Variations in bacterial profiles associated with semen collection timing and bull breed

Semen samples were collected from 20 dairy bulls (8 Holstein Friesian and 12 Swedish Red). Each bull provided three ejaculates within a week: the first after a 96-hour since previous collection (T1), the second 48 hours later (T2), and the third 24 hours after the second (T3). Bacterial species were identified through culturing on cattle blood agar, followed by MALDI-TOF MS identification. Additionally, 16S rRNA sequencing was performed to determine bacterial diversity after DNA extraction. MALDI-TOF analysis identified 33 bacterial species across 60 semen samples. Six species were exclusive to Holstein Friesian (VH) bulls, while 12 were specific to Swedish Red (VR) bulls, with Proteus spp. found only in VR samples. Certain bacterial species were present only at specific time points: three in the first ejaculate, seven in the second, and five in the third.