INVESTIGATION OF OPERATING PARAMETERS FOR A

SEMEN QUALITY ANALYSIS SYSTEM

S. Atherton, C. R. Evans, P. Roach, D. C. Hughes, G. McHale and M. I. Newton

School of Science and Technology

Nottingham Trent University, Clifton Lane, Nottingham

NG11 8NS, U.K.

Keywords: Sperm, Semen, Motility, Acoustic wave, QCM, Time of flight.

Abstract: To increase the success rate of Artificial Insemination (AI) in animals, it is important that the semen sample

is of a high quality. The quality is related to both the number and motility of sperm present. Numerous

methods of analysing semen samples exist, but these are generally expensive and/or laboratory based. A

useful alternative would be an inexpensive simple system that could be used in the field immediately prior

to insemination. We present a time of flight (ToF) technique using a quartz crystal microbalance (QCM). In

this system the sperm are introduced at one end of a liquid filled swim channel and self propel to a QCM

sensor at the other end. A chemical coating is applied to the QCM to bind the sperm and from the frequency

change the number of attached sperm and their ToF can be measured. We report the effect of temperature

and the introduction of small quantities of progesterone into the swim channel on the sperm ToF. Results

show the QCM can be used to detect the arrival of the sperm and that increasing temperature and the

presence of progesterone are both shown to decrease the ToF.

1 INTRODUCTION

Within the Artificial Insemination (AI) industry it is

important to be able to measure the concentration of

viable sperm in a sample. AI is a common procedure

in farm animals, more than 100 million inseminations

are performed globally every year. It is not only the

number of sperm in a semen sample that is crucial to

the success of the insemination process, but also the

motility of the sperm. Whilst having a large number

of motile sperm in a sample is not a guarantee of

fertility, it is an excellent indicator of semen quality.

Two optical methods of performing sperm

counts are the haemocytometer and counting

chambers. The drawback of these methods is that

multiple measurements are needed to achieve an

acceptable level of precision, resulting in a more time

consuming procedure. The reason for the relative

inaccuracy of these methods is due to the rapid

movement of the sperm under high magnification

and the tedious nature of the work for the human

operator. It is possible to perform a more objective

assessment of sperm motility using a computer

assisted semen analyzer (Mortimer, 2000), which is a

laboratory based instrument that can measure

different aspects of the sperm movement. To further

increase precision, a combination of fluorescent

staining and flow cytometry can be used to analyze

thousands of sperm in a sample (Christensen et al,

2005). The common drawback with all of the above

techniques is the price of the equipment itself and the

need for a skilled operator.

In some species the sample may be successfully

frozen and thawed before use however this is not

always the case. The sperm of other species cannot

be successfully frozen and so a fresh sample, with a

shelf life of only a few days, must be used.

Increasingly, AI is being used for equine applications

and recent changes in UK legislation (Artificial

Insemination of Mares Order 2004) mean that lay

(non-Veterinary) and farm workers are now being

trained to perform such inseminations. For sport

equine applications the cost of semen for a single

mare to be covered may exceed £1000. Semen

analysis is currently a specialist, subjective and

skilled process that is normally carried out under

laboratory conditions. Given these trends, a low cost,

simple to use and objective technique to assess the

quality of the semen, particularly under field

conditions just before insemination, would greatly

Atherton S., R. Evans C., Roach P., C. Hughes D., McHale G. and I. Newton M. (2009).

INVESTIGATION OF OPERATING PARAMETERS FOR A SEMEN QUALITY ANALYSIS SYSTEM.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 13-16

DOI: 10.5220/0001429600130016

 SciTePress

improve the quality and practice of artificial

insemination in animals. It would also provide an

easier and more cost effective method for monitoring

male animal fertility and breeding male welfare.

Acoustic wave sensors detect very small changes

in mass attached to their surface and often contain a

sensitizing layer that can recognize and bind the

species to be detected onto the mass sensitive

surface. The quartz crystal microbalance (QCM) is

the most widely used acoustic wave device for sensor

applications.

Δf = -2.26 x 10

-6

Δm/A (1)

The Sauerbrey equation (Sauerbrey, 1959) relates

the change of the crystals resonant frequency to the

change in rigid mass on the crystal surface; this is

shown for AT cut quartz in equation 1 where Δf (in

Hz) is the change in frequency that occurs for an

increase in mass Δm (in grams) on the surface of area

A (in cm

) with a crystal resonant frequency of f (in

Hz) and the constant comes from the crystal

materials properties. A well-designed oscillator

circuit can still resonate a crystal even under the high

damping caused by immersion in a liquid. The

change in mass rigidly attached to the surface still

causes a proportional change in frequency although

changes in other parameters such as the liquids

viscosity and density will also cause changes in

frequency. The acoustic wave will only sense mass

changes within a short distance into the liquid called

the penetration depth. (Kanazawa & Gordon, 1985)

Previous studies have shown up to 70% of the sperm

mass to be made up of water (Da Silva et al, 1992) so

it is not obvious how the attachment of a sperm will

change the QCM response. In a preliminary report

(Newton et al, 2007) we have fitted the resonance

curves of 5MHz QCM to the Butterworth van Dyke

model and this has shown that the sperm may be

treated a rigid mass and so a model based on the

Sauerbrey equation is appropriate when using an

effective mass of around 5pg. For other operating

frequencies or other species sperm this effective mass

would be different.

In this report we extend this preliminary work to

investigate the effect of environmental parameters on

the time of flight (ToF). For any practical

measurement technique it is essential the time the

measurement takes is sufficiently short to be usable.

For a portable field instrument then power

consumption may also be an issue therefore the first

parameter we investigate is operating temperature

and we consider a range from room temperature to

body temperature.

Progesterone is a steroid hormone involved in

female menstrual cycle, pregnancy and

embryogenesis of humans and other species. It is one

of a number of substances said to cause

hyperactivation of mammalian spermatozoa and its

presence may therefore affect the time of flight; the

effect of adding progesterone to the swim medium is

reported.

Figure 1: Magnified view of a boar sperm.

2 EXPERIMENTAL

Figure 2 shows a schematic diagram of the

experiment. This consists of an inlet port to a channel

filled with phosphate buffered saline (PBS) buffer.

At the other end of the channel is a quartz crystal

followed by a vent to air preventing pressure changes

being recorded in the QCM response when a semen

sample is added. Sperm are introduced at the inlet

port and are self propelled through the channel to the

QCM where they are detected. A volume of 20μl of

the semen was used and added using a Gilson pipette.

The channel length was set to approximately 14.5 cm

and contained 4ml of PBS; note that for any practical

field instrument the swim channel length could be

considerably reduced to give an analysis time under 5

minutes. The sensing element in the experiments was

a 5MHz AT-cut quartz crystal (Testbourne 149211-

1). A Maxtek PLO-10 phase lock oscillator was used

to drive the crystal and the resonant frequency was

measured with an Agilent universal frequency

counter interfaced to a computer.

To sense the sperm it was necessary to get them

to adhere to the surface of the QCM. To achieve

sperm adhesion to the crystals they were coated in

either Poly-L-Lysine (Sigma-Aldrich) or cysteamine

(Sigma-Aldrich). Crystals were initially cleaning

with ethanol then ozone treated for 30 minutes. They

were then placed in either poly-L-lysine (as

supplied), or a cysteamine solution of 1mmol in

toluene and left overnight. The devices were then

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

washed in PBS buffer to remove any excess. The

cysteamine coating were found to be the most

reliable method of binding the sperm to the surface as

poly-L-lysine shows significant variability from

batch to batch.

Side view

Top view

sample inlet port

buffer filled

channel

Quartz

crystal

inlet port

buffer filled

channel

vent

Sperm detection region

Figure 2: Schematic diagram of swim channel.

To provide temperature control to the

experiments the swim channel was housed inside an

Octagon 10 incubator. This allowed the temperature

to be controlled so as to investigate the effect this

would have on the sperm ToF. For these the

temperature was varied from 23-45

C. The incubator

also allowed the temperature to be kept constant

across all the progesterone experiments.

When looking at the effects of progesterone,

(Sigma-Aldrich) different concentration were added

to the PBS in the swim channel. Firstly 15.7mg of

progesterone was dissolved in 50ml of ethanol. This

mixture was diluted in PBS at concentration of 20-

90μmol.

The porcine semen was supplied by a

commercial artificial insemination centre (JSR

Genetics, Driffield, UK). The semen was received

already mixed with a dilutant (Androhep), cooled to

a temperature of 17

C and packaged in plastic

bottles. The androhep allowed the semen to be

stored for up to 5 days at ambient temperature.

However, this does result in the concentration of

semen in the mixture being quite low. To get a more

concentrated sample a centrifuge was used to

separate the sperm from the androhep. To achieve

this, sealable microtubes were filled with 50μl of the

androhep and semen mixture and these were

centrifuged for 40 seconds. This resulted in the

sperm being concentrated at the bottom of the tube.

The androhep was removed with the pipette and

50μl of PBS was added to one of the tubes. The PBS

and sperm were mixed together, this mixture was

removed with the pipette and added to the next tube

and the process was repeated for all 15 tubes. What

was left was a more concentrated sperm sample

mixed with 50μl of PBS.

3 RESULTS AND DISCUSSION

Figure 3 shows the QCM frequency response to

sperm binding on the surface. The arrow shows the

time at which the semen sample was introduce to the

inlet of the swim channel. The arrival of the sperm is

signified by a decrease in the frequency of the sensor

with the fastest ToF of approximately 20 minutes.

-45

-35

-25

-15

-5

0 50 100 150

time (minutes)

frequency change (Hz

)

Figure 3: Graph showing the frequency decrease

indicating sperm arrival.

The frequency continues to drop as more sperm

make their way to the QCM and bind to the surface.

This continues for another 120 minutes until further

arrival of motile sperm finishes.

Using the previously determined sperm effective

mass and taking the rate of frequency change from

figure 1, the Sauerbrey equation can be used to

derive the rate of sperm arrival and this is shown in

figure 3. For use in a screening application, a simple

threshold number of detected sperm would be

required however this demonstrates that quantitative

analysis is also possible with this instrument.

Figure 4: Number of sperm arriving at the QCM over the

course of the experiment.

Figure 5 is a plot of the ToF of the sperm against the

temperature of the environment. The results show a

decrease in the ToF as temperature increases with

almost a 50% fall between room temperature and

body temperature. The scatter observed can be

attributed mainly to the experiments being

performed a differing lengths of time from the

INVESTIGATION OF OPERATING PARAMETERS FOR A SEMEN QUALITY ANALYSIS SYSTEM

receipt of the samples and the quality of the semen

degrades over time.

22 27 32 37 42

Temperature (Deg C)

ToF (min)

Figure 5: The time of flight for fastest sperm arrival as a

function of temperature.

To further speed up the measurement,

progesterone was used to cause hyperactivation in an

attempt to decrease the sperm ToF; the results of this

are shown in figure 6. Comparing the non-

progesterone experiments with the progesterone

ones we see a significant decrease in the ToF of the

sperm however for the full range investigated there

was little effect from the progesterone concentration.

300

400

500

600

700

800

900

0.0 20.0 40.0 60.0 80.0 100.0

Progesterone concentration (μmol)

Time of flight (s)

Progesterone

No progesterone

Figure 6: Time of flight as a function of progesterone

concentration.

4 CONCLUSIONS

We have demonstrated that a time of flight

technique with an acoustic wave sensor provides a

viable method for determining the quality of a

semen sample both as a screening technique and as

an analytical tool. The cysteamine coating on the

QCM proved to be the more reliable method of

binding the sperm to the surface. Experiments

varying the temperature showed a general decrease

in ToF as temperature is increased suggesting that

body temperature would be the optimum value. The

presence of progesterone also reduces the ToF

however this was not concentration dependent over

the range investigated. Whilst the laboratory based

instrument reported here used commercial sensor

crystals, the cost of quartz crystals employed more

generally in electronic oscillator circuits are

inexpensive and are still offer a mass sensitive

surface. Using such crystals, pre-treated with

cysteamine, would reduce costs sufficiently to offer

the possibility of a disposable element. With a

modification to the swim channel length to bring

down the measurement time, this technique then

becomes a powerful tool for routine monitoring of

animal reproductive health and investigating factors

that affect the semen motility of animal.

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Mortimer, S. T., 2000, CASA - Practical Aspects, J.ournal

Andrology, 21, p.515-524

Christensen, P., Boelling, D., Pedersen, K. M., Korsgaard,

I. R., & Jensen, J., 2005, Journal of Andrology, 26

Sauerbrey, G., 1959, Zeitschrift für Physik, p.155-206

Da Silva, L. B., Trebes, J. E., Balhorn, R., Mrowaka, S.,

Anderson, E., Attwood, D. T., Barbee, T. W., Brase, J.,

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Kanazawa, K., & Gordon, J. G., 1985, Frequency of a

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90(15)

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