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The truck's driver, Emily Woodrum of Missouri, said she was speeding in the passing lane and lost control of the vehicle, causing it to go up the shoulder of the road and overturn.
She was on her way to deliver the truckload of puppies to local pet stores.
Troopers Mark Bernard, Jordan Sheehan and Pete Peters helped gather up all of the puppies and remove them from the truck.
Five of the puppies sustained minor injuries and were taken to Steuben County SPCA, state police told WHEC.
The SPCA said on its website it had since been caring for "a large number of puppies" from the accident until they are set to be returned to their owner out of state.