The defence pathologist, who found there were no injuries to three-year-old Courtney Pieters' genital area, could not explain how semen was found in her vagina.
He was testifying on Tuesday in the Western Cape High Court trial of Mortimer Saunders, the man accused of murdering and raping the toddler.
Saunders denied that he raped her while she was alive. In his plea explanation, Saunders admitted to the murder and claimed that he used his fingers to penetrate her vagina after she had already died.
During cross-examination, Dr Segeran Naidoo stood by his earlier testimony that the fissures he initially identified in his report were, in fact, the folds of her vaginal wall.
When prosecutor Esmerelda Cecil questioned whether he was aware that forensic analyst Lukhanyo Tiya had found semen in the little girl's vagina, Naidoo indicated that he had not been aware of this fact.
"My focus was not on the DNA. It has been on my field - pathology and medicine," he said.
On Monday, Naidoo said that, if the insertion of the fingers had occurred, there would be abrasions on the full circumference of the vagina.
Naidoo had earlier admitted that he was at a disadvantage because had not examined the body.
He used the post mortem photographs, the histology report, post mortem report and various affidavits as the basis for his findings.
Courtney's body was found in Epping Industria in May last year, nine days after she after she went missing from her Elsies River home.
Saunders admitted to feeding her ant poison to make her sick, before beating her and using a towel to cover her mouth.
He claims that he murdered her because he had "ill feelings" towards Courtney's mother, Juanita.