WO2013029625A1

WO2013029625A1 - Apparatus for measuring the metabolic rate of motile organisms

Info

Publication number: WO2013029625A1
Application number: PCT/DK2012/050318
Authority: WO
Inventors: Niels B. Ramsing; Jens K. Gundersen; Bjørn Molt PETERSEN
Original assignee: Unisense Respirometry A/S
Priority date: 2011-08-29
Filing date: 2012-08-29
Publication date: 2013-03-07

Abstract

The present invention relates to an apparatus and a method for measuring the metabolic rate of motile organisms, such as sperm cells or some forms of plankton. One embodiment of the invention discloses an apparatus for measurement of the metabolic rate of motile organisms in a medium comprising at least one compartment capable of accommodating at least a part of the medium, one or more capillary tubes in fluid communication with said compartment, and at least one metabolite detector for measuring the concentration of metabolite inside the compartment. One embodiment of the invention relates to measurement of the oxygen consumption of sperm cells, thereby obtaining a semen quality parameter.

Description

APPARATUS FOR MEASURING THE METABOLIC RATE

OF MOTILE ORGANISMS

The present invention relates to an apparatus and a method for measuring the metabolic rate of motile organisms, such as sperm cells or some forms of plankton. One embodiment of the invention relates to measurement of the oxygen consumption of sperm cells thereby obtaining a semen quality parameter.

Background of invention

Semen quality is a measure of the ability of semen to accomplish fertilization. Thus, it is a measure of fertility in a man. It is the sperm in the semen that are of importance, and therefore semen quality typically involves both sperm quantity and quality. Decreased semen quality is a major factor of male infertility and unfortunately the quality of human semen shows a deteriorating trend. A successful artificial fertilization, be it in vivo or in vitro, depends highly on the semen quality, and hence there is an increasing need for rapid methods for quantitative evaluation of semen.

Summary of invention

A semen analysis typically measures the number of sperm cells per millilitre of ejaculate and possibly also the motility and/or the morphology of the sperm cells. A number of factors may influence the accuracy of semen analysis results and results for a single man may have a large amount of natural variation over time. For this reason, a sub-fertile result must typically be confirmed with at least two further analyses. One object of the invention is therefore to improve the quality of a semen analysis. This can be achieved by an apparatus for measurement of the metabolic rate of motile organisms in a medium comprising at least one compartment capable of

accommodating at least a part of the medium, one or more capillary tubes in fluid communication with said compartment, and at least one metabolite detector for measuring the concentration of metabolite inside the compartment.

The invention further relates to a method for measuring the metabolic rate of motile organisms in a medium, the method comprising the steps of • injecting and/or arranging the medium into a compartment through a capillary inlet,

• measuring a first metabolite concentration in the medium,

• measuring at least a second metabolite concentration after a period of time, and · obtaining a metabolic rate of the motile organisms by correlating the metabolite concentration measures.

The invention further relates to a method for measuring the metabolic rate of motile organisms in a medium, the method comprising the steps of

· providing at least one device as mentioned above,

• injecting and/or arranging a medium containing motile organisms in the

compartment,

• measuring a metabolite concentration in the medium obtaining a metabolite concentration measure, and

· correlating said metabolite concentration measure to a metabolic rate of the motile organisms.

The viability of a sample of semen is an important parameter in order to determine suitable male donors or for selecting a particular sample of semen. The respiration rate of sperm cells may prove to be a good candidate for an objective viability indicator. The invention thus further relates to a method for selecting a viable sample of semen comprising

• determining the metabolic rate of at least a part of the semen sample in a

device as described above, and

· selecting the semen sample having an optimal metabolic rate, such as the highest metabolic rate.

Definitions

Motile organism: A motile organism is a living organism that is able to move

spontaneously and actively, consuming energy in the process. Examples of motile organisms are sperm cells, plankton, zooplankton, autonomously moving organisms, autonomously moving aquatic organisms and microalgae. Amperometric oxygen sensor: A Clarck type electrochemical sensor with a gold cathode polarized against an internal reference, where oxygen is reduced on the cathode surface. A current meter converts the resulting reduction current to a signal. Diffusion: The process whereby particles of liquids, gases, or solids intermingle as the result of random molecular motions caused by thermal agitation, resulting in a net transport of dissolved substances from a region of higher to one of lower concentration.

Impermeable material: In the present context an "impermeable material" or

"substantially impermeable material" means a material with markedly reduced permeability for the metabolite in question as compared to water, preferably the permeability is reduced to < 1 % for the metabolite in question as compared to water, more preferably reduced to < 0.2% or < 0.05%, so that the area integrated flux through this material to the metabolizing object is much lower than the flux through the permeable material (e.g. opening, permeable membrane and/or diffusion barrier). The area integrated flux through the impermeable or substantially impermeable material should be <10%, preferably < 1 % or most preferably < 0.01 % of the total area integrated flux to the metabolizing cells. Metabolite. In the present context the term "metabolite" means a compound that is either taken up or released by the motile organisms. Examples of metabolites include oxygen, carbon dioxide, amino acids, glucose, ions, such as Ca⁺⁺ ions and H₃0⁺ ions.

Metabolic rate: The rate at which the metabolite in question is consumed or released by the motile organisms. The metabolic rate is dependent on both the metabolite in question and on the level of activity of the organism.

Metabolising: In the present context the term "metabolising" refer to the process of taking up or releasing metabolite. A preferred metabolite which is being metabolised is oxygen which is taken up and consumed by respiration.

Metabolizing organism or metabolizing cell: in the present context the term

"metabolizing organism" means an organism taking up or releasing metabolites during a period of time. A preferred type of metabolizing organism is a respiring organism which consumes oxygen by respiration. The metabolizing organism may also be a synthetic organism consuming oxygen.

Noninvasive method: A method, which without any destructive disturbance, or without requiring insertion of an instrument or device through the skin or body orifice can measure a parameter related to a body of interest.

Respiration rate: Most living organisms consume oxygen in their energy metabolism by a process called respiration. The oxygen consumption rate of a respiring organism is also named the respiration rate.

Response time: The time from initiating a measurement until a response or signal adequate for the measurement is obtained, and the measurement can be considered successful.

Stagnant liquid: A liquid without any flow, turbulence or movement. Transport of dissolved substances primarily takes place by diffusion.

Steady state: A situation where consumption and transport are in equilibrium such that gas partial pressure, or concentration gradients of dissolved substances, is stable and no partial pressure change or concentration change takes place over time.

Detailed description of the invention

The present invention relates to establishing the metabolic rate of motile organisms, i.e. a plurality of motile organisms. The metabolic rate can be established non-invasively in order not to disturb the motile organisms. The invention is based on the finding that the rate of metabolization of motile organisms in a medium may be determined fast and non-invasively by measuring the concentration of a predetermined metabolite when the medium is located in a compartment, preferably a sealed compartment, which is constructed to be pressure equalized with the surrounding environment and adapted to effectively prevent diffusion of said metabolite to or from the motile organisms. By such a construction the predetermined metabolite decreases in the medium and by measuring the concentration of the predetermined metabolite two times (or more) it is possible to calculate the metabolic rate of the motile organisms.

The present invention relates to motile organisms. Motile organisms of interest to the present invention include human sperm cells or animal sperm cells. Other motile organisms of interest to the present invention are e.g. plankton, zooplankton, autonomously moving organisms, autonomously moving aquatic organisms and microalgae. If the motile organisms are arranged in an environment wherein replenishment and removal of metabolites is made unhindered by spherical diffusion effective with moderate metabolic rates, then the concentration of these metabolites are only marginally affected. However, if the motile organisms are placed in a compartment, which effectively prevents the diffusive re-supply or removal of metabolites, then measurable changes in the concentration inside the compartment of these metabolites can be detected. The apparatus according to the present invention accomplish this by minimizing the volume through which the metabolites can pass by molecular diffusion by impermeable (or substantially impermeable) surfaces. These surfaces (or walls) almost entirely surround the metabolizing cells, but leaves one or more capillary openings, through which the pressure with the surroundings is equalized but the capillary opening(s) effectively limits diffusion of the metabolite. The design of the apparatus/compartment may serve three purposes:

1) Keeping a substantially constant pressure inside the compartment.

2) Enabling determination of the metabolic rate for the motile organisms inside the compartment based on the detected change in metabolite concentration.

3) Preventing transport of metabolite to and from the metabolizing motile

organisms by turbulent flow.

The latter purpose of the diffusion barrier is usually accomplished by confining the medium between surfaces that are positioned so close to each other that the liquid cannot mix by turbulent flow between the surfaces.

Compartment

The medium, or at least a part of the medium, comprising motile organisms is arranged in a compartment, e.g. a compartment with predefined dimensions. Furthermore, it is preferred that the compartment is in fluid communication with the outside of the compartment allowing pressure equalization with the surroundings, but at the same time diffusion of the metabolite to and from the compartment should be prevented such that a change in the concentration of the metabolite is taking place over time. The medium inside the compartment should preferably be kept stagnant.

In one embodiment of the invention the volume of the compartment, preferably the inner volume is known, predefined and/or predetermined. In one embodiment of the invention the compartment comprises an inlet adapted for insertion/injection of said medium into the compartment. Said inlet may be adapted for direct connection with an injection device, such as a syringe. The medium can thereby easily be injected into the compartment directly from a syringe. Preferably said inlet is connecting with the compartment at or near the bottom of said compartment. This may help to reduce the formation of bubbles in the medium when the medium is injected into the compartment.

In one embodiment of the invention one capillary tube is an inlet. I.e. the medium is injected/inserted into the compartment through the capillary inlet. Preferably at least one capillary tube, such as an inlet capillary tube, is connecting with the compartment at or near the bottom of said compartment.

After metabolic analysis the compartment may be emptied the same way as the medium entered the compartment, e.g. through an inlet. In one embodiment of the invention one capillary tube is an outlet. The outlet may thus be provided as an exit for the medium after metabolic analysis. Preferably at least one capillary tube, such as an outlet capillary tube, is connecting with the compartment at or near the top of said compartment. In one embodiment of the invention the apparatus comprises means for pressure equalizing the compartment. Thus, the apparatus may be adapted for pressure equalizing with the surroundings. Preferably at least one of said capillary tube(s), such as an outlet capillary tube, is adapted for pressure equalizing the compartment. If a capillary tube is located at or near the top of the compartment pressure equalizing may be provided through said capillary tube. If the compartment is provided with both inlet and outlet capillary tubes a flow-through compartment may be provided. This is explained in further detail below. In one embodiment of the invention at least one of said capillary tube(s) is configured to effectively prevent diffusion of said metabolite to and from the compartment.

In one embodiment of the invention at least one of said capillary tube(s) is configured for pressure equalizing the compartment and configured to effectively prevent diffusion of said metabolite to and from the compartment.

In one embodiment of the invention the compartment wall and the capillary tube(s) are produced from a substantially metabolite impermeable material, such as glass. Thus, the compartment wall and/or the capillary tube(s) may be at least partly transparent or translucent.

The medium inside the compartment accommodating the sperm cells should preferably be kept stagnant, such that transport of substances dissolved in the medium can take place by diffusion only.

Furthermore, the compartment should be designed so that the medium inside is kept stagnant, and furthermore so that the transport of the predetermined metabolite to the compartment is effectively prevented or at least minimized. The compartment may in principle exhibit every suitable shape for measuring a concentration for the metabolite(s) in question. However, the shape of the compartment should preferably also facilitate the handling of the medium with motile organisms, in particular in relation to insertion and withdrawal of the medium. In the present context the shape refers to the inner dimensions of the compartment. The outer dimensions of the compartment may attain any practical shape. Thus, in one embodiment of the invention the inner shape of the compartment is selected from the group of a cylinder, a polyhedron, a cone, a hemisphere or a combination thereof. Thus, the general shape of the compartment may be a cylinder, such as a circular cylinder, an elliptic cylinder, a parabolic cylinder, or a hyperbolic cylinder. The (inner) volume of the compartment must be selected such that there are sufficient medium and motile organisms to detect a metabolite concentration and in particular detect a change in that concentration. However, in case of e.g. semen analysis only a fraction of a semen sample should be necessary to use for the analysis, because the semen which has been analysed is typically thrown away after analysis. If the motile organisms are also analysed with respect to motility, concentration and also visual inspection the compartment must be adapted to make room for all these

measurements. Thus, in one embodiment of the invention the (inner) volume of the compartment is less than 5 milliliters (mL), such as less than 4 mL, such as less than 3 mL, such as less than 2 mL, such as less than 1 mL, such as less than 0.8 mL, such as less than 0.7 mL, such as less than 0.6 mL, such as less than 0.5 mL, such as less than 0.4 mL, such as less than 0.3 mL, such as less than 0.2 mL, such as less than 0.1 mL.

In a further embodiment of the invention the (inner) volume of the compartment is between 0.1 and 5 milliliters (mL), such as between 0.5 and 2 mL, such as between 0.5 and 1.5 mL, such as between 0.75 and 1.25 mL, such as between 0.9 and 1.1 mL. As diffusion of a metabolite across a barrier is proportional to the metabolite

concentration gradient across the barrier and the cross-sectional area of the barrier and inverse proportional to the length of the barrier, the length and cross sectional area of the capillary tube(s), which is the barrier in this case, must be selected to at least minimize diffusion of the metabolite, preferably effectively prevent diffusion of the metabolite through the tube(s). As these tube(s) may function as inlet and/or outlet the configuration of the tube(s) must also be practical for that purpose. Thus, in one embodiment of the invention the maximum inner diameter of the capillary tube(s) is less than 1 mm, more preferably less than 0.8 mm, more preferably less than 0.6 mm, more preferably less than 0.5 mm, more preferably less than 0.4 mm, such as less than 0.35 mm, such as less than 0.3 mm, such as less than 0.25 mm, such as less than 0.2 mm, such as less than 0.15 mm, such as less than 0.1 mm. Further, the total length of the capillary tube(s) may be between 10 and 100 mm, such as between 10 and 20 mm, such as between 10 and 20 mm, such as between 20 and 30 mm, such as between 30 and 40 mm, such as between 40 and 50 mm, such as between 50 and 60 mm, such as between 60 and 70 mm, such as between 70 and 80 mm, such as between 80 and 90 mm, such as between 90 and 100 mm.

And further it is preferred that most of the medium containing the motile organisms end up in the compartment, i.e. the inner volume of the compartment is preferably large compared to the inner volume of the capillary tube(s). Thus, in one embodiment of the invention the total (inner) volume of the capillary tube(s) is less than 10% of the volume of the compartment, more preferably less than 5%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1 %, more preferably less than 0.5%, more preferably less than 0.1 %, such as less than 0.8%, such as less than 0.7%, such as less than 0.6%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.2%, such as less than 0.1 %, such as less than 0.05%, such as less than 0.01 % of the (inner) volume of the compartment.

Metabolites

The metabolite(s) measured according to the present invention may be any metabolite relevant to be either taken up by the motile organisms or released from said motile organisms. In one embodiment the metabolite is a gas, such as oxygen that may be detected by several methods as described below, or the metabolite may be e.g. carbon dioxide, glucose or lactose.

Thus, in a preferred embodiment the present invention relates to determination of the respiration rate of the motile organisms by measuring the gas partial pressure of oxygen and/or carbon dioxide.

Apparatus In one embodiment of the invention the apparatus comprises means for determining the concentration of the motile organisms in the medium. Further, the apparatus may comprise at least one motion detector for determining the motility of the motile organisms. Means for determining concentration and/or motility of e.g. sperm cells in a medium is known in the art, e.g. Computer Assisted Sperm Analysis (CASA) or by using microscopy or fluorescent microscopy.

In one embodiment of the invention the motion detector comprises at least a first light source, such as a laser, for illuminating at least a part of the medium. The motion detector may further accommodate at least one sensor for measuring light scattered from motile organisms (moving) in the illuminated part of the medium, i.e. cells moving in the light creating diffraction and/or scattering of the light which can be detected.

In a further embodiment of the invention the motion detector is based on a laser beam with diffractive optical elements to provide for beam splitting of the laser beam (e.g. known from Kajanto et al., 1989; Johansson & Bengtsson, 2000). This may be in the form of simple bars, a grid or more complex beam splitting with individual directions and intensities of the beamlets. Divergence or convergence of the beamlets may furthermore be provided. This way of measuring motion has previously been applied within measurement of liquid flow. However, it may also be applied within

measurement of motility of individual objects, such as motile organisms, in a medium because the structuring of the beamlets will enable measurements of velocities, concentration and size of the motile organisms. Post processing of the captured time- series data when applying this method is known in the art (Johansson & Bengtsson, 2000; Ripoll et al., 2004; Engstrom et al., 2009).

As described above temperature induced movement of the medium is minimized, turbulent flow in the compartment is minimized and thus the medium inside the compartment is preferably kept stagnant during measurement. Under these conditions the motile organisms will dominate any motility measurement. However, a controlled movement of the medium in the form of e.g. controlled stirring may advantageously be introduced, e.g. by applying a temperature gradient across the compartment. This may e.g. be provided by means of two Peltier elements mounted on opposite sides of the compartment, one of them cooling and the other one heating. By appropriate control of the induced current this temperature gradient may provide a very gentle stirring of the entire medium in the compartment. It is important that no "foreign" particles in the medium impair the contained motile organisms. This stirring will further allow for an assessment of the total number of contained organisms, objects and/or particles by the motility sensor, and not only the motile organisms. When measuring motility random noise from non-motile organisms, objects and particles may be a problem. When applying a controlled movement of the medium the random noise may be converted to a constant noise that is straightforward to filter out, because the non-motile organisms, objects and particles all move in substantially the same direction whereas only the motile organisms will move in other directions.

In a further embodiment of the invention inspection means for visually inspecting the motile organisms are provided. The inspection means may comprise at least one camera for imaging at least a part of the medium. Further, the inspection means may comprise at least a second light source for illuminating at least a part of the medium. The visual inspection may be supplemented with computer assisted sperm analysis (CASA) software, for counting and tracking sperm cells.

The apparatus is preferably under some kind of temperature control. Thus, in a further embodiment of the invention the apparatus comprises thermostatic means for controlling the temperature of the compartment. However, the apparatus may merely be placed in a temperature controlled environment.

In one embodiment of the invention the volume of the medium is known, predefined and/or predetermined.

In one embodiment of the invention the medium is a fluid medium, such as a liquid medium. The medium may be semen, such as human or animal semen. The medium may be a dilution medium, such as a balanced salt solution, comprising semen, such as human or animal semen. The volume of semen in the medium may be known, predefined and/or predetermined. The sperm cells may be human or animal sperm cells. The medium can also be a water sample from either fresh- or saltwater. The water sample may be undiluted or diluted by a salt and nutrient solution. The medium may be a salt and nutrient solution containing one or several cultured plankton species.

In the preferred embodiment of the invention the metabolite is a gas, such as oxygen or carbon dioxide. Thus, the metabolite detector is preferably an oxygen detector for measuring the oxygen concentration in the medium. The oxygen detector may be an optode or a Clark-type sensor. Thus, the respiration of the motile organisms may be determined.

Method of determining the metabolizing rate

• injecting and/or arranging the medium into a compartment through a capillary inlet,

· measuring a first metabolite concentration in the medium,

• measuring at least a second metabolite concentration after a period of time, and

• obtaining a metabolic rate of the motile organisms by correlating the metabolite concentration measures. The invention further relates to a method for measuring the metabolic rate of motile organisms in a medium, the method comprising the steps of

• providing at least one device as mentioned above,

• injecting and/or arranging a medium containing motile organisms in the

compartment,

· measuring a metabolite concentration in the medium obtaining a metabolite concentration measure, and

• correlating said metabolite concentration measure to a metabolic rate of the motile organisms. The metabolite may be as described above, e.g. oxygen or carbon dioxide. The metabolic rate may be determined by two or more measurements of the metabolite concentration.

In one embodiment of the invention the compartment is pressure equalized. Further, the compartment is preferably configured to minimize diffusion, preferably effectively prevent diffusion, of said metabolite to and from said compartment. Preferably at least two measurements of the metabolite concentration are performed.

In one embodiment of the invention diffusion of said metabolite to and from the compartment is minimized, preferably effectively prevented, during and between the measurements of the metabolite concentration.

In one embodiment of the invention the medium is injected and/or arranged into the compartment from the bottom of said compartment.

Furthermore, the concentration of cells in the medium may be determined, such as by counting number of organisms in a predefined volume of medium. And further the motility of said motile organisms may be determined, such as determined by measuring the deflections in a light path created by movement of a motile organism.

In a further embodiment of the invention at least a part of the motile organisms in the compartment are visually inspected.

The medium is preferably a liquid medium, such as semen, such as human or animal semen. The medium may be a dilution medium, such as a balanced salt solution, comprising semen, such as human or animal semen. The volume of semen in the medium may be known, predefined and/or predetermined. The sperm cells may be human or animal sperm cells. Further, the medium may be a water sample from either fresh- or saltwater. The sample may be undiluted or diluted by a salt and nutrient solution. The amount of plankton organisms and their species composition in the medium may be known or unknown. The medium may be a salt and nutrient solution, with one or several cultured plankton species.

Semen viability

The present invention may be applied within semen analysis as the metabolic rate of the sperm cells may be a good indicator of semen quality. Thus, a further embodiment of the invention relates to a method for determining the quality of a sample of semen by determining the metabolic rate of at least a part of the semen sample in a device as described above and/or or by a method as described above, thereby obtaining a semen quality measure. In a further embodiment of the invention the concentration of sperm cells in the medium is determined whereby the individual metabolic rate of the sperm cells in the medium is determined, thereby obtaining another semen quality measure. Further, the motility of sperm cells in the medium may be determined, thereby obtaining yet another semen quality measure.

The viability of a sample of semen is an important parameter in order to determine suitable male donors or a selecting a particular sample of semen. The respiration rate of sperm cells may prove to be a good candidate for an objective viability indicator. The invention thus further relates to a method for selecting a viable sample of semen comprising

• determining the metabolic rate of at least a part of the semen sample in a

device as described above, and

• selecting the semen sample having an optimal metabolic rate, such as the highest metabolic rate.

Toxicity assessment Environmental impairments, such as the presence of toxic substances or radioactive irradiation may have a measureable impact on the turnover of metabolites by autonomously moving aquatic organisms, such as microalgae and zooplankton (Alonzo et al., 2006; Jensen et al., 2006). A further embodiment of the present invention therefore relates to toxicity assessment of motile organisms by application of the herein described apparatus or method. E.g. a toxic substance could be added to the medium containing the motile organisms and the present invention facilitates measurement of the metabolic impact, e.g. by monitoring the metabolic impact over time with a predefined amount of the toxic substance or by monitoring the metabolic impact when increasing amounts of the toxic substance are added. Measurements of the metabolic impact may be supplemented with measurement of the impact on the motility of the motile organisms.

Description of Drawings The invention will now be explained in more detail with reference to the drawings in which

Fig. 1 shows an exemplary embodiment of the apparatus according to the

invention, and

Fig. 2 shows another exemplary embodiment of the apparatus according to the invention.

Detailed description of the drawings

In the following a number of different embodiments of the present invention will be described with reference to the accompanying drawings, but it is to be understood that these embodiments only constitute examples of the general inventive idea and that other embodiments may be conceivable by a person skilled in the art.

The embodiment of the invention shown in figure 1 for measuring respiration of motile organisms illustrates a longitudinal compartment 1 which is arranged with the longitudinal axis in a vertical direction. The bottom of the compartment, which could be cylindrical, connects to an inlet capillary tube 4 which is connected to a syringe 13 by means of a syringe adapted connection 6. The inlet capillary tube 4 connects to the compartment 1 at the inlet 2 which is located at the bottom of said compartment 1. An outlet capillary tube 5 connects to the compartment 1 at the outlet 3 which is located at the top of said compartment. The walls of the compartment 1 and the capillary tubes 4, 5 are made of a gas impermeable and transparent material, like glass or plastic, which allows visual inspection of the motile organisms in the medium under magnification. The only openings in the compartment are therefore the capillary inlet 2, 4 and the capillary outlet 3, 5. In this case the outlet provides pressure equilibrium with the surroundings but the length of the outlet tube 5 and the small cross sectional area of the tube 5 effectively prevents diffusion of oxygen to and from the compartment 1.

Oxygen concentration can be measured by means of a microsensor 7, such as an optode, with the sensor tip located inside the compartment 1. Knowing the initial oxygen concentration and its time related decay, e.g. caused by spermatozoon consumption in the case of sperm cells, precise oxygen consumption measurements are facilitated by the apparatus and method according to the invention.

The apparatus in fig. 1 is further provided with means for measuring motility of motile organisms inside the compartment. The motility measurements may for example be provided by means of motile organisms interacting with an electromagnetic field generated by e.g. laser.

Motile organisms inside the compartment may further be visually inspected by means of a micro-camera 11 and a (second) light source 10 illuminating the inside of the compartment 1.

As seen from fig. 1 the apparatus is a kind of a flow-through cell. The medium containing motile organisms, e.g. semen diluted with a buffer solution, is arranged in the syringe 13 and injected into the compartment 1 through the capillary inlet 4. Due to the thin capillary in- and outlets 4, 5 the evaporation during analysis is reduced to a minimum. If the total volume of medium in the syringe 13 is bigger than the total volume of the compartment 1 and the capillary tubes 4, 5 the compartment 1 may be completely filled and the medium will thus continue through the capillary outlet 5 and into the collection chamber 12. A steady state inside the compartment 1 can now be obtained because the compartment 1 and the capillary tubes 4, 5 are filled with medium. Diffusion of oxygen to and from the compartment is effectively prevented due to the capillary tubes 4, 5 and the respiration rate of the motile organisms can now be determined by measuring the change in oxygen concentration per time unit.

Measurements of the motility of the motile organisms and visual inspection etc. can be performed concurrently. In the case of semen analysis the outcome may thus be a time series of spermatozoon motility and metabolic activity given by the oxygen

consumption. This may be supplemented by the visual inspection. A semen sample which has been subject to measurement is typically not intended for further use. After the semen analysis the medium containing the semen sample can be easily discharged from the compartment 1 and the tubes 4, 5 by applying pressure and/or a cleaning solute through the inlet capillary tube 4. Fig. 2 shows an alternative embodiment of the apparatus according to the invention where the compartment V is "lying down", i.e. arranged horizontally, and the measurement units 7, 8, 9, 10, 11 are arranged vertically whereas they were arranged horizontally in fig. 1. The basic flow-through principle is the same, i.e. the medium containing sperm cells are injected from the syringe 13 into the compartment V through the capillary tube 4' and the inlet 2', which is again located near the bottom of the compartment V to minimize air bubbles during filling of the compartment V. The medium can exit the compartment V through the outlet 3' and the outlet capillary tube 5'.

References

Alonzo, F., Gilbin, R., Bourrachot, S., Floriani, M., Morello, M. & Garnier-Laplace, J. 2006. Effects of chronic internal alpha irradiation on physiology, growth and

reproductive success of Daphnia magna. Aquat. Toxicol. 80, 228-236.

Engstrom, D., Frank, A., Backsten, J., Goksor, M. & Bengtsson, J. Grid-free 3D multiple spot generation with an efficient single-plane FFT-based algorithm. 2009. Optics Express, vol. 17, 9989- 10000.

Kajanto, M., Byckling, E., Fagerholm, J., Heikonen, J., Turunen, J., Vasara, A. & Salin, A. 1989. Photolithographic fabrication method of computer-generated holographic interferograms. Appl. Opt. 28, 778-784.

Jensen, T. C, Anderson, T.R., Daufresne, M. & Hessen, D.O. 2006. Does excess carbon affect respiration of the rotifer Brachionus calyciflorus Pallas? Freshwater Biology 52, 2320-2333. Johansson, M. & Bengtsson, J. 2000. Robust design method for highly efficient beam- shaping diffractive optical elements using an iterative-Fourier-transform algorithm with soft operations. Journal of Modern Optics 47, 1385-1398.

Ripoll, V., Kettunen, H., & Herzig, H. P. (2004). Review of iterative Fourier-transform algorithms for beam shaping applications, Optical Engineering, 43, 2549-2556.

Claims

An apparatus for measurement of the metabolic rate of motile organisms in a medium comprising

• at least one compartment capable of accommodating at least a part of the medium,

• one or more capillary tubes in fluid communication with said

compartment, and

• at least one metabolite detector for measuring the concentration of metabolite inside the compartment.

The apparatus according to any of preceding claims, wherein the (inner) volume of the compartment is known, predefined and/or predetermined.

The apparatus according to any of preceding claims, further comprising an inlet adapted for injection of said medium into the compartment.

The apparatus according to claim 3, wherein said inlet is connecting with the compartment at or near the bottom of said compartment.

The apparatus according to any of claims 3 to 4, wherein said inlet is adapted for direct connection with an injection device, such as a syringe.

The apparatus according to any of preceding claims, wherein one capillary tube is an inlet.

The apparatus according to any of preceding claims, wherein at least one capillary tube, such as an inlet capillary tube, is connecting with the compartment at or near the bottom of said compartment.

8. The apparatus according to any of preceding claims, wherein at least one of said one or more capillary tubes is adapted for connection with an injection device, such as a syringe.

9. The apparatus according to any of preceding claims, wherein one capillary tube is an outlet.

10. The apparatus according to any of preceding claims, wherein at least one i capillary tube, such as an outlet capillary tube, is connecting with the

compartment at or near the top of said compartment.

1 1. The apparatus according to any of preceding claims, further comprising means for pressure equalizing the compartment.

12. The apparatus according to any of preceding claims, wherein at least one of said capillary tube(s) is adapted for pressure equalizing the compartment.

13. The apparatus according to any of preceding claims, wherein at least one of said capillary tube(s) is configured to effectively prevent diffusion of said metabolite to and from the compartment.

14. The apparatus according to any of preceding claims, wherein at least one of said capillary tube(s) is configured for pressure equalizing the compartment and configured to effectively prevent diffusion of said metabolite to and from the compartment.

15. The apparatus according to any of preceding claims, wherein the compartment wall and the capillary tube(s) are produced from a

substantially metabolite impermeable material, such as glass.

16. The apparatus according to any of preceding claims, wherein the compartment wall and/or the capillary tube(s) is at least partly transparent or translucent.

17. The apparatus according to any of the preceding claims, wherein the shape of the compartment is selected from the group of a cylinder, a polyhedron, a cone, a hemisphere or a combination thereof.

18. The apparatus according to any of preceding claims, further comprising means for determining the concentration of the motile organisms in the medium.

19. The apparatus according to any of preceding claims, further comprising at least one motion detector for determining the motility of the motile organisms.

20. The apparatus according to any of preceding claims, further comprising means for applying a temperature gradient across the compartment, such as a horizontal temperature gradient across the compartment.

21. The apparatus according to any of preceding claims, further comprising a heating element and a cooling element, said elements mounted on opposite sides of the compartment, and said elements configured to apply a temperature gradient across the compartment.

22. The apparatus according to any of preceding claims, further comprising two Peltier elements mounted on opposite sides of the compartment, said Peltier elements configured to apply a temperature gradient across the compartment.

23. The apparatus according to any of preceding claims 20 to 22, wherein said temperature gradient is adapted to induce a controlled movement of the medium, such as a controlled stirring of the medium.

24. The apparatus according to any of preceding claims, further comprising inspection means for visually inspecting the motile organisms.

25. The apparatus according to claim 24, wherein the inspection means

comprises at least one light source for illuminating at least a part of the medium.

26. The apparatus according to any of claims 24 to 25, wherein the inspection means comprises at least one camera for imaging at least a part of the medium.

27. The apparatus according to any of the preceding claims, wherein the

volume of the medium is known and/or predetermined.

28. The apparatus according to any of the preceding claims, wherein the

medium is a liquid medium.

29. The apparatus according to any of the preceding claims, wherein the

medium is the medium is a water sample, such as a sample from freshwater or saltwater.

30. The apparatus according to any of the preceding claims, wherein the

medium is a dilution medium, such as a balanced salt solution, comprising plankton organisms or microalgae.

31. The apparatus according to any of the preceding claims, wherein the

medium is semen, such as human or animal semen.

32. The apparatus according to any of the preceding claims, wherein the

medium is a dilution medium, such as a balanced salt solution, comprising semen, such as human or animal semen.

33. The apparatus according to claim 32, wherein the volume of semen in the medium is known and/or predetermined.

34. The apparatus according to any of the preceding claims, wherein the motile organisms are human or animal sperm cells.

35. The apparatus according to any of the preceding claims, wherein the motile organisms are plankton such as zooplankton, autonomously moving aquatic organisms or microalgae.

36. The apparatus according to any of the preceding claims, wherein the metabolite is a gas, such as oxygen or carbon dioxide..

37. The apparatus according to any of the preceding claims, wherein the i metabolite detector is an oxygen detector for measuring the oxygen

concentration in the medium.

38. The apparatus according to claim 37, wherein the oxygen detector is an optode or a Clark-type sensor.

39. The apparatus according to any of the preceding claims, further comprising thermostatic means for controlling the temperature of the compartment.

40. The apparatus according to any of the preceding claims, wherein the (inner) volume of the compartment is less than 5 milliliters (ml_), such as less than 4 ml_, such as less than 3 ml_, such as less than 2 ml_, such as less than 1 ml_, such as less than 0.8 ml_, such as less than 0.7 ml_, such as less than 0.6 ml_, such as less than 0.5 ml_, such as less than 0.4 ml_, such as less than 0.3 ml_, such as less than 0.2 ml_, such as less than 0.1 ml_.

41. The apparatus according to any of the preceding claims, wherein the (inner) volume of the compartment is between 0.1 and 5 milliliters (ml_), such as between 0.5 and 2 ml_, such as between 0.5 and 1.5 ml_, such as between 0.75 and 1.25 ml_, such as between 0.9 and 1.1 ml_.

42. The apparatus according to any of preceding claims, wherein the maximum inner diameter of the capillary tube(s) is less than 1 mm, more preferably less than 0.8 mm, more preferably less than 0.6 mm, more preferably less than 0.5 mm, more preferably less than 0.4 mm, such as less than 0.35 mm, such as less than 0.3 mm, such as less than 0.25 mm, such as less than 0.2 mm, such as less than 0.15 mm, such as less than 0.1 mm.

43. The apparatus according to any of preceding claims, wherein total length of the capillary tube(s) is between 10 and 100 mm, such as between 10 and 20 mm, such as between 10 and 20 mm, such as between 20 and 30 mm, such as between 30 and 40 mm, such as between 40 and 50 mm, such as between 50 and 60 mm, such as between 60 and 70 mm, such as between 70 and 80 mm, such as between 80 and 90 mm, such as between 90 and 100 mm.

44. The apparatus according to any of preceding claims, wherein the total (inner) volume of the capillary tube(s) is less than 10% of the volume of the compartment, more preferably less than 5%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1 %, more preferably less than 0.5%, more preferably less than 0.1 %, such as less than 0.8%, such as less than 0.7%, such as less than 0.6%, such as less than 0.5%, such as less than 0.4%, such as less than 0.3%, such as less than 0.2%, such as less than 0.1 %, such as less than 0.05%, such as less than 0.01 % of the (inner) volume of the compartment.

45. A method for measuring the metabolic rate of motile organisms in a

medium, the method comprising the steps of

a. injecting and/or arranging the medium into a compartment through a capillary inlet,

b. measuring a first metabolite concentration in the medium, c. measuring at least a second metabolite concentration after a period of time, and

d. obtaining a metabolic rate of the motile organisms by correlating the metabolite concentration measures.

46. A method for measuring the metabolic rate of motile organisms in a

medium, the method comprising the steps of

a. providing at least one device as defined in any of claims 1-44, b. injecting and/or arranging a medium containing motile organisms in the compartment,

c. measuring a metabolite concentration in the medium obtaining a metabolite concentration measure, and

d. correlating said metabolite concentration measure to a metabolic rate of the motile organisms.

47. The method according to any of the claims 45 to 46, wherein the compartment is pressure equalized, such as in pressure equilibrium with the surrounding environment.

48. The method according to any of the claims 45 to 47, wherein at least two measurements of the metabolite concentration are performed.

49. The method according to any of the claims 45 to 48, wherein at least two, three, four, five, six, seven, eight, nine or at least ten measurements of the metabolite concentration are performed.

50. The method according to any of the claims 45 to 49, wherein diffusion of said metabolite to and from the compartment is minimized, preferably effectively prevented, during and between the measurements of the metabolite concentration.

51. The method according to any of the claims 45 to 50, wherein the medium is injected and/or arranged into the compartment from the bottom of said compartment.

52. The method according to any of the claims 45 to 51 , wherein the

concentration of motile organisms in the medium is determined, such as by counting number of motile organisms in a predefined volume of medium.

53. The method according to any of the claims 45 to 52, wherein the motility of said motile organisms is determined.

54. The method according to any of the claims 45 to 53, wherein at least a part of the motile organisms in the compartment are visually inspected.

55. The method according to any of the claims 45 to 54, wherein the motile organisms are sperm cells, such as human or animal sperm cells.

56. The method according to any of the claims 45 to 55, wherein the motile organisms are plankton such as zooplankton, autonomously moving aquatic organisms or microalgae.

57. A method for determining the quality of a sample of semen by determining the metabolic rate of at least a part of the semen sample in a device as defined by any of the claims 1-44, thereby obtaining a semen quality measure.

58. A method for determining the quality of a sample of semen by determining the metabolic rate of at least a part of the semen sample by the method according to any of the claims 45-56, thereby obtaining a semen quality measure.

59. The method according to claim 57 to 58, wherein the concentration of sperm cells in the medium is determined whereby the individual metabolic rate of the sperm cells in the medium is determined, thereby obtaining a semen quality measure.

60. The method according to any of claims 57 to 59, whereby the motility of sperm cells in the medium is determined, thereby obtaining a semen quality measure.

61. A method for selecting a viable sample of semen comprising

a. determining the metabolic rate of at least a part of the semen sample in a device as defined by any of the claims 1-44, and

b. selecting the semen sample having an optimal metabolic rate, such as the highest metabolic rate.