An in ovo jet injection apparatus and related methods for treating live eggs. The jet injection apparatus includes one or more jet injection delivery devices configured to deliver one or more treatment substances to predetermined areas of eggs using a high pressure stream of the treatment substance(s). Multiple treatment substances can be delivered so that they are spatially and/or temporally separate. The devices and methods of the invention enable the effective use of a plurality of treatment substances, including those that are effective when used alone but can be noxious if mixed. The methods and apparatus for the jet injection of substances into embryonic chicks reduce the risk of mechanical injury to the developing birds that would be caused by injection needles. The methods and apparatus of the invention can also reduce the introduction of an infection into the chicks.