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The run-up to Saturday's Grand National was overshadowed on Friday after the death of a horse following a fall on the Aintree course, the second such case in successive days, British media reported.
Animal rights campaigners slammed Grand National organisers after Battlefront became the 23rd horse to die on the Liverpool course since 2000.
Little Josh became the second horse to die at this year's meeting at the rnowned venue after the 11-year-old, ridden by Sam Twiston-Davies and trained by his father Nigel, fell during the Topham Chase, held on the recently redesigned Grand National course.
Little Josh suffered a fractured shoulder and had to be put down by veterinary surgeons.
Aintree has altered the Grand National fences, making them of a more flexible plastic rather than wood, as part of a bid to focus on horse welfare at the meeting.
Last year's race was marred by the death of two horses, According To Pete and Synchronised.
There were also two fatalities in the 2011 race - Ornais and Dooney's Gate.
Battlefront was pulled up by jockey Katie Walsh during the fourth race on the course on Thursday.
It came after Walsh defended the sport earlier this week, saying in a magazine interview that the horses were treated better than "many children".
The BBC quoted Professor Chris Proudman, veterinary advisor to Aintree, as saying: "We are sad to confirm that is has been necessary to humanely put down Little Josh on welfare grounds, as a result of his fall at the 15th fence in the John Smith's Topham Chase."
He added the injury was not treatable.
John Baker, Aintree and North West Regional Director for Jockey Club Racecourses, expressed his regret but added: "We have made significant improvements in safety at the course, but we also recognise that jump racing carries risk you can never completely remove from the sport."