The objective of the study was to investigate the effects of an in ovo injection of CuNano and the timing of injection on metabolic rate (O consumption and heat production, HP) and development of layer hatchlings. On day 1 of incubation, 192 fertile eggs from 29-week-old Lohmann breeder strain chickens were distributed into four groups that were administered colloidal CuNano on: day 1 and/or 10. Gaseous exchange was measured in an open-air-circuit respiration unit, and HP was calculated for 16- and 19-day-old embryos. Yolk free body weight (YFBW) at 24h after hatching and the relative organ weights were used as a measure of hatchling development. In ovo injection of CuNano on different days during incubation significantly decreased O consumption and HP compared with the control group. The residual yolk sac weight in the treated groups was significantly higher than in the control group (P0.05). Furthermore, the plasma concentrations of IgM and IgG and the mRNA expression of NF-kB and TNF-α were not affected (both; P>0.05), indicating the absence of inflammatory modulation by CuNano. These preliminary results demonstrated that CuNano, regardless of the day of injection, altered the metabolic rate of embryos and depressed the development of organs; however, it did not affect YFBW, immunoglobulin concentrations and the expression of immuno-related genes.