Two horses and a pony were killed when they bolted in front of a car on the A24 in West Sussex.
A couple in the Volkswagen Polo were taken to hospital after hitting the animals just north of the Washington roundabout, near Storrington, yesterday.
Fire crews had to erect screens to hide the aftermath which left one horse dead and forced vets to put down another and a pony, both of which had been seriously injured. One pony survived.
The impact blocked the northbound lane of the dual carriageway, leading away from Worthing, for two hours during the rush hour from 5.30pm.
RSPCA inspector Tony Pritchard said: "It was pretty nasty. When I arrived, it was like a war zone. One of the dead horses' legs had been amputated in the accident.
"The other pony was standing with a member of the fire brigade. It had hit its leg and had a cut which was bleeding profusely. Shooting was the only option for that pony."
After a further inspection, the damage to a joint on the third horse's leg was considered too severe and it also had to be shot.
The animals had broken free from a field near Mitchbourne Farm.
Reports suggested they had been spooked and galloped off down a country track which led to the carriageway.
The owners were said to have been devastated by the mysterious deaths.
Mr Pritchard said the surviving pony was left because it was not as fast as the other animals, which has stopped the traffic by the time it arrived at the road.
The injured man and woman were taken to Worthing Hospital. No details about their condition have been released.
A West Sussex Fire Brigade spokesman said the crash resulted in major tailbacks.
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