I almost sounds as if Leonardo operated on living animals and people to get the info he got. How could he understand valve motion and blood flow otherwise? A dead heart doesn't seem like it would give up a lot of details.
A living heart would be pretty hard to study, actually, it'd be shaking and squirting blood all over, obscuring the examination. And cutting a living heart enough to get a good view of the valve in action would result in abnormal or non-functional operation.
Da Vinci could have cut the valve area out of a fresh human or animal heart, and then poured clear liquids through it from different directions to study how it operated.