Organic trace elements are structurally stable and do not dissociate easily before absorption, and after entering the digestive tract, they can avoid being absorbed by precipitates in the intestinal lumen (e.g., phytic acid, phosphoric acid, oxalic acid, etc.) [
25]. In addition, small peptide-chelated organic trace elements are transported and absorbed in the form of amino acids, which reduces antagonism and competition for binding sites with other inorganic trace elements, thus demonstrating advantages over inorganic trace elements [
12,
26]. Our study indicates that low levels of organic trace elements added to diets in place of inorganic micronutrients do not negatively impact the production performance. Specifically, Organic trace elements at 20%, 30%, and 40% commercial levels were effective in maintaining production indicators, such as egg production rate, similar to those of inorganic trace elements at 100% commercial levels, which is in line with previous research reports [
8]. Serum levels of UA, GLU, T-CHO, and TG were indicators of animal health. Trace elements are associated with lipid metabolism, and deficiencies may cause disorders of lipid metabolism, which are usually manifested by elevated serum lipid metabolism-related markers [
27]. In this study, low levels of organic trace elements replacement did not significantly affect these indicators. The results of Aksu’s study equally showed that lower levels of organic trace elements did not negatively affect serum biochemical indices in broilers [
27]. When tissue cells are damaged or necrotic, Alanine aminotransferase (ALT) and Aspartate transaminase (AST) are released and their activity in the serum is elevated [
28,
29]. It has been reported that 1/3 NRC recommended levels of organic trace elements do not significantly affect serum ALT and AST activities in livestock [
27], which is consistent with the results obtained in our experiment. Immunoglobulin A (IgA), Immunoglobulin M (IgM) and Immunoglobulin G (IgG), are immune response antibodies produced by bone marrow-dependent lymphocyte. Trace elements act as activators that indirectly influence the content of immune proteins [
30]. There was no significant difference in the immunological indices of all treatments in this experiment. In addition, there were no significant differences in serum antioxidant indicators such as T-SOD, Cu/Zn-SOD, Mn-SOD, GSH-Px, and MDA among the groups. However, compared with the CON group, the serum CAT level was improved more significantly after organic trace elements supplement in the diet. In fact, serum CAT activity, as the only antioxidant indicator that differed in this trial, is not comprehensive enough to reflect the ability of organic trace elements’ replacement to improve antioxidant capacity in late laying hens. Trace elements are the active components of many antioxidant enzymes, and the lack of any one element will lead to the reduction of enzyme activity, thus affecting the biological body’s function of scavenging superoxide free radicals and reducing its resistance to oxidative toxicity [
31,
32,
33]. Therefore, the results of this experiment suggest that different levels of organic trace elements replacing inorganic trace elements have no negative effects on the normal physiological and metabolic functions of laying hens.
The egg quality index is related to the quality of the eggshell [
34]. When the eggshell quality is at a low level, the protective effect of the eggshell on the egg is weakened due to the decrease in the effective thickness of the eggshell, the egg becoming fragile, and the risk of harmful bacteria infection increasing [
35]. In our experiment, the effect of egg quality traits was more pronounced with increasing concentrations of organic trace elements. Briefly, we found that organic trace elements supplement had no effect on the yolk color, while the MOT and HOT supplement in diet significantly increased albumen height, Haugh unit, shell strength, and eggshell thickness, which delayed the quality deterioration of eggs. These findings are in accordance with the findings reported by Stefanello [
12]. Further analysis of the eggshell submicroscopic structure revealed that, compared with the CON group, the mammillary layer thickness was lower in MOT and HOT groups while total thickness, palisade layer thickness, mammillary knob width, and the ratio of mammillary layer to palisade layer of the eggshell were greater. However, the LOT group was not significantly different in terms of improving the ultrastructure of the eggshell than the CON group. Relevant studies have shown that the total thickness of the eggshell, the ratio of the fenestrae to the papillae, and the density and width of the papillae nodes affect the strength of the eggshell, with the longer the length of the fenestrae and the shorter the length of the papillae, the greater the ability of the eggshell to resist external forces, and the greater the strength of the eggshell [
36,
37]. Therefore, these results confirm that 30% and 40% commercial levels of organic trace elements improve eggshell quality by modulating the eggshell submicroscopic structure. However, the effect of the 20% commercial level of organic trace elements supplementation was similar to that of the CON group, and even worse than that of the CON group in terms of eggshell strength, which might be caused by the low level of organic trace elements supplementation in the LOT group. Copper, manganese and zinc are promoters or components of enzymes such as Lysyloxidase (LOX) and Carbonicanhydrase (CA), respectively, which are involved in the formation of eggshells [
38,
39,
40]. LOX is a copper-containing enzyme involved in the conversion of lysine to a variety of proteins within the eggshell membrane and plays a key role in the formation of the eggshell membrane [
38,
41]. CA is a zinc-containing enzyme that catalyzes the hydrolysis of carbon dioxide to form the calcium carbonate precursor of the eggshell, which in turn leads to the deposition of calcium carbonate crystals to form eggshells [
42,
43]. Compared with the CON group, the serum LOX concentration was lower in the LOT group, while this in the HOT group was greater compared with the CON group. However, the MOT and HOT group showed a higher CA level in the serum compared with the CON group. Estrogen (E) is a steroid hormone produced primarily by the ovaries that exerts its biological effects by binding to receptors and influencing the metabolism [
44]. Alkaline phosphatase (ALP) is a zinc-containing enzyme, activated by manganese, associated with eggshell calcification [
45]. In our study, the serum ALP level was significantly higher in the HOT group than in the CON group and significantly lower in the LOT group than in the MOT and HOT groups. The concentration of E in the serum had no changes in the experiment. Then, we examined the calcium and phosphorus levels in serum and eggshells and found that the calcium levels in eggshells in the MOT and HOT groups were significantly increased, which is consistent with the enzyme activity results. It is worth noting that uterine homeostasis underlies eggshell biomineralisation [
46]. Previous studies have reported that, with increasing age, the length of the uterine cilia becomes long and sparse and the number of tubular gland cells in the uterine tissue decreases [
47]. Histological analysis of the uterus in the present study suggested that the MOT and HOT groups delayed the decline of uterine tissues and that the uterine cilia length of these two groups was significantly lower than that of the CON and LOT groups. To further explore how organic micronutrients affect eggshell quality, we determined the expression of genes involved in eggshell deposition in uterine tissues. Previous studies have shown that, ovocleidin-17 (OC17) [
48], ovocalyxin-32 (OCX32) [
49], ovocleidin-116 (OC116) [
50], and CA2 are the main matrix proteins secreted from uterine fluid, which are involved in eggshell mineralization [
51,
52]. Similar to the change of CA concentration, the MOT group showed a higher CA2 level in the serum compared with the CON and LOT groups. Moreover, the organic trace elements supplement significantly increased the gene expression of OC116 and OCX32 in the oviduct, while the gene expression of OC17 did not change in all groups. Therefore, organic trace elements, as the substitute for inorganic micronutrients, added to the diets of laying hens in the late laying stage can improve eggshell quality by increasing the activity of relevant enzymes, maintaining the morphology of uterine tissues, and increasing the expression of relevant genes.
Deposition of trace elements in animal tissues and organs is often the main indicator for assessing the bio-efficiency of trace elements in feed [
53]. In this study, we measured the contents of trace element in the serum, yolk, shell and feces of laying hens to find low levels of organic trace elements substituted for inorganic trace elements added to the diets significantly reduced the excretion of Fe, Cu, Zn and Mn in the feces. Similar to the change of trace elements deposition patterns in feces, the LOT group had the lowest level of organic trace elements concentrations in the serum. In addition, the MOT and HOT groups had no effect on the Cu and Mn concentrations the in serum compared to the CON group. Furthermore, with the exception of the MOT group which reduced the Cu concentration in the eggshell, the other indicators showed that organic trace elements had no effect on yolk and eggshell deposition. The concentration of iron in the serum was reduced after organic trace elements were supplied in the diet. Notably, iron and copper existed in the serum in both the bound and free states [
20,
54]. The transferrin and ceruloplasmin in the serum reflected the binding activity of iron and copper, respectively [
55,
56]. Our study suggests that the MOT group significantly increased the activity of transferrin in the serum, while the ceruloplasmin concentration did not change in any of the groups, which may be due to the lower serum Fe concentration in the LOT, MOT and HOT groups than that in the CON group.