RESULTS During the Mn depletion phase (0–8 wk), the average egg production of all the birds declined from 85.6 to 82.3% (data not shown in the table). During 9 to 12 wk, the egg production was not affected by dietary supplementation of Mn. Within the whole experimental phase, dietary Mn deficiency did not influence bird mortality (1.32%). Layers fed with 25 mg/kg of Mn had the highest egg production, but no differences were observed between the control group and the Mn-supplemented treatments in egg production, feed efficiency, or feed intake (Table 3; P > 0.05). The eggshell quality data are shown in Table 4. Dietary Mn supplementation at 100 mg/kg significantly increased eggshell breaking strength, thickness, and fracture toughness (P < 0.05) compared with the control group, whereas dietary Mn supplementation at 25 mg/kg had no significant effect on these properties (P > 0.05). Dietary Mn supplementation of 100 mg/kg significantly increased the eggshell breaking strength by 15.4%, the shell thickness by 9.72%, and the fracture toughness by 12.2% compared with the control group (P < 0.01). There were no differences in breaking strength, thickness, elastic modulus, or fracture toughness between the control and the dietary Mn supplementation of 25 mg/kg (P > 0.05). Moreover, there were no differences in egg weight, shell ratio, eggshell shape index, and stiffness between the control and the Mn-added groups (P > 0.05). Scanning electronic microscopy photographs (Figure 1) showed that dietary Mn deficiency caused a different eggshell ultrastructure compared with the Mn-added group. Manganese deficiency gave rise to larger and abnormally arranged mammillary cones, and there were obvious cracks on the outer surface in the control group. The thickness of the mammillary layer was 56.5% greater (Table 5) and the width of mammillary cones was 23.6% greater in the control group relative to the Mn-added group (100 mg/kg). Dietary supplementation of Mn at 100 mg/kg significantly reduced the thickness of the mammillary layer and the width of mammillary cones (P < 0.01). Furthermore, dietary Mn addition decreased the cuticle cracks on the outer surface of the eggshell. The content of uronic acids and GAG in the eggshell membrane, but not in the calcified eggshell, responded significantly to dietary Mn addition (P < 0.05). The mRNA expression of GlcAT-I in eggshell glands was also enhanced by dietary Mn supplementation (P < 0.05). There was no difference in mRNA expression of GlcAT-I between the control and dietary supplementation of 25 mg/kg Mn (P > 0.05). Dietary Mn supplementation of 25 mg/kg increased uronic acids and GAG in the eggshell membrane by 19.0 and 17.6% (Table 6), respectively, compared with the control group, but no significant differences were observed (P > 0.05). There were no differences in GAG and uronic acids in the calcified eggshell between the Mn-supplemented group and the control (P > 0.05). Western blot analysis indicated that the GlcAT-I synthesis was reduced because of Mn deficiency (Figure 2).