Albert Barnes Commentary 1 Samuel 6:12

"And the kine took the straight way by the way to Beth-shemesh; they went along the highway, lowing as they went, and turned not aside to the right hand or to the left; and the lords of the Philistines went after them unto the border of Beth-shemesh." — 1 Samuel 6:12 (ASV)

Lowing as they went - Milking cows were chosen specifically to make the sign more significant. Nature would obviously incline the cows to go toward their calves, so their going in the opposite direction was clearly a divine impulse overruling their natural instinct. This is made even more distinct by the mention of their lowing, which was caused by their remembering their calves.

And the lords - The fact that the five lords of the Philistines accompanied the ark in person made it impossible for the Israelites to practice any deception . It also serves as a striking testimony to the turmoil caused among the Philistines by the plagues inflicted on them while the ark was in their country.