The invention relates to the field of positioning a device in an egg of a bird.

For example, the invention relates to the positioning of a device at the surface of the yolk of such an egg, which may be fertilized.

As shown on figure 1 , a fertilized egg 100 of a bird comprises a shell 101 , albumen 102, an inner membrane 103 between the shell 101 and the albumen 102, a yolk 104, a vitelline membrane 105 between the albumen 102 and the yolk 104 and an embryo 106 which develops in the yolk 104.

The egg 100 may be a poultry egg, and more particularly a chicken egg.

The positioning of a device in an egg of a bird may, for example, find an application for precisely injecting a liquid, such as a vaccine, in an embryo during maturation.

The device may also be operated so as to determine the gender of the bird at an early stage of egg maturation. Generally, the gender determination occurs once the young has hatched from the egg For industrial reasons, it would be valuable to determine the gender of the embryo at an early stage of egg maturation not to incubate the egg further if the gender is not the one to be produced. For example, one may consider that an embryo is at its earliest stage of maturation between 3 and 7 days following the fertilization of the egg.

In order to determine the gender of the bird at an early stage of maturation sampling the yolk or the embryo, without breaking the vitelline membrane, may be required. Alternatively, injecting a liquid into the embryo without breaking the vitelline membrane may also be required.

The known industrial devices do not allow this operation so far because they do not precisely localize the position of the embryo.

The known devices may precisely localize the position of the embryo in a plane which is perpendicular to the axis passing through a hole made in the shell of the egg and a given point in the egg, such as the embryo. In this purpose, a camera is often used, such as a CCD camera watching through the hole of the shell in order to localize the embryo in that plane.

However, the known devices are not accurate enough for precisely determining the distance between the hole in the shell and a given point along this axis, such as the embryo at its earliest stage of maturation. For example, one may consider that an embryo is at its earliest stage of maturation between 3 and 7 days following the fertilization of the egg.

This is due to many factors such as the low optical properties of the albumen, the low optical properties of the vitelline membrane and, the tiny size of the embryo in its first week of maturation which occupies a volume less than 1mm ³ .

Moreover, even though the known devices were able to precisely determine the distance between the hole made in the shell and a given point in the egg, such as the embryo, these devices are not themselves accurate enough to be positioned right to this point.

Thus, there exists a need to overcome the inaccuracies of the known devices.

An aim of the invention is to solve at least one of the drawbacks of the known devices mentioned hereinabove.

To reach this aim, the invention proposes a device intended for being in contact with the vitelline membrane of an egg of a bird, comprising:

a microtube ;

a first means connected to a first end of the microtube for translating said microtube into the egg ;

a second means for measuring the force applied on a second end of said microtube.

Thus, when a needle is provided with the device, the needle may pass through the vitelline membrane to be introduced with certainty in the yolk of the egg or in the embryo developing in the egg.

To reach this aim, one also proposes a method for positioning said device in contact with the vitelline membrane of an egg of a bird, comprising the following steps:

(a) making at least one hole through the shell of the egg ; (b) translating the microtube into the egg through the hole, towards the vitelline membrane of the egg, while measuring the force applied on the second end of the microtube ;

(c) stopping the translation of the microtube once a parameter depending on the force applied on said second end of the microtube exceeds a threshold value, said second end then touching the vitelline membrane.

To reach this aim, one also proposes an other method for positioning said device in contact with the vitelline membrane of an egg of a bird, comprising the following steps:

(a) making at least one hole through the shell of the egg ;

(b) translating the microtube into the egg through the hole, towards the vitelline membrane of the egg, while measuring the force applied on the second end of the microtube ;

(c) stopping the translation of the microtube once the distance D travelled by the microtube from said hole has reached a predetermined value D _p , said second end of the microtube then touching the vitelline membrane.

The method allows positioning the device according to the invention in contact with the vitelline membrane of the egg in such a way that there is no need to determine a precise value of the real distance separating the hole made in the shell of the egg and the vitelline membrane.

The device may also have of the following technical features, alone or in combination:

the microtube comprises an external diameter comprised between 200pm and 5mm ;

the wall of the microtube has a width comprised between 50μηι and 500pm, preferably between 100pm and 500pm ;

the microtube has a length comprised between 1cm and 20cm, preferably between 3cm and 10cm ;

said second means include a balance, connected to the first end of the microtube, which sensitivity is equal or less than 0.005g ;

said second means include a pressure sensor mounted on the second end of the microtube ;

a camera, such as a CCD camera ; a hollow needle mounted in the microtube ;

the needle has an external diameter comprised between 10 m and 500μπν,

a means for injecting and/or pumping a fluid in the needle ;

a first end of the needle is connected to a second means for translating said needle in the microtube, from a first position in which the needle is inside the microtube to a second position in which a second end of the needle extends from the second end of the microtube and conversely ; the second end of the needle in its second position and the second end of the microtube ranges between 100pm and 1mm;

the needle is rigidly mounted inside the microtube, the second end of the needle extending from the second end of the microtube ;

the distance d between the second end of the needle and the second end of the microtube ranges between 100μιη and 1mm.

The method may also have of the following technical features, alone or in combination:

the device comprising a hollow needle mounted within the microtube which is able to translate with respect to the microtube, a step (d) of translating the needle from a first position in which the needle is inside the microtube to a second position in which a tip of the needle extends from the second end of the microtube, so that the tip of needle penetrates the yolk of the egg ;

it further comprises a step (e) of pumping the yolk of the egg ;

it comprises the following steps applied to a fertilized egg comprising an embryo :

(A) making a first hole through the shell of the egg, along a first axis, said first hole being preferably drilled on the top of the egg along a vertical axis ;

(B) viewing the position of the embryo through the first hole, namely determining the position of the embryo in a plane, said plane being perpendicular to the first axis;

(C) selecting a second axis and making a second hole through the shell, along said second axis, said second hole being located by projecting said position of the embryo in the plane on the shell along said second axis;

(D) translating the microtube of the device into the egg through said second hole, towards the vitelline membrane in the direction of said second axis, while measuring the force applied on the second end of the microtube ;

(E) performing step (c).

- the step of translation of the microtube into the egg is such that no step of penetration of an embryo, which may be in the egg, occurs.

The invention shall be better understood, and other aims, advantages and features will appear by reading the following description, written in regard of the accompanying drawings, on which:

- figure 1 illustrates an egg of a bird;

- figure 2 illustrates a device according to the invention, which comprises a microtube intended to be introduced into the egg;

- figure 3 comprises figures 3(a) and 3(b), which both show the evolution of the weight measured by dedicated means, as a function of the distance travelled by the microtube within the egg;

- figure 4 comprises figures 4(a) to 4(c), which respectively illustrate an image acquisition made by a camera of the device, an image segmentation on the embryo of the fertilized egg and a localization of the embryo;

- figure 5 is a partial cut-off view of the lower part of the microtube in an alternative embodiment to the microtube shown on figure 2.

The device 1 comprises a microtube 10 connected by a first end 11 to a means 30, 31 for measuring the force applied on a second end 12 of the microtube.

In a preferred embodiment, as shown on figure 2, said means for measuring the force applied on the second end 12 of the microtube 10 is a balance 31. The microtube 10 and the balance 31 are linked to first means 20 for translating said microtube 10 and said balance 31. The translation of both microtube 10 and balance 31 is carried out so that a second end 12 of the microtube 10 translates within an egg, towards the yolk. The force applied to the second end 12 of the microtube 10 is measured by the balance 31 , as both of the microtube 10 and the balance 31 are rigidly linked.

The balance 31 , or weighing system, may have a sensitivity equal or less than 0.005g. The balance 31 provides a signal which depends on the force F applied on the second end 12. This force depends on the medium in contact with the second end 12.

In this preferred embodiment, said means 30, 31 include a balance, but it could be any system measuring a force, such as a dynamometer.

Alternatively, in another embodiment, said means 30, 31 may include a pressure sensor 30 mounted on the second end 12 of the microtube 10 for measuring the pressure applied to this second end 12. The pressure sensor may cover all the surface of this second end 12, or only part of it. In this particular case, depending on the way the pressure sensor is operated, a readout system 32 may be connected to the microtube. However, in such embodiment, the pressure sensor may become rapidly contaminated by the medium, which may imply decontamination operations.

The device 1 may also comprise a hollow needle 40, a first end 41 of which being connected to a second translation means 50 for translating said needle 40 into the microtube 10.

This second translation means 50 allows the needle to be translated between a first position in which the needle 40 is inside the microtube 10 to a second position in which the second end 42 of the needle 40 extends from the second end 12 of the microtube 10 and vice versa. In other words, the needle 40 may translate within the tube, so that its second end 42 opens in and out from the second end 12 of the microtube 10. By second end of the needle, it is meant the tip of the needle.

The distance between the tip of the needle 42 in its second position and the second end 12 of the microtube 10 is referred by the extension distance d, as mentioned on figure 2.

Alternatively, the needle 40 may be rigidly mounted inside the microtube 10, its second end 42 always extending from the second end 12 of the microtube, the distance between the second end 12 and the second end being referred by the extension distance d. In both cases, the distance d may range between 100pm and

1mm.

The microtube 10 has an external diameter ranging between 200pm and 5mm. The thickness of the wall of the microtube 10 ranges between 50pm and 500pm, preferably between 100μιη and 500pm. The internal diameter of the microtube may range between 100 μηη and 5mm. The microtube 10 has a length ranging between 1cm and 20cm, preferably between 3cm and 10cm.

The needle 40 has an external diameter ranging between 10pm and 500pm.

The second end 12 of the microtube 10 may be rounded to limit the risks for the microtube 10 to break the vitelline membrane 105 of the egg 100 when the microtube 10 comes into contact with the vitelline membrane 105.

The microtube 10, as the hollow needle 40, may be made of a biocompatible material, such as a biocompatible polymer or a biocompatible metal.

The device 1 may also include a means 70 for injecting a fluid in the egg 100, and more particularly in the embryo 106, through the needle 40. The fluid may be a vaccine or a fluid for affecting sex determination of the embryo 106.

Alternatively, said means 70 may have a function of pumping of a fluid or may be designed for either injecting a fluid or pumping a fluid.

Alternatively, the needle 40 may be replaced or associated to one or many fiber optics, said fiber optics being connected to remote display means. In this particular case, the device 1 may also include display tools as well as an image processing unit.

The device 1 may also comprise a camera 80 so as to see within the egg 100.

All the means 20, 70, 50, 31 and 80 are connected to a control means 90, such as a computer.

A method for positioning the device 1 in contact with the vitelline membrane 105 of the egg 100 comprises the following steps:

(a) making at least one hole through the shell 101 of the egg 100; (b) translating the microtube 10 of the device 1 into the egg 100 through the hole, towards the vitelline membrane 105, while measuring the force applied on the second end 12 of the microtube 10;

(c) stopping the translation of the microtube 10 once the second end 12 of the microtube 10 comes into contact with the vitelline membrane 105.

Step (b) may be performed blindly by one skilled in the art as the yolk 104 of the egg, wrapped by the vitelline membrane 105, fills a great part of the egg and is generally disposed in the centre of the egg.

It should be noted that, if the egg comprises an embryo, the translation of the microtube 10 into the egg may be such that no step of penetration of the embryo occurs. Alternatively, in some cases, the translation may be such that the needle 40 of the device 1 finally penetrates the embryo.

Step (c) may occur when said means 30, 31 detects that a parameter exceeding a threshold value. By parameter, it is meant a data which is related to the weight measured by said means 30, 31 for measuring the force applied on the second end of the microtube 10.

For example, this parameter may be the force applied on the second end 12, or a derivative of this force with respect to the distance (or depth) D between the hole and the second end 12 of the microtube 10.

Indeed, it is possible to determine where the second end 12 of the microtube 10 is in the egg 100 (albumen, vitelline membrane, yolk, etc.), as shown on Figure 3. In other words, the distance between the second end 12 and a reference point, for example the hole, can be monitored. The monitoring means may be either included or being connected to the translation means.

Figure 3 shows the evolution of the force applied on the second end of a microtube with respect to the distance D between the hole of the shell and the second end of the microtube. This graph was plotted using an experimental set-up which is similar to the device 1 described above.

In this experimental set-up, the microtube is made of stainless steel. The first means for translating said microtube into the egg is a Cyberstar® bench control (model Oxypuller®) which allows a displacement with an accuracy of 0.01mm. The microtube has an external diameter of 40Όμιτι and an internal diameter of 150μιη, so that the surface of the second end 12 of the microtube 10 is about 0.1mm ² .

The second end 12 of the microtube 10 is rigidly linked to the means 30, 31 so as to continuously monitor the force applied to the second end 12. In this example, the means includes a balance 31 , being a Sartorius® weighing system (model L6200S®), which has a sensitivity of 0.001g

Five points Pi to P5 are identified on figure 3.

Pi corresponds to the break of the inner membrane 103 of the egg 100. P ₂ corresponds to the detection of the contact between the microtube and the vitelline membrane 105. P ₃ corresponds to the break of the vitelline membrane 105. P ₄ corresponds to the break of the vitelline membrane 105, once the microtube has crossed the yolk 104. Finally, P ₅ corresponds to the break of the shell 101 once the microtube has crossed the egg 100.

As it can be clearly seen from these drawings, as the second end 12 translates through the albumen, it is possible to monitor the following steps: breakage of the inner membrane (Pi), contact the vitelline membrane and the microtube (P ₂ ), breakage of the vitelline membrane by the microtube (P ₃ ). Between P ₂ and P3, the second end 12 of the tube is very likely to have reached the vitelline membrane.

An interesting point on this curve is the point P ₂ , which a close- up is shown on figure 3(b). It can be seen from this figure 3(b) that, when the second end 12 comes into contact with the vitelline membrane, a significant evolution, i-e a slope, of the derivative of the force applied to the second end 12, can be detected. By detecting this slope, one may detect the contact with the vitelline membrane 105 of the egg 100.

According to another embodiment,, step (c) may occur when the distance D travelled by the microtube 10 from the hole made in the shell of the egg has reached a predetermined value D _p . Unlike in the previous embodiment, the translation distance of the second end 12 within the egg might not be set up on a case by case basis, but is a fixed translation distance D _p .

For example, for a chicken egg, it is well known that the fixed translation distance D _p ranges between 10mm and 30mm. This translation distance D _p may change, within this range, from an egg to the other, ^" in particular depending on where the hole was made through the shell of the egg.

To determine the value of this fixed translation distance D _p , it is possible to carry on experimental tests on many, for example some tens or some hundreds of chicken eggs with an experimental set-up prior to any industrialization, so as to get relevant statistical data. Consequently, the value of Dp is predetermined before performing steps (a) to (c) with the device 1 on an industrial set-up.

The experimental set-up may be the one shortly described above with which the results of figure 3 have been obtained.

In the particular case of a chicken egg, the value for D _p is initially set up within the range from 10mm to 30mm.

Then, for a great quantity of chicken eggs, it may be checked whether the chosen distance allows the second end of the microtube contacting the vitelline membrane 105 of the egg 100, without breaking the vitelline membrane 105. The fixed translation distance D _p chosen may be considered as acceptable if the device has contacted the vitelline membrane 105 of the egg 100, without breaking the vitelline membrane 105, for more than a given percentage, say 95%, of the chicken eggs tested.

During these tests, the position of the hole made through the egg shell may be arbitrary chosen, so that the distance D _p finally obtained is less dependent from the relative position of the device with respect to the egg.

The effective contact between the second end of the microtube and the vitelline membrane 105 is checked thanks to the means 30, 31 .

In the experimental test shown on this figure, P ₂ is located at a distance of about 15mm from the hole and the two points P ₂ and P ₃ are separated from a distance of about 15mm. It means that the distance D _p may be chosen between about 15mm and about 30mm. If the vitelline membrane is broken, the egg is wasted; therefore, it is of prime importance to put the microtube 10 in contact with the vitelline membrane, while being highly confident on the fact that this membrane is not broken.

From this test, it can be understood that once the device 1 is in contact with the vitelline membrane 105 (point P ₂ for the experimental test), the device 1 may push the yolk 104 within the albumen 101 of the egg 100 without breaking the vitelline membrane 105 (point P ₃ for the experimental test).

Consequently, determining the fixed translation distance D _p to which the device 1 is in contact with the vitelline membrane 105 is quite easy for one skilled in the art.

However, should experimental tests yield to a given percentage, say more than 5%, of the eggs having their vitelline membrane broken, the distance D _p might be decreased. In that case, new tests may be carried out with a lower value of D _p , which is however still in the range from 10mm to 30mm in the particular case of a chicken egg. And if, after testing, the microtube has not reached the vitelline membrane 105 for more than 5% of the eggs tested, it means that the distance D _p is too weak. In that case, new tests have to be carried out with a higher value of D _p , which is however still in the range 10mm to 30mm.

It should be noted that the alternative approach for step (c) do not require a precise value of the distance separating the shell hole and the vitelline membrane to be precisely known. Only the knowledge of the fixed translation distance D _p is required to make the second end of the device 1 reaching the vitelline membrane, while unlikely breaking it.

Indeed, in the first case, the microtube is translated through the egg until a parameter measured by said means for measuring the force applied on the second end of the microtube reaches a threshold value. In the second case, the translation distance D _p of the microtube in the egg is fixed and may be predetermined through previously described procedures.

If the device 1 comprises a hollow needle 40 extending from the second end 12 of the microtube 10, the needle 40 penetrates the yolk 104 of the egg 100 when step (c) occurs.

Alternatively, if the device 1 comprises a hollow needle 40 mounted in the microtube 10 which is able to translate with respect to the microtube 10, an additional step (d) of translating the needle 40 from its first position to its second position is performed, so that the needle 40 extends from the second end. In that case, the needle 40 penetrates the yolk 104 of the egg 100 when step (d) occurs.

Once the needle 40 has penetrated the yolk 104 of the egg 100, a final step (e) of pumping yolk may be performed, for example to be analyzed. This final step may be useful, for example, for biological analyses, such as detection of bacteria or proteomic analyses.

If one wants to put the device 1 in contact with the vitelline membrane 105 closer to the embryo 106, one may operate in the following way:

(A) making a first hole through the shell 101 of the egg 100, along a first axis, said first hole being preferably drilled on the top of the egg along a vertical axis ;

(B) viewing the position of the embryo 106 through the first hole, i-e determining the position (coordinates (x,y)) of the embryo in a plane, said plane being perpendicular to the first axis,

(C) selecting a second axis and making a second hole through the shell, along said second axis, said second hole being located by projecting said position of the embryo in the plane on the shell along said second axis. In a preferred embodiment, said second axis is also vertical.

(D) translating the microtube 10 of the device 1 into the egg 100 through said second hole, towards the vitelline membrane 105 in the direction of said second axis, while measuring the force applied on the second end 12 of the microtube 10;

(E) stopping the translation of the microtube 10 once the second end 12 of the microtube 10 is in contact with the vitelline membrane 105.

Stopping in step (E) occurs when the second end 12 of the microtube 10 comes into contact with the vitelline membrane 05 according to one of the possibilities mentioned hereinabove for step (c), namely :

- when the balance 31 (or weighing device or any system measuring a force, such as a dynamometer or a pressure sensor) detects that a parameter depending on the force applied on the second end 12 exceeds a threshold value; or - when the distance D travelled by the microtube 10 from the second hole made in the shell of the egg has reached the predetermined depth D _p .

It should be noted that for putting the device 1 in contact with the vitelline membrane 105 closer to the embryo 106, one also may make only one hole if the second axis turns to be similar to the first axis.

The fixed translation depth D _p may still be predetermined by the experimental tests described hereinabove.

Then, the following additional step may be performed: (G) a liquid is injected into the embryo 106.

Alternatively, as previously described, a hollow needle 40 may be connected to a second translation means 50, so that the needle can translate between a first position wherein the needle 40 is located inside the microtube 10 to a second position wherein the second end 42 of the needle 40 extends from the second end 12 of the microtube 10. In this case, a step (F) is performed following step (E), wherein the needle 40 is translated from its first position to its second position.

In that case, the needle 40 penetrates the embryo during step

(F).

Then, step (G) hereinabove mentioned may be performed.

Steps (A) to (G) finally allow injecting a liquid in the embryo 106, the embryo being at its earliest stages of maturation.

The extension distance d over which the needle 40 extends from the second 12 of the microtube 10 or, alternatively, this extension distance d between the needle 40 ,in its extended (or second) position, from the second end 12 of the microtube 10 may be determined thanks to experimental tests. Such tests may be carried out on many chicken eggs, prior to industrialization.

Actually, this extension distance d mostly depends on the distance between the embryo 106 and the vitelline membrane 105. According to past experiences, it is assumed that this extension distance may range between 100 pm and 1 mm.

In this purpose, experimental tests may be carried out for many chicken eggs. The experimental set-up may be the same as that one used for determining D _p .

In particular, it comprises a microtube made of stainless steel, and having an external diameter of 400μιη and an internal diameter of 150pm. The first means for translating said microtube into the egg is a Cyberstar® bench control (model Oxypuller®) which allows a displacement with an accuracy of 0.01 mm. Said means 30, 31 for measuring the force applied on the microtube is a weighing system 31 , such as a Sartorius® balance (model L6200S®), which has a sensitivity of 0.001g.

In addition, the experimental set-up comprises a high-sensitivity camera for viewing the embryo through the hole, together with an illumination system. The set-up also comprises a peristaltic pump for pumping blood from the embryo.

The tests for determining the extension distance d may be carried out after or during the tests for determining the fixed translation distance Dp.

For each egg, the camera views the embryo 106 through a hole made in the shell 101 of the egg 1 1 , for example along a vertical axis. The image obtained by the camera is illustrated on figure 4(a). The position of the embryo 106 in the horizontal plane is determined by means of image processing which is well-known by one skilled in the art and will not be further described. A second hole is drilled through the shell 101 of the egg 100 along the vertical axis passing through of the embryo 106.

Then, the microtube is translated along the said vertical axis, until its second end comes into contact with the vitelline membrane 105.

The contact between the microtube and the vitelline membrane 105 is determined thanks to one of the methods discussed hereinabove.

To determine if the needle 40 tip is in the embryo 106, one may activate a pump, such as peristaltic pump, mounted at the first end of the needle.

If blood is extracted through the needle, it means that the needle 40 is in the embryo 106, the distance d being thus accurate. Should some yolk be extracted from the needle, the distance would need to be adjusted.

Once such test has been carried out on many chicken eggs, the extension distance d would be considered to be correct if blood has been extracted for more than a given percentage, say 95% of the eggs tested.

Alternatively, we may inject ink or another contrast liquid in the embryo 106 through needle 40 to see if the colour of the embryo 106 changes. This can be performed using a peristaltic pump in reverse mode.