The aimof this study was to assess the effect of in ovo injection with saline, vitamins (D₃ and B₁₂) and zinc and L-carnitine on the hatching rate and blood constituents in newly hatched chicks. A total of 1170 eggs from broiler breeders were divided into sixtreatmentgroupsof eggs:a control group without injectionwhich served as sham-operatedor a negative control group and five groups were injected with saline, vitamin D₃, zinc, vitamin B₁₂or L-carnitine, respectively. All eggs were set at a temperaturerange between 24-26°C for 6 hours before exposure to the incubation temperature. Then the injection process occurred at the 18^thday and 12 hours of the embryonicdevelopment by depositing the test materials into the air sac at the wide end of the eggs, then the hole was closed with wax. The highest value of fertile hatchability was recorded in eggs injected with vitamin D₃ and zinc, respectively, followed by vitamin B₁₂ and L-carnitine. The injected test materials (vitamin D₃, zinc, vitamin B₁₂and L-carnitine) had a positive effect on the percentage of late dead embryos compared to negative control group. All injected materials except zinc led to an increase in plasma levels of cholesterol and low-density lipoprotein but levels of glucose, triglyceride, high-density lipoprotein, total protein, albumin and globulin were not affected. In conclusion, in ovo injection of vitamins (D₃ and B₁₂), zincand L-carnitine may be suggested as an effective technique for increasing hatchabilityand profitability in aged broiler breeders.