Chicken spermatozoa protein extract analyzed by Top-down HRMS

Currently, evaluation of quality semen is based on traditional in vitro sperm tests as measures of motility, viability and acrosomic reaction. However, the results are not always well correlated with real male fertility. In the present study, we evaluated Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) method as diagnostic molecular tool to substitute in vitro tests in order to phenotype semen with more robustness. For that, we investigated a large male population of known fertility that included two highly different genetic lines (meat and egg laying lines). Our optimized ICM-MS-based method through automation and the construction of fertility-predictive mathematical models was able to discriminate individuals on their reproductive capacity in the two lines studied. Furthermore, we showed a better diagnostic accuracy than traditional in vitro sperm quality tests and demonstrated that differential markers presenting a high discriminating power between fertile and subfertile sperm cells in sperm were implicated in fertility processes. To characterize m/z peaks observed in ICM-MS spectra, we performed high-throughput top-down protein identification of sperm cell extracts. Therefore, we identified more than 75% of the m/z peaks, revealing that these comprises mostly degradation products of testis-specific proteins implicated in the most important functional pathways in sperm cells such as energy metabolism and structure/movement.