Sperm-FISH Analysis for Validating Sexed Italian Mediterranean River Buffalo (Bubalus bubalis) Semen

In both cattle and Italian Mediterranean River Buffalo breeding, sexed semen plays a vital role in economic and reproductive management. The most recent technology employed for semen sexing is laser ablation, where female sperm, containing more DNA chromosomes, are typically larger than male sperm. This technique involves passing the semen through a laser that can identify stained X chromosomes and use ablation to remove the Y chromosomes, resulting in female-sexed semen. This study aims to assess the accuracy of this sexed semen technology through Fluorescence in Situ Hybridization of Spermatozoa (Sperm FISH). For the analysis, we utilized two different probes containing sequences complementary to those of the buffalo X and Y chromosomes from CHORI BAC libraries 240 (cattle) and validated on river buffalo metaphases. We counted 1000 spermatozoa in six bulls under the fluorescence microscope, both in the total and sexed semen fractions of each animal. The total sperm fraction showed an X signal percentage close to 50%, while sexed semen ranged between 70% and 80%. Only one sexed semen sample showed an X signal percentage identical to the total fraction, highlighting the importance of FISH analysis for sexed semen validation. Moreover, it emphasizes the need for cytogenetic methods to assess X chromosome-carrying sperm concentration, a fundamental aspect of dairy farming. Additionally, validating sexed semen is crucial as it can cost up to 10 times more than unsorted semen, underscoring the economic significance of accurate validation procedures. This study highlights practical implications for the dairy industry and buffalo breeding, stressing the need for further research to optimize semen sexed validation methods.