Four Victorian racehorse trainers and a prominent veterinary surgeon have been charged over the use of the banned substance cobalt.

Stewards laid a total of 29 charges against trainers Danny O'Brien, Mark Kavanagh and Lee and Shannon Hope on Thursday, ending a long-running Racing Victoria investigation.

Dr Tom Brennan, employed by O'Brien and Kavanagh to provide veterinary services, faces 20 charges over supplying and administering cobalt to racehorses in the leading stables.

Brennan has emerged as a central figure in a Racing NSW cobalt inquiry involving Sam Kavanagh, the son of Mark Kavanagh.

"Today's issuing of charges against five persons follows exhaustive and complex investigations by Racing Victoria's integrity services team into elevated cobalt samples," Racing Victoria spokesman Dayle Brown said.

"We have dedicated considerable resources to undertake these investigations as swiftly as possible whilst ensuring they were thorough.

"During the course of the investigations we have collected a considerable amount of evidence and have undertaken extensive forensic analysis and testing."

No date has been set for the hearings to take place before the Racing Appeals and Disciplinary Board.

An investigation into the presence of cobalt in a horse trained by Peter Moody remains ongoing.

Moody's stayer Lidari returned an illegal cobalt level after finishing second in the Turnbull Stakes during last year's spring carnival.

Cobalt is a substance found naturally in horses but a marked increase in levels is said to improve racing performance.

Only trace amounts of cobalt are needed by horses and it is toxic when administered at high levels.

Victorian authorities introduced a threshold of 200 micrograms per litre in April last year and this was adopted as a national threshold from January 1.

Top three-year-old Bondeiger is among four O'Brien-trained horses to exceed the threshold, returning levels of 330 micrograms and 370 micrograms per litre in two samples after finishing second in last year's Victoria Derby.

The Mark Kavanagh-trained Magicool tested positive after winning the UCI Stakes.

Magicool, winner of the Queensland Derby last Saturday, returned a level of 670 micrograms per litre in his Flemington victory.

Three horses out of the stable of the father-and-son training partnership of Lee and Shannon Hope - Windy Citi Bear, Best Suggestion and Choose - returned unacceptable levels.

Brennan, a partner in the Flemington Equine Clinic, denied supplying Sam Kavanagh with two bottles of a substance labelled "Vitamin Complex" when the suspended trainer's cobalt hearing opened in Sydney on Tuesday.