Blood samples were collected from the hens at seven different stages during the breeding cycle and are expressed as follows: Day 0 represents the control sample which was collected when the hens were moved to the outdoor aviaries ; Day 1 represents the day of start of incubation which was identified by the unwillingness to leave her eggs in the nest and to peck when a hand approaches; Day 11 represents the sample collected during the mid-incubation period which was 11 days after start of incubation; Day 19 represents the sample collected two days before the expected day of hatching; Day 21 represents the sample collected on the day of hatching; Day 35 represents the sample collected two weeks post-hatch; Day 49 represents the sample collected four weeks post-hatch.

The traits considered to measure the development of the brood patch are de-feathering, vascularization, edema and contour feathers. The traits were scored on a scale of 0 to 4 where 0 indicates the absence of the trait and 4 indicates the complete development of the trait. This scoring was done at five different locations in the hen’s body namely: left lateral patch, right lateral patch, median patch, thigh and neck. The brood patch was scored every time the hen was handled to collect a blood sample in their respective stages during the breeding cycle.

The steroids were first extracted by means of precipitation using 100% ethanol. The corticosterone levels were determined using a double antibody radioimmunoassay kit. Sandwich ELISA technique was used to determine the prolactin concentration.