MXPA06008407A - A method for determination of dna fragmentation in animal sperm cell - Google Patents

Abstract

The present invention describes a method for the determination of DNA fragmentation in animal sperm cells. Specially it is related to a method to evaluate the integrity of the chromatin/DNA of the sperm cells by means of the sample treatment with a denaturing DNA solution followed, optionally, of a staining;a later treatment with a lysing solution which does not contain protein denaturing detergent, followed optionally by a staining;and an evaluation of the integrity of the chromatin/DNA. The present invention is furthermore related to a kit to evaluate the quality of the animal sperm cells which includes a denaturing DNA solution and a lysing solution which does not contain protein denaturing detergent.

Description

A PROCEDURE FOR THE DETERMINATION OF DNA FRAGMENTATION IN SPERMATOZOIDS OF ANIMALS FIELD OF THE INVENTION This invention has its field of application within the health sector, mainly that related to the biology of reproduction, especially it is directed to procedures and methods for determining the quality of semen in animals.

BACKGROUND OF THE INVENTION Currently 6% of men in Western countries, of childbearing age, have some kind of pathology that prevents them from normal reproduction. To this end, the World Health Organization (WHO) has combined in a single protocol a series of laboratory procedures that standardize the analysis of semen quality internationally. These studies focus on the determination of the concentration, morphology and motility of sperm, complemented by the possible evaluation of certain functional tests, as well as certain biochemical and enzymatic parameters of semen (WHO, 1999). With this set of tests it is possible to estimate the total volume of the same and the concentration of sperm per milliliter and can be diagnosed if male infertility is due to an absence (azoospermia) or a clear decrease (oligospermia) of the number of sperm in the ejaculate. Likewise, the possible existence of motility problems (asthenozoospermia) is determined, which makes it impossible for these cells to cross the uterine cavity and successfully reach the external third of the tubes. It is also analyzed if they present serious problems of morphology of its components (head, neck, tail) (teratozoospermia) since these variations affect the capacity for an effective fertilization of the female ovule. In addition, the participation of glands such as prostate and seminal vesicles (infections, agenesis) is also explored. Finally, functional tests such as the HOS test (cell membrane ionic permeability) or the ability of sperm to progress in vitro, give an idea of the fertility of semen. Finally, these laboratory studies occasionally need to be completed with hormonal profiles, testis biopsy and / or karyotype determination (chromosomal study that defines the inherited condition of the male or female sex of an individual) and / or molecular genetic tests. Despite clinical and laboratory studies, the cause of infertility can not be determined in about 30-50% of infertile males, being labeled as idiopathic infertility. Recently, it has been recognized that the DNA damage of sperm can be the explanation for a high percentage of these idiopathic cases (Evenson et al., 1999, Larson et al., 2000), so that the study of fragmentation The DNA of spermatozoa is a subject of active research with ongoing publications in specialized journals (Evenson et al., 2002). Chromatin anomalies or damage to the nuclear DNA of sperm could take place or be the result of anomalies in DNA packaging that occurs during spermiogenesis (Sailer et al., 1995). It is also possible that they are the result of damage produced by free radicals that cause oxidative stress (Ait et al., 1998), or as a consequence of a possible process of apoptosis (Gorczyca et al., 1993). There are different methodologies to evaluate the integrity of the chromatin / DNA of human sperm. These include the labeling of DNA breaks in situ introducing nucleotides marked on them using enzymes such as terminal transferase (TUNNEL) or DNA polymerase (in situ nick translation ISNT) (Gorczyca et al., 1993). These methodologies are based on the use of enzymes on sperm fixed on slides. For this reason its efficiency is not very high, only those breaks accessible to the enzyme are marked, which translates into a relatively low reproducibility of the results. In addition, the reagents are expensive, so these techniques are only applin research studies, not being possible to use them for the clinical assessment of semen. Another technique is the comet test (Hughes et al., 1996). The spermatozoa are included in an agarose microgel on a slide and are subjected to using solutions to extract the membranes and proteins. Nucleoids are thus obtained, that is, deproteinized nuclei, in which the loops of DNA have been relaxed by decompaction. The nucleoids are subjected to an electrophoresis in a bucket filled with buffer solution, in such a way that the DNA fibers migrate towards the anode, constituting a comet image, with a head and a tail in the direction of electrophoretic migration. These comets are stained with a fluorescent dye, to be observed by fluorescence microscopy. If the nucleus presents DNA fragmentation, a large number of fragments of it will have migrated, concentrating on the tail of the comet. It is a very sensitive test, but relatively expensive and complicated for a conventional clinical laboratory. In fact, it requires certain non-common instruments: electrophoresis source and cuvette, fluorescence microscopy, and a system for capturing the images and analyzing them. Therefore, it is not applicable to the clinical study of semen and is only used for research purposes. The current reference technique for the study of sperm DNA fragmentation is the Evenson chromatin structure assay (SCSA: Sperm Chromatin Structure Assay; Evenson et al., 1980; 2000; Evenson and Jost, 1994). In this technique, sperm in suspension are subjected to an acid denaturing solution. Those sperm without breaks in their DNA are resistant to such denaturation, remaining as double-stranded DNA. However, sperm with fragmented DNA do denature their DNA, transforming into single-stranded DNA. Afterwards, they are stained with acridine orange. This dye emits green fluorescence when it binds to double-stranded DNA. However, in spermatozoa with denatured DNA, in single chain, this fluorochrome emits red fluorescence. Sperm with fragmented DNA are quantified using a flow cytometer, to discriminate both types of fluorescence. The SCSA is a technique with great clinical projection, having been evaluated profusely in patient samples. Using this system, it has been established that when the individual presents 30% or more of the sperm with fragmented DNA, their probability of achieving a pregnancy at term is less than 1%, both in natural fertilization and assisted reproduction techniques. (Evenson et al., 1999; Larson et al., 2000). The percentage of sperm with fragmented DNA can be more or less constant in the different spermatogenic cycles of the individual, but it can also vary as a result of exogenous factors, or for example after an intense febrile episode, such as influenza (Evenson et al., 2000 ). In this way, serial studies can be made, selecting those samples with a lower level of fragmentation, to be used later in the techniques of assisted reproduction. It is important to bear in mind that the freezing of semen samples in liquid nitrogen does not modify the levels of DNA fragmentation, so the test can be performed on frozen samples, which can then be used in insemination, IVF (in vitro fertilization). ) or ICSI (Intracytoplasmic Sperm Injection). This supposes a great operative advantage for the patient and for the laboratory. The SCSA technique, although robust and highly reproducible, is a very expensive system, difficult to implement, and not very accessible to the basic laboratory (De Jonge, 2002). In this way, the DNA quality of the sperm continues to be evaluated routinely, despite its proven clinical value in the study of infertility. Recently, our research group described in a preliminary way a technique that allowed to disperse in situ the chromatin of human spermatozoa, demonstrating that those spermatozoa unable to disperse chromatin contained fragmented DNA (Fernández, JL et al., Journal of Andrology, 2003, vol. 24, No. 1 p.59-66: "The sperm Chromatin Dispersion Test: a simple method for the determination of sperm DNA fragmentation"). By means of this method, semen samples are treated sequentially in agarose microgel with a denaturing acid solution, with two lysis solutions and with one of washing, to be then dried and stained. This technique, which was called the Sperm Chromatin Dispersion (SCD: Sperm Chromatin Dispersion) test, uses excessively aggressive reagents and conditions. The method described does not give consistent results, which makes repeated evaluation difficult. On the other hand, the quality and contrast of the images obtained and the reproducibility of the results are not good enough for it to be commercially applicable. In addition, the structure of the sperm is affected and the tail is no longer visible in the samples. This problem is important, since sperm can not easily be distinguished from other types of cells present in the sample, with the consequent error in the quantification of the number of sperm with damaged chromatin / DNA. Therefore, there is still a need for a reliable process that can be used routinely and simply for the study of semen quality of animals and in particular to assess the integrity of chromatin / DNA. The process has to be robust, easy to implement, cheap and accessible to the basic laboratory. You have to solve the aforementioned problems. In addition, it must give homogeneous results between different laboratories and be susceptible to automation.

OBJECT OF THE INVENTION The object of the invention is a method for evaluating the integrity of the chromatin / DNA of sperm from animals quickly and accurately and that can be incorporated in the routine activity of any laboratory for analysis, veterinary or specific for human reproduction. Thus, an object of the invention is a method for evaluating the integrity of the chromatin / DNA and of the sperm of an animal comprising: a) a stage of treatment of the sample containing the sperm, with a DNA denaturing solution, b ) a single stage of treatment with a lysis solution to extract nuclear proteins, c) a stage of evaluation of the integrity of the chromatin / DNA of the spermatozoa, characterized in that the lysis solution does not contain denaturing protein detergent and essentially does not destroy the tail of the sperm. In general, it is preferred that step a) precede b). As indicated, the selection of the lysis solution is critical to achieve the objectives of the invention. Among the denaturing detergents of proteins that should not be used we have anionic or cationic detergents such as SDS, dodecylsulfate, alkylbenzene sulfonate, hydrated salt of glycocholic acid, etc. They are detergents that provoke a great disruption of membranes, with lysis effects and at the same time they are active denaturing proteins. They are used in denaturing electrophoresis in which proteins are subjected to migration ensuring complete denaturation (loss of three-dimensional structure). They are active above all at acidic pH, preferably on Gram-positive bacteria. Its activity inside the detergents is high. In the process of the invention preferably a non-ionic non-denaturing protein detergent is used, ie a detergent that solubilizes the proteins but does not denature them. Among them, toctylphenoxypolyethoxyethanol (Triton X-100), N, N-Bis (3-D-gluconamidopropyl) cholamide (BigCHAP), Brij (r) 35 P, N-decanoyl-N-methylglucamine, digitonin, dodecanoyl-N are preferred. -methylglucamide, heptanoyl-N-methylglucamide, octylphenoxy poly (ethyleneoxy) branched ethanol (Igepal CA-630), N-Nonanoyl-N-methylglucamine, Nonidet P 40, N-Octanoyl-N-methylglucamine, Span 20 solution, polysorbate 20 ( Tween 20). Particularly preferred is the Triton X-100 for the good results it gives and its easy availability. It is preferred that the lysis solution has sufficient ionic strength to facilitate the lysis process without denaturation. We have found that an effective solution is one containing sodium chloride between 1 and 3 M, dithiothreitol (DTT) between 0.001 and 2M, 2-amino-2- (hydroxymetyl) -1, 3-propanediol (Tris) 0.001 and 2M and Triton X-100 between 0.1 and 3%. Particularly suitable is a solution containing about 2.5M NaCl, about 0.2M DTT, about 0.2M Tris, about 100% Triton X-100 and about 7.5 pH. The denaturing solution of the DNA is preferably acidic, for example of an acid selected from the group hydrochloric, acetic, nitric acid or mixtures thereof. It is preferably a hydrochloric acid solution. The method according to the invention has a step of evaluating the integrity of the chromatin / DNA of the spermatozoa after steps a) and b). Although there are several alternatives for this evaluation, it is preferred that it be visual. For this purpose preferably the process includes a step of staining the sample after steps a) and b). A stain that gives excellent results and allows to visualize both the tail of the sperm and the characteristic halo formed is a Wright type solution. In a preferred variant the sperm are included in a medium similar to a suspension, preferably in a microgel, especially in an agarose microgel. The invention is also directed to a kit for the evaluation of the sperm quality of animals comprising: a) a DNA denaturing solution, b) a lysis solution for extracting nuclear proteins, characterized in that the lysis solution does not contain detergent denaturing protein and essentially does not destroy the tail of sperm. The kit allows carrying out the process according to the invention that has just been described.

BRIEF DESCRIPTION OF THE FIGURES Figures 1a-1 c. Parameters used for the definition of the size of halos in human sperm, according to the methodology of the invention. Figure 1a: The nucleoid, which corresponds to the massively deproteinized sperm nucleus, is composed of two parts: silhouette of the nucleus of the sperm, called "core", in the central position, and a peripheral halo of chromatin / DNA dispersion. The tail of the sperm is visible. Figure 1 b: Relief filter for a better visualization and establishment of the limits between the halo and the "core". Figure 1c: Lower diameter of the "core" (a) and thickness of the halo (b), as a sample of the measurements used to establish the different sizes of the haloes, as explained in the methodology of the invention. Figures 2a-2f. Different types of sperm defined according to the size of the halo that is generated after applying the methodology of the invention. Figure 2a: Spermatozoon with large halo. Figure 2b: Sperm with halo of medium size. Figure 2c: Spermatozoon with halo of small size. Figure 2d: Sperm without halo. 2e: Sperm without halo and degraded. Figure 2f: General field in which the different types of sperm described above are observed. Figures 3a-3e and 3a'-3e. Correlation between the different halo sizes visualized after staining with DAPI (ae, blue fluorescence) and the in situ hybridization signal using a total human genomic DNA probe, according to the DBD-FISH methodology (figures 3 a'- 3e ' , red fluorescence) to visualize the level of DNA fragmentation. Figure 3 a: Sperm with halo and low hybridization signal (Figure 3 a '). Figure 3b: Sperm with medium halo and low hybridization signal, although slightly higher than in the previous case (figure 3b '). Figure 3 c: Spermatozoon with small halo and remarkable increase in the level of hybridization (Figure 3 c '). Figure 3 d: Sperm without halo and high level of hybridization (figure 3 d '). Figure 3 e: Sperm degraded and showing an irregular distribution of hybridization (figure 3 e '). Figures 4a-4f. Application of the method of the invention to sperm samples of the following species: mouse (Mus musculus), bull (Bos taurus), turbot (Scophthalmus maximus) and earthworm (Lombrícus terrestrís). Figure 4a corresponds to bull; Figures 4b, 4 c, 4 d correspond to mouse; Figure 4 e corresponds to the earthworm; Figure 4 corresponds to the turbot. Figure 5. Patient sample with presence of high levels of leukocytospermia. The absence of glue in the leukocytes can be seen, which makes it possible to differentiate them from the rest of the cell types.

DETAILED DESCRIPTION OF THE INVENTION As will be detailed, the method and kit of the invention are a simple and reliable system for determining the frequency of sperm with fragmented DNA. The methodology is applicable in the andrology laboratories and assisted reproduction clinics and animal reproduction laboratories. In addition, it is a very versatile system since it is possible to freeze the samples and analyze them when interested, without inducing changes in the results of the analysis. The method of the invention, which allows evaluating the integrity of the chromatin / DNA and sperm of an animal comprises: a) a stage of treatment of the sample containing the sperm, with a DNA denaturing solution; b) a single stage of treatment with a lysis solution to extract nuclear proteins, which does not contain protein denaturing detergent and essentially does not destroy the tail of the sperm; c) a step of evaluating the integrity of the chromatin / DNA of the sperm. In addition to others, the main differences of the method of the invention, with respect to the state of the art and specifically with respect to Fernández, J.L. et al. Journal of Andrology, 2003, vol. 24, no. 1 p.59-66, reside primarily in the field of lysis and staining. Thus, a single lysis solution is used, instead of two sequential ones. The composition is different since it does not contain SDS (denaturing protein anionic detergent) or EDTA (chelating agent). It can incorporate a relatively mild, non-denaturing neutral detergent, such as Triton X-100. Technically these differences translate into the conservation of sperm tails. It is a crucial improvement, because the detection of it is an indispensable morphological data to be able to discriminate if the images of nucleoids come from spermatozoa or correspond to other cell types that could be present, for example, cells desquamated from the genitourinary pathway, cells inflammatory, blood, etc. This persistence is achieved through a much less aggressive cell lysis, as well as discarding the use of SDS. In addition, the smoother lysis achieves the unfolding of the chromatin loops, maintaining better the morphology of the previous head, or "core", and achieving dispersion halos with greater density of chromatin material, resulting in a more intense staining of the same. . As a result, the contrast and visual discrimination of the different halo s is greatly improved, especially when using Wright's stain. Another significant advantage is that the absence of denaturing protein detergent, such as SDS, allows the sequential use of the technique described here with others that allow the visualization of other cellular components. Thus, on the nucleoids obtained, according to the methodology described, immunodetection techniques of proteins of the laminin type and other nuclear proteins can be applied, as well as detection of RNA associated with the nuclear matrix, since the DNA is extended maintaining the highest amount of possible nuclear structure. This is important in certain research topics of the structure of the sperm nucleus. Another additional advantage is that a smaller quantity of reagents is used and, consequently, there is a lower economic expense. For example, the DTT is especially expensive, and the reduction to the concentration described in the examples (a quarter of that described in the article) is important for the cost. From the foregoing it is clear that the process to be patented results in images of sperm nucleoids that are highly improved and more reproducible with respect to the state of the art. It is possible to discriminate whether the nucleoids come from mature sperm or from other cell types, and the categorization of the of the halo is much more precise and safe. Consequently, with the procedure to be patented, the determination of the fragmentation levels of the DNA of the sample is much more reliable, which means that it can be used routinely, simply and at a low cost. Its application is relevant in different laboratories, both clinical and over human samples, and in veterinary laboratories for the study of animal samples. This is very important, because it is a test of possible clinical application to patients. The sequence of the treatment steps of the sample can be done in any order, first with a DNA denaturing solution, followed by the treatment step with a lysis solution or vice versa. However, it is preferable to treat the sample with the denaturing solution of the DNA and then with the lysis solution, since it gives better results. In the other variant (lysis followed by DNA denaturation) sperm with fragmented DNA behave differently. In this case, they scatter chromatin / DNA fragments, giving rise to larger halos. Even a single treatment with a lysis solution may be sufficient to observe this behavior, although the discrimination of the halo s is not very precise. The procedure of the invention is detailed below, along with some variants and optional stages. The person skilled in the art will understand that there are other modes of realization and possibilities as long as the fundamental aspects described are maintained. The first step is the preparation of the sample. Through the usual procedures in this field. the sperm concentration of the sample is obtained and checked. The proper concentration for the analysis ranges from 0.1 to 20 million cells per milliliter. If the sample is excessively concentrated, adjust it to the appropriate concentration by diluting it with culture medium or with saline / buffered phosphate (PBS) or similar. The semen sample must be placed on a support for processing according to the method of the invention and to facilitate its evaluation. The support is preferably a glass slide that can be coated with a standard agarose film. For this, a standard agarose solution is prepared between 0.2 and 1% in distilled water in a Coplin jar or similar. It is covered with a perforated plastic sheet and placed in a microwave oven. The microwave oven is regulated at a power between 300-1000W, for example 500W, stirring the container occasionally for better dissolution of the agarose, leaving it until it boils. This procedure can also be done using a thermostatic bath. When the agarose solution becomes completely transparent, you will be ready to deposit it in vertical containers with a content between 10 and 250 ml. These containers should be previously tempered in a bath between 60-100 ° C, for example 70 ° C, to keep the agarose solution in a liquid state. Before introducing the slides in the agarose solution, these are cleaned by rubbing with a cloth to remove possible impurities. The slides are immersed vertically, holding them with tweezers in the ground area, between 1-60 seconds, removing them and submerging them once or ten times, until a homogenous film is formed on the slide. These are deposited horizontally on a smooth and cold surface between 1 and 15 ° C, preferably 4 ° C, for example, glass or metal. This plate, with the slides, is placed in the refrigerator at 4 ° C for a minimum of 30 min, until it is verified that the agarose solution has gelled on the surface of the slide. The trays are removed from the refrigerator and the surface of the slides that were in contact with the plate is wiped with a blotting paper. Next, the slides are placed horizontally in an oven in a temperature range of 37-100 ° C, until the agarose completely dries and forms a thin film adhering to the glass. The slides thus treated can be used immediately or stored in a well-sealed box at room temperature for several months. To facilitate the processing of the sample containing the sperm, it can be included in a medium with characteristics similar to those of a suspension such as, for example, an agarose microgel. In this case, a low melting point agarose solution is prepared at a concentration between 0.5 and 2% in distilled water. The fusion of this agarose is carried out using a microwave oven or a thermostatted bath, and it is subsequently maintained between 30 and 37 ° C in a tube introduced in a thermostatted bath or stove. In an Eppendorf tube or similar, carefully mix the semen and the agarose solution, so that the latter is at a concentration between 0.3 and 1%. For example, 70 microliters of the agarose solution + 30 microliters of the sample. It is important that the temperature of the agarose does not exceed 37 °, so as not to damage the cells. Finally, to obtain the sample on support, place the coated slides on a smooth and cold glass or metal surface, with a temperature that oscillates between 1 and 15 ° C, avoiding to form bubbles of air. It is recommended to deposit with a micropipette a drop between 5-200 microliters of the mixture, placing a coverslip on top of the drop. As a precaution, it is recommended to process each sample in duplicate, and use a control sample each time the technique is applied. The plate with the slides is placed in a refrigerator at 4 ° C, between 2 to 30 minutes until suitable gelling of the agarose occurs. Once the gelation has occurred, the coverslips are removed very gently, inside the same refrigerator and avoiding damage to the microgel. Once the samples have been adequately prepared for easy and repeated handling, they are treated according to the method of the invention with a treatment step with a DNA denaturing solution and a lysis treatment step to extract nuclear proteins. In a preferred variant, the slides are first introduced with the sample in a horizontal position in a container containing the denaturing solution. The denaturing solution of the DNA can be acidic, for example a solution of acetic acid, nitric acid, sulfuric acid, or alkaline such as, for example, a solution of sodium hydroxide, barium hydroxide, potassium hydroxide, at mild concentrations. In a preferred variant a hydrochloric acid solution is used whose concentration can vary between 0.01 and 0.5 N, especially between 0.1 and 0.3 N, particularly preferred is a concentration around 0.2 N. It is recommended that this solution be prepared on the same day as the carrying out the test and keeping the slides in incubation in the DNA denaturing solution between 1 and 15 minutes at a temperature between 1 ° C and 37 ° C, preferably 18 ° C-25 ° C, especially 20-22 ° C.

Once this part of the process is finished, the samples should be lysed with a single lysis solution that is gentle enough so that the tails of the sperm are not destroyed. To do this, each slide is submerged, horizontally, in another container that contains it. As mentioned above, the lysis solution is selected in such a way that it achieves the unfolding of the chromatin loops, maintaining better the morphology of the starting head and therefore the formation of the characteristic halos with greater density of chromatin material. It must also be soft enough for the conservation of sperm tails. This is achieved by regulating avoiding aggressive detergents, protein denaturants. Additionally, the regulation of the ionic concentration also allows to modulate this process. In a preferred variant, this solution is composed of: sodium chloride between 1 and 3 M, preferably between 2 and 3 M; dithiothreitol (DTT) between 0.001 and 2M, preferably between 0.01 and 0.8M; 2-amino-2- (hydroxymetyl) -l, 3-propanediol (Tris) between 0.001 and 2M, preferably between 0.01-0.4M; and Triton X-100 between 0.1 and 3%, preferably between 0.5-1.5%. This solution is adjusted to pH between 6.5 and 8.5, preferably 7-7.5. There are other alternative lysis solutions, or the concentrations and times and temperatures of incubation of the described solution can be varied as long as their fundamental functional characteristics are maintained. Thus, as alternatives to DTT, there are compounds such as beta-mercaptoethanol and other reducing agents. As alternatives to Tris, other buffer solutions may be employed, such as Hepes, Mops, Pipes. As an alternative to Triton X-100, other neutral detergents may be used as mentioned above. Depending on the solution used and the type of sample, the preparations are incubated in the lysis solution between 1 and 60 minutes, preferably between 15 and 35 minutes, especially preferably around 25 minutes; and at a temperature between 1 and 37 ° C, preferably 18 ° C-25 ° C, especially preferred is a temperature of 20-22 ° C. As a global alternative to the sequence of processes described above, the order of incubation in the denaturing and lysis solutions can be reversed. The effects on the chromatin of the sperm also allows to discriminate the spermatozoa with chromatin / DNA damaged from the rest of the sperm. The details of the differences obtained will be described in example number 6. After treatment with denaturing DNA solution and with lysis solution, the preparations can be washed to remove the remains of these solutions. For this, a washing solution is used as smooth as possible, avoiding chelating agents or detergents. For example, they are immersed in a horizontal position in a container containing abundant distilled water or a buffer solution or physiological saline for a time between 1 and 60 minutes.

The dehydration of the sample is then carried out. For this you can use solutions of increasing concentration of alcohol. For example, the slides are lifted and submerged in a horizontal position, in containers with increasing concentration series of ethanol, between 5 and 100%, for 30 seconds to 60 minutes each and then the preparations are allowed to air dry. As alternatives to incubations in series of ethanol, the preparations can be dehydrated by incubating in solutions of different alcohols such as methanol, or by allowing them to dry in the air or in an oven. Once dry, the slides already processed containing the semen sample can be stored in storage boxes at room temperature for months. This facilitates the separation of the treatment process according to the invention and the subsequent step of evaluating the integrity of the chromatin / DNA of the sperm. The archiving allows a repeated evaluation at different intervals of several samples of the same individual. Once treated the samples according to the invention, you go to the evaluation stage. There are several possible processes to evaluate the integrity of the chromatin / DNA of the sperm as indicated above. The advantage is that the samples treated according to the invention have a much clearer halo of visualization and have maintained the structure of the sperm, especially the integrity of the tails, which makes it possible to clearly distinguish sperm from other cell types.

In a preferred variant, the sample is stained, which facilitates visual evaluation. By conveniently choosing the staining conditions, a high quality of the images and a high consistency of the evaluation results can be obtained. There are several strategies for staining, depending on whether conventional clear field microscopy or fluorescence microscopy is used. Staining for observation in a light field microscope: In this case, Wright, Giemsa, Orcein, Schiff reagent, Carmine acetic, thiazine type and Romanowsky type mixtures or derivatives of the aforementioned ones can be used (see Chromosome banding by AT Sumner, pp.90-91). Dyes such as Wright's are preferred for the most intense staining of the sample and especially of the haloes. With these dyes the contrast and visual discrimination of the different sizes of haloes is significantly improved. In addition, they have the advantage of low cost and easy availability for all types of laboratories. Its use allows to visualize the tails, since these are not usually visible in DNA stains with fluorochromes used for fluorescence microscopy. It is important to emphasize that this staining is very easily manipulated to achieve the adequate level of staining, which is not feasible with Diff-Quik or similar. Other stains, such as Diff-Quik, described in Fernández, J.L. et al. Journal of Andrology, 2003, vol. 24, no. 1 p.59-66, they are considerably weaker and do not achieve an adequate contrast of the halo with respect to the background. Consequently, when the halo is very dispersed, it is usually difficult to visualize its peripheral tip, which can be considered as a small halo, assigning the category of fragmentation to a sperm that has intact DNA. That is, the publication procedure tends to overestimate the levels of fragmentation, especially in light field staining. This is relatively compromised for a test of possible application to individuals. Consequently, it is obvious that this improvement is of enormous relevance in the reliability of the technique. In a variant of the sample coloration, Wright's solution (Merck 1.01383.0500) is mixed with phosphate buffer, for example, at pH 6.88 (Merck 1.07294.1000) in proportions between 1: 30 and 30: 1 (v / v). A layer of dye is placed, horizontally, covering the dried microgel. The staining time to achieve optimal contrast ranges from 30 seconds to 60 minutes. It is recommended to blow on the dye layer occasionally. The excess dye is decanted, the slide is washed gently with tap water and allowed to dry. If the staining is excessive, it can be washed, with more intensity, in water. Another possibility is to bleach in ethanol, dry and re-dye. If the staining is weak, especially in the region of chromatin dispersion halos, it can be re-stained directly with more Wright's solution. As alternatives, other dyes such as Hemacolor 2 (Merck 1.11956) and Hemacolor 3 (Merck 1.11957), Giemsa, as well as other dyeing solutions of the same family can be used.

Staining for fluorescence microscope observation: Depending on the availability of fluorescence filters, samples can be stained with specific fluorochromes for DAPI-type DNA, Hoechst 33258, Ethidium Bromide, Propidium Iodide, etc., in an "antifading" medium "(for example Vectashield, Vector H-1000). In case permanent preparations are desired, processed and stained slides can be included in mounting media (eg, Entellan, Merck 1.07961). Finally, the integrity of the chromatin / DNA of the spermatozoa proceeds to the distinction of cell types. As already mentioned, the method of the invention greatly facilitates this evaluation with respect to the state of the art. The obtained images can be studied by means of direct visual analysis or by applying analysis software of digitalized images, obtained by analogue or digital cameras, coupled to the microscopy platforms. Initially, the study of a minimum of 500 spermatozoa per sample is recommended, adopting the following basic criteria (see Figures 1a-1c and Figures 2a-2f): 1. Spermatozoa without chromatin dispersion halo (Figures 1a-1c). 2. Spermatozoa without chromatin dispersion halo and degraded: those without showing halo, have the head fragmented into granules or show a very weak staining (Figures 1a-1c). 3. Spermatozoa with dispersion halo of small size: the thickness of the halo is equal to or less than 1/3 of the smaller diameter of the "core" (Figures 1a-1c). 4. Sperm with dispersion halo of medium size: the thickness of the halo is between: greater than 1/3 of the smaller diameter of the "core" and smaller than the smaller diameter of the "core" (Figures 1 a-1c). 5. Spermatozoa with large dispersion halo: spermatozoa whose halo is equal to or greater than the smaller diameter of the "core" (Figures 1 a-1c). 6. "Others": nuclei of cells that do not correspond to sperm. One of the morphological characteristics that distinguish them is the absence of tail. Spermatozoa with fragmented DNA are considered to be those without chromatin 1 dispersion halo, those that are presented without chromatin dispersion halo and degraded 2 and those with dispersion halo of small size 3. Those spermatozoa with chromatin dispersion halo of large and medium size, it is considered that they do not have fragmented DNA. The criteria to establish the correlation between the size of the halos and DNA fragmentation derives from the results obtained using the DBD-FISH technique (DNA Breakage Detection-Fluorescence In Situ Hybridization, Fernández et al., 1998; 2000; 2002; Fernández and Gosálvez, 2002). This procedure allows the detection and quantification of DNA breaks in nuclei of deproteinized cells and subjected to a controlled denaturation of DNA. This denaturation generates strands of single-stranded DNA from the ends of rupture, which are detected by in situ hybridization using a total genomic DNA probe labeled with a fluorochrome, visible by fluorescence microscopy. The higher the level of breaks in the cellular DNA, the greater the amount of single chain DNA generated by the denaturing solution, the greater the amount of hybridized probe and the greater the fluorescence observed. The samples processed according to the methodology described in the present invention, contain single-stranded DNA, generated by the denaturing solution, from the possible ends of breakage that exist in the DNA. Therefore, the intensity of hybridization using a total genomic DNA probe will be related to the number of breaks present in the sperm nucleus. In this way, it has been confirmed that the nucleoids without halo, or with a halo of very small size, show an intense labeling with DBD-FISH, which shows the intense fragmentation of their DNA (Figures 2a-2f). The rest of the nucleoids show very low levels of labeling with this probe, which correspond to the hybridization background generated by the chromatin treatment itself.

The invention also contemplates a Kit for the assessment of the chromatin / DNA integrity of animal sperm. This Kit contains a DNA denaturing solution and a lysis solution to extract nuclear proteins, which is characterized in that the lysis solution does not contain protein denaturing detergent and does not essentially destroy the tail of the sperm. The DNA denaturing solutions and the preferred lysis solutions are described above. Optionally, the Kit can also contain the pre-treated support, for example with agarose, as well as a solution for the preparation of a medium with characteristics similar to those of a suspension that will contain the sample. For example, a low melting point agarose solution that allows the preparation of a microgel. The contents and manner of use of a Kit according to a variant of the invention are detailed below: Description of the contents of the kit Pretreated slides * Eppendorf tubes containing low melting agarose tube (A) Tube with 37% HCl, tube (B) Tubes with lysis solution, tube (C) *. Composition: NaCl 2.5M, DTT 0.2M, Tris 0.2M, Triton X-100 1%, pH 7.5.

Processing vessels for the denaturing solution and for lysis solution Lancet Floats for Eppendorf tubes * Preparation as referred to above in the description Material and equipment required Clear field or fluorescence microscope (immersion objective recommended) Refrigerator at 4 ° C Incubation bath at 37 ° C Plastic gloves Glass coverslips (18x18 mm, 22x22 mm or 24x60 mm) Micropipettes 4 boxes for incubations in horizontal Distilled water Ethanol 70%, 90%, 100% Instructions for use Preparation of a sample by slide 1) Take a bottle C to place the lysis solution at room temperature (22 ° C) 2) Dilute the sample of semen in culture medium or PBS, at a concentration of 5-10 millions per milliliter. Both fresh and frozen samples can be used directly in liquid nitrogen.

Preparation of the randomized micro-cartridge 3) Lightly tap an Eppendorf tube with low-melting agarose (Tube A), vertically, to deposit the agarose at the bottom of the tube. 4) Add 140 microliters of distilled water, avoiding bubbles, and resuspend. 5) Insert the tube A into the float, leaving it at the level of the lid, and let float for 5 minutes in water at 90-100 ° C, until the agarose dissolves. The fusion of the agarose can be carried out alternatively in a microwave oven. 6) Transfer tube A with the float to a thermostatic bath to 37 ° C, and leave for 5 minutes until the temperature is equalized. 7) Add 60 microliters of the semen sample to the contents of tube A and resuspend. 8) Place a pre-treated slide on a cold surface at 4 ° C (for example, a metal or glass sheet). 9) Once the slide is cooled, deposit the cell suspension of tube A and put on a glass coverslip, avoiding the formation of air bubbles. It is recommended to deposit a drop of 11, 17 and 50 microliters, for a coverslip of 18x18mm, 22x22mm, or 24x60mm, respectively. 10) Introduce the cold slide with the slide in the refrigerator and let the sample gel for 5 minutes.

Processing of the samples 11) Prepare the denaturing solution. To do this, add 80 microliters of the content of tube B in 10 milliliters of distilled water, mix and deposit in the green box. 12) Remove the coverslip, sliding it gently, and immediately insert the slide, horizontally, into the denaturing solution and leave to incubate for 7 minutes, at room temperature (22 ° C). 13) Lift the slide with the help of the lancet, using gloves. Hold it horizontally, and place it horizontally, in the white container containing 10 ml of lysis solution (tempered C tube). Incubate for 25 minutes. 14) Lift the slide and place it horizontally in a box containing plenty of distilled water to wash the lysis solution. Leave to incubate for 5 minutes. 15) Insert the slide, horizontally, in a box with 70% ethanol (2 minutes), then in 90% ethanol (2 minutes), and finally in 100% ethanol (2 minutes). 16) Allow to air dry. Once the processed slides are dry, they can be stored in filing boxes at room temperature for months.

Staining the samples Staining for observation in a light field microscope - Mix Wright's solution with phosphate buffer (1: 1), and deposit a layer of colorant, horizontally, covering the dried microgel. Leave staining for 5-10 minutes, blowing over occasionally. Decant, wash gently under running water and let dry. If the staining is excessive, it can be destained in ethanol, dried and re-stained. If the stain is weak, especially in the halos, it can be re-stained directly with more Wright's solution. - Another possibility is the incubation 5 minutes, vertically, in a coplin with Hemacolor 2 solution (Merck 1.11956), let it drain vertically for 10 seconds and then incubate in another coplin, in vertical, with Hemacolor 3 solution (Merck 1.11957), 5 minutes. Finally wash gently in distilled water and let dry. If a permanent preparation is desired, it can be installed in Entellán.

Staining for fluorescence microscope observation Depending on the availability of fluorescence filters, samples can be stained with specific fluorochromes for DAPI type DNA, Hoechst 33258, Ethidium Bromide, Propidium Iodide, etc., in an antifading medium (for Vectashield example, Vector, ref: H-1000).

Security and environment Avoid inhalation and contact with the solutions provided Solutions B and C contain Hydrochloric acid, Dithiothreitol and Triton X-100. Consult the specifications supplied by the manufacturers. Do not dispense used products to the environment. Comply with the regulations of Centers for the storage and removal of toxic products. Biological samples should be handled as potentially infectious.

Storage and stability Store at room temperature, except for solution C, which should be stored at 4 ° C. Expiration: reagents and materials are stable for a minimum period of 6 months. It is recommended that solutions B and C remain vertical and tightly closed.

The present invention has different areas where its application is relevant. Its usefulness in application to humans is evident. For example, in samples of infertile individuals whose semen parameters are normal, in pairs with repeat abortions, in samples used for assisted reproduction, in samples that are going to be frozen (cryopreservation) for their future use in assisted reproduction techniques due to a subsequent extirpation of the testicle. Also in patients undergoing chemo- and / or radiotherapy for oncological pathologies, and before performing a vasectomy. The study carried out with the procedure and Kit of the invention can improve the selection criteria of aspirants to semen donor, as well as complement the periodic evaluation of donor samples, in semen banks. It is also possible to analyze the effect of advanced age on semen quality and fertility. Its application is interesting for the evaluation of patients with pathologies that can affect the integrity of sperm: fever, infections, varicocele, stress, exposure to genotoxic agents in a work or accidental way (pesticides, radiation, environmental estrogens, etc.). hormonal treatments, or repeated exposure to high heat (professions related to blast furnaces, ceramics, glass, or vehicle drivers). These individuals can also be evaluated on a periodic basis. Finally, the invention is useful in basic and clinical research.

Similarly, the invention is also useful in veterinary laboratories. It is possible to study the level of sperm DNA fragmentation in different animal species, for example in breeding males, in stored samples, in pathological processes, in males of species in danger of extinction and in the evaluation of the damage generated by toxic agents.

EXAMPLES The invention will now be described on the basis of some examples which will illustrate in more detail some of the characteristics described above.

EXAMPLE 1 In a fresh semen sample, the described methodology was applied to produce the chromatin dispersion halos. For this, the sample diluted to a concentration of 10 million per milliliter, in PBS, was mixed with liquid agarose of low melting point to 1%, to obtain a final concentration of the latter, of 0.7%. After gelifying the microgel on the slide, the sample was incubated at 22 ° C, for 8 minutes, in the denaturing solution composed of 0.08M HCl, and then in the lysis solution constituted by NaCl 2.5M, DTT 0.2M, Tris 0.2 M, Triton X-100 1%, pH 7.5, for 25 minutes, at 22 ° C. The slides were washed in distilled water for 5 minutes, dehydrated in ethanol baths, and air-dried. Subsequently, sequentially and on the same cells, we proceeded to perform the DBD-FISH (DNA Breakage Detection-Fluorescence In Situ Hybridization; Fernández et al., 1998; 2000; 2002; Fernández and Gosálvez, 2002) using a total genomic DNA probe. This procedure allows the detection and quantification of DNA breaks in nuclei of cells immersed in agarose microgels, deproteinized and subjected to a controlled denaturation of DNA. This denaturation generates strands of single-stranded DNA from the ends of rupture, which are detected by in situ hybridization using a total genomic DNA probe labeled with a fluorochrome that emits red fluorescence (Cy3). The higher the level of breaks in the cellular DNA, the greater the amount of single chain DNA generated by the denaturing solution, the greater the amount of hybridized probe and the greater the red fluorescence obtained. The samples processed according to the method of the present invention contain single-stranded DNA, generated by the denaturing solution, from the possible ends of breakage that exist in the DNA. Therefore, the intensity of hybridization using a total genomic DNA probe will be related to the number of breaks present in the sperm nucleus. We counted 250 cells obtained at random. The DAPI staining images of the chromatin dispersion halos were captured using a refrigerated CCD camera using two filters for the staneous display of scattering haloes, visible in blue, and the Hybridization signal, visible in red. The ultimate goal was to establish a correlation between the size of the dispersion halos of the chromatin and the level of DNA breaks. The results showed a Inverse correlation between the relative area of the dispersion halos of the chromatin and the intensity of labeling of DNA breaks by DBD-FISH (Table 1).

CUADR0 1 HALO GRANDE Area halo / total DMotal Total 0.85 13.07 Standard deviation 0.05 7.35 Account 154 154 HALO MEDIAN Area halo / total DMotal Total 0.73 24.85 Standard deviation 0.07 12.52 Account 38 38 SMALL HALO Area halo / total DM total Total 0.48 180.28 Standard deviation 0.14 117.82 Account 22 22 NO HALO Area halo / total DM total Total 407.34 Standard deviation 252.69 Account 29 29 DEGRADED Halo / total area DMotal Total - 101 Standard deviation - 86 Count 7 7 Note that as the relative area of the halo decreases, there is an increase in the total average density (DM) of the hybridization. Consequently, the simple determination of the size of the chromatin dispersion halos, obtained by our method, offers a simple and direct estimation of the integrity of the chromatin / DNA of human sperm.

EXAMPLE 2 Complementary method for the evaluation of donor semen used in assisted reproduction techniques. In an assisted reproduction clinic, 10 samples of semen donors were taken. As a complement to the usual spermiogram, we proceeded to determine the level of DNA fragmentation in these samples. 500 cells were counted per individual. In this case, the results were obtained by applying the chromosine dispersion halos test of the invention. The samples were included in the agarose microgel, incubated in the acid and lysis solutions, washed, dehydrated and allowed to dry, as described in example 1. The staining, in this case, was not performed with DAPI but with Wright's dye, for light field microscopy. For this, Wright's solution was mixed with phosphate buffer solution (1: 1), and a layer of dye was deposited, horizontally, covering the dried microgel. It was dyed for 5-10 minutes, blowing over occasionally. After a wash in running water, it was allowed to dry and the nucleoids were visualized. The results are shown in table 2. The mean fragmentation level estimated in this group was less than 20% in all cases (15.4 +/- 3.1).

TABLE 2 % Cells% Cells% Cells% Cells without% Cells% Cells n ° sample Halo large medium small halo degraded fragments n1 76.2 7.4 10.2 5.0 1.2 16.4 n2 74.4 7.0 11.2 7.4 - 18.6 n3 72.6 16.4 6.2 4.8 - 11.0 n4 78.6 6.0 7.4 6.6 1.4 15.4 n5 81.6 5.8 7.0 5.4 0.2 12.6 n6 69.4 11.0 12.6 6.0 1.0 19.6 n7 73.2 7.2 9.6 8.8 1.2 19.6 n8 80.2 5.4 8.4 5.0 1.0 14.4 n9 85.2 2.6 5.0 6.8 0.4 12.2 n10 79.8 5.6 6.8 7.0 0.8 14.6 Distribution of the percentages of halo size categories obtained in 10 semen donors. The percentage of sperm with fragmented DNA comprises the sum of the categories of spermatozoa with small halo, without halo and without halo-degraded.

EXAMPLE 3 Clinical evaluation of infertile patients In an assisted reproduction clinic, 17 samples of semen donors were taken. As a complement to the usual spermiogram, we proceeded to determine the level of DNA fragmentation in these samples. 500 cells were counted per individual. As in the previous example, the results were obtained by applying the chromatin dispersion halos test of the invention. The results are shown in table 3. The mean fragmentation level estimated in this group was greater than 20% in all cases (49.9 +/- 20.7). In some patients levels close to 90% of sperm have been obtained with fragmentation in their DNA.

TABLE 3 % Cells% Cells% Cells% Cells Cells Halo Halo Cells < fragmented degradation Small large Halo sample without Halo ss medium P1 47.0 12.0 16.2 22.4 2.4 41.0 P2 38.4 3.2 15.2 42.2 1.0 58.4 p3 39.6 1.6 13.2 44.6 1.0 58.8 p4 53.4 10.6 11.0 17.6 7.4 36.0 p5 42.4 5.2 15.2 35.8 1.4 52.4 p6 50.0 5.8 10.0 32.9 1.2 44.2 P7 39.0 11.2 22.6 21.2 6.0 49.8 p8 60.6 4.8 9.4 22.8 2.4 34.6 p9 69.4 3.2 7.4 19.0 1.0 27.4 p10 60.6 4.4 10.4 24.0 0.6 35.0 p11 11.3 3.0 6.0 75.8 4.0 85.8 p12 65.6 4.2 2.4 24.4 3.4 30.2 p13 16.7 11.9 24.3 46.8 0.3 71.4 p14 8.4 4.4 18.6 67.0 1.6 87.2 p15 64.7 8.2 11.2 13.7 2.2 27.1 p16 14.6 5.0 10.6 63.4 6.4 80.4 p17 65.8 5.8 9.4 17.4 1.6 28.4 Distribution of the percentages of halo size categories obtained in 17 patients. The percentage of sperm with fragmented DNA comprises the sum of the categories of spermatozoa with small halo, without halo and without halo-degraded.

EXAMPLE 4 Use of the invention for the evaluation of toxicological damage that affects human sperm. Damage due to exogenous and endogenous agents. As an illustrative example, a study analyzing DNA damage induced by a nitric oxide (NO) chemical donor agent is presented. For this, aliquots of 50 microliters of a fresh total semen sample from a normal individual were incubated for 1 hour, at room temperature, with different doses of sodium nitroprusside (SNP). Subsequently, the treated samples were centrifuged gently removing the supernatant, to wash the SNP. After resuspending in PBS, the samples were processed according to the method described in the present invention. The results are presented in table 4. It was observed that as the concentration of the NO donor increased, the percentage of sperm with damaged chromatin / DNA increased.

TABLE 4 Concentration%%%%%% of SNP Cells Cells Cells Cells Cells Cells Halo Halo Halo. ,,. degrades fragment (microM) .. _ without Halo 5 _? large medium small, n adas 0.0 61.8 14.1 12.0 12.1 0.0 24.1 62.5 38.0 23.2 19.6 19.2 0.0 38.8 125 21.8 24.2 32.6 23.1 0.0 55.7 250 14.9 19.0 40.3 30.1 0.0 70.4 500 3.8 22.5 39.6 34.1 0.0 73.7 Distribution of the percentages of the halo size categories obtained in a semen sample treated with different concentrations of a nitric oxide donor (SNP), capable of producing DNA damage. The percentage of sperm with fragmented DNA comprises the sum of the categories of spermatozoa with small halo, without halo and without halo-degraded.

EXAMPLE 5 Reproducibility of the assay using frozen semen samples. Two factors that could affect the quality of the samples, such as freezing and dilution, were studied using the methodology of analysis of the degree of dispersion of chromatin halos. For this, 4 samples of different fresh donors were analyzed and aliquots were frozen in liquid nitrogen. The analysis of the samples was made by direct visual counting of the different types of nucleoids (500 cells), on two different slides and a minimum of two times, per sample and experimental point. Reproducibility of counts. The intraclass correlation coefficient (R) was calculated for the different measurements that were made on each slide. The results of the indices for each cell type and calculated with the average of two counts oscillate between values of 0.78 and 0.92, and taking into account that the values of R vary between 0 and 1, it is demonstrated that a high reproducibility is obtained (table 5).

TABLE 5 Mean confidence interval 95% Difference% large halo cells -0.78 (-1.98; 0.42) Difference% medium halo cells -0.56 (-0.17; 0.29) Difference% small halo cells -0.36 (-0.48; 0.50) Difference% cells without halo -0.78 (-0.88; 0.50) Difference% cells without halo degradation -0.19 (-0.35; 0.43) Difference% cells with fragmented DNA 0.04 (-0.67; 1.09) Freezing. The results obtained were contrasted using a two-factor analysis of variance (conservation method and sample). It was shown that there are no significant differences (p> 0.05) in the level of fragmentation of samples processed fresh and frozen in liquid nitrogen. The freezing time does not seem to affect the proportion of sperm with fragmented DNA (Table 6).

TABLE 6 Sample Sample status Mean Standard deviation 1 E. Fresh 19.58 0.22 E. Frozen 20.20 1.48 2 E. Fresh 13.38 1.87 E. Frozen 13.39 1.86 3 E. Fresh 12.75 3.04 E. Frozen 21.56 1.92 4 E. Fresh 22.13 0.74 E. Frozen 21.56 2.68 In conclusion, the reproducibility of the results obtained after the direct visual analysis is revealed.

EXAMPLE 6 Analysis of the integrity of the chromatin / DNA of the human sperm using the variant in the order of incubation in denaturing and lysis solutions. In this variant, after including the spermatozoa in the agarose microgels, they are incubated, in a first stage, in the lysis solution described in the kit, for 25 minutes, at 22 ° C. Subsequently, the slides are immersed in the denaturing solution composed of 0.08 M HCl, for 8 minutes, at 22 ° C. Finally, after washing in distilled water, the slides are dehydrated and stained with Wright's solution, being observed by light field microscopy. Using this technical variant, sperm with fragmented DNA behave differently. In this case, they scatter chromatin / DNA fragments, giving rise to larger halos.

EXAMPLE 7 Results of the application of the methodology on samples of sperm from different animals. In order to evaluate the universal character of the proposed methodology, male individuals of different species were selected to carry out a study of the DNA fragmentation levels in the spermatozoa and the parallel visualization of their tail as a distinctive cellular element. Sperm samples were taken from the following species: mouse (Mus musculus), bull (Bos taurus), turbot (Scophthalmus maximus) and earthworm (Lombricus terrestris). In all the species, the application of the technique generates halos of dispersion of the chromatin in the spermatozoa and the tail of the same can be recognized, as much of which they have their normal DNA as of those that present it fragmented (Figures 4a-4f) . The shape of the sperm and the type of halo it generates are characteristic of each species (figure 4a corresponds to bull, figures 4b, 4c, 4d correspond to the mouse, figure 4e corresponds to the earthworm, figure 4f corresponds to the turbot) . The morphology of the sperm, both the one that contains fragmented DNA and the one that does not, is different between the species and different in turn from that found in humans. In all cases, the dispersion of the chromatin is parallel and comparable to that found in the case of human sperm samples. That is, chromosine dispersion halos of different sizes are produced and the tail of the sperm can be visualized. The morphology and size of the halos in the different species were studied and the results obtained were as follows. In the case of the bull, four independent samples were analyzed using 5 different observers. In this case, differences were found in the percentage of sperm nuclei with DNA fragmented by each individual, but no differences were observed between percentages obtained by each observer (table 7) TABLE 7 % TOTAL% GRANDE% MEDIUM% SMALL% WITHOUT HALO% FRAGMENTA DEGRADADAS DAS Ob1-500 75.8 9.8 7.2 7.2 0 14.4 Ob2-500 77 7.2 12 3.6 0.2 15.8 Ob3-500 77 8 9.8 5 0.2 15 Ob4-500 72.8 11.4 8.8 7 0 15.8 Ob5-500 73 11.2 8.2 7.6 0 15.8 500 75.1 9.4 9.1 5.9 0.1 15.3 Ob1-500 80.6 10 6.2 3.2 0 9.4 Ob3-500 82.2 8 6 3.8 0 9.8 Ob4-500 82.4 8.4 6.2 3 0 9.2 500 81.7 8.8 6.1 3.3 - 9.5 Ob1-500 71.2 6.2 10.8 11.4 0.4 22.6 Ob-2-500 70.8 4.6 14 10.6 0 24.6 Ob3-500 75 5.6 12 7.2 0.2 19.4 Ob5-500 72.2 6.8 14.2 6.4 0.4 21 500 72.3 5.7 12.7 8.6 0.3 21.8 Ob2-500 85.2 8.4 3.8 2.6 0 6.4 Ob3-500 84.4 10 3.2 2.4 0 5.6 Ob4-500 80.8 12.8 4 2.4 0 6.4 500 83.4 10.2 3.7 2.5 0 6.1 In the case of the mouse, two different strains were used: one normal (M1-32NNC and another consanguineous one (M2-32BC) .The percentages obtained for the different halo types show clear differences between a normal strain (7.1) and the inbred strain (25.1). ) (table 8).

TABLE 8 WITHOUT% TOTAL% BIG%%% DEGRADED% MEDIUM SMALL HALO FRAGMENTADAS S M2-32BC 63 11.5 18.1 6.9 0.1 25.1 M1-32NNC 86.2 6.5 4.6 2.5 0.2 7.1 In the specific case of turbot, spermatozoa could be distinguished that had a dispersion halo of the large chromatin and a small "core", compared to those that had a halo of small dispersion in front of a large "core" and finally others without a halo. dispersion. The results are shown in table 9.

TABLE 9 % Halo Large /% Halo Small / Core% No Halo Only Head Core Small Large 94 5.6 0.4 92.8 7 0.2 95.6 4 0.4 94.1 5.4 0.3 72.2 25.2 2.6 71.6 25.4 3 77 20.2 2.8 73.6 23.5 2.8 75.2 18.4 6.4 71.2 22 6.8 78 15 7 74.7 18.2 6.7 In the case of the earthworm, there is a halo generation dynamic similar to that described in the previous cases and in this case the head of the sperm and its tail can also be perfectly distinguished. The estimated percentage of spermatozoa containing fragmented DNA in 2 studied individuals (one young and one mature) was 15% and 22%, respectively. In this case, sperm heads appear with a partial formation of halos, whose meaning is currently under investigation (see Figure 4 e).

EXAMPLE 8 Visualization on the same cytological preparation of dispersion halos of chromatin, tail of spermatozoon and leukocytes. Evaluation of the effect of leukocytospermia on DNA integrity in sperm samples. Leukocytospermia is an unwanted invasive process that refers to the abnormal increase of leukocytes in seminal fluid samples (> 5 x 106 / ml). It has been detected that between 10 and 20% of infertile men present leukocytospermia. It seems that both neutrophils and macrophages present in semen can generate ROS (Reactive Oxygen Species) that would facilitate oxidative stress and consequently damage to sperm DNA (Omu et al., 1999; Erenpreiss et al. , 2002; Henkes et al., 2003). In fact, leukocytospermia has been associated with different abnormalities of the classical parameters used in the analysis of sperm quality. For example, while the incidence of abnormal sperm is present in only 47% of the samples of individuals without leukocytospermia, the percentage increases to 88% when this circumstance occurs (www.clevelandclinic.org). In a sample of 5 patients, with presence of high levels of leukocytospermia of different etiology (prostatitis and Chlamydomonas infections and bacterial agents), the response of the technique was studied to unequivocally differentiate the percentage of leukocytes present in the sample. of semen and the levels of DNA fragmentation in the spermatozoa of those same samples. The difference between both cell types, when the sample is subjected to the same treatment, is obvious given that both the sperm with fragmented DNA and those that do not have it, show the tail that characterizes them. The absence of glue in the leukocytes allows us to differentiate them from the rest of the cell types (Figure 5). In this way, a direct correlation can be established between the number of leukocytes per sample and levels of DNA fragmentation in the spermatozoa. Table 10 shows the level of leukocytes in semen samples in 5 patients affected by leukocytospermia of different etiology and the percentages of sperm with normal DNA (large and medium G / M halo) and fragmented (small halo and without P / SH halo) .

TABLE 10 % Leukocytes% Halo cells (G / M)%% Halo cells (P / SH) Sample 1 6.1 73.9 20 Sample 2 15.5 55.3 29.2 Sample 3 17 48.2 34.8 Sample 4 22 31.7 46.3 Sample 5 25.6 30.5 43.9 REFERENCES Aitken RJ, Gordon E, Harkiss D (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol. Reprod. 59: 1037-1046. De Jonge C (2002) The clinical value of nuclear sperm DNA assessment Hum. Fertile. 5: 51-53. Erenpreiss J, Hlevicka S, Zalkalns J and Erenpreisa JJ (2002) Effect of Leukocytospermia on Sperm DNA Integrity: A Negative Effect in Abnormal Semen Samples. Journal of Andrology 23: 5. Evenson DP, Darzynkiewicz Z, and Melamed, MR (1980) Relation of mammalian sperm heterogenity to fertility. Science. 210: 1131-1133. Evenson DP and Jost, LK (1994) Sperm chromatin structure assay: DNA denaturability. In: Darzynkiewicz Z, Robinson JP.Crissman HA, eds. Methods in Cell Biology. Vol 42. Flow Cytometry. 2nd ed. Orlando, Fia: Academic Press; 42: 159-176. Evenson DP, Jost LK, Corzett M, Balhorn R (2000) Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study. J. Androl. 21: 739-746. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, Angelis P, Claussen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod. 14: 1039-1049. Evenson DP, Larson KJ, Jost LK (2002) Sperm Chromatin Structure Assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J. Androl. 23: 25-43. Fernández JL, Goyanes VJ, Ramiro-Díaz J, Gosálvez J (1998) Application of FISH for the detection and quantification of DNA breakage. Cytogenet Cell Genet. 82: 251-256. Fernández JL, Vázquez-Gundín F, Delgado A, Goyanes VJ, Ramiro-Díaz J, from Torre J, Gosálvez J (2000) DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features. Mutat. Res. 453: 77-82. Fernández JL, Gosálvez J (2002) Application of FISH to detect DNA damage: DNA Breakage Detection-FISH (DBD-FISH). Methods Mol. Biol. 203: 203-216. Fernández JL, Goyanes V, Gosálvez J (2002) DNA Breakage Detection-FISH (DBD-FISH). In: Rautenstrauss B, Liehr T, eds. FISH technology-Springer manual lab. Heidelberg: Springer-Verlag; 282-290. Fernández JL, Muriel L, Rivero MT, Goyanes V, Vázquez R, Alvarez JG (2003) The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J. Androl. 24: 59-66.

Henkel R, Maass G, Hajimohammad M, Menkveld R, Stalf T, Villegas J, Sanchez R, Kruger TF, Schill WB. (2003) Urogenital inflammation: changes of leucocytes and ROS. Andrology 35: 309-13. Larson KL, DeJonge C, Barnes A, Jost L, and Evenson DP (2000) Relationship between assisted reproductive techniques (ART) outcome and status of chromatin integrity as measured by the Sperm Chromatin Structure Assay (SCSA). Hum. Reprod. 15: 1717-1722. Gorczyca W, Gong J, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53: 945-951. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W (1996) A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile using a modified comet assay. Mol Hum Reprod. 2: 613-619. Omu AE, Al-Qattan F, Al-Abdul-Hadi FM, Fatinikun MT, Fernandes S. (1999) Seminal immune response in nfertile men with leukocytospermia: effect on antioxidant activity. Eur J Obstet Gynecol Reprod Biol. 86: 195-202 (1999) Sailer BL, Jost LK, Evenson DP (1995) Mammalian sperm DNA susceptibility to in-situ denaturation associated with the presence of DNA strand breaks as measured by the deoxynucleotidyl transferase assay . J. Androl Sumner AT (1990) Chromosome banding. Unwin Hyman, London. World Health Organization (1999) WHO laboratory manual for the examination of the human semen and semen-cervical mucus interaction. Fourth Edition, Cambridge University Press, Cambridge, UK.

Claims (15)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for evaluating the integrity of the chromatin / DNA and of the sperm of an animal comprising: a) a stage of treatment of the sample containing the sperm, with a denaturing solution of the DNA, b) a single stage of treatment with a lysis solution for extracting nuclear proteins, c) a step of evaluating the integrity of the chromatin / DNA of the sperm, characterized in that the lysis solution does not contain denaturing protein detergent and essentially does not destroy the tail of the sperm.

2. The method according to claim 1, further characterized in that step a) precedes to b), or only proceed to b) and c).

3. The process according to claim 1 or 2, further characterized in that the lysis solution comprises a non-ionic non-denaturing protein detergent.

4. The process according to claims 1-3, further characterized in that the non-ionic detergent is a detergent selected from the group toctylfenoxipoliethoxyethanol (Triton-X100), N, N-Bis (3-D-gluconamidopropyl) cholamide (BigCHAP) ), Brij (r) 35 P, N-decanoyl-N-methylglucamine, digitonin, dodecanoyl-N-methylglucamide, heptanoyl-N-methylglucamide, octylphenoxy poly (ethyleneneoxy) branched ethanol (Igepal CA-630), N-Nonanoil-N -methylglucamine, Nonidet P 40, N-Octanoyl-N-methylglucamine, Span 20 solution, polysorbate 20 (Tween 20) and mixtures thereof, preferably Triton-X 100.

5. The process according to claims 1-4, characterized also because the lysis solution comprises sodium chloride between 1 and 3 M, dithiothreitol (DTT) between 0.001 and 2M, 2-amino-2 (hydroxymetyl) -1,3-propanediol (Tris) 0.001 and 2M and Triton X-100 between 0.1 and 3%.

6. The process according to claims 1-5, further characterized in that the lysis solution comprises sodium chloride about 2.5M, DTT about 0.2M, Tris about 0.2M, Triton-X about 1% and pH around 7.5.

7. The process according to claims 1-6, further characterized in that the DNA denaturing solution is acidic.

8. The process according to claim 7, characterized in that the DNA denaturing solution comprises an acid selected from the group hydrochloric, acetic, nitric acid or mixtures thereof.

9. The process according to claim 8, further characterized in that the DNA denaturing solution comprises hydrochloric acid.

10. - The method according to claims 1-9, further characterized in that after stages a) and b) there is a staining step of the sample.

11. The method according to claim 10, further characterized in that the staining is done with a Wright type solution.

12. The method according to claims 1-11, characterized in that the sample containing the sperm is included in a medium similar to a suspension, preferably in a microgel.

13. The method according to claim 12, characterized in that the sample containing the sperm is included in an agarose microgel.

14. A kit for the evaluation of the quality of the sperm of animals that includes: a) a DNA denaturing solution, b) a lysis solution to extract nuclear proteins, characterized in that the lysis solution does not contain protein denaturing detergent and essentially does not destroy the tail of the sperm.

15. The kit according to claim 14, further characterized in that the lysis solution comprises sodium chloride between 1 and 3 M, dithiothreitol (DTT) between 0.001 and 2M, 2-amino-2- (hydroxymetyl) -1, 3 -propanediol (Tris) 0.001 and 2M and Triton X-100 between 0.1 and 3%.