THE RSPCA said it was "appalled" as the badger cull spread to Gloucestershire.
The animal welfare charity stressed it remained committed to persuading the Government to stop what it called "this misguided, unethical and unscientific attempt to control bovine TB in cattle".
It said the cull would not solve problems caused by the devastating disease or benefit cattle, badgers or dairy farmers and rural communities.
RSPCA chief executive Gavin Grant said: "We have real doubts about how humane the methods being used to kill the badgers are – and it is very possible that many of these iconic British mammals could be suffering and seriously injured, rather than killed."
The public and the media descended on parts of Gloucestershire on Tuesday night.
Vet Mark Jones, the UK director of the Humane Society International, was out with about 40 anti-cull campaigners as part of Wounded Badger Patrols.
He said: "I have no doubt that many thousands of badgers are now set to suffer in the county I call home, for the sake of a policy that has no scientific basis, will at best make a marginal difference to TB in cattle, and which could very well make things worse."
Wildlife film-maker and TV presenter Simon King has called on the Government to put pressure on Europe to allow a trial of TB vaccines for cattle, warning the controversial badger cull could do more harm than good.
Mr King, president of the Wildlife Trusts, accused ministers of selectively picking facts from a major 10-year study in order to back up the cull to appease farmers, even though the research concluded culling could not make a meaningful contribution to tackling bovine TB in cattle.
A Defra spokesman said: "Scientists have agreed that culling badgers where bovine TB is widespread reduces the level of disease. Many species can be infected with bovine TB, but only cattle and badgers are effective in spreading the disease. If we had a vaccine for cattle we would use it, but it is currently illegal under European law. We are working with the European Commission to make a vaccine available but we know this is at least 10 years away and we cannot wait while this disease continues to march across England."
