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Trolley Troubles - Oswald the Lucky Rabbit - 1,927
Before he created Mickey Mouse, Walt Disney drew cartoons of Oswald the Lucky Rabbit for Universal Pictures and Charles Mintz. This was the first of these films which was released to theaters.
Other animators who were involved included Ub Iwerks, Hugh Harman, Friz Freleng, Les Clark, Ben Clopton, Norm Blackburn and Rollin "Ham" Hamilton.
Note that the original film was silent. The soundtrack was added when it was rereleased a few years later.
Note that I am now moderating the comments because of some bad apples.