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Acting Senior Sergeant Trevor Thomson, of Dunedin, said the alleged attack happened overnight on Saturday. An estimated $20,000 worth of Salmon had been lost, he said.
In January, the hatchery was broken into and the aerators switched off, killing about 200 salmon worth $30,000. Police were still investigating the first incident and had made no arrests.
Trust chairman Steve Bennett said about 45 male fish died in the latest attack, which meant the hatchery would not have fish to fertilise the females.
There was "no rhyme or reason" as to why anyone would commit the crime, he said.
"It's a not-for-profit organisation run by volunteers, so I am baffled as to why someone would do such a thing.
"The place was finger-printed but nothing has come to light. After last time we changed locks and took other measures. We're doing as much as we can but we can't know when these things will happen."