Chinese swimmer Chen Xinyi was suspended for failing a doping test and a Polish weightlifter and Bulgarian runner were both kicked out for drugs transgressions.
In shock results, defending champion and world record-holder Sun Yang flopped dismally in his 1,500m freestyle heat and the USA, who have won the last three Olympic competitions, were knocked out of the women's football.
Athletics took centre stage in Rio needing a lift after the Russia doping storm, but there were immediate questions about Ayana's 10,000m run, which broke a 23-year-old world record.
Her winning time was nearly 14 seconds quicker than the previous world best of 29:31.78 set by Wang Junxia in Beijing in 1993.
Ayana's stunning performance set tongues wagging but the Ethiopian smiled as she insisted she was "crystal clear".
"My doping is my training, my doping is Jesus -- otherwise I'm crystal clear," she said.
The world sports tribunal had earlier expelled European champion weightlifter Tomasz Zielinksi and Bulgarian 3,000m steeplechase runner Silvia Danekova over doping as the Games registered its first major cases.
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Chen, 18, a rising star in the Chinese swimming team, was suspended after she tested positive for a banned diuretic following Sunday's women's 100m butterfly, where she placed fourth.
Poland's Zielinski tested positive for the anabolic steroid nandrolone -- just three days after his brother Tomasz suffered the same fate.
Damekova, a 3,000m steeplechase athlete, admitted she had tested positive for the blood booster EPO.
David Rudisha cruised into the semi-finals of the men's 800m in Rio after a Kenyan coach was sent home in disgrace for allegedly trying to impersonate an athlete for a doping test.
British star Jessica Ennis-Hill made a smooth start to the defence of her heptathlon crown, landing early blows on rival Brianne Theisen-Eaton.
Ennis-Hill, 30, won the 100m hurdles and placed third in the high jump as she bids to become only the third woman to retain an Olympic athletics title after giving birth.
In rowing, there were two more golds for Britain with Helen Glover and Heather Stanning taking the women's pair. Mohammed Sbihi, George Nash, Constantine Louloudis and London 2012 winner Alex Gregory claimed the men's four.
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French duo Jeremie Azou and Pierre Houin won the men's lightweight double sculls. Ilse Paulis and Maalke Head of the Netherlands won the women's sculls race.
Egyptian judoka Islam El Shehaby was jeered after refusing to shake hands with his Israeli opponent at the end of their heavyweight first round encounter.
As he left the mat area, El Shehaby was called back to the centre by the referee to bow to Israel's Or Sasson. But he was then loudly jeered out of the arena by angry supporters.
The USA have won the last three Olympic women's football titles and are the reigning world champions, but they crashed out to Sweden on penalties in the quarter-finals.
In swimming, Sun's unhappy Olympics came to a shuddering halt when he finished seventh in his heat and 16th overall in qualifying for the 1,500m, an event in which he has been untouchable.
Sun won the 200m freestyle but lost out to Mack Horton in the 400m race -- and then was at the centre of a storm when the Australian called him a "drugs cheat", referring to his doping suspension in 2014.
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