ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

A preliminary study on the rheological properties of human ejaculate and changes during liquefaction

Yong-De Shi¹, Lan-Feng Pan¹, Fei-Kun Yang¹, Si-Qi Wang²

Asian J Androl  2004 Dec; 6: 299-304           

Keywords: human semen; rheology; hysteresis loops; yield stress

Abstract

Aim: To study the changes in rheological properties, namely the parameters of the hysteresis loops and yield stress versus time for human semen after ejaculation. Methods: Ejaculates were obtained from volunteers and immediately put into the test cup of a Brookfield Programmable DV-11 Rheometer, by which the hysteresis loops and yield stress were determined. Results: (1) Yield stress values dropped down from more than 3000 mPa to 60 mPa in about 5 minutes after ejaculation; (2) The shape of the hysteresis loops of shear stress versus shear rate was changed from the counter-clockwise direction, that enclosed a large area, into the clockwise direction, that enclosed a very small area. Conclusion: Human ejaculate originally possesses semi-solid or visco-elastic body behavior and in 5 minutes after liquefaction, it becomes a thixotropic fluid or shearing thinning fluid with very low viscosity.

1 Introduction

Recently, it has been shown that the quality of human semen tends to decline with time [1], and the reasons may be connected to factors from two perspectives: on the in vivo side, changes in the biochemical, biophysical and cytological conditions during the courses of maturation and transportation of spermatozoa and seminal plasma, such as sperm surface protein modification [2, 3] and rearrangement [4], functional processing of fertilin in epididymis [5, 6], the density and distribution changes in sperm surface charge [7], the hepatocyte growth factor effect [8], the level of zinc [9], the reactive oxygen species [10], etc., especially for chromosome deletion [11, 12] or normal or abnormal gene expression [13-15]; and on the environmental side, variations such as those due to different geographic regions and latitudes [16, 17], seasons [18], electromagnetic radiation effects, occupational risk factors, etc [19].

The quality of human semen is routinely tested by ejaculate volume, sperm density, and observation on morphology and motility, among which the motility test is a very popular parameter. From the points of view of biorheology, the motility should be determined on spermatozoa themselves, and the bulk fluidity, with the semen suspended in seminal plasma. It has been reported that the viscosity values and the thinning with time depends on the time after ejaculation, the preliminary thinning taking about 5 - 10 minutes and total thinning taking 20 - 40 minutes. The thinning semen viscosity could be estimated by the length (2 - 3 cm) of the spinning filament with a syringe or a grass rod, or from the flow time (less than 120 sec, middle value as 50 sec) through a special viscometer with 0.5 mL volume of the thinning semen [20]. The accuracy of the above methods seemed not very satisfactory and standardized, therefore, it is necessary to study the relationship between the fluidity behavior and the ejaculation time in order to describe sperm motility and to characterize the rheological property of semen. In this paper the characteristics of yield stress and hysteresis loops, the two basic parameters of rheology, for the human semen, were investigated.

2 Methods

Healthy fertile men (fathered one or more children) were enrolled. Ejaculates were taken by masturbation after at least 5 days of abstinence. The whole ejaculate was immediately and directly put into the rheometer test cup, avoiding mechanical stirring as far as possible; a timer was started to read the time in seconds. Every sample was tested at 20 points at 1, 2, 3, ..., 20 minutes, respectively. The zero time was defined as just the end point of semen emission.

A type DV-II+ Programmable Rheometer (Brookfield Enginnering Laboratories, Inc., USA) was used for determining the yield stress and hysteresis loops for human semen samples. The computer programs for real time sampling and treating data were made with software (Brookfield, Inc.) The detailed methods were mentioned in our previous report [21]. The temperature was controlled automatically at 28.5 ± 0.1 ℃.

2.3 Semen samples

We tested 5 samples from 5 men, the results for each being basically the same. Because of some differences in data sampling design for stress, shear rate and time, it was not appropriate to calculate the mean and standard deviation. The data from one sample were the most comprehensive, thus we report those results in this paper.

The results are shown in Figure 1. Yield stress was defined as the least stress for any substance to deform or flow. The computer read the shear stress value by real time sampling with decreasing shear rate in the sequence of 1 s^-1, 0.8 s^-1, 0.4 s^-1, 0.2 s^-1, 0 s^-1 on the rheometer. Yield stress was an objective criterium for fluid or solid: if the yield stress value was increasing with time, it would become solidified, and conversely, if the yield stress was decreasing with time, it would become liquefied. Figure 1 shows the yield stress of human semen dropping from more than 3 000 mPa to about 60 mPa within 5 minutes. This means that human semen rapidly changed from a solid-like material into a fluid-like one, which is an important physiological characteristics for human semen. From 5 minutes after ejaculation onward, the liquefied semen was still thinning by a slow decreasing rate.

Figure 1. Relationship between yield stress (mPa) and time (minute) after ejaculation (Regression analysis: two straight line equations were obtained: Equation 1, involved 5 points, from 1 to 5 minutes, to be regressed as Y₁=-1154.X₁+5063.5, its regression coefficient was R₁= -0.90, P<0.05. Equation 2, involved 16 points, from 5 - 20 min, to be regressed as Y₂ = -3.22647X₂ + 87.58088, its regression coefficient was R₂= -0.97, P<0.001. these two straight lines intersected at yield stress of 73.225323 mPa, and time of 4.449 minutes (=267 seconds).

The relationship between yield stress and the time was described by two lines, the 1 - 5 min points obeyed a linear equation and the 5 - 20 min points obeyed another one (Figure 1). These two regression lines intersected at the point of yield stress of 73.23 mPa, and time of 4.449 minutes (267 seconds). It meant a sudden transition where a semi-solid or visco-elastic body changed into a fluid. According to our linear regression, before the transition point its slope (the rate of yield stress decreasing) was 1154.2 mPa per minute and after that point, its slope was 3.22647 mPa per minute. These two slopes were strikingly different. They gave semen liquefaction an objective biophysical description.

Figure 2 shows 20 hysteresis loop curve shapes at 1 to 20 minutes after ejaculation. Because of the great differences among the 20 curve shapes and the crowding among some curves, it was necessary to show them in the following ways:

From Figure 2, two types of typical hysteresis loop shapes were selected and presented in Figure 3. one belongs to type 1, which was the curve at 3 minutes after ejaculation, it was a counter-clockwise hysteresis loop enclosing a considerable area. The other one belonged to type 2, which was the curve at 4 minute after ejaculation, it was a figure of 8-like hysteresis loop enclosing two ring areas, the lower-left ring ran counter-clockwise and the upper-right ring clockwise. At the earliest times after ejaculation, semen showed the hysteresis loop in type 1 shape. Four minutes after ejacula-tion, the semen all show the hysteresis loop in type 2 shape. In Figure 3, we also supposed that there would exist a hysteresis loop as type T, which should be a transition state from type 1 to type 2 at a critical time (we did not obtain it, because to get such an instantaneous time was very difficult). From the points of views of rheology, counter-clockwise hysteresis loops indicate an absorbing energy course, showing solid or visco-elastic behavior, its enclosing area showed the energy values per volume per time; the clockwise hysteresis loop indicates a dissipating energy course, showing thinning property or liquefaction effect. Thus according to changes and their digital analysis of the hysteresis loop shapes, we could obtain useful information on semen parameters.

Figure 3. Three human semen hysteresis loop shapes (Type 1, the curve at 3 minutes after ejaculation; Type 2, the curve at 4 minutes after ejaculation; Type T, the curve at a critical time between 3 and 4 minutes after ejaculation).

In order to illustrate the detailed changes for the hysteresis loops, we displayed the loops collected at 2, 3, 4, 5, 10 and 20 minutes in Figure 4. Figure 2 and 4 showed the hysteresis loop shapes at 1, 2 and 3 minutes after ejaculation running in a counter-clockwise type and enclosing a considerable area with the vertical axis.

Figure 4. Comparisons among human semen hysteresis loop shapes at 2, 3, 4, 5, 10, 15 and 20 minutes after ejaculation (M=minutes).

Figuer 5 showed the hysteresis loop shapes which belonged to figure 8-like shapes at 4 minutes or longer, which contained two rings, the lower-left ring was counter-clockwise, the upper-right ring was a clockwise. In order to compare the figure 8-like shapes for the hysteresis loops at 4 - 20 minutes, we specially displayed loops at 5, 10 and 20 minutes in Figuer 5. It showed that all the loop shapes belonged to figure 8-like shapes, each one with two rings, the lower-left ring ran counter-clockwise and the upper-right clockwise.

Figuer 5. Comparison among the human hysteresis loop shapes among 5, 10 and 20 minutes after ejaculation (M=minutes).

From the points of rheology, we made the digital analysis in the Table 1 for all the hysteresis loops. The biophysical meanings of the parameters on Table 1 were explained in the foot notes. These data showed that semen was a semi-solid tissue when it was first ejaculated, but in only 4 or 5 minutes, lost the solid property and became liquid.

Table 1. Digital analysis for the human semen hysteresisi loops at 1-20 minutes after ejaculation.

Notes: (1) Counter-clockwise ring enclosing area: the area was integrated by computer, its physical meaning was energy per volume per time, the unit is 10^-2 erg/cm³/s, this energy was absorbed by the system from rheometer's rotational energy. The increasing value means solid behavior strengthening.
(2) Clockwise ring enclosing area: the area was also integrated by computer, its physical meaning was also energy per volume per time, the unit is 10^-2 erg/cm³/s, this energy was dissipated from rheometer's rotational energy into thinning semen sample itself. The increasing value means liquid behavior strengthening.
(3) The difference of (1) minus (2), the positive and higher values in this column showed solid behavior increasing; the negative and higher values showed liquid behavior increasing;
(4) The ratio of solid behavior over liquid behavior, values more than 1 in this column indicate the predominance of solid behavior; values much high than 1 mean more solid. The values less than 1 in this column indicate the predominance of liquid behavior; values much less than 1 mean more liquid.
(5) "No" means a pure "visco-elastic body" or "liquid body". "Yes" means a complex body with "some visco-elastic property" or "some thixotropic liquid property".
(6) and (7), is a series of critical points with vertical shear stress value and horizontal shear rate value, any point at the hysteresis loop curve, if locates at right-up side, the system has "thixotropic liquid property" if locates at left-down side, it has "visco-elasticity".
(8) This column series means "apparent viscosity" at shear rate 1 s^-1 , higher values mean solid behavior, lower valuse mean liquid behavior.

4 Discussion

Human semen upon ejaculation possesses a yield stress of more than 3 000 mPa and belongs to special semi-solid or visco-elastic tissue. It takes a stress about 40000 Pa for man to ejaculate it. Before ejaculation, spermatozoa are stored and protected in the epididymal fluid in the epidimymis. The semen emitted is composed of spermatozoa and seminal plasma, and the latter includes secretions from the epididymis, ductus deferens, ampulla, seminal vesicles and prostate, etc. Seminal plasma is a mixture chemically and a complex body physically. In the epdidymal fluid sperm will get nutrition, but their motility may be limited, the sperm taking quiet rest and waiting for the event of ejaculation, motility initiation and capicitation. Shortly after ejaculation, the semen suddenly changed its physical properties from semi-solid into thinning fluid, lost its high yield stress and its visco-elastic counter-clockwise hysteresis loop. After ejaculation it is beneficial for sperm to make active motility and to penatrate cervical mucus to reach ovum for fertilization.

From the rheological points of view, we establish a series of parameters to describe the semen changes after ejaculation, such as yield stress and two stress regressive equations versus time, and the intersection point for these two equations, hysteresis loop shapes versus time, counter-clockwise ring integrated area, clockwise ring integrated area, the above two areas difference and ratio, hysteresis loop curve intersection and its site, the apparent viscosity at a certain shear rate. The above parameters coincidentally reflected semen thinning course from visco-elastic or semi-solid body and the relationship versus time. Among them we can select some more practicable and meaningful ones to serve semen quality tests in the future.

Our results showed that at about 4.45 minutes the regressive linear straight lines of yield stress versus time intersected (it means here its yield stress decreasing rate changed from a high value into a very low value). Almost the same time the hysteresis loop shape changed from the counter-clockwise ring to the figure 8-like one (it means the change of semen from visco-elastic or semi-solid state into fluid state). These two series of data show natural coincidence, and perhaps reflect the possible configuration change for some protein molecules in seminal plasma before its liquefaction, they may be as a 3-dimensional network structure, and after its lique-faction, the network may be dissociated into the mono-molecules. We think the former possesses high yield stress and the remarkable counter-clockwise hysteresis loop, and the latter has very low yield stress and loses its remarkable counter-clockwise hysteresis loop to become the figure 8-like loop. After liquefaction, how do we explain the reason why the semen still retains some visco-elasticity (with a certain yield stress, and counter-clock ring in the left-down site of their figure 8-like loops), we suggest that sperm possess response to stress, any stress acts on them, they will response to it. For the existence of such a property, semen shows figure 8-like loop (not only with clockwise ring) rheologically, and show sperm possess their activity and motility to swim in a viscous media as cervical mucus of vagina, and to penetrate through the cumulus mass and zona pellucida of the oocyte to achieve fertilization.

The rotational cone-plate rheometer used in this paper, possesses enough accuracy to reflect the semen properties, however for semen analysis it is necessary especially to design a new test cup which will not be affected by semen volume during immediately loading of the whole fresh ejaculation for rheological determination.

We think during the course of testing, some errors in the data can not be avoided, because the fresh semen sample may be heterogeneous, and its behavior may be affected by the stress from cone-plate rotation, its thinning extent may also be heterogeneous in test cup space. Since the semen heterogeneity would be random, such errors should not influence our general observation.

This paper is only a preliminary study. The most previous papers on semen were involved in biochemistry, molecular and cell biology, histology, immunology, etc, but with few reports in the field on biophysics, mechanics, and rheology. Moreover, We find it is necessary to study in this field and at the same time we hope more clinicians and scientists collect more cases and data in this field, perhaps the concepts and parameters suggested by this paper might be helpful in productive medicine in the future.

The work was supported by grants from DLR, Germany. The rheological instrument and computer software were provided by Brookfield Engineering Laboratory, Inc., USA. The authors thank to Academician Zhang Yong-Lian, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy; Prof. Artmann Gerhard, University of Applied Sciences Aachen, Germany; Prof. Cokelet Giles, University of Montana, USA; Prof. Rampling Michael W Imperial College School of Medicine, London, UK; and Prof. Xu Shi-Xiong, Fudan University, Shanghai, for reviewing the manuscript and giving advice.

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Correspondence to: Prof. Yong-De Shi, Fudan University Shanghai Medical College, 138 Yi Xue Yuan Road, Shanghai 200032, China. 
Tel/Fax: +86-21-6427 5720
E-mail: sswwl@sh163.net
Received 2004-02-09 Accepted 2004-07-21