Simple SummaryThe contributions to the development and application of artificial insemination (AI) in beef cattle are essential to improve genetics and increasing farm productivity. Many factors affect sperm viability and fertilization capacity whether fresh, refrigerated or frozen–thawed semen doses are used. The aim of this study was to elucidate how different in vitro storage conditions affected semen quality in the first two hours after ejaculation. For this purpose, we studied three evaluation times from collection (<75, 75–105 and 105–120 min), two storage temperatures (refrigeration and room temperature), and two extenders (AndroMed®and BIOXcell®) as well as the interaction of these factors on classical sperm parameters, CASA results and microbial growth on ejaculates collected under different farm conditions. We found that both extenders were suitable for seminal sample storage at both temperatures during these times. However, AndroMed®induced a more curvilinear sperm movement, while BIOXcell®stimulated straighter sperm motility, regardless of storage temperature.AbstractSperm quality decreases over time, so bull semen may need to be preserved after field collection. However, the effect of handling such semen samples from commercial farms and placing them in very short–term storage has not been elucidated. Therefore, ejaculate from 25 bulls from 1 dairy and 14 beef cattle farms were collected under farm conditions and evaluated for semen quality during the first two hours after collection. Two commercial extenders (AndroMed®and BIOXcell®) and two different storage temperatures (5 °C and room temperature) were used to evaluate the influence on semen quality and sperm kinetics in ejaculates grouped into three evaluation times, based on time since collection (Time 1: <75 min,n= 7; Time 2: 75–105 min,n= 11; and Time 3: 105–120 min,n= 7). Classical semen parameters, sperm motion kinetics by CASA and colony-forming units were assessed. The differences between both extenders in curvilinear and straight–line velocities (VCL and VSL) for the different time groups (Time 2 and Time 3) were statistically significant forp< 0.05. AndroMed®showed lower VSL, straightness and linearity in sperm compared to BIOXcell®(p< 0.05). In conclusion, AndroMed®induced more curvilinear movement, while BIOXcell®stimulated straighter motility.