effect-of-non-newtonian-fluid-properties-on-bovine-sperm-motility_2015_journal-of-biomechanics.pdf

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

a r t i c l e i n f o

Article history:

Accepted 3 August 2015

Keywords:

Bovine sperm

Viscosity

Non-Newtonian uid

Flagellum

Image analysis

a b s t r a c t

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 rheo-

logical 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 methylcel-

lulose 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-New-

tonianuid, 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

agellumapped. 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.

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 inter-

nal 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 sur-

rounding uid environment is extremely important.

In this study, we consider the sperm motility of mammals. As

mentioned above, mammal sperm participates in internal fertili-

zation. When the sperm moves toward the ovary through the

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 inu-

enced 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

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Journal of Biomechanics

http://dx.doi.org/10.1016/j.jbiomech.2015.08.005

0021-9290/&2015 Elsevier Ltd. All rights reserved.

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

E-mail address: [email protected] (T. Hyakutake).

Journal of Biomechanics 48 (2015) 29412947
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agellum and suggested that bovine sperm rotates in three

dimensions. Rikmenspoel (1965) distinguished between non-

rotating 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 con-

tinuously 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 variousspecies, 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 three-

dimensional 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 vis-

coelastic uid that contains gelatinous materials and macro-

molecules.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 theeffect 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; Rikmen-

spoel 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 high-

viscosity 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 non-

Newtonian 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

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 sus-

pension was warmed in a water bath at 38.5 C, and the temperature of the sus-

pension was maintained using a thermoplate during the observation. The sus-

pension 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 (WakoPure 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 con-

centrations, 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 Newtonianuid. An increase in the PVP-

K90 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 concentra-

tions of 10% and 15%. The viscosity when the shear rate was 1.0 s1 was approxi-

mately three times that when shear rate was 100 s1. 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 three-

dimensional observation is very difcult. Therefore, we obtained the two-dimen-

sional 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,

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

MC100 0.6 %MC100 2.0 %MC4000 1.0 %

Shear rate [1/s]

Viscosity[Pa*s]

1 10 10010

-3

10-2

10-1

100

Fig. 1. Relation between the shear rate and viscosity of PVP-K90, MC100, and

MC4000. Thelled triangle represents PVP-K90 4.0%, the lled diamond represents

PVP-K90 10%, thelled 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%.

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 294129472942


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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 tra-

jectory,A. First, we obtained the progress axis of the sperm trajectory by using the

least-squares method from the obtained trajectory point (red line inFig. 2(a)). Next,

we obtained the furthest two points on both sides of the progress axis and de ned

half the distance between these two points as the amplitude of the sperm trajec-

tory. 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 visc-

osity. In this case, 17 sperm samples were taken.

3. Results

Fig. 3shows the relation between the straight-line velocity VSTand 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 PVP-

K90 concentrations of 4%, the experimental data were mostly

distributed in the area above the line passing through the origin

with a slope of VSP/VST3.0. 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 thePVP-K90 concentrations of 10% and 15%, the decreases in VSP and

VST were signicant. The experimental data for these concentra-

tions were mostly distributed in the area above the line passing

through the origin with a slope ofVSP/VST1.0; this slope corres-

ponds 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 ten-

dencies than that of PVP solutions. In the MC100 solutions, both

velocities decreased as the concentration increased. Conversely,

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

inFig. 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 sig-

nicantly 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.

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.)

VST [m/s]

VSP

[m/s]

Diluted solutionPVP-K90 4.0 %PVP-K90 10 %PVP-K90 15 %MC100 0.6 %MC100 2.0 %

MC4000 1.0 %

0 20 40 60 80

50

100

150

200

250

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 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%.

T. Hyakutake et al. / Journal of Biomechanics 48 (2015) 29412947 2943


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To investigate these different tendencies in more detail, we

dened the linearity as the reciprocal of the slope in Fig. 3.

L V V/ 1ST SP = ( )

L1 means that the sperm moves in a straight line, and L0

means that the sperm does not progress at all. Fig. 4 shows the

relation between the linearityLand the amplitude Adof 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

(seeFig. 2(b)).

Fig. 5summarizes the variation in the average values ofVSPand

VSTfor 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 straight-

line velocity decreased from 40 to 3.7m/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 37m/s, adecrease 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 move-

ment.Fig. 6compares 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

linearityL(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

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 indi-

cates the agellum. From this gure, in the case of diluted solution,

at which the viscosity is near that of water, the entire agellum

L [-]

Ad[-]

Diluted solutionPVP-K90 4.0 %PVP-K90 10 %PVP-K90 15 %

MC100 0.6 %MC100 2.0 %MC4000 1.0 %

0 0.2 0.4 0.6 0.8 1

1

2

3

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

50

100

150

200

250

Dilutedsolution

PVP-K904.0%

PVP-K9010%

PVP-K9015%

MC1000.6%

MC1002.0%

MC40001.0%

m/s

V_SP V_ST

Fig. 5. Comparison of average sperm and straight-line velocities ( VSP, VST) for dif-

ferent solutions. The error bar indicates the standard deviation of the average value.

0

2

4

6

8

10

12

Dilutedsolution

PVP-K904.0%

PVP-K9010%

PVP-K9015%

MC1000.6%

MC1002.0%

MC40001.0%

Hz

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.



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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 a-

gellum moves in a curved apping motion. The portion of the

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 solutionsare Newtonian uids, and the MC4000 solution is a non-New-

tonian 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 New-

tonian 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 non-

Newtonian 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 spermmotion 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-New-

tonian 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 poly-

acrylamide (high viscoelasticity). Furthermore, they investigated

the change in velocities with hyperactivation. When the present

L[-]

VST

/f[m]

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

0 0.2 0.4 0.6 0.8 1

2

4

6

8

10

12

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%.

Fig. 8. Comparison ofagellar 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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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 dif-

ferent. 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 spe-cies. 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. Con-

sequently, 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 fur-

thermore 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 con-

stant as viscosity increased was because of the inuence of non-

Newtonian properties. In a high-viscosity Newtonian uid envir-

onment, the straight-line velocity of the sperm decreases. There-

fore, 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 character-

istics 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 environ-

ment. These results suggest that the sperm agellum has evolved

to be able to effectively travel through the non-Newtonian ovi-

ductal 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. Addi-

tionally, with the present experimental apparatus, we conducted

only two-dimensional observations. Investigating the three-

dimensional motion of agella in a high-viscosity uid environ-

ment 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 hyper-

activated sperm is characterized by a sharp bend in the proximalmidpiece 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, ampli-

tude 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 muchlower; as a result, the linearity increased signicantly. The beat

frequency in a non-Newtonian uid was greatly increased com-

pared with that in a Newtonian uid with comparable viscosity.

Additionally, changes in the rheological properties of the sur-

rounding 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 inu-

enced 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.



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