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Highland Reel, winning his second Group One race inside a month, also brought relief to the 47-year-old trainer after his hotly-fancied Churchill had failed to fire and finished only fourth in Tuesday's feature, the St James's Palace Stakes.
The five-year-old - who has won six Group Ones, including in England, Hong Kong and the United States - looked beaten at the furlong pole but somehow found the reserves to storm back and win by a length and a quarter to take his prize money to just under the £6 million ($7.6m, 6.8m euros) mark.
O'Brien hailed Highland Reel as 'unbelievably courageous'.
"If it comes anywhere near courage, then he will be there fighting," said O'Brien.
"He has danced every dance. I'm not sure we have ever had a horse with the constitution he has."
O'Brien was typically humble about his own landmark of 300 Group and Grade One wins.
"I'm just a small part in a massive team," he said.
"It's a huge chain of people involved and I feel privileged to be part of it."
Highland Reel also gave jockey Ryan Moore his 44th Royal Ascot winner.
"Highland Reel has been everywhere and keeps coming back," said Moore.
"We knew he would see it out real well (the race distance), and the last furlong was his best.
"I'd say it was probably a career-best from him today."
Queen Elizabeth II resolutely turned up in her carriage at the meeting, despite having her husband Prince Philip taken to hospital on Tuesday and then having to attend the State Opening of Parliament.
However, bedecked in a buttercup yellow dress, and a matching coloured hat, she was accompanied by her eldest son Prince Charles and his wife Camilla.
There was a French flavour to the winners' enclosure on two occasions.
Jean Claude Rouget's smart filly Qemah won the Group Two Duke of Cambridge Stakes to record her second win in as many visits, having won the Group One Coronation Stakes last year.
"Qemah ran like she did last year, and it was reassuring as she was very calm before the off," said jockey Gregory Benoist.
"She is a good filly and can go anywhere."
However, Mikael Barzalona will not look back on the race with any great pride having delivered the Andre Fabre-trained Usherette far too late and only secured third.
Fabre, never one to hide his emotions, didn't dally long when Barzalona returned, but the French master trainer had been wearing a far happier expression when Le Brivido prevailed in the opening Group Three Jersey Stakes.
"He looked beaten with a furlong to go and showed his class and courage to come back and beat the other horse (Spirit of Valor)," said 71-year-old Fabre, who recorded his ninth success at the Royal meeting.
Adam Kirby rode Clive Cox's pessimistically-named filly Heartache to victory in the Group Two Queen Mary Stakes to put American odds-on favourite Happy Like A Fool - like last year's winner Lady Aurelia, trained by Wesley Ward - in her place.
Her victory was a great reward for the 75-strong Hot to Trot syndicate who put up just £2000 to get a share in the seven horses that make up their roster.
"I was 100 per cent confident that we would pass the American filly; she was beat from halfway," said Kirby, who was riding his seventh Royal Ascot winner.
"I am delighted for Clive. He is a great trainer and doesn't get enough credit really in my view."
Ward, 49, gained some compensation when he landed the last on the card, the Listed Sandringham Handicap Stakes, with Con Te Partiro for his ninth Royal Ascot winner.
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