Yes, it was a final gag but on another level it was much more than that. It set into motion once again the push and pull element of nature vs. nuture and asks the audience which matters most.
The mice have now learned all of these human skill sets (i.e. dining with correct forks; strategizing better escape routes to evade predators; writing the letters of the alphabet; etc.) that would serve them well in a world to which they were well-suited. But they are not...because they are mice and they function properly in their natural habitat which is the wild.
As for Puff, he is now a functioning member of society with a cultural intelligence that could rival most. But even with this new wealth of knowledge and experience the somberness in his eyes while looking through the car window at the forest reveals that he longs to be with nature and live in the wild even though, on the surface of things, it would not be a wise choice.
"Nobody can hear you. Nobody cares about you. Nothing will come of this."
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