A lethal injection has been given to a 16-year-old bullock which tested positive for bovine TB at a religious community in Carmarthenshire.
Assembly government officials took the water buffalo away
A nine-month-old water buffalo was also put down after being taken away by Welsh Assembly Government officials.
Last month, the temple's bullock Shambo was killed after testing positive for the disease at the Skanda Vale site.
Monks lost a long-running legal challenge to save Shambo and staged a protest ceremony as he was taken away.
However, despite the two further slaughters, the monks said there would be no repeat of the ceremony which took place on the day the six-year-old Friesian was killed.
After Shambo's death, more tests were carried out on animals at the community.
Monks chanted while the officials were on site
On Thursday, an assembly government vet and 10 officials visited the community and it was decided to slaughter the older bullock, called Bhakti, on site as it was too ill with arthritis to travel.
The young water buffalo, called Dakshini, was then taken away for slaughter.
Rural affairs minister Elin Jones said: "Formal notices have been issued to the community at Skanda Vale for two more bovines at the community to be put down to bring the outbreak of bovine TB there under control."
Brother Alex of Skanda Vale said the community was "very disappointed" by the new slaughters but they had "exhausted all possibilities in terms of legal challenges".
And Brother Michael said the slaughter was another "unacceptable desecration" of the community's way of life.
He said: "Life is sacred, and we have repeatedly asked the assembly government to work with us to find a solution which doesn't involve killing."
Shambo first tested positive for bovine TB during a routine screening on 27 April, but was not killed until 26 July.
Shambo was kept in a special enclosure for weeks at the temple
Brother Michael said he did not accept that allowing the bullock to live so long had put the rest of the community's animals at risk.
He said: "Shambo was isolated. There was no way any problem he had could have been passed to any of the other cattle.
"He was never in contact with any of the other cattle, so we don't accept that."
However, the move has been welcomed by farming unions.
Farmers' Union of Wales president Gareth Vaughan said: "This proves that what has happened at Skanda Vale has followed a classic trend of TB transmission with an initial infection apparently spreading to other animals."
Llama and deer
The fate of Shambo became the focus of worldwide attention after it first tested positive for bovine TB in April.
It culminated three months later when supporters from around the UK and overseas joined a religious ceremony as officials, backed by police, took the bullock away to be put down.
The Skanda Vale community has a wide range of livestock including an elephant, ponies, water buffalo, cows, bulls, llama and deer.
More tests on the 54-strong herd are due to be carried out within 60 days.