At age of one day, 82 male chickens were marked wings with an ID and assigned to three diffrent groups, stress, and control and enrichment group.
On the age of 4 days, birds from stress group were exposed to social isolation as a stress treatment.  The treatment lasted for three week with increasing the time an hour per week to avoid habituation. During the treatment, the birds were placed individually in the social isolation mesh boxes (25x28x18) where they had vocal contact with limited sight and no physical contact with other birds.
Simultaneously with the stress treatment, the enrichment group was provided with a combination of positive treatments, that were represented in three levels perches and some wooden cubes that were distributed all over the pen. All birds from all groups were weighed weekly.

At the age of four weeks, after the three weeks treatment, eight birds per group were culled in order to check the short term effects of stress. I collected blood samples in addition to testis dissection. A body weight measurement was obtained as well.
At the age of six months when birds reached sexual maturation, sperms were collected in order to check the effect of early stress on the germline. Blood samples and body weighs were obtained.
For the analysis, birds from age of one day until the age of 4 weeks (i.e. culling week) were classified as young group. On the other hand sexually mature birds at the age of six months, were classified as adults group.

To access cytokine concentration in both young and adult birds, Chicken Interleukin 6 (IL-6) ELISA kit (CUSABIO) instructions were followed and applied on the plasma samples of the three groups (stress, control, enrichment) from two age stages young and adults.

In order to check DNA methylation change that might arise from social isolation in different types of cells (blood &sperms), I combined two methods Genotyping by sequencing (GBS) and Methylated DNA immuno-precipitation (MedIP) for the first time. Furthermore, I developed a protocol to extract sperms DNA from frozen testis.