A total of 144 ostrich eggs (24 per group) were injected with 4 mL of physiological saline solution [positive control (PC)], carbohydrates (CHO — 20% maltose, 2.5% sucrose, and 2.5% dextrin), β-hydroxy-β-methylbutyrate (HMB), vitamin B₆ solution, and vitamin D₃ (vit D₃) solution at 38 d of incubation. Injection of 4 mL physiological saline decreased the hatchability compared with negative control (NC) group (not injected eggs) (68.5% vs. 71.7%; P < 0.05). There was also lower hatchability and more number of embryonic deaths before piping in vitamin-injected group (72.2%). Heart, gizzard, pancreas, spleen and thymus, ileum, cecum, and rectum relative weight were not significantly different among treatments at hatch (P > 0.05). Carbohydrates group had higher duodenum and jejunum relative weight, whereas NC and PC groups had the least duodenum, jejunum relative weight (P < 0.05). Vit D₃ injection increased bone strength, fresh and dry bone relative weight (P < 0.01). Chicks from HMB and CHO group had the highest levels of glycogen in the livers, whereas PC, NC, and vit D₃ exhibited very low levels of glycogen in their liver (P = 0.014). Vitamins in ovo injected groups had the least duodenum villus height and also vit D₃ group had the least jejunum villus height (P < 0.01). In conclusion, the use of CHO for the in ovo injection of ostrich hatching eggs would be practical in industry due to improvement in hatchability, bone characteristics, small intestine villus height and capacity, and also liver glycogen sources.