Uterine inflammation status modulates eggshell mineralization via calcium transport and matrix protein synthesis in laying hens

Abstract

This study explored the effects of uterine inflammation on eggshell mineralization, ultrastructure and mechanical properties in laying hens modified by a lipopolysaccharide (LPS) challenge or dietary essential oil (EO) addition. In trial 1, a total of 72 Hy-line Brown layers at 36 wk of age were randomly assigned to 3 treatment groups (n = 8), where they were intravenously injected with phosphate buffered saline, LPS at 1 mg/kg body weight, or LPS 3 times at 24-h intervals. In trial 2, a total of 288 Hy-line Brown layers at 60 wk of age were randomly divided into 4 groups (n = 8), where they were fed basal diets supplemented with EO at 0, 50, 100 and 200 mg/kg for 12 wk. A uterine inflammation model was constructed with LPS treatment, indicated by the elevated expression of IL-1β and TNF-α (P < 0.05) and lymphocyte infiltration. Uterine inflammation caused remarkable decreases in eggshell thickness and mechanical properties with structure deteriorations (P < 0.05). Uterine inflammation stimulated the expression of matrix proteins ovotransferrin (TF) and ovalbumin (OVAL), while depressing the mRNA levels of calbindin-1 (CALB1) and osteopontin in uterine mucosa (P < 0.05). In contrast, EO addition alleviated uterine inflammation, evidenced by depressed levels of IL-1β and IL-6 (P < 0.05). There was a significant elevation in shell thickness and breaking strength following EO intervention (P < 0.05), and these effects were maximized at addition of 100 mg/kg. Further, EO improved shell ultrastructure including more early fusion, less type B mammillae, and increased effective thickness (P < 0.05). The alleviated inflammation decreased the expression of OVAL and TF, whereas ion transport genes like CALB1 and solute carrier family 26 member 9 were upregulated (P < 0.05). Our findings suggest that inflammatory status can impact uterine functions in calcium transport and the synthesis of matrix proteins especially such as OVAL and TF, which in turn modulates calcium precipitation and ultrastructure formation, thereby determining eggshell mechanical properties. These findings provide a novel insight into the uterine inflammation-mediated modifications of eggshell quality.

Keywords: Eggshell ultrastructure; Essential oil; Laying hen; Lipopolysaccharide challenge; Mechanical property; Uterine inflammation.

Fig. 1

Lipopolysaccharide (LPS) challenge induced deteriorations in eggshell quality. Evolution of egg weight (A), eggshell weight (B), shape index (C), shell ratio (D), shell thickness (E), breaking strength (F), stiffness (G), fracture toughness (H) and elastic modules (I) of eggshell from the control and LPS injection groups.

Fig. 2

Lipopolysaccharide (LPS) challenge modified ultrastructure characteristics and chemical composition of eggshell. (A) Hierarchical cluster analysis of eggshell quality attributes during the post-LPS challenge period. Control means eggshell samples from control laying hens without LPS challenge; M1, M2 and M3 mean eggshell samples in multiple-injection groups were divided into 3 clusters, including M1 (d 5 to 8), M2 (d 9 to 12) and M3 (d 13 to 14). Ultrastructure observation (magnification 200× ) of eggshell from the control (B), M1 (C), M2 (D) and M3 (E) groups. Ultrastructure observation in the mammillary layer of eggshell from control (F, magnification 200× ; G, magnification 500× ) and M1 (H, magnification 200× ; I, magnification 500× ) groups. Ultrastructure parameters, including mammillary thickness (J), effective thickness (K), total thickness (L) and mammillary width (M) of eggshell collected from laying hens following LPS challenge. The contents of calcium (N), phosphorus (O) and matrix protein (P) in eggshell collected from laying hens following LPS challenge. Values are presented as mean ± SD, n = 8. ^a–c Means with different letters indicate a significant difference (P < 0.05).

Fig. 3

Lipopolysaccharide (LPS) challenge induced uterine damage and altered mRNA expression of genes related to immune response and tight junction proteins in the uterus of laying hens. Morphological observation of the uterus from laying hens in the control (A, magnification 1.8× ; D, magnification 10× ), SI (B, magnification 1.8× ; E, magnification 10× ) and MI (C, magnification 1.8× ; F, magnification 10× ) groups. SI means single injection with LPS 1 mg/kg body weight (BW); MI means multiple injection with LPS. Injection program: LPS injection with 24 h intervals, 1 mg/kg of BW on d 0 and 0.5 mg/kg of BW on d 1 and 2 at 3 h after oviposition. The lower-case letters in Fig. 3E and F, “x” means edema or dissolution of tubular glands, “y” means degeneration, necrosis or exfoliation of epithelial cells, “z” means lymphocytic infiltration. Morphology parameters included villus length (G), the height (H), width (I) and area (J) of mucosal folds, and ratio of EDTG (K) in uterus from laying hens following LPS challenge. EDTG = edema or dissolution of tubular glands. Values are presented as mean ± SD, n = 8 (6 measured values for each sample). The villus length of the uterus was measured from the top of the villus to the top of the lamina propria. The height of mucosal folds was defined as the vertical length from the epithelial to the fold, and the width of mucosal folds was measured by drawing a perpendicular line across the widest part of mucosal folds. The outline of mucosal folds was traced and their areas were measured. The mRNA expression of immune response (L) and tight junction protein (M) related genes in uterus of LPS-challenged laying hens. ZO-1 = zonula occludens-1 Values are presented as mean ± SEM, n = 8. ^a–c Means with different letters indicate a significant difference (P < 0.05).

Fig. 4

Lipopolysaccharide (LPS) challenge altered the relative mRNA expression of key genes related to eggshell biomineralization in (A and B) the uterus of laying hens. SI means single injection with LPS 1 mg/kg body weight (BW); MI means multiple injection with LPS. Injection program: LPS injection with 24 h intervals, 1 mg/kg of BW on d 0 and 0.5 mg/kg of BW on d 1 and 2 at 3 h after oviposition. TF = ovotransferrin; OVAL = ovalbumin; OC-17 = ovocleidin-17; OPN = osteopontin; CALB1 = calbindin 1; CA2 = carbonic anhydrase 2; ATP2B1 = ATPase plasma membrane Ca²⁺ transporting 1; ATP2B2 = ATPase plasma membrane Ca²⁺ transporting 2; SLC26A9 = solute carrier family 26 member 9. Values are presented as mean ± SEM, n = 8. ^{a, b} Means with different letters indicate a significant difference (P < 0.05).

Fig. 5

Dietary essential oil supplementation modulated eggshell ultrastructure characteristics. Eggshell ultrastructure observation in the control (A) and essential oil-supplemented (B) groups. Effects of dietary essential oil supplementation on ultrastructure characteristics of eggshell (C–H). Control means laying hens fed with the basal diet; M50, M100 and M200 mean laying hens fed with the basal diets supplemented with 50, 100 and 200 mg/kg essential oil, respectively. Values are presented as mean ± SD, n = 8. ^{a, b} Means with different letters indicate a significant difference (P < 0.05). TT = total thickness; ET = effective thickness; MT = mammillary layer thickness; MW = width of mammillary knobs. Ultrastructural variations in the mammillary layer of eggshell. (I) Normal structure of mammillary knobs and (L) cuffing (yellow arrow) and early fusion of mammillary knobs (blue arrow; magnification 500× ) in eggshell from essential oil-supplemented groups; (J) Type B mammillae (blue arrow; magnification 200× ) and (K) late fusion of mammillary knobs (blue arrow; magnification 500× ) in eggshell from the control.

Fig. 6

Dietary essential oil supplementation modulated uterine morphology and the mRNA expression of genes related to immune response and tight junction proteins in uterus of laying hens. Morphological observation of the uterus from laying hens in the control (A and C) and EO (B and D) groups. Morphology parameters included villus length (E), the height (F), width (G) and area (H) of mucosal folds, and ratio of EDTG (I) in uterus of laying hens in the control and EO groups. The mRNA expression of immune response (J) and tight junction protein (K) related genes in uterus of laying hens in the control and EO groups. The villus length of uterus was measured from the top of the villus to the top of the lamina propria. The height of mucosal folds was defined as the vertical length from the epithelium to the fold, and the width of mucosal folds was measured by drawing a perpendicular line across the widest part of mucosal folds. The outline of mucosal folds was traced and their areas were measured. Control means laying hens fed with the basal diet. EO means laying hens fed with the basal diet supplemented with 100 mg/kg essential oil. EDTG = edema or dissolution of tubular glands. ZO-1 = zonula occludens-1. Values are presented as mean ± SD, n = 8 (6 measured values for each sample). Blue arrows indicate the inflammatory cell infiltration in the sub-mucosa of the uterus. Asterisks indicate a significant difference between groups (P < 0.05).

Fig. 7

Dietary essential oil supplementation regulated the mRNA expression of key genes related to eggshell biomineralization (A and B) in the uterus of laying hens. Control means laying hens fed with the basal diet. EO means laying hens fed with the basal diet supplemented with 100 mg/kg essential oil. OVAL = ovalbumin; TF = ovotransferrin; OPN = osteopontin; OC-17 = ovocleidin-17; CALB1 = calbindin 1; CA2 = carbonic anhydrase 2; ATP2B1 = ATPase plasma membrane Ca²⁺ transporting 1; ATP2B2 = ATPase plasma membrane Ca²⁺ transporting 2; SLC26A9 = solute carrier family 26 member 9. Values are presented as mean ± SEM, n = 8. Asterisks indicate a significant difference between groups (P < 0.05).

Fig. 8

A schematic model displaying the potential mechanism by uterine inflammation-mediated modification of eggshell quality. ↑Up arrows indicate the effects of stimulation; ↓down arrows indicate the effects of suppression. IL = interleukin; TNF = tumor necrosis factor; CALB1 = calbindin 1; OVAL = ovalbumin; TF = ovotransferrin; OPN = osteopontin.