Reivindicaciones

1. Procedure for the quantification of DNA of theN. caninum parasite in bovine semen, characterised because it requires the extraction of DNA from the parasite by means of the following stages: a). Separation of seminal fluid (fraction F1) and cellular fraction (F2) of the semen by centrifugation. b). Isolation of the DNA of each fraction by means of the use of a commercial test, subjecting the samples to digestion with proteinase K at 60°C for 1 h and a treatment with an RNAse at 37° C for 30 min. 2. Procedure for the quantification of DNA of theN. caninum parasite in bovine semen, according to claim 1, characterised in that for samples of semen frozen with diluent, the extraction of DNA from the parasite requires: - The passage through a chromatography column of Sephacryl S-400 before proceeding to the separation of the F1 and F2 fractions. - The precipitation of the DNA with isopropanol with glycogen. 3. Procedure for the quantification of DNA of theN. caninum parasite in bovine semen, according to the previous claims, characterised by employing a quantitative real time PCR technique using the SYBR Green I system, which comprises of the following stages: a). Amplification of the DNA of the parasite employing as oligonucleotides those corresponding to a fragment of 76 pb of the Nc-5 region of theN. caninum region, and quantification by interpolation of the Ct value of the sample on a standard curve constructed with known concentrations of the DNA of the parasite. b). Amplification of the DNA of the host employing as oligonucleotides those corresponding to a fragment of 71 pb of the 28S rRNA gene, and quantification by interpolation of the Ct value of the sample on a standard curve constructed with known concentrations of the genome DNA. 4. Procedure to evaluate the viability of theN. caninum parasite in bovine semen samples, analysed according to a previous claim, characterised by its carrying out by means of a bioassay in mice of thenude or athymic strain, which comprises of the following stages: a). Separation of the seminal fluid and cellular fraction of the semen. b). Inoculation of the cellular fraction in mice of thenude or athymic strain. c.) Follow up of the inoculated mice for the detection of symptoms compatible with neosporosis and/or subsequent detection-quantification of the DNA of the parasite from the brain by means of using quantitative PCR in real time. 5. Procedure for the evaluation of the viability of theN. caninum parasite in samples of bovine semen, according to claims 3 and 4, characterised in that the sample is frozen semen with diluent. 6. Procedure for the evaluation of the viability of theN. caninum parasite in samples of bovine semen, according to claims 4 and 5, characterised in that the animals used in the bioassay are of the BALB/cnu /nu strain.