An indirect method was developed. This dictates that an indicator must be able to pass through GI tract at the same flow rate as other nutrients, and it must not be digested and absorbed by animals. Chromic oxide and some rare earth elements are commonly used external indicators. Lignin has been used as an internal indicator. Not all the nutrients absorbed are retained in the animals.
Major loss is in the urine.
Collection of urine and subtract nutrient loss from total nutrient intake provide us with even more precise estimate of nutrient retention by an animal. This can be achieved by a metabolic trial. In order to establish the requirement of certain nutrient for animals, it is necessary to conduct a balance trial, which must consider other losses such as nutrient loss in hair, sweat and other body excretions. Use your imagination, please spare me on this one.