It's spritesheeting correctly, but it previously didn't pad out the last frame if it was by itself. This meant the last image only is a non-power-of-two size and doesn't get a mipmap, affecting the downscaling quality. Since you have 'Downscaling' set to 'High quality' I guess you want it to preserve this case, so when that option is chosen it will also pad out the last animation frame to a power-of-two size, which fixes this case.
BTW your animations are really wasteful. The image is very large, but only a tiny part of it changes. The "farm" object now comes out as 3 2048x2048 spritesheets, using 50mb of memory for that single object alone. However such a tiny part of the image changes that you could easily have a static background with just a small segment changing, using probably only ~1.3mb for the changing segment.