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The presence of N. caninum in bovine semen is quantified by means of quantitative PCR in real time, which uses the SYBR Green I system. For the extraction of the DNA of the semen parasite, the seminal fluid is separated from the cellular fraction and the DNA is extracted from each one of the fractions using a commercial test. If the semen is frozen with diluent, the sample is passed through a chromatography column of Sephacryl S-400 and the DNA is precipitated with isopropanol and glycogen. The quantification of N. caninum is carried out by means of interpolation of the Ct (threshold cycle: cycle in which a sample is considered positive) on a standard curve constructed with known concentration of the DNA of the parasite against their respective Ct. For the quantification of the DNA of the host it is carried out with another standard curve with known concentrations of genomic DNA. The viability of the parasite is confirmed by means of bioassay, inoculating BALBc nu / nu mice with the cellular fractions of the samples of bovine semen. The appearance of symptoms compatible with the infection and/or the quantification of the DNA of the parasite by quantitative PCR in samples of brain demonstrate the viability of the parasite in the seminal sample.

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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.

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