In this study, a new comparison was tested in Mediterranean Italian buffaloes (Bubalus bubalis), between two different strategies for obtaining sperm cells of the desired sex. In addition, a control group of animals was included in the study, where only ordinary semen straws, containing unselected sper cells were used. Sexed and control straws were then used for Artificial Insemination (AI) in order to confront pregnancy and gender rates. The first of the two strategies to preselect sperm cells relies on the physical determination of DNA content in single spermatozoa though cell sorting machines. The second strategy instead, relies on the coincubation of either fresh or frozen/thawed semen with a specific solution specifically designed for the species of interest. The trial involved a total of 206 heifers and 211 pluriparous buffaloes belonging to a buffalo farm in the south of the Lazio Region in Italy. In this study, no significant differences between treated and control groups were detected in terms of pregnancy rates both on heifers (P=0.6) and pluriparous buffaloes (P=7). With regard to gender rates, pregnancy rates were significantly different among the three groups of heifers (P=0.001) and the three groups of pluriparous buffaloes (P=0.002). Sperm cells preselected for gender determination through Cell Sorting (CST) resulted effective in shifting the gender ratio towards the female, when compared to both the Coinbubation Technique (COiT) and control group of animals.