 6 compares the frequency for each reagent. For PVP-K90
and MC100, increasing the viscosity decreased the frequency. For
MC4000, the beat frequency was highest with every reagent.
Furthermore, we investigated the relationship between the value
of the straight-line velocity divided by the beat frequency and
linearity L (Fig. 7). In the case of low viscosity (diluted solution and
4%, and MC100 0.6 and 2%), the results were concentrated in the
near region. Therefore, for a region with such low viscosity, the
distance by which the sperm progresses in one stroke and the
linearity are not largely affected by the viscosity. On the other

MC100
2.0%

MC4000
1.0%

V_ST

Fig. 5. Comparison of average sperm and straight-line velocities (VSP, VST) for different solutions. The error bar indicates the standard deviation of the average value.

12

10

Hz

L = VST /VSP

MC100
0.6%

0
Diluted
solution

PVP-K90
4.0%

PVP-K90
10%

PVP-K90
15%

MC100
0.6%

MC100
2.0%

MC4000
1.0%

frequency
Fig. 6. Comparison of average frequency of the sperm agellum for different
solutions. The error bar indicates the standard deviation of the average value.

hand, as the viscosity increased, VST/f decreased, and conversely

the linearity increased. The linearity increased further in the case
of MC4000.
Finally, we investigated the effect of the rheological properties
of the surrounding uid on the agellar shape of the sperm. Fig. 8
shows the agellar shape for several reagents. The red line indicates the agellum. From this gure, in the case of diluted solution,
at which the viscosity is near that of water, the entire agellum

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

from the root to the tip moved in a curved apping motion.

However, when the PVP-K90 concentration was 10%, at which the
viscosity is approximately 80 times that of water, the curvature of
the agellum near the midpiece was very small, and primarily only
the half of the agellum toward the tip moved. With a further
increase in the viscosity, the portion of the agellum near the
midpiece moves minimally, and primarily only the tip of the agellum moves in a curved apping motion. The portion of the

12
Diluted solution
PVP-K90 4.0 %
PVP-K90 10 %
PVP-K90 15 %
MC100 0.6 %
MC100 2.0 %
MC4000 1.0 %

V ST / f [m]

10
8
6
4
2

0.2

0.4

0.6

0.8

L [-]
Fig. 7. Linearity (L) versus progress distance par one stloke (VST / f). The lled
square represents the diluted solution, the lled triangle represents PVP-K90 4.0%,
the lled diamond represents PVP-K90 10%, the lled inverted triangle represents
PVP-K90 20%, the open triangle represents MC100 0.6%, the open diamond
represents MC100 2.0%, and the open inverted triangle represents MC4000 1.0%.

2945

agellum that moved in the 15% PVP-K90 and MC4000 solution

was shorter than that in the 10% PVP-K90 solution.

4. Discussion
In the present study, we experimentally observed the motion
characteristics of bovine sperm swimming in solutions made with
two different solutes, PVP and MC. The PVP and MC100 solutions
are Newtonian uids, and the MC4000 solution is a non-Newtonian uid. We observed several parameters involved in sperm
motile capability (sperm and straight-line velocities and trajectory
amplitude) and determined how these parameters differ in Newtonian and non-Newtonian uid environments. In the Newtonian
uid environment, as the concentration of the solutes increased,
both the sperm and straight-line velocities and beat frequency
decreased, and the sperm motility declined. However, in the nonNewtonian uid environment, as the concentration of the solute
increased, the sperm velocity decreased, but the straight-line
velocity was almost constant. As a result, the linearity signicantly
increased. The beat frequency of the agellum also increased. We
also investigated the effect of different solutions on the agellar
shape of the sperm. The experimental results indicate that sperm
motion characteristics are signicantly affected by the rheological
properties of the uid surrounding the sperm. Previous studies
have primarily discussed the motion characteristics of sperm in
diluted semen. The sperm motility in a high-viscosity non-Newtonian uid whose rheological properties are similar to those of
oviductal mucus is drastically different from that in Newtonian
uid. This is a signicant result in understanding the mechanics of
sperm motility in the actual environment.
Suarez and Dai (1992) measured the sperm velocity and
straight-line velocity of the mouse sperm for three solutions: the
diluted solution, methylcellulose (high viscosity), and polyacrylamide (high viscoelasticity). Furthermore, they investigated
the change in velocities with hyperactivation. When the present

Fig. 8. Comparison of agellar shape for several reagents. The red line indicates the agellum. (a) Diluted solution, (b) PVP-K90 10%, (c) PVP-K90 15%, and (d) MC4000 1%.

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T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

results were compared to the non-hyperactivated case, the

decrease in the sperm velocity due to the high viscosity reected
the same results. However, the observed agellar shape was different. Two potential reasons were considered: the difference in
species (i.e., mouse and bovine), and the difference in medium
(i.e., methylcellulose and polyacrylamide). Consequently, for the
mouse sperm used by Suarez et al. the viscoelasticity did not
increase the linearity. It is very interesting that the effect of the
viscoelasticity on the sperm velocity differs according to the species. Furthermore, Smith et al. (2009) experimentally investigated
the effect of viscosity on human sperm motion characteristics
using MC as the uid surrounding the sperm. They compared
sperm movement in two solutions, a low-viscosity saline medium
with a viscosity of approximately 0.0007 Pa s and a high-viscosity
saline-methylcellulose medium with a viscosity of 0.14 Pa s. Consequently, their experimental results indicated that a difference in
the viscosity signicantly inuences the trajectory and agellar
shape of the sperm but has little inuence on the straight-line
velocity, which they call progressive velocity. In this experiment,
we made smaller incremental changes to the viscosity and furthermore compared the results of Newtonian and non-Newtonian
uids. Consequently, our study has claried that the phenomenon
Smith et al. reported of the straight-line velocity remaining constant as viscosity increased was because of the inuence of nonNewtonian properties. In a high-viscosity Newtonian uid environment, the straight-line velocity of the sperm decreases. Therefore, to understand the mechanics of sperm motility in the actual
environment in more detail, it is important to examine the effect
of a non-Newtonian uid environment on the motion characteristics of the sperm.
Interestingly, differences in the agellar shape were observed
depending on the rheological properties of the surrounding uid.
As the viscosity increased, the agellar shape changed from the
entire agellum apping to only a portion of it near the tip. This
difference was similarly observed by Smith et al. (2009). The
thickness of the agellum is nonuniform, and it gradually tapers
from the midpiece to the endpiece. In the low-viscosity uids, the
thickness of the agellum minimally affected its curvature,
whereas it had a much larger effect on curvature as viscosity
increased. Consequently, the change in the agellar shape led to an
increase in the linearity of the sperm. The agellar shape of 15%
PVP-K90 was similar to that of MC4000. Therefore, the change in
the agellar shape was assumed to be affected by the increase in
the viscosity rather than by the non-Newtonian uid. On the other
hand, the difference between the Newtonian and non-Newtonian
uids appeared to cause changes in the sperm velocity and beat
frequency (Figs. 5 and 6). These results mean that the sperm has
progressive motility in a high-viscosity non-Newtonian environment. These results suggest that the sperm agellum has evolved
to be able to effectively travel through the non-Newtonian oviductal uid and reach the ovum as quickly as possible.
In the present experiment, the maximal viscosity was 0.45 Pa s
(Fig. 1). However, the viscosity of oviductal mucus is in the range of
0.11.0 Pa s, according to the measurements of Lai et al. (2007).
Additionally, the change in the viscosity for the shear rates shown
in Fig. 1 is larger than that of the MC4000 solution; that is, the
non-Newtonian properties of the actual environment are much
stronger than those in this experiment. Therefore, it may be
necessary to investigate sperm motility in an environment with a
greater viscosity and stronger non-Newtonian properties. Additionally, with the present experimental apparatus, we conducted
only two-dimensional observations. Investigating the threedimensional motion of agella in a high-viscosity uid environment may also lead to a better understanding of the mechanics of
sperm motility in the actual environment. Furthermore, since we
rst diluted the semen to facilitate observations, interactions

between spermatozoa were not observed. Under actual conditions,

spermatozoa will move in a group in the oviduct. Therefore, the
effect of the interaction between spermatozoaon sperm motility in
a high-viscosity non-Newtonian uid may also be important.
When a mammalian spermatozoon moves toward the ovary
through the oviduct, changes in sperm motility occur because of
the increase in the concentration of the intracellular calcium ions,
which is called hyperactivation. The motion pattern of the hyperactivated sperm is characterized by a sharp bend in the proximal
midpiece of the sperm agellum because of the penetration of the
sperm through the zona pellucida (Stauss et al., 1995). As a result,
the linearity of sperm motion has been experimentally observed to
almost disappear; however, these motion characteristics have only
been observed in diluted solutions. Since this decrease in linearity
conicts with the sperm reaching the ovary as quickly as possible,
the trajectory of the hyperactivated sperm motion in the actual
environment may differ from that in the diluted solution. Future
work should include an investigation of the motion characteristics
of hyperactivated sperm in a high-viscosity non-Newtonian uid.

5. Conclusions
In this study, we experimentally observed the motion of bovine
sperm in uid environments with different rheological properties
using PVP and MC as solutes. This study particularly focused on
the impact of varying the viscosity and the differences between
Newtonian and non-Newtonian uid environments. First, we
investigated the relationship between the sperm velocity, the
straight-line velocity, the amplitude from the sperm trajectory,
and beat frequency. In the Newtonian uid environment, when the
viscosity increased, the sperm and straight-line velocities, amplitude and beat frequency decreased, and the linearity of the sperm
movement increased. In the non-Newtonian uid environment,
the straight-line velocity was nearly the same as that in the
Newtonian uid environment, but the sperm velocity was much
lower; as a result, the linearity increased signicantly. The beat
frequency in a non-Newtonian uid was greatly increased compared with that in a Newtonian uid with comparable viscosity.
Additionally, changes in the rheological properties of the surrounding uid resulted in different agellar shapes of the sperm,
which is relevant to the promotion of the sperms progressive
motion. These results suggest that the sperm agellum has
evolved to be able to effectively travel through the non-Newtonian
oviductal uid and reach the ovum as quickly as possible. The
results of this study will be helpful in better understanding sperm
motion characteristics in the actual high-viscosity non-Newtonian
environment.

Conict of interest statement

The authors conrmed that there are no known conicts of
interest associated with this publication and there has been no
signicant nancial support for this work that could have inuenced its outcome.

Ackowledgment
This research was supported by a Grant-in-Aid for Challenging
Exploratory Research (No. 26560204) from the Japan Society for
the Promotion of Science.

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947

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