Calcium Carbonate Nanoparticles-Toxicity and Effect of In Ovo Inoculation on Chicken Embryo Development, Broiler Performance and Bone Status

Abstract

The use of intensive selection procedure in modern broiler chicken lines has led to the development of several skeletal disorders in broiler chickens. Therefore, current research is focused on methods to improve the bone quality in birds. In ovo technology, using nanoparticles with a high specificity to bones, is a potential approach. The present study aimed to evaluate the effect of in ovo inoculation (IOI) of calcium carbonate nanoparticles (CCN) on chicken embryo development, health status, bone characteristics, and on broiler production results and bone quality. After assessing in vitro cell viability, the IOI procedure was performed with an injection of 500 μg/mL CCN. The control group was not inoculated with CCN. Hatchability, weight, and selected bone and serum parameters were measured in embryos. Part of hatchlings were reared under standard conditions until 42 days, and production results, meat quality, and bone quality of broilers were determined. CCN did not show cytotoxicity to cells and chicken embryo and positively influenced bone parameters of the embryos and of broilers later (calcification) without negatively affecting the production results. Thus, the IOI of CCN could modify the molecular responses at the stage of embryogenesis, resulting in better mineralization, and could provide a sustained effect, thereby improving bone quality in adult birds.

Keywords: bone quality; broiler; calcium carbonate; chicken embryo; nanoparticles.

Figure 1

Representative zeta potential of CCN at the concentration of 50 µg/mL (three peaks) (A). Transmission electron microscope image of CCN. Scale bar represents 1 µm (B).

Figure 2

Alizarin red staining for mineralization. The calcified nodules appeared bright red color (original magnification ×100). Cells in control group, without CNN (A). Cells incubated with CCN at 5 μg/mL (B). Cells incubated with CCN at 25 μg/mL (C). Cells incubated with CNN at 100 μg/mL (D). Cell viability in groups with increasing CCN concentration determined by the XTT assay after 24 h of incubation (E). The error lines represent standard error of mean.

Figure 3

Hatchability (A), BW (B), liver weight (C), breast muscle weight (D) of chicken embryos on day 20 after IOI with 500 μg/mL of CCN. * Value on bars differs significantly at p ≤ 0.05. The error lines represent standard error of mean.

Figure 4

Selected parameters of chicken embryo bones on 20 day after IOI with CNN. Femur and tibia weight (A,B), femur and tibia length (C,D), femur and tibia middle diaphysis diameter (E,F), femur and tibia maximum resistance to breaking (G,H), femur and tibia Ca, P and ash content (I). * Value on bars differs significantly at p ≤ 0.05. The error lines represent standard error of mean.

Figure 5

Production results of broiler chickens after 42 d of rearing. Average BW in groups on days 1, 10, 35 and 42. SEM values for average BW on days 1, 10, 35, and 42: 0.239, 3.496, 35.48, and 44.49, respectively (A). FCR and mortality of broiler chickens after the rearing period (B).

Figure 6

Selected parameters of broiler chicken male femoral bone from different groups. Femur weight (A), femur length (B), femur maximum resistance to breaking (C), femur Ca, P and ash content (D), femur micromineral content (E), femur average relative mineral density (F), average femur volume and average femur volume for 500 and 1000 HU (G). * Value on bars differs significantly at p ≤ 0.05. The error lines represent standard error of mean.

Figure 7

Histological cross sections from proximal metaphysis of broiler chicken male femoral bone. Femur after H&E staining (A). Alizarin red staining for mineralization (calcium deposits) for broiler chicken after IOI with CCN (B) and without IOI (C). Higher red colour intensity value suggests better calcification of the bone (D). TB: trabecular bone; CB: compact bone. * The value next to the averages differs significantly at p ≤ 0.05.

Figure 8

The OC concentration in serum and femoral bone from chicken embryo on 20 day after IOI with CNN (A) and from broiler chicken male after 42 d of rearing period (B). * Value on bars differs significantly at p ≤ 0.05. The error lines represent SEM.