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Animal rights groups say they are alarmed at the increasing number of animals being "manipulated" in research, testing and teaching.
The 341,520 animals used last year was the highest number since records began in 1989 and Hans Kriek, of Save Animals from Exploitation (Safe), said the rise was shocking.
Nearly 40 per cent of the animals died or were euthanased, including 1 per cent of the 2327 reptiles used, 23 per cent of the 31,053 birds, 4 per cent of the 78,093 sheep, 4 per cent of the 804 cats, and 5 per cent of the 792 dogs.
Sue Kedgley, of the Green Party, said: "The sharp increase in the number of animals experimented upon shows that the Animal Welfare Act is not working and the commitment to reducing or replacing the number of animals used in experiments is simply spin."
The National Animal Ethics Advisory Committee's latest statistics showed 38.5 per cent more animals were used last year than in 2007, which in turn was down 22.6 per cent on the 318,489 used in 2006. The 2003 total was 320,911 and the 2000 figure 324,395.
The number of animals manipulated "for animal husbandry purposes" soared by 233 per cent, from 18,419 in 2007, to 61,297 last year, and animals sourced from farms rose 250 per cent.
Committee chairman John Martin said fluctuations were largely due to a three-year reporting cycle for long-term projects, and possibly more scientific work being conducted.
Reports for animals used in long-term projects are not required every year but usually when the project is completed or ethics approval expires.
But the committee's annual report noted that Agriculture Minister David Carter still has "under consideration" its repeated call in recent years for the definition of "manipulation" to include those animals killed for research purposes, or fetuses operated on in the first half of gestation - when genetic engineering is usually done.
The committee said rodents made up most of the animals judged to have suffered moderate (85.4 per cent) high (84.4 per cent) and very high (99.9 per cent) impacts. Two other species were 18 sheep used for veterinary research, and a shark used for basic biological research.