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Background: Genetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations（AI） are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3（CMA3） has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups（highly fertile and subfertile）.Results: Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters（morphology, viability, total and progressive motility） were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve（AUC） of 0.54, 0.64 and 0.68, respectively（P > 0.05）, and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73（P = 0.05）, CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78（P = 0.02）.Conclusions: Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.