Leading John Smith's Grand National contender Tidal Bay has suffered a "tiny stress fracture" and will not run at Aintree on April 6.
Allotted top-weight of 11st 10lb in the Liverpool showpiece, the Paul Nicholls-trained 12-year-old has not ran since winning the Lexus Chase at Leopardstown over Christmas.
He was forced to miss the Argento Chase at Cheltenham last month when reported to be lame, and Nicholls said in his Betfair blog: "As previously mentioned, he was slightly lame before the Argento and we couldn't get him ready in time for that race, or the Irish Hennessy.
"He was out cantering as usual on Monday and Tuesday, but on Wednesday we decided to step up his work.
"But his work rider came back up and said he didn't feel right.
"So we immediately called in Buffy (Shirley-Beavan) and, after discussions, we got him down to Newmarket today for an MRI scan.
"The news through from Buffy is that Tidal Bay has a tiny stress fracture of the lower cannon bone on his right hind leg. This is something that the X-rays we took before his problem in the Argento didn't pick up; only today's MRI did.
"He needs a month's box rest, so that rules out the National. As soon as I got the news, I immediately rang Mr (Graham) Wylie and we agreed to release this statement as soon as possible."
Bookmaker reaction to the news was to push out Nicholls to 4/1 from 3/1 to retain his champion trainers' title.
Last year's National success of Neptune Collonges was a first for Nicholls and also sealed the title in his long-running duel with Nicky Henderson, who is long odds-on this season.
Coral spokesman David Stevens said: "Despite being given top weight, Paul Nicholls felt Tidal Bay had been handed a chance in the National, and punters agreed, backing him from 25/1 down to 14/1.
"So news of his injury has come as a huge blow to backers, as well as weakening his trainer's chances of holding on to the championship."
Tidal Bay had been a 7/1 chance for the World Hurdle and 14/1 for the National with William Hill.
Owner Wylie said: "He's out for the season, I'm afraid. Paul took him down to Newmarket, the X-ray didn't suggest there was anything wrong with him but the MRI showed that there was a very small fracture.
"We need to do our best for the horse's welfare and he'll have plenty of box rest before we can hopefully get him back next season."
Wylie still has two strong National chances with the Willie Mullins-trained pair of On His Own and Prince De Beauchene vying for favouritism.