Role of zinc-methionine chelate on bone health and eggshell quality in late–phase laying hens

Abstract

This experiment aimed to assessing bone status and eggshell properties of laying hens at the end of a production cycle in response to inclusion of zinc-methionine chelate and zinc sulfate to diet. In a completely randomized design, 125 Leghorn laying hens (w36) 80 weeks old were divided into five treatment groups with five replications.Treatments were control (without zinc supplementation), zinc sulfate treatments (30 and 45 mg/kg), and zinc-methionine chelate (30 and 45 mg/kg). There was a significant increase in the feed intake in zinc sulfate and zinc -methionine chelate treatments campare the control treatment (P < 0.05). Tibia ash was significantly higher in treatments containing sulfate and zinc methionine chelate than in the control treatment (P < 0.05). The tibia's cortical thickness was improved in laying hens receiving 30 mg/kg zinc-methionine chelate. The supplementation of zinc could lead to an increase of mineralization in bone tissue. According to the results, zinc supplementation can improve feed intake, egg weight and the optimal zinc absorption in tissues. Zinc-methionine chelate boosts tibial mechanical properties without compromising eggshell quality of laying hens at the end of a production cycle.

KEYWORDS:

• Bone health
• Feed intake
• Eggshell
• late-phase of laying hens
• zinc-methionine chelate

Introduction

Some reports indicated that supplementation of Zn in the diets of old laying hens, could alleviate the negative effects of age on egg quality in late laying hens. The metabolism of laying hens and maintain effective egg production is highly dependent on the bioavailability of the minerals used in the formation of the eggshell. Bones, especially medullary bone, formed on the endosteal surface of the long bones of maturating hens, are primary mineral storage organs (Muszyński et al. 2022). However, information regarding compensating for the adverse effects of hen age on health and zinc deposition in the body is limited. Zinc is fundamental for growth and development, bone health, egg quality and immune function in laying hens (Yu et al. 2020). Cell growth, differentiation, reproduction, carbohydrate and protein metabolism involve Zn, which has a significant role in the isthmus during the formation of the eggshell membranes and in the magnum during the albumen deposition. Zn is considered a cofactor for carbonic anhydrase supplying carbonic ions during shell formation. Carbonic anhydrase inhibitors cause an enhancement in the production of shell-less eggs and a reduction in bicarbonate ion secretion. Zn deficiency results in economic loss in the poultry industry, reduction or cessation of egg production and poor hatchability (Ogbuewu and Mbajiorgu 2022). Dietary zinc supplementation improved egg weight, eggshell thickness and blood zinc concentrations in laying hens; however, it had no significant impact on eggshell weight and feed intake. Subgroup analysis revealed a significantly higher hen daily egg production in laying hens fed a diet supplemented with zinc at 100 mg/kg feed. The subgroup findings indicate that investigated moderators (zinc form, hen age at the start of the trial, supplementation duration, and inclusion level) affected aspects of the response variables. Compared with the controls, the observed significant improvement in egg production and quality traits in laying hens fed Zn-supplemented diets will assist in sustainable use of zinc and policy advancements in the egg production industry. It is recommended to use the regression function in determining zinc inclusion levels for egg production and optimal quality in laying hens (Ogbuewu and Mbajiorgu 2022).

Many authors have discussed the bioavailability and zinc supplement reserve, and the difference in absorption of organic and inorganic supplements is one of the most interesting discussions (Suttle 2010). Zinc can be supplemented in different forms, including organic and inorganic forms. The mineral zinc can be generated as sulfates, oxides, carbonates, or chlorides, while organic zinc is available as the metal ion complexes attached to organic molecules (Attia et al. 2013). Zinc methionine, zinc lysine and zinc threoninate are zinc amino acid chelates improving absorption and availability by reducing antagonism compared to the inorganic form, which may react with no absorbable compounds in the gastrointestinal tract (Neto et al. 2020). Generally, chelated organic elements are rare elements with higher tissue availability (Mondal et al. 2009). Mabe et al. (2003) found the basic differences between the mineral and organic zinc reserves on three predetermined occasions; however, regardless of the origin, the zinc, manganese, and copper supplements have increased the eggshell breaking strength in aged laying hens. In addition, it has been suggested that organically generated zinc has been prioritized in the cases in which the dietary levels of trace elements are pushed to the margins. Also, organic zinc (as the complex of metal amino acids) has been very effective in improving feed conversion ratio, broken eggs percentage, shell thickness, and Haugh Unit in the young laying hens.

Swiatkiewicz and Koreleski (2008) observed no differences in bone quality and eggshell thickness when replacing inorganic zinc with zinc amino acid complexes (Swiatkiewicz and Koreleski 2008; Gheisari et al. 2011). Various investigations have examined and compared the effects of different forms of mineral and organic zinc; however, there are controversial results in the scientific community regarding the origin of zinc supplementation when feeding laying hens. The disparity in the results of the investigations can be attributed to differences in the employed research models in terms of the strain of chicken used, hen age, experimental and basal diet composition, relative substitution, and the origin of the organic mineral used (Gheisari et al. 2011; Ogbuewu and Mbajiorgu 2022). On the other hand, dietary supplementation of organic Zn could alleviate the negative effects of age on egg quality characteristics in laying hens (Behjatian Esfahani et al. 2021). Since there is no consensus on whether zinc supplementation can improve zinc deposition in the body tissues and compensate for the adverse effects of hen age on health, prompted us to investigate mineral deposition in the body tissues and bone health of Leghorn laying hens in their last laying phase when the bone mineral reserves would be significantly required.

Materials and methods

Experimental design and measurements

The trial was conducted in a windowless house at Kashmar Research Station according to the Animal Care Committee of Islamic Azad University (Kashmar Branch, Kashmar, Iran) from March 2020 to July 2020. 125 80-weeks old commercial White Leghorn laying hens (W-36 Hy-Line strain) were used in a completely randomized design experiment. The hens were kept in wire cages 52 cm long, 34 cm wide, and 30 cm high on three floors with a maximum temperature of 25°C and a humidity of 56%. A daily photoperiod of 16:8 hrs lightness: darkness was applied throughout the experiment. There were two nipple drinkers in each cage, and the feed was freely available to the hens. A diet was formulated based on corn-soybean meal to supplement adequate levels of all nutrients as recommended by the Hyline international, 2020, except for zinc.

To conduct this research, the mineral supplement of zinc sulfate (5H₂O) with 34.12% pure zinc as the mineral base in the diet and - zinc-methionine chelate supplement (Zinpro Company) with 12% pure zinc as an organic source in the diet were used.

The diets were adjusted based on the requirements recommended for the strain (Hyline International, 2020) and after reviewing the research conducted in this field according to Table 1.

Table 1. Basal Diet Ingredients and Chemical Analysis.

Ingredients
Yellow Corn%	53
Soybean meal %	25
Calcium carbonate%	10
Vegetable oil%	1.40
Wheat bran%	6.08
Dicalcium phosphate%	2.20
Salt%	0.25
Sodium Bicarbonat %	0.15
^1Vitamin supplement%	0.25
^2Mineral supplement%	0.25
DL-Methionine%	0.14
Lysine%	0.06
Chemical analysis
Metabolizable energy (kcal / kg)	2810
Crude protein %	15
Calcium %	4.65
Available phosphorus %	0.40
Sodium %	0.18
Methionine%	0.32
Methionine-cysteine %	0.65
Lysine %	0.66
Arginine%	0.90
Threonine %	0.59
^3Zn (mg/kg)	33.58

¹ Vitamin supplement composition for each kilogram of diet includes: 3200000 U/kg of vitamin A, 1320000 U/kg of vitamin D3, 8000 U/kg of vitamin E, 1000 mg / kg of vitamin K3, 1000 mg / Kg of vitamin B1, 2200 mg / kg of vitamin B2, 3200 mg / kg of vitamin Kalban, 12000mg / kg of niacin, 1600 mg / kg of B6, 360 mg / kg of B9, 9 mg / kg of B12, 30 mg G / kg biotin, 44000 mg / kg choline, antioxidant 3000 mg / kg. ²Mineral supplement without Zn for each kilogram of basic diet includes: 79.01 mg of Mn, 3200 mg of copper, 480 mg of iodine, 88 mg of selenium, 16000 mg of iron. ³The amount of Zn in the basic diet measured in the laboratory.

The feed provided as a mash form. During the experiment, no antimicrobial drugs and vaccines were used. All vaccination program was applied before starting of experiment.

Hens were assigned to five dietary treatments with five replications. Each replication consisted of cages (five hens per cage), with 25 hens in each treatment. The hens under the experiment were first weighed, and those with the same weight were selected for treatment to standardize the experiment conditions. In addition, for adaptation, the control diet was fed to the hens for two weeks. These diets, which contained different levels of inorganic and organic chelating agents, were prepared as control (basal diet), a basal diet with 30 mg/Kg of zinc sulfate, a basal diet with 45 mg/Kg of zinc sulfate, a basal diet with 30 mg/Kg of zinc-methionine chelate and a basal diet with 45 mg/Kg of zinc-methionine chelate.Total zinc (mg/Kg) were 33.58,63.58,78.58,63.58 and 78.58 respectively. The amounts (percentage) of the zinc element provided in zinc sulfate supplement 9.06 and 13.58 and in zinc methionine chelate supplement 25 and 37.7 used for the experiment. The amount of basal diet analyzed in the laboratory was 33.58 mg/Kg. The Hy-Line strain need is 80 mg, according to the strain breeding guide (2020).

Measurements of bone mechanical properties

In brief, ten hens/treatments were selected and sacrificed by rapid cervical dislocation, and the right tibia was removed to analyze tibia breaking strength using an Instron device (Model H5KS, Tinius Olsen Company).

Measurements of mineral deposition in body tissues

Two hens were selected from each replication to evaluate the level of zinc reserves in body tissues at the end of the experiment, and 10 mL of blood was taken from each hen during slaughter. Before slaughter, all experimental hens were fasted for 10 h to improve the accuracy of blood factor measurements. Serum samples were centrifuged at 3000 rpm for 10 min to separate the serum and were stored at −20°C until analysis. Moreover, to evaluate the amount of zinc in the bones, the tibia was dried in an oven for 24 h and then placed in an oven at 550°C to prepare the ash. The blood sample, tibia, yolk, and liver of two replication samples were chosen and sent to the Laboratory to determine the zinc level. The zinc levels of the serum and other tissues were analyzed by Inductively coupled plasma-optical emission spectrometry (ICP-OES, Optima 8300, PerkinElmer, Waltham, MA, USA) according to the method suggested by Qiu et al. (2020).

Egg properties and performance

All hens were weighed as individually at the onset and end of the experiment. Mortality was calculated during the experiment. Egg weight was recorded daily from 80 to 92 weeks of age. All eggs were weighed by an electronic balance (Sartorius, accuracy 0.001). Feed intake was recorded weekly. The feed conversion ratio was calculated as the ratio of the mass of feed consumed per egg mass-produced. At the end of the trial, 2 egg/cage randomly were selected to determine of some quality parameters of egg After measurement of egg weight, the eggshell thickness was measured using an ESTG-1, ultrasonic eggshell thickness gauge. The shell thickness was measured using a thickness gauge (OSK13469) with an accuracy of 0.01 by measuring at three points (two ends and center in millimeters). In order to determine the fracture resistance of the shell, the digital egg shell force Gauge model-II was used in terms of the kilogram of force required to break the shell in a cross section of one square centimeter.

Statistical analysis

This experiment was conducted using simple CRD (Performance traits) and CRD with several observations in each replication using the following models. $\begin{array}{rl}Yij& =\mu +Ti+eij\end{array}$ $\begin{array}{rl}Yijk& =\mu +Ti+eijk+ϵijk\end{array}$where µ demonstrates the mean trait, Ti indicates the treatment effect, and ε ijk and eijk illustrate the effect of sampling and experimental error, respectively. The data were analyzed by using GLM in SAS statistical software. Duncan's test was utilized to compare the mean of various treatments when the mean difference was significant.

Results and discussion

Bone mechanical properties

The results regarding the effects of inorganic and organic zinc supplementation on tibia bone status are presented in Table 2. According to these results, no significant differences were observed between the experimental treatments (P > 0.05). However, the cortical thickness of the bone and deformation with 30 g of chelated organic zinc (T6) was significantly increased compared to that in the control and the other experimental treatments.

Table 2. Effect of feeding supplemental zinc sulfate and zinc methionine chelate on tibial mechanical properties at the end of the laying cycle.

Treatment	Cracking Force (N)	Work to Cracking (J)	Bend to breakpoint (mm)	Cortical thickness (mm)	Deformation (%)
control (basal diet)	212.25	223.30	0.15	2.00^b	1.39^a
basal diet with 30 mg/Kg of zinc sulfate	193.80	200.80	0.12	3.10^b	1.39^a
basal diet with 45 mg/Kg of zinc sulfate	148.55	179.30	0.15	3.85^b	1.46^ab
basal diet with 30 mg/Kg of zinc methionine chelate	131.60	197.95	0.19	5.94^a	1.55^b
basal diet with 45 mg/Kg of zinc methionine chelate zinc	212.30	224.80	0.14	1.47^ab	4.82^b
SEM*	28.81	33.56	0.02	0.21	0.05
P-Value	0.09	0.91	0.35	0.01	0.35

It might be assumed that the observed effect of treatments on egg deformation might have been due to changes in the mammillary layer and eggshell microstructure (Taylor et al. 2016). Due to the lack of information in this fregard, it is recommended to consider this issue in the next experiment.

Zinc deficiency (10 mg/kg) in young hens Lead to decrease of bone information has a limiting effect on bone formation. In broilers, an increase in the zinc level up to 100 mg/kg resulted to a significant improvement in bone strength and lowered injury risks. Manangi et al. (2015) reported no significant association between chelated trace minerals (CTM) diet and the mineral source on this parameter. However, they showed that the use of CTM resulted in the strongest bone strength values. Substitution of inorganic Zn and Mn with amino acid complexes in birds aged up to 70 weeks (Stefanello et al. 2014) had no significant effect on the tibia bone, and the authors speculated that bone properties in older birds might be less sensitive to the availability of Zn and Mn in the diet than eggshells.

In terms of tibia status, the treatment group receiving 30 mg/kg zinc methionine chelate showed a significant increase in the level of flexion and the thickness of the bone tissue (P < 0.05); therefore, it can be concluded that the hens in this treatment group had higher bone strength than the other treatments. Moreover, the results related to the shell thickness revealed that the eggshell thickness in the treatment group receiving 30 mg/kg zinc methionine chelate zinc was increased. However, this difference was not significant, which can be due to the different bioavailability of zinc in the shell and bone. The antagonist relationships between zinc and calcium and how the carbonic enzyme anhydrase works in response to these two elements must be investigated.

Cufadar et al. (2020) stated that different levels of zinc and their interactions had no effects on the tibia weight, tibia pressure, and tibia breaking strength as the mechanical parameters of the tibia (P > 0.05). Replacement of zinc and manganese oxides with micronutrient amino acid complexes also did not affect the physical and geometric parameters of the tibia and ash content in the tibia and toes (Swiatkiewicz and Koreleski 2008). Manangi et al. (2015) showed that mineral elements such as calcium, zinc, copper, and manganese led to the highest bone strength, although not statistically significant (P = 0.01). These results indicate other advantages of using zinc methionine chelate sources of zinc over inorganic sources.

Nevertheless, adding Zn has been declared to enhance Ca's utilization in hens and better the eggshell's qualitative parameters (Klecker et al. 2002). It has also been reported that zinc supplementation increases eggshell quality since it is an element of the carbonic anhydrase enzyme, supplying the carbonate ions during eggshell formation (Ogbuewu and Mbajiorgu 2022). Moreover, it has been indicated that the secretion of alkaline phosphatase is reduced under stress conditions, and this enzyme interacts with zinc in the bone's calcium storage (Rajabi and Torki 2021).

Mineral deposition in body tissues

Table 3 presents tibia ash and the zinc deposition in the aged laying hens’ body tissues. The measurement of tibia ash showed that bone mineralization was significantly higher in treatments containing sulfate and zinc methionine chelate than in the control treatment (P < 0.05). Bone ash percentage can be used as indicators of bone mineralization.

Table 3. Mineral Reserves in Tibia and the Body Tissues at late phase of laying hens.

	Tibia Ash (%)	Bone	Eggshell	Serum	Liver	Yolk
control (basal diet)	29.9^a	173/38^b	0.56	6.47^b	83.46^b	17.35^b
basal diet with 30 mg/Kg of zinc sulfate	34.7^b	216.45^a	0.63	7.34^a	81.63^ab	40.65^a
basal diet with 45 mg/Kg of zinc sulfate	31.7^ab	205.02^a	0.56	5.38^b	137.99^a	54.23^a
basal diet with 30 mg/Kg of zinc methionine chelate	31.7^ab	193.15^a	1.01	6.16^ab	74.39^ab	37.58^a
basal diet with 45 mg/Kg of zinc methionine chelate	32^ab	210.04^a	1.18	7.36^a	64.16^ab	65.27^a
SEM	0.7	3.56	0.20	0.20	5.11	3.08
P-Value	0.003	0.09	0.35	0.01	0.03	0.04

*per ppm/ml. ^a.b The means with dissimilar letters in each column are statistically significantly different (P<0.05), SEM: Standard Error of Mean

The zinc reserves was remarkably different between the yolk, tibia, liver, and serum treatments.Abedini et al. (2018) investigated three sources of zinc, namely zinc oxide, zinc oxide nanoparticles, methionine-zinc, and reported that zinc deposition in the tibia, liver, pancreas, eggs, and feces was significantly different between the treatments (P < 0.01). Zinc has a protective effect on pancreatic tissue against oxidative damage, and it may help the pancreas function properly, including secretion of digestive enzymes, thus improving the digestibility of nutrients (Onderci et al., 2003).

The measurement of zinc in the tibia showed that reserve was significantly higher in treatments containing sulfate and zinc methionine chelate than in the control treatment (P < 0.05). Li et al. (2019b), in a study on 576 Lingnan parent broiler hens aged 58 weeks, reported that the hens receiving 24–48 mg/kg zinc had a higher tibia breaking strength than those in the other experimental treatments. In addition, they reported that the increase in Zn-Met-induced calcium deposition might be due to the increase in zinc content in the serum and tissues, which is attributed to the rise in serum calcium and albumin concentrations and the concentration of calcium, albumin, and CaBP-D28k mRNA in the eggshell gland (ESG) Li et al. (2019c). Cufadar et al. (2020) examined three sources of zinc (zinc oxide, zinc proteins, and zinc oxide nanoparticles) at 20, 40, 60, 80, and 100 mg/kg levels in laying hens’ diet. They indicated that different sources of zinc had a significant effect on the zinc levels, and their reactions did not significantly affect the calcium and phosphorus content of the tibia (P > 0.05).

In addition, the zinc deposition in the yolk in sulfate and zinc methionine chelate supplement treatments had a significant increase compared to the control treatment (P < 0.05). Li et al. (2019a) compared three levels of 0, 40, and 80 mg of zinc sulfate and methionine-zinc. They stated that the treatment containing 80 mg/kg methionine-zinc led to a higher calcium deposition on the transverse section of the eggshell and had a higher zinc content in the yolk and serum (P < 0.05). Saleh et al. (2020) evaluated laying hens receiving a mixture of 0.5 and 1 g of sulfate and organic zinc, manganese, and copper compared to the control treatment and reported that the yolk's zinc was significantly increased compared to the control treatment (P > 0.05).

Based on the obtained results, there were no significant differences between the control treatment and the treatments containing sulfate and zinc methionine chelate supplements in terms of the shell's zinc deposition (P > 0.05). However, hens receiving 30 and 45 mg/kg Zinc methionine chelate zinc treatments had the highest amounts of zinc in their shells.

In the current study, serum zinc was enhanced in the treatment group receiving 45 mg/kg zinc methionine chelate, while there was no increase in the other treatments. Abd El-Hack et al. (2018) reported that the ZnO or Zn-Met supplement increased serum zinc compared to the control group, and there were no differences between the supplemental doses of zinc.

Diet zinc sources significantly affected the liver zinc deposition (P < 0.05). The zinc deposition in body tissues is a part of the digested and absorbed elements used in animal metabolism. Therefore, selecting a precise criterion is of great importance in estimating bioavailability. The bioavailability of different mineral combinations changes when added to Zinc methionine chelate or inorganic carriers (Ammerman et al. 1995).

Furthermore, it should be noted that there are some interactions between high-consumption and low-consumption mineral elements that can lead to antagonism or cooperation between them. However, similar results regarding the zinc reserves in body tissues both in sulfate and organic forms of zinc. Yu et al. (2020) studied the effects of two organic sources of amino acid-zinc and zinc sulfate at two concentrations of 35 and 70 mg/kg. They reported that the zinc content of the eggs was linearly increased for both sources.

Evaluating the minerals’ storage or deposition into chosen tissues is the utmost common variable evaluated in trace mineral RBV experiments (Richards et al. 2015). Zinc in chelate creates a soluble complex with zinc, and as a result, it is readily available to the animal. An alternative possibility is that organic Zn sources are absorbed through amino acid or peptide transport systems, leading to higher bioavailability and digestibility (Behjatian Esfahani et al. 2021). Consequently, higher bioavailability Zn probably has a significant role in enhancing the absorption of nutrients and digestion, improving the egg quality and performance of laying hens in the present experiment.

Egg properties and perfrmance

Feed intake and Egg properties of laying hens showed (Table 4) a significant difference in aged laying hens’ egg weight. Shell weight, thickness and egg shell breaking strength show that supplementation of Zn-sulfate and AA-Zn had no significant effect on the shell weight, thickness and breaking strength (P>0.05). Manangi et al. (2015) observed that birds fed chelated minerals at 40-10-40 (Zn-Cu-Mn) had a lower egg weight than those fed the chelates at 20-5-20 (P = 0.03). One of the factors affecting eggshell quality is micronutrients such as zinc and manganese. These minerals act as cofactors in enzymes involved in eggshell formation (Park et al. 2004). One of the most critical issues in the poultry industry is the decrease in eggshell quality of aged laying hens. Although most nutrition research on shell quality has focused on macronutrients such as calcium, phosphorus, and vitamin D₃, several enzymes associated with trace elements are calcified and involved in eggshell formation. These trace elements include zinc and manganese, which are responsible for producing carbonate and mucopolysaccharides as cofactors of metalloenzymes and play an important role in forming eggshells (Swiatkiewicz and Koreleski 2008). Based on the results of our previously published paper on higher egg production of layers, the feed intake, egg production (%), egg weight, egg mass, and conversion ratio were affected by the experimental treatments (P < 0.05). The results regarding the performance traits in this experiment are contrary to the findings of Stefanello et al. (2014), who reported that mineral trace elements in laying hens’ diet had no effects on the feed intake, feed conversion ratio, and egg production. Manangi et al. (2015) also reported no effects for rare organic minerals. In a study on 96 Hy-Line W-36 Leghorn hens, the effects of adding zinc to the diet of laying hens on their performance and behavior were evaluated for ten weeks using four treatments (40, 70, 100, and 130 mg/kg zinc per diet). The results indicated that egg production in the hens receiving 130 mg/kg zinc was significantly higher than in the hens receiving 40 and 70 mg/kg zinc (P < 0.05). Moreover, the hens receiving 130 mg/kg zinc had a lower feed intake than the other two treatments (P < 0.05).

Table 4. Comparison between the Increasing Levels of Zinc in terms of Feed intake and Egg shell Properties at the end of the laying cycle.

Treatment	Feed intake (g)	Egg weight (g)	Egg Shell percentage (%)	Thickness (mm)	Shell strength (Kg/cm3)
control (basal diet)	107.56^b	65.38^b	9.29	0.25	2.14
basal diet with 30 mg/Kg of zinc sulfate	108.68^ab	66.59^ab	9.81	0.27	2.23
basal diet with 45 mg/Kg of zinc sulfate	109.32^a	67.45^a	9.44	0.27	2.05
basal diet with 30 mg/Kg of zinc methionine chelate	109.02^a	68.36^a	9.69	0.28	2.16
basal diet with 45 mg/Kg of zinc methionine chelate -	108.92^a	68.40^a	9.52	0.27	2.21
SEM	0.27	0.39	0.25	0.01	0.29
P-value	<0.01	<0.01	<0.27	<0.45	<0.27

^a,b The means with dissimilar letters in each column are statistically significantly different (P<0.05), SEM: Standard Error Mean.

The cause of enhancement in egg production can be ascribed to the impact of zinc on egg production as a result of the impact of zinc in the albumin deposition in the formation of eggshell layers in the isthmus, the magnum, and eggshell formation in the uterus, and the impact of zinc on the enhancement in FSH, and LH hormones, progesterone, and estrogen (Park et al. 2004). Egg production improvement is probably because of the impact of zinc on the secretion of reproductive hormones. A considerable enhancement in the concentration was observed in the experiment on broiler estrogen and progesterone breeders by adding different zinc levels to the diet. Ovarian estrogen releases oviduct growth and enhances proteins, vitamins, blood calcium, fats, and nutrients required for egg formation.

The mean egg weight at the end of the experimental period was significantly higher in Treatments containing chelated organic zinc (P < 0.05) than the control treatment and other treatments (P < 0.05). Li et al. (2019c) stated that the treatment containing 80 mg/kg of zinc in the diet led to higher egg weight.

Conclusion

In conclusion, the current study’s results imply the importance of supplementary dietary Zn from different sources that improve feed intake in older laying hens. Zinc methionine chelate supplementation to these diets is also recommended as it results in improvements in bone mechanical properties, without compromising eggshell quality at the end of the laying period, indicating its positive effect in the overall maintenance of bone mineral reserves during the end egg laying cycle. The use of 30 mg/kg Zinc methionine chelate in the last phase of laying improved the cortical thickness of the bone in older laying hens. On the other hand, dietary supplementation of Zn could alleviate the negative effects of age on bone characteristics in laying hens. It is suggested that the additional expenses of purchasing supplementations be thoroughly investigated.

Authors’ contribution

Study concept and design: R.Vakili.

Acquisition of data: A.D. Niknia.

Analysis and interpretation of data: A.M. Tahmasbi.

Drafting of the manuscript: R. Vakili.

Critical revision of the manuscript for important intellectual content: R. Vakili.

Statistical analysis: A.D. Niknia.

Administrative, technical, and material support: Study supervision: R. Vakili.

Study supervision: R. Vakili.

Data availability statement (DAS)

The data that support the findings of this study are openly available in ‘figshare’ at doi:10.6084/m9.figshare.20326641.

Disclosure statement

No potential conflict of interest was reported by the author(s).