Advances in understanding ICE horizontal transfer in ruminant mycoplasmas have been facilitated by the establishment of axenic, optimal conditions. However, these provided only limited information on the influence of host factors on this event which is known to play an important role in chromosomal transfer. To address this issue, ICE-transfer frequencies were analyzed in M. agalactiae under replicative environments of increasing complexity, from axenic and cell culture growth conditions to ex vivo infections using Precision-Cut Lung Slices (PCLS). Remarkably, co-incubation of mating partners with epithelial cells resulted in increasing ICE-transfer frequency by ca 200-fold when compared to axenic, optimized mating conditions. ICEs are known to integrate randomly in the mycoplasma chromosome and a similar number of different integration sites were found in axenic and cell conditions by whole-genome sequencing of transconjugants, thus ruling out the amplification of a few individual transconjugants with high fitness values in cell culture conditions. The positive effect of host-related environmental factors on the conjugative behavior of M. agalactiae has also been observed upon ex vivo infections of bovine PCLS. As expected, only sporadic transconjugants could be selected when mating partners were incubated alone in the culture medium without host cells. This study reveals that the conjugative properties of mycoplasma cells may be influenced by their replicative environment, with enhanced mating frequency upon co-incubation with host cells. Most importantly, these results also suggest that the frequency of horizontal gene transfer in vivo is likely to be underestimated.< Réduire