WO2007107956A2

WO2007107956A2 - Method and apparatus for detecting chromosome contents in spermatozoa of domestic animals, in particular for sorting such spermatozoa

Info

Publication number: WO2007107956A2
Application number: PCT/IB2007/050973
Authority: WO
Inventors: Maurizio Grego; Bruno Raso; Riccardo Aleandri; Andrea Galli
Original assignee: Istituto Sperimentale Italiano 'lazzaro Spallanzani'
Priority date: 2006-03-22
Filing date: 2007-03-20
Publication date: 2007-09-27
Also published as: ITCN20060005A1; WO2007107956A3

Abstract

A method and an apparatus for detecting chromosome contents in spermatozoa of domestic animals, in particular for sexing such spermatozoa, are described. For such a determination, a source (1) of electromagnetic radiations having frequencies in the Terahertz range illuminates a flow of spermatozoa (5), and detection and analysis means (6, 8) directly measure the vibrational response of DNA in a spermatozoon when the latter is illuminated by the radiation. The measure is performed by evaluating the variations of predetermined characteristics of the radiation, such as power and polarisation, induced by the interaction with the spermatozoon.

Description

METHOD AND APPARATUS FOR DETECTING CHROMOSOME CONTENTS IN SPERMATOZOA OF DOMESTIC ANIMALS, IN PARTICULAR FOR SORTING SUCH SPERMATOZOA Description The present invention relates to a method of and an apparatus for detecting chromosome contents in spermatozoa of domestic animals, in particular for sexing such spermatozoa.

Sperm sexing is the operation allowing predetermination of the sex of offspring. This practice is used since long time especially in zootechnical breeding, in order to surely determine the sex of offspring when using artificial insemination.

The broad diffusion of artificial insemination (AI) techniques has allowed zootechnical industry to increase efficiency of systems for producing heads of livestock selected according to useful characteristics. Sex preselection is a further optimisation of that process, whose benefits may concern farmers interested in milk production and/or meat production, or in programming the births of cows for in-farm covering or bulls to be introduced into genetic centres.

Industrial development of methods of preselection of spermatozoa has been made possible by flow cytofluorometry, which nowadays is the best technique to put said methods into practice. Flow cytofluorometer is a system consisting of: sources of one or more laser beams passing through the specimen; a hydrodynamic system introducing the cells of the specimen to be analysed into a flow of a liquid isotonic with the specimen; a set of light detectors (photodiodes and/or photomultipliers); and a processor for real time analysis of data. The analysis is based on the evaluation of the amount of light emitted by the cells under test when they intercept the laser beam along their path. Such cells are previously stained with fluorochromes, i.e. substances emitting a particular fluorescence when hit by the coherent laser light. Fluorescence is amplified by the photomultipliers and analysed by a software capable of optimising the output signal and to make it interpretable. The very high sensitivity of cytofluorometers enables measuring the different amount of DNA present in X spermatozoa with respect to Y spermatozoa (a difference of about 3 - 4 %, varying depending on the species), by using dyes such as Bisbenzimide Hoechst 33342, which binds to DNA regions rich in adenine and timine.

Subsequently, sorting enables physically separating cells that pass through the flow and that are positively or negatively charged, depending on the characteristics they have, which are set when the sorting procedures are started. Actually, two plates attract the particles depending on their charge and deflect the particles to two collectors. The collected and sorted material can be subsequently analysed again by reintroducing it into the flow to check the separation efficiency. By defining the sorting as a function of the bimodality of the frequency distribution of the DNA contents of spermatozoa (Johnson et al., 1989, Biol. Reprod. 41 :199-203), it is possible to both quantify X and Y spermatozoa and divide them into two populations, namely X and Y populations.

Flow cytofluorometry has therefore proven valid not only for analysis, but also for physical separation of spermatozoa (Garner et al, 1983, Biol. Reprod. 28:312-321; Johnson, 1991, Reprod. Dom. Anim. 26:309-314; Johnson et al., 1989, Biol. Reprod.

41:199-203; Morell et al., 1988, Vet. Rec. 122:322-324), but its low yield (107 sperm/hour are separable) limits it use in AI, since a whole day's sorting would be necessary to obtain a so called "paillette" of frozen cattle sperm, with a suitable concentration for use in AI.

For studying DNA of spermatozoa, cytometers with epi-illumination (front illumination relative to the cells) are favoured (Pinkel et al., 1982, Cytometry 1:1-9), whereas cytometers with orthogonal illumination should be suitably modified so that they are able to measure light diffracted also at 0° (Johnson and Pinkel, 1986, Cytometry 7:268-273) besides light refracted at 90°, as they usually do. Indeed, in cytofluorometers with orthogonal illumination, the amount of light refracted at 90°, containing fluorescence, is greater if the laser beam impinges on the wider surface of a spermatozoon, and is smaller if the laser beam impinges on the narrower surface of a spermatozoon. Since cells introduced into a flow acquire a rotational movement directly proportional to the flow velocity, it has been demonstrated that the heads of spermatozoa, at the instant of intersection with the laser beam, are prevailingly oriented with the thin part directed towards the detector at 0°, thereby producing a smaller amount of light refracted at 90° and thus a lower fluorescence (Pinkel et al., 1982, supra). Since the cytofluorometric reading is based on fluorescence levels, this entails that the mere intersection of cells in a certain position with the laser beam can compensate by defect a different level of fluorochromatisation. Such a problem does not exist for cytometers using epi-illumination (with incandescent arc light source). Actually, in such case, spermatozoa are longitudinally instead of transversally illuminated, so that the problem due to the flat shape thereof does not arise (Pinkel et al., 1982, supra).

French patent FR 2699678 discloses a sexing method where cell sorting takes place without electrically charging the cells, hence under conditions conceptually different from what described above, depending on the specific features of the employed cytofluorometer (PAS III, Partec). UK Patent GB 2145112 discloses a sexing method using flow cytofiuorometry and exploiting electrical charging of spermatozoa carried out in a suitable transport liquid. A method of sexing semen by flow cytofiuorometry, based on the same principle of separation according to the charge, is described in WO 90/13303, which uses a flow cytofluorometer EPICS V (RTM, Coulter). The sexing technique proposed in that patent is based on a number of improvements to the cytofluorometer (concerning e.g. the needle of the flow cell), such as to create a flow of cells having their major surfaces perpendicular to the laser beam. Moreover, the orthogonal illumination instrument is modified so that fluorescence of light diffracted at 0° - 18° (usually exploited only for particle size evaluation) can also be acquired, by applying a photomultiplier in place of the conventional photodiodes, which are not capable of detecting low florescence levels. Such modifications correspond to the proposals of Johnson and Pinkel (1986, Cytometry 7:268- 273). The modifications carried out make fluorescence measurement more sensitive and hence make standardisation of the technique (especially the modification concerning the flow cell needle) more problematic. Therefore, a set of protocols have been developed in an attempt to minimise detection variability, e.g. by eliminating (or further limiting) rotation of spermatozoa. To this end, a new flow cell has been developed (Johnson and Welch, 1999,

Theriog. 52:1323-1341), which enables increasing the percentage of correctly oriented cells from 25% (obtained by using the modified needle) to 70%. Applying such a cell to a cytofluorometer with high sorting speed, obtained by applying pressures up to 60 PSI to the cells in the flow ((MoFlo® by Cytomation Inc. - high speed cell sorter), has enabled obtaining an instrument capable of sorting and separating up to 20 millions sperms/hour with a purity of 75 - 80%. By using doses of sexed frozen cattle semen in AI (Seidel et al., 1999, Theriog. 52:1407-1420), values of fertility have been obtained that are similar to those obtained with non-sexed frozen semen.

However, cytofluorometric methods have a number of drawbacks. A first problem is related with orientation of the spermatozoon, whose head has a shape comparable to a parallelepiped, when it is illuminated by the laser. Actually, the wavelength employed in the measurement is comparable with the spermatozoon head size, and this alters the absorption of exciting photons, thereby changing the amount of reflected radiation. The response will thus depend not only on the DNA amount in the nucleus, but also on the spatial orientation of the spermatozoon at the measurement time. The systems developed till now failed to wholly solve that problem. Moreover, use of fluorescent substances makes the procedure relatively complex.

It is an object of the invention to provide a method of and a device for determining the chromosome contents of spermatozoa, which method and device are not sensitive to the spatial orientation of the spermatozoa at the measurement time and do not require use of fluorescent substances and photomultipliers.

Specifically, according to a first aspect of the invention, there is provided a method of detecting the chromosome contents of spermatozoa, wherein a flow of spermatozoa in which spermatozoa individually follow each other at predetermined intervals is illuminated with an electromagnetic radiation and the radiation having passed through the flow is collected and analysed, characterised in that said chromosome contents are determined by illuminating the flow with a radiation having a frequency in the Terahertz range and by measuring the variations of predetermined characteristics of the radiation having passed through the flow, which variations are induced in the radiation due to the interaction with a spermatozoon, in order to obtain e.g. the vibrational response to said radiation of DNA in a spermatozoon.

The invention exploits the strong interactions the base research has shown to exist between radiations having frequencies in the Terahertz range and the molecular chain forming DNA. See in this respect the document "Far-infrared vibrational modes of DNA components studied by Terahertz time-domain spectroscopy", by B.M. Fischer, M. Walther and P. Uhd Jepsen, available at Internet site staks.iop.org/PMB/47/3807. More particularly, the fact is exploited that spermatozoa having different DNA amounts (hence different chromosome contents) have different absorption spectra due to the different molecular vibrational modes related with the different length of the DNA molecule and hence exhibit a differentiated absorption in respect of the exciting radiation in the Terahertz range. Measuring the amount of radiation reflected/transmitted by the spermatozoa enables therefore a measurement of the vibrational response of DNA, and hence a direct measurement of the chromosome contents, instead of an indirect measurement like that performed in cytofluorometers, which determine the amount of a fluorescent marker differently binding with spermatozoa having different DNA amounts.

Moreover, radiations in the Terahertz range have wavelengths exceeding by about two orders of magnitude the wavelengths currently used in cytofluorometers, thereby enabling a measurement that is not affected by the spatial orientation of the spermatozoon head.

In a second aspect, the invention provides an apparatus for carrying out the method, comprising:

- a source of electromagnetic radiations; - means for conveying past the source a flow of spermatozoa in which spermatozoa individually follow each other at predetermined intervals;

- detection and analysis means for collecting and analysing the radiation having passed through the flow; the apparatus being characterised in that the source is a source of radiations having frequencies in the Terahertz range, and the analysis means are arranged to determine the chromosome contents of each spermatozoon in the flow by evaluating the vibrational response of DNA present in the spermatozoon when the latter is illuminated by the radiation having a frequency in the Terahertz range, wherein the analysis means are arranged to determine said vibrational response by measuring variations of predetermined characteristics of the radiation having passed through the flow, which variations are induced in the radiation due to the interaction with a spermatozoon.

Preferred features are claimed in claims 2 to 10 in respect of the method, and 12 to 23 in respect of the apparatus.

The invention will now be described in greater detail with reference to the accompanying drawings, which show some embodiments given by way of non-limiting examples, and in which:

- Fig. 1 is a schematic representation, in side view, of a first example of an apparatus carrying out the method of the invention;

- Fig, 2 is enlarged view of a detail of Fig. 1; - Fig. 3 is a schematic representation of the optical arrangement of the different parts of the apparatus; and

- Fig. 4 is a schematic representation, in plan view, of a second example of an apparatus carrying out the method of the invention.

Referring to Fig. 1, the measurement apparatus comprises a source 1 of radiations having frequencies in the Terahertz range, which source illuminates, through a suitable optics 2, a capillary tube 3 transparent to radiations in the Terahertz range. Capillary tube 3 conveys, past source 1, a flow of physiologic solution 4, the central region of which in turn contains a flow of biologic material 5 containing the spermatozoa to be measured that individually follow each other at predetermined intervals. Radiations transmitted or reflected by the specimen are then collected by a detector 6, onto which they are focused by a focusing optics 7.

Detector 6 is then followed by a processing device 8 that, depending on the amount of reflected/transmitted radiation and possibly on the state of polarisation, if polarised radiations are used, detects the presence of a spermatozoon and the DNA amount present therein, and hence the chromosome content.

In the particular application to sexing, processing device 8 will control means for physical separation of the spermatozoa, located downstream of capillary tube 3 containing specimen 4, 5. The separation means can be of any kind known in the art and are not explicitly shown, since they are not part of the invention.

Advantageously, source 1, e.g. a laser, generates a radiation with a frequency ranging from some tenths of Terahertz to some ten Terahertz (corresponding to wavelengths ranging from some ten to some hundred micrometres), more particularly a frequency ranging from about 0.5 to about 30 THz. The radiation may have fixed or variable frequency. Detector 6 is for instance a bolometer.

The translation speed of the liquid is variable, i.e. adjustable for instance from 10 cm/sec to 40 m/sec depending on the choices made by the operator (for instance, at a speed of 40 m/sec, with serialised spermatozoa spaced apart by 2 mm, each metre contains

500 individuals, so that 20,000 specimens per second, i.e. in total 72,000,000 specimens per hour, are measured).

Source 1 continuously illuminates a portion of the capillary tube with variable length, e.g. in the order of 500 μm, such length being in any case shorter than the spacing between two successive spermatozoa (usually a few millimetres). In this way, the reflected (or transmitted) signal has a constant average value, defined by the characteristics of the transport liquid, whereas the passage of a spermatozoon produces a discontinuity generating a variation in the reflected (or transmitted) signals. Such variations are detected by detector 6 and analysed by processing device 8.

In order to optimise the system response, optics 2 is to focus the radiation emitted by source 1 onto a region 9 whose transverse size is similar to that of flow 4 carrying the biological material, e.g. 80 μm, or, if longer wavelengths are used, is in the order of one wavelength. In order to maximise the response, measurement area 9 may be focused with elliptical shape, as shown in Fig. 2.

Referring to Fig. 3, in order to optimise both the illumination and the detection, in a system implementation, illuminator or source 1 may be located in a first focus of an ellipsoidal mirror 10, whereas the specimen, schematised here simply by capillary tube 3, is located in the other focus of ellipsoidal mirror 10. Detector 6 will be located between the two foci and directed towards the specimen. The mirror may consist of a pair of separate elements, whose reflecting surfaces are parts of a same ellipsoidal surface 10. Of course, mirror 10 may extend over a certain height (in which case the Figure would show only the mirror cross section), thereby enabling use of a source array and a detector array that will track the specimen while the latter is moving along the longitudinal axis of mirror 10 (orthogonal to the drawing plane). In this manner an individual spermatozoon is measured multiple times, and the different readings can then be integrated to reduce the error margin.

A further information easily attainable through a repeated measurement is the exact translation speed of spermatozoa.

Fig. 4 shows an alternative measurement arrangement that enables refining the measurement at will. Elements similar to those of Fig. 1 are denoted by similar reference numerals, beginning with digit 2.

The arrangement of Fig. 4 may consist of an array 21 (e.g. an array 100x10,000) of Josephson junction elements operating in a.c. (generators), which emit a radiation with frequency in the THz range and are located on one side of transport capillary tube 23 (possibly with a flat external surface), whereas a similar array of detectors 26 are located on the opposite side. The detectors may include HEBs (Hot Electron Bolometers), CEBs (Cold Electron Bolometers), pyroelectric detectors, or an array of Josephson junction elements similar to those of array 21 but used as receivers, and so on.

By such an arrangement, the measurement is distributed over a region of variable length with such a size that it enables refining the measurement as desired. Josephson junction elements, or in any case the emitters mentioned hereinbefore, can operate either in a frequency range set for a possible evaluation of the spectrometric response of the material being illuminated, or at fixed frequency, should this be sufficient.

The same considerations apply to the measurement of a possible variation in the plane of polarisation of the reflected/transmitted signal, if a polarised source is used. In the latter case, use of external magnetic field with suitable strength (e.g. > 10 gauss) applied to the measurement region may be useful.

It is clear that the above description has been given only by way of non-limiting example and that changes and modifications are possible without departing from the scope of the invention.

Claims

Patent claims

1. A method of detecting the chromosome contents of spermatozoa, in which a flow of spermatozoa (5) is illuminated with an electromagnetic radiation, and the radiation having passed through the flow is collected and analysed, characterised in that said chromosome contents are determined by illuminating the flow with a radiation having a frequency in the Terahertz range and by measuring the variations of predetermined characteristics of the radiation having passed through the flow, which variations are induced in the radiation due to the interaction with a spermatozoon, in order to obtain the response to said radiation of DNA in a spermatozoon.

2. The method as claimed in claim 1, characterised in that said radiation is a fixed frequency radiation.

3. The method as claimed in claim 1, characterised in that said radiation is a variable frequency radiation.

4. The method as claimed in claim 2 or 3, characterised in that said radiation has a frequency ranging from some tenths of Terahertz to some ten Terahertz, more particularly from about 0.5 to about 30 THz.

5. The method as claimed in any preceding claim, characterised in that the absorption of the radiation by spermatozoa is determined for measuring said vibrational response.

6. The method as claimed in any preceding claim, characterised in that said radiation is a polarised radiation, and in that the variation of the state of polarisation due to the interaction with the spermatozoa is determined.

7. The method as claimed in claim 6, characterised in that the flow of spermatozoa is made to pass through a magnetic field provided in correspondence of the region (9) where the flow is illuminated by the radiation in the THz range.

8. The method as claimed in any preceding claim, characterised in that the radiation is focused onto a substantially elliptical region (9), of which the major axis is shorter than the spacing between two subsequent spermatozoa, and the minor axis is of the same order of magnitude as the transverse flow size or, if the wavelength of the radiation used exceeds such a transverse size, of the order of magnitude of the wavelength.

9. The method as claimed in any preceding claim, characterised in that the analysis of the radiation having interacted with a spermatozoon is repeated several times for each spermatozoon, and the different measures obtained are integrated.

10. The method as claimed in any preceding claim, characterised in that said spermatozoa. are spermatozoa of animals, to be subjected to a sexing operation.

11. An apparatus for detecting the chromosome contents of spermatozoa, comprising:

- a source of electromagnetic radiations (1 ); - means (3) for conveying past the source (1) a flow of spermatozoa (5), where said flow is illuminated by said electromagnetic radiations;

- detection and analysis means (6, 8) for collecting and analysing the radiation having passed through the flow; characterised in that said source (1) and said detection means (6) are a source and detection means, respectively, of radiations having frequencies in the Terahertz range, and the analysis means (8) are arranged to determine the chromosome contents of each spermatozoon in the flow by evaluating the response of DNA in the spermatozoon when the latter is illuminated by the radiation having a frequency in the Terahertz range, wherein the analysis means (8) are arranged to obtain said vibrational response by measuring variations of predetermined characteristics of the radiation having passed through the flow, which variations are induced in the radiation by virtue of its interaction with a spermatozoon.

12. The apparatus as claimed in claim 11, characterised in that said source (1) is a source of radiations with frequencies ranging from some tenths of Terahertz to some ten Terahertz, more particularly from about 0.5 to about 30 THz.

13. The apparatus as claimed in claim 11 or 12, characterised in that said source (1) is a source of fixed frequency radiations.

14. The apparatus as claimed in claim 11 or 12, characterised in that said source (1) is associated with means for varying the frequency of the radiations generated by the source (1) itself.

15. The apparatus as claimed in any of claims 11 to 14, characterised in that the detection and analysis means (6, 8) are arranged to determine the absorption of the radiation by a spermatozoon.

16. The apparatus as claimed in any of claims 11 to 15, characterised in that said source (1) is a source of polarised radiations, and the detection and analysis means (6, 8) analyse the variation of the plane of polarisation of the radiation due to the interaction with a spermatozoon.

17. The apparatus as claimed in claim 16, characterised in that it comprises means for generating a magnetic field in correspondence with a region (9) of the flow illuminated by the source (1).

18. The apparatus as claimed in any of claims 11 to 17, characterised in that the source (1) is associated with an optical system for focusing the radiation onto a substantially elliptical region (9), of which the major axis is shorter than the spacing between two subsequent spermatozoa, and the minor axis is of the same order of magnitude as the transverse flow size or, if the wavelength of the radiation used exceeds such a transverse size, of the same order of magnitude as the wavelength.

19. The apparatus as claimed in any of claims 11 to 18, characterised in that the source (1) and the flow conveying means (3) are located in respective foci of an ellipsoidal reflecting surface.

20. The apparatus as claimed in any of claim 11 to 19, characterised in that the source (1) comprises one or more generators chosen out of lasers and Josephson junction generators.

21. The apparatus as claimed in any of claims 11 to 20, characterised in that the detection means (6) comprise one or more detectors chosen out of bolometers, Josephson junction detectors and pyroelectric detectors.

22. The apparatus as claimed in any of claim 11 to 21, characterised in that the source (1) comprises an array of fixed-frequency or variable-frequency generators, and the detecting means (6) comprise a corresponding array of detectors, where the linear sizes of both arrays, in the direction of transportation of the spermatozoa, are such that the detection and analysis means (6, 8) collect and analyse multiple times the radiation reflected and/or transmitted by an individual spermatozoon, the analysis means (8) averaging the responses obtained for that spermatozoon.

23. The apparatus as claimed in any of claims 11 to 22, characterised in that it is associated with means, controlled by said analysis means (8), for physically separating the spermatozoa according to their chromosome contents, for sexing the same spermatozoa.