
==== Front
Vet Med Sci
Vet Med Sci
10.1002/(ISSN)2053-1095
VMS3
Veterinary Medicine and Science
2053-1095
John Wiley and Sons Inc. Hoboken

10.1002/vms3.70035
VMS370035
Original Article
POULTRY
Original Article
Determining the optimal level and the effect of different zinc sources on performance, egg quality and the immune system of laying hens at the end of the production period
ALIREZAEI et al.
Alirezaei Shahraki Parva 1
Kheiri Farshid https://orcid.org/0000-0001-5517-4524
1 farshid_kheiri@yahoo.com
f.kheiri@gmail.com

Amanlou Hamid 2
Faghani Mostafa https://orcid.org/0000-0003-1215-419X
1
Jalali Sayed Mohammad Ali https://orcid.org/0000-0002-9261-4776
3
1 Department of Animal Science Shahrekord Branch Islamic Azad University Shahrekord Iran
2 Department of Animal Science, Faculty of Agriculture University of Zanjan Zanjan Iran
3 Research Center of Nutrition and Organic Products (RCNOP) Shahrekord Branch, Islamic Azad University Shahrekord Iran
* Correspondence
Farshid Kheiri, Department of Animal Science, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Email: farshid_kheiri@yahoo.com; f.kheiri@gmail.com

11 9 2024
9 2024
10 5 10.1002/vms3.v10.5 e7003511 7 2024
15 2 2024
23 8 2024
© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Background

Trace elements, such as zinc, magnesium and copper, are essential for improving the performance and health of broiler breeders and the development of chicken embryos. These elements are integral to various proteins involved in metabolism, hormone secretion and the immune system, necessitating their inclusion in small amounts in poultry diets.

Objectives

This study aimed to determine the optimal level and effect of different zinc sources on performance, egg quality and the immune system of laying hens at the end of the production period.

Methods

The experiment involved 520 Lohmann LSL laying hens, aged 80 weeks, divided into 13 treatments with 5 replications and 8 birds per replication. The hens were fed diets supplemented with 40, 60 and 80 mg/kg of zinc from various sources: mineral zinc oxide, mineral zinc sulphate, organic zinc chelated with glycine and organic zinc chelated with an organic acid. Key parameters measured included body weight, egg weight and immune response.

Results

The basal diet contained 63.58 mg/kg of zinc, with the requirement per the Lohmann LSL guideline being 80 mg/kg. Zinc supplementation significantly increased body weight in the second month, with 80 mg/kg being the optimal dose. Zinc oxide notably increased egg weight compared to the control. The hens utilized zinc from all sources, resulting in weight gain and improved parameters such as egg quality. Immune parameters were also positively influenced by zinc supplementation.

Conclusions

Zinc supplementation at appropriate levels enhances the performance and egg quality of laying hens, particularly at the end of the production period. It improves bioavailability, enriches eggs and mitigates age‐related declines in productivity.

Zinc supplementation at 80 mg/kg, especially from zinc oxide, optimizes body weight and egg quality in laying hens at the end of their production period. Enhanced zinc intake significantly improves immune response and overall performance, with 80 mg/kg being the optimal level. Organic and inorganic zinc sources both positively affect egg quality and productivity, demonstrating the importance of adequate zinc in poultry diets.

diet
egg production
egg quality
performance
zinc
source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:11.09.2024
Alirezaei Shahraki, P. , Kheiri, F. , Amanlou, H. , Faghani, M. , & Jalali, S. M. A. (2024). Determining the optimal level and the effect of different zinc sources on performance, egg quality and the immune system of laying hens at the end of the production period. Veterinary Medicine and Science, 10 , e70035. 10.1002/vms3.70035
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pmc1 INTRODUCTION

The growth of the population and advancements in science and technology have led to a significant increase in egg production through poultry farming, accompanied by a trend of declining egg quality. Trace elements, such as zinc, magnesium and copper, are essential components for improving broiler breeder performance and chicken embryo development (Abd El‐Hack et al., 2017; Ay et al., 2023; Behjatian Esfahani et al., 2021; Ekizoğlu et al., 2020; Ogbuewu & Mbajiorgu, 2022; Ülger et al., 2023). These trace elements are constituents of several proteins involved in intermediary metabolism, hormone secretion and the immune system (Chand et al., 2020; Huang et al., 2020) and are required in small amounts in the diet. Deficiencies in Zn, Mn and Cu have well‐documented effects on breeder performance and embryo development, leading to low egg production, reduced shell strength, poor hatchability, reduced fertility, increased bone abnormalities in embryos, poor plumage and dermatitis (Chen et al., 2017; Ülger & Mahmood, 2023; Yu et al., 2020; Zhang et al., 2020).

The mineral concentration of conventional feeds can fluctuate considerably due to the trace element composition of soils, geographic area, climate and yield. Therefore, nutritionists typically include a wide margin of safety for trace minerals in poultry formulations to ensure proper bird growth and maximize performance (Abbasi et al., 2022; Aghaei et al., 2017; Kaya et al., 2001; Kheiri & Toghyani, 2009). Zinc is one of the essential trace elements required for normal chicken function, including physical growth, muscle development (Sun et al., 2012), reproduction and egg production (Fan et al., 2022). Zinc plays a crucial role in cell division and the production of healthy sperm. It is vital for testosterone metabolism, testicular growth, sperm production, motility and count, as well as the reduction of excess oestrogen in male reproductive tissues (Gholizadeh et al., 2022; Kidd et al., 1993).

There is substantial evidence that trace elements can affect shell quality through their catalytic properties as key enzymes involved in membrane and eggshell formation processes, or by directly interacting with calcite crystals during eggshell formation. Zinc is a component of the enzyme carbonic anhydrase; a deficiency of this enzyme can reduce bicarbonate ion secretion, ultimately leading to decreased eggshell quality (Gholizadeh et al., 2022; Hudson et al., 2005). To counteract these problems, zinc supplementation is often added to poultry diets.

Zinc supplementation is commonly used in broiler and layer diets because most dietary ingredients are zinc‐deficient. Zinc is a component of over 300 enzymes involved in the metabolism of energy, proteins, carbohydrates and nucleic acids (Zaghari et al., 2015). For instance, zinc acts as a cofactor for the enzyme carbonic anhydrase, which is essential for calcium carbonate deposition during eggshell formation (Zhang et al., 2017). Additionally, zinc plays a significant role in albumin deposition in the magnum and the production of eggshell membranes in the isthmus (El‐Hack et al., 2018). Zinc is also part of the structure of RNA and DNA polymerase enzymes and is required for the activity of hormones, such as glucagon, insulin and growth hormone, playing an important role in immunity and the metabolism of vitamins such as vitamin A (Ibs & Rink, 2003; Marek et al., 2017; Rahman et al., 2002).

Given the sensitivity of zinc levels in the body and the factors that can lead to zinc deficiency, dietary zinc deficiency is associated with stunted growth, decreased egg production and quality and increased mortality in poultry (Naz et al., 2016). Therefore, it is crucial to provide zinc in the diet at levels that meet the birds’ needs. Previous studies have shown that zinc intake from organic and mineral sources affects bird performance and egg production differently (Abedini et al., 2018; Jahanian & Rasouli, 2015; Jahanian & Yaghoubi, 2010; Min et al., 2019). Organic zinc sources, such as zinc methionine or zinc proteinate, are expected to lead to increased bioavailability and more notable positive effects on laying hen performance, egg quality and immune system function compared to inorganic zinc sources (Bao et al., 2007; Lima et al., 2000; Park et al., 2004; Qiao et al., 2008; Sandoval et al., 1997).

Inorganic zinc is often used as oxides and sulphates; however, most zinc supplements in broiler and layer diets are in oxide form, which is less bioavailable than the sulphate form. Studies have shown that organic zinc sources are more bioavailable than inorganic zinc sources (Bahakaim et al., 2014; Cao et al., 2000; Cao et al., 2002; Gayathri & Panda, 2018; Paik, 2001; Stefanello et al., 2014). For example, El‐Hack et al. (2020) concluded that supplementing 75 mg/kg of zinc from a mineral source increases antioxidant activity and performance in laying hens (El‐Hack et al., 2020). However, according to the National Research Council (1994), the optimal level of zinc in poultry diets is 40 mg/kg, regardless of the source. Despite numerous studies, none has precisely determined the optimal level of zinc intake in laying hen diets that would affect performance, egg quality and production.

The hypothesis of this study is that different sources and levels of zinc supplementation will differentially affect the performance, egg quality and immune function of laying hens at the end of their production period. Investigating this is crucial as it addresses the nutritional needs of ageing laying hens, which can enhance their productivity and overall health, thereby improving economic outcomes and animal welfare.

The overall objective of this study was to determine the optimal level and effect of different zinc sources on performance, egg quality and the immune system of laying hens at the end of the production period.

By addressing these gaps, this study aims to provide insights into the effective management of zinc supplementation in poultry diets, enhancing the productivity and health of laying hens, particularly towards the end of their production cycle.

2 MATERIALS AND METHODS

The experimental diets were formulated and produced in accordance with established nutritional guidelines, ensuring variations in zinc levels and sources. The diet composition encompassed essential nutrients, with specific attention to zinc content, in order to assess its influence on laying hen performance, egg quality and physiological parameters. The laying hens, initially weighed and randomly assigned to treatment groups, were subjected to controlled feeding conditions throughout the production period.

To accomplish this experiment, 520 laying hens of the Lohmann LSL strain at the age of 80 weeks were used, which were divided into 13 treatments and 5 replications and 8 birds in each repetition. Experimental treatments include as follows: (1) a basic diet (control) containing the mineral supplement without zinc; (2) control + 40 mg/kg mineral zinc oxide supplement; (3) control + 60 mg/kg mineral zinc oxide supplement; (4) control + 80 mg/kg mineral zinc oxide supplement; (5) control + 40 mg/kg mineral zinc sulphate supplement; (6) control + 60 mg/kg mineral zinc sulphate supplement; (7) control + 80 mg/kg mineral zinc sulphate supplement; (8) control + 40 mg/kg organic zinc supplement chelated with glycine amino acid; (9) control + 60 mg/kg organic zinc supplement chelated with the glycine amino acid; (10) control + 80 mg/kg organic zinc supplement chelated with the glycine amino acid; (11) control + 40 mg/kg organic zinc supplement chelated with the organic acid; (12) control + 60 mg/kg organic zinc supplement chelated with the organic acid and (13) control + 80 mg/kg organic zinc supplement chelated with the organic acid. The amount of zinc in the basic diet analysed in the laboratory was 63.58 mg/kg, and the strain's requirement, according to the Lohmann LSL strain breeding guideline is estimated to be 80 mg/kg (Lohmann Tierzucht, 2016) The mineral premix does not have any source of zinc. Different kinds of zinc sources were added to the basal diet; because chelated zinc was added to the basal diet, the usage of synthetic methionine has been decreased by the addition of zinc methionine; thus, all dietary treatments were equal in terms of crude protein and total sulphur amino acids. The experimental diet was prepared in the form of the mesh and based on the recommendation for Lohmann LSL strain breeding (Table 1).

TABLE 1 Ingredients and composition of the experimental diets.

Ingredient (g/kg)	Masse (g/kg)	
Corn	602.15	
Soybean meal	240	
Soybean oil	8	
Wheat bran	40	
Carbonate calcium	83.3	
Dicalcium phosphate	14.9	
NaHCO3	1.6	
Salt	2.4	
Mineral premix a	2.5	
Vitamin premix b	2.5	
dl‐Methionine	1.9	
Colin chloride	0.75	
Nutrient composition		
Metabolizable energy (kcal/kg)	2690	
Crude protein (%)	16.09	
Lysine (%)	0.79	
Methionine + cysteine (%)	0.7	
Threonine (%)	0.61	
Calcium (%)	3.75	
Available phosphorous (%)	0.37	
Potassium (%)	0.70	
a The mineral premix supplied the following per kilogramme of feed: Mn: 123.3 mg; Fe: 77 mg; Zn: 0 mg; Se: 0.42 mg; Cu: 7.7 mg; Iodine: 2.38 mg.

b The vitamin premix supplied the following per kilogramme of feed: vitamin A: 12,320 IU; vitamin D3: 4620 IU; vitamin E: 23.1 IU; vitamin K3: 3.08 mg; vitamin B1: 2.38 mg; vitamin B2: 7.7 mg; niacin: 39.2 mg; pantothenic acid: 9.24 mg; pyridoxine: 4.62 mg; folic acid: 0.84 mg; cobalamin: 0.031 mg; biotin: 0.08 mg; choline: 154 mg; antioxidant: 1.4 mg.

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The body weight at the beginning of each month, the average feed consumption and the conversion factor were measured and calculated weekly. The eggs produced in each repetition were counted and weighed daily. The weight and floating weight of the eggs were also calculated daily in each repetition. To evaluate the qualitative traits (white weight, yolk weight, height and diameter, shell weight and shell thickness), four eggs from each replication were randomly selected and evaluated. The shell thickness was measured using a digital calliper. Then the shell was washed and dried at 40°C for 24 h and weighed.

The quantification of immunoglobulins IgG and IgM in the blood serum of avian subjects was carried out with meticulous details. The chosen method involved the utilization of the Adit commercial kit, as outlined by Vieira (2008). This commercial kit employs a calorimetric method, relying on the principles of enzyme‐linked immunosorbent assay. Simultaneously, the investigation delved into the antioxidant status of the subjects by evaluating the activity of the serum superoxide dismutase (SOD) enzyme. This was accomplished through the application of a diagnostic kit, specifically Randsel SOD from Randox.

2.1 Statistical analysis

The resulting data were statistically analysed using the GLM procedure 9.1 SAS statistical software in the form of a completely random design. Tukey's test was used to compare the mean of the treatments at a significance level of 0.05.

3 RESULTS

The effects of adding zinc to the diets of laying hens on their body weight are presented in Table 2. It was found that the levels of zinc added to the diets significantly increased the body weight of the laying hens in the second month of feeding, with the optimal dose of zinc being 80 mg/kg.

TABLE 2 Effect of the different levels of zinc on body weight (kg) at the different months of production.

Treatments	First month	Second month	
C	1.50	1.52b	
ZnO‐40	1.53	1.56ab	
ZnO‐60	1.56	1.52b	
ZnO‐80	1.51	1.53ab	
ZnS‐40	1.54	1.52b	
ZnS‐60	1.55	1.59ab	
ZnS‐80	1.55	1.64a	
ZnG‐40	1.56	1.59ab	
ZnG‐60	1.50	1.57ab	
ZnG‐80	1.54	1.62ab	
ZnOA‐40	1.56	1.61ab	
ZnOA‐60	1.53	1.60ab	
ZnOA‐80	1.54	1.61ab	
SEM	0.069	0.78	
p value	0.87	0.02	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a, b) in each effect are statistically significant (p ˂ 0.05).

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Table 3 shows the effect of different levels of zinc intake on the feed intake of laying hens during the 8 weeks of experimentation. Significant variations were observed among different weeks. The feed conversion ratio (FCR) of laying hens, as presented in Table 4, showed significant variations only during the sixth week. The FCRs generally ranged from 2.17 to 2.41.

TABLE 3 Effect of different levels of zinc on feed intake (g/day/bird) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	117.85a	109.21b	101.14ab	102.43b	99.92b	101.97b	104.89b	104.30b	104.61b	
ZnO‐40	119.46a	108.71b	103.10ab	111.24ab	110.54ab	111.97ab	109.69ab	111.22ab	110.24a	
ZnO‐60	110.71b	108.49b	100.01ab	104.27b	115.51a	115.49a	114.11a	118.85a	110.51a	
ZnO‐80	115.17a	111.36ab	120.33a	116.10a	88.25b	118.37a	113.59a	111.49ab	111.58a	
ZnS‐40	110.36b	105.53c	105.81ab	100.30b	92.10b	109.28ab	106.23b	114.88a	105.16b	
ZnS‐60	117.14a	112.82ab	101.08ab	113.33ab	114.97a	109.10ab	109.87ab	107.66b	109.89ab	
ZnS‐80	113.01ab	112.98ab	109.95ab	116.67a	113.83a	104.29b	109.20ab	108.92b	110.53a	
ZnG‐40	111.31ab	114.27ab	114.63a	111.41ab	118.22a	113.53a	109.70ab	111.35ab	112.67a	
ZnG‐60	112.24ab	111.36b	113.40a	117.03a	91.97b	104.76b	115.09a	103.32b	108.24ab	
ZnG‐80	109.19b	118.36a	110.15ab	114.64a	110.20ab	104.65b	116.62a	112.56ab	111.64a	
ZnOA‐40	118.39a	115.45a	104.36ab	113.75ab	117.48a	114.62a	103.30b	98.39c	110.22a	
ZnOA‐60	117.29a	116.47a	110.68ab	111.76ab	115.08a	113.69a	108.37ab	104.44b	111.72a	
ZnOA‐80	117.85a	117.56a	90.49b	105.03b	108.59b	117.59a	116.96a	110.63ab	110.08a	
SEM	6.47	8.39	4.30	9.59	9.01	7.66	7.46	4.24	1.27	
p value	0.03	0.007	0.01	0.008	0.001	0.001	0.001	0.001	0.008	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–c) in each effect are statistically significant (p ˂ 0.05).

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TABLE 4 Effect of different levels of zinc on feed conversion ratio at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	2.34	2.28	2.36	2.38	2.36	2.38a	2.40	2.41	2.35	
ZnO‐40	2.18	2.23	2.24	2.32	2.30	2.34a	2.32	2.36	2.32	
ZnO‐60	2.27	2.30	2.26	2.30	2.32	2.32a	2.33	2.35	2.30	
ZnO‐80	2.29	2.35	2.25	2.29	2.34	2.35a	2.35	2.38	2.31	
ZnS‐40	2.30	2.34	2.32	2.27	2.33	2.35a	2.34	2.37	2.32	
ZnS‐60	2.19	2.31	2.31	2.33	2.35	2.30a	2.36	2.36	2.32	
ZnS‐80	2.17	2.25	2.28	2.30	2.30	2.13b	2.35	2.35	2.30	
ZnG‐40	2.20	2.27	2.22	2.32	2.31	2.32a	2.33	2.34	2.33	
ZnG‐60	2.25	2.33	2.26	2.31	2.29	2.31a	2.34	2.37	2.31	
ZnG‐80	2.27	2.35	2.29	2.26	2.28	2.14b	2.32	2.36	2.32	
ZnOA‐40	2.23	2.24	2.21	2.28	2.33	2.14b	2.31	2.38	2.30	
ZnOA‐60	2.28	2.22	2.27	2.27	2.31	2.30a	2.33	2.35	2.30	
ZnOA‐80	2.17	2.29	2.25	2.29	2.30	2.30a	2.32	2.37	2.31	
SEM	0.07	0.09	0.04	0.05	0.03	0.06	0.08	0.05	0.06	
p value	0.18	0.23	0.14	0.32	0.10	0.15	0.09	0.08	0.12	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a, b) in each effect are statistically significant (p ˂ 0.05).

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From the analysis of Table 5, it appears that different levels of zinc in the diet resulted in a slight increase in egg production, with variations by week. Table 6 demonstrates significant variations in egg weights across different production weeks, ranging from 56.70 to 64.15 g.

TABLE 5 Effect of different levels of zinc on egg production (%) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	90.15	91.42	90.25	89.67	89.65	88.45b	88.04b	87.82b	89.14	
ZnO‐40	91.57	90.58	92.17	90.14	92.44	93.53a	91.68ab	91.68ab	91.27	
ZnO‐60	90.42	92.12	91.26	92.35	92.78	92.49ab	92.47ab	90.46ab	91.39	
ZnO‐80	92.89	89.49	90.65	89.58	90.47	90.67ab	89.58ab	92.37a	90.12	
ZnS‐40	89.36	90.16	91.93	91.94	91.36	92.47ab	90.14ab	89.46ab	90.39	
ZnS‐60	91.12	91.57	89.79	92.59	92.57	91.63ab	89.79ab	90.57ab	90.64	
ZnS‐80	92.67	92.78	93.40	91.86	90.79	93.45a	92.44ab	91.32ab	91.77	
ZnG‐40	90.57	91.69	89.14	93.76	92.24	93.37a	93.76a	93.11a	91.72	
ZnG‐60	91.61	89.76	92.47	92.57	92.37	92.58ab	93.37a	92.89a	91.62	
ZnG‐80	90.23	93.42	91.17	90.88	91.58	93.12a	91.28ab	93.95a	91.51	
ZnOA‐40	89.97	92.41	90.47	92.56	92.69	93.89a	92.27ab	93.56a	91.89	
ZnOA‐60	91.58	91.17	90.33	91.17	90.69	92.67ab	93.97a	92.49a	91.28	
ZnOA‐80	92.14	90.78	92.95	92.08	91.78	92.89ab	92.78a	93.18a	91.79	
SEM	2.76	3.23	1.65	2.89	3.09	3.18	2.15	2.80	1.47	
p value	0.28	0.34	0.09	0.16	0.07	0.02	0.01	0.02	0.26	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a, b) in each effect are statistically significant (p ˂ 0.05).

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TABLE 6 Effect of different levels of zinc on egg buoyancy weight (g) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	56.70b	58.11	62.71ab	58.85c	59.93	59.63c	61.45	59.04ab	59.55b	
ZnO‐40	58.07b	61.53	60.97ab	61.13abc	60.90	59.37c	57.45	57.41ab	59.6b	
ZnO‐60	59.01b	61.13	61.87ab	60.91abc	62.93	60.61bc	58.36	58.29ab	60.39ab	
ZnO‐80	59.17b	58.14	58.49b	59.17bc	63.15	61.31abc	59.47	57.93ab	59.53b	
ZnS‐40	58.94b	59.16	58.85b	58.04c	62.91	61.68abc	59.18	53.64b	59.05b	
ZnS‐60	58.56b	60.97	61.29ab	59.39bc	59.76	61.85abc	61.26	58.22ab	60.16ab	
ZnS‐80	58.84b	58.98	59.89ab	61.35abc	62.55	64.32a	61.92	60.77ab	61.08ab	
ZnG‐40	59.60ab	61.58	61.47ab	62.93ab	60.82	61.75abc	61.63	60.57ab	61.29a	
ZnG‐60	63.64a	60.65	64.15a	64.02a	62.86	62.26abc	64.51	61.01ab	62.88a	
ZnG‐80	60.81ab	60.01	61.12ab	61.35abc	62.87	62.85ab	61.20	62.22a	61.55a	
ZnOA‐40	59.92ab	58.85	62.82ab	60.09abc	61.53	63.12ab	61.24	59.44ab	60.88ab	
ZnOA‐60	59.28b	58.07	61.59ab	60.29abc	60.53	61.92abc	58.69	61.65ab	60.25ab	
ZnOA‐80	58.46b	58.46	60.93ab	60.94abc	62.72	60.61bc	61.44	59.31ab	60.36ab	
SEM	4.74	3.57	3.31	3.69	4.34	4.18	4.29	3.37	3.14	
p value	0.02	0.18	0.01	0.01	0.09	0.02	0.14	0.03	0.04	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–c) in each effect are statistically significant (p ˂ 0.05).

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Table 7 indicates that egg albumen weight was significantly increased at Week 8 for all zinc doses tested. Table 8 shows significant variations in yolk weights during different production weeks, with weights ranging from 15.50 to 18.22 g.

TABLE 7 Effect of different levels of zinc on egg albumen weight (g) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	35.97a	32.65ab	36.03ab	36.54	36.35	35.03bc	37.34	34.63	35.38	
ZnO‐40	32.67abc	33.54ab	34.15ab	34.81	34.72	33.83c	32.36	32.14	33.51	
ZnO‐60	34.40abc	34.28ab	35.61ab	33.94	35.89	35.51bc	32.63	31.34	34.17	
ZnO‐80	34.09abc	32.27ab	32.49b	33.58	36.52	36.21bc	33.70	33.71	34.21	
ZnS‐40	33.09abc	36.50a	32.87b	33.13	35.46	35.49bc	32.71	33.64	34.09	
ZnS‐60	33.44abc	34.34ab	35.77ab	34.84	33.56	36.64ab	35.50	34.1	34.50	
ZnS‐80	34.02abc	32.01ab	33.66ab	36.25	35.29	38.83a	36.02	34.13	34.99	
ZnG‐40	34.24abc	34.69ab	35.72ab	36.85	34.47	36.53ab	36.11	35.07	35.40	
ZnG‐60	31.73bc	31.14b	37.12a	36.12	34.25	34.88bc	35.56	34.81	34.05	
ZnG‐80	34.29abc	33.48ab	34.52ab	35.74	35.42	37.09ab	35.39	35.74	35.32	
ZnOA‐40	34.80ab	32.75ab	37.12a	34.03	34.84	36.97ab	36.47	31.45	34.80	
ZnOA‐60	31.04c	32.44ab	35.33ab	36.92	33.75	34.92bc	33.10	35.69	34.39	
ZnOA‐80	32.76abc	32.76ab	34.95ab	34.86	35.60	34.53bc	35.61	34.13	34.26	
SEM	1.07	1.44	1.13	2.14	1.02	0.78	1.52	1.36	0.5	
p value	0.04	0.045	0.04	0.46	0.56	0.001	0.3	0.34	0.19	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–c) in each effect are statistically significant (p ˂ 0.05).

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TABLE 8 Effect of different levels of zinc on egg yolk weight (g) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	19.02a	16.32ab	17.26a	17.64a	17.15	17.81a	17.06a	16.87	17.41ab	
ZnO‐40	18.22abc	16.77ab	17.00ab	17.33ab	16.72	17.26ab	16.29ab	16.86	17.07ab	
ZnO‐60	17.49bcde	16.21ab	16.44ab	16.98abc	16.99	17.06ab	15.72b	16.57	16.70ab	
ZnO‐80	16.97cde	17.06a	15.94abc	17.03abc	16.78	16.63ab	16.50ab	16.88	16.78ab	
ZnS‐40	17.58bcde	16.53ab	15.78bc	15.87cde	16.57	17.62ab	17.09a	16.08	16.61ab	
ZnS‐60	16.41de	17.19a	15.68bc	15.50e	16.41	16.45c	16.81ab	15.71	16.28b	
ZnS‐80	17.85abcd	15.79ab	16.02abc	16.73abcd	16.75	16.94ab	16.70ab	17.64	16.79ab	
ZnG‐40	17.69abcde	16.62ab	15.72bc	16.73abcd	16.10	17.19ab	16.66ab	17.29	17.88a	
ZnG‐60	17.41bcde	17.10a	14.89c	15.50de	16.93	16.83ab	16.66ab	16.49	16.57ab	
ZnG‐80	18.22abc	15.64ab	16.09abc	16.28bcde	16.73	16.88ab	16.03ab	17.08	16.68ab	
ZnOA‐40	16.91cde	15.18b	16.13abc	16.11cde	16.47	17.19ab	16.80ab	16.50	16.45ab	
ZnOA‐60	18.73ab	15.22b	16.12abc	17.07abc	16.51	17.23ab	16.74ab	17.49	16.94ab	
ZnOA‐80	16.31e	16.31ab	15.89abc	16.14abcd	16.82	17.06ab	16.92a	16.26	16.52ab	
SEM	1.09	1.54	1.18	0.97	0.87	1.27	1.47	1.56	0.41	
p value	0.01	0.01	0.03	0.01	0.23	0.001	0.03	0.15	0.041	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–e) in each effect are statistically significant (p ˂ 0.05).

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Yolk height varied significantly during the production weeks as shown in Table 9, with higher heights recorded from the sixth week onwards. Table 10 reveals significant variations in yolk diameter from Weeks 1 to 5, with no significant variation noted from the sixth to the eighth week.

TABLE 9 Effect of different levels of zinc on yolk height (mm) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	14.01d	15.00bcd	15.31bc	16.46a	15.88ab	16.17	16.70a	16.46	15.73	
ZnO‐40	14.79cd	14.79cd	15.60abc	16.27ab	15.48ab	1612	16.36ab	16.19	15.69	
ZnO‐60	14.94cd	14.48d	15.38abc	15.92abcd	15.68ab	16.18	16.12ab	16.86	15.66	
ZnO‐80	15.39bc	14.46d	16.32a	16.20ab	15.89ab	16.34	15.66b	16.26	15.82	
ZnS‐40	15.46bc	15.80abcd	15.64abc	15.72abcd	16.36a	16.57	15.82ab	16.10	15.95	
ZnS‐60	16.65a	15.69abcd	15.50abc	15.49cde	15.10b	16.28	16.06ab	16.33	15.90	
ZnS‐80	15.12bcd	15.48abcd	15.53abc	15.75abcd	16.12ab	16.41	16.04ab	16.21	15.87	
ZnG‐40	14.47cd	16.13abc	15.56abc	15.75abcd	15.69ab	16.81	15.69ab	16.69	15.81	
ZnG‐60	14.10d	16.35ab	15.96ab	15.91abcd	16.22a	16.39	16.11ab	16.64	15.94	
ZnG‐80	15.16bcd	15.83abcd	15.66abc	15.95abc	15.83ab	16.25	16.46ab	16.04	15.82	
ZnOA‐40	14.77cd	15.86abcd	14.79c	15.13cde	16.35a	16.39	16.29ab	16.50	15.73	
ZnOA‐60	15.11bcd	16.83a	15.44abc	15.05de	16.25a	16.80	15.89ab	16.48	16.04	
ZnOA‐80	16.16ab	16.16abc	15.47abc	14.66e	15.95ab	16.37	16.29ab	16.43	15.87	
SEM	0.78	0.89	0.67	1.20	1.43	0.90	1.61	1.21	0.12	
p value	0.01	0.03	0.04	0.001	0.01	0.23	0.01	0.20	0.59	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–e) in each effect are statistically significant (p ˂ 0.05).

John Wiley & Sons, Ltd.

TABLE 10 Effect of different levels of zinc on yolk diameter (mm) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	41.89a	40.11a	41.91	41.21ab	40.65a	40.96	41.32	40.66	41.08a	
ZnO‐40	40.79a	41.31a	41.43	40.75abcd	40.76a	40.57	41.36	40.27	41.02ab	
ZnO‐60	39.98ab	40.50a	41.72	41.00abc	40.50a	40.41	39.51	41.07	40.56abc	
ZnO‐80	38.66abc	40.36a	41.39	41.74a	40.96a	41.56	41.74	41.26	40.92ab	
ZnS‐40	38.93abc	38.51abc	39.62	40.04bcd	39.82a	40.50	40.38	40.47	39.80cde	
ZnS‐60	39.35abc	36.46c	40.81	39.89bcd	35.83b	40.35	41.12	40.29	39.02e	
ZnS‐80	40.08ab	36.12c	39.34	40.49abcd	41.15a	40.29	41.15	40.33	40.00bcd	
ZnG‐40	41.03a	37.91bc	40.55	39.77cd	40.73a	40.46	39.59	40.46	40.09abcd	
ZnG‐60	41.72a	37.69bc	40.74	39.42d	40.09a	41.17	40.84	41.17	40.28abcd	
ZnG‐80	40.76a	37.13c	39.01	39.96bcd	39.83a	41.37	40.26	42.13	40.02bcde	
ZnOA‐40	36.45c	35.62c	39.78	40.90abc	40.50a	40.60	41.93	40.13	39.48de	
ZnOA‐60	40.23ab	36.12c	39.86	41.30ab	40.27a	40.78	41.25	41.50	40.17abcd	
ZnOA‐80	37.01bc	37.01c	39.57	39.97bcd	40.76a	40.37	40.27	40.84	39.34de	
SEM	0.1	0.92	1.7	0.43	0.98	0.55	0.7	0.71	0.32	
p value	0.02	0.001	0.46	0.003	0.041	0.86	0.46	0.76	0.001	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–e) in each effect are statistically significant (p ˂ 0.05).

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Eggshell weights, as shown in Table 11, varied significantly over the weeks except at Week 5, where no significant variation was observed for the different zinc doses.

TABLE 11 Effect of different levels of zinc on egg shell weight (g) at different weeks of production.

Treatments	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Total	
C	7.53ab	8.34a	7.84abc	8.44a	8.03	8.34a	8.24ab	8.34ab	8.13a	
ZnO‐40	7.14abc	8.22ab	8.11abc	7.70bc	7.88	7.75b	8.34ab	8.00abc	7.89b	
ZnO‐60	6.97abc	7.82abc	8.42a	8.06ab	7.90	7.73b	8.07ab	7.94abc	7.85b	
ZnO‐80	7.22abc	7.59bcd	8.00abc	7.36c	8.00	8.09ab	8.10ab	7.65c	7.80b	
ZnS‐40	7.53ab	7.10def	8.10abc	7.56bc	7.60	8.28a	8.55ab	8.51a	7.85b	
ZnS‐60	7.59ab	7.13def	7.59bc	7.50cb	7.86	8.01ab	8.28ab	8.55a	7.88b	
ZnS‐80	7.40ab	7.51cde	7.33c	8.11ab	8.00	7.70b	7.85b	8.30ab	7.82b	
ZnG‐40	7.20abc	6.85efg	8.24ab	8.11ab	7.99	7.72b	8.30ab	7.75bc	7.77b	
ZnG‐60	6.90bc	6.60fg	7.64abc	7.56bc	7.86	8.11ab	7.99ab	8.33ab	7.64b	
ZnG‐80	7.57ab	6.21g	7.67abc	7.84abc	7.85	8.39a	8.32ab	8.36ab	7.74b	
ZnOA‐40	7.17abc	6.98def	7.54bc	7.57bc	8.10	8.05ab	8.63a	8.31ab	7.75b	
ZnOA‐60	7.64a	6.48fg	7.57bc	7.98abc	8.07	8.34a	8.19ab	8.37ab	7.85b	
ZnOA‐80	6.63c	6.62fg	7.73abc	7.95abc	7.93	8.28a	8.02ab	8.53a	7.70b	
SEM	0.21	0.22	0.24	0.19	0.18	0.16	0.21	0.19	0.08	
p value	0.02	0.001	0.05	0.004	0.9	0.003	0.042	0.015	0.02	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–g) in each effect are statistically significant (p ˂ 0.05).

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Table 12 presents the effect of different zinc levels on IgG, IgM immunoglobulins and SOD concentrations. Significant variations were observed for IgG and IgM immunoglobulins across all zinc concentrations tested.

TABLE 12 Effect of different levels of zinc on IgG, IgM and superoxide dismutase (SOD) serum concentrations.

Treatments	IgM (mg/dL)	IgG (mg/dL)	SOD (U/mL)	
C	89.27bc	205.70c	226.69	
ZnO‐40	69.77gf	148.76h	285.91	
ZnO‐60	64.82g	151.65h	193.30	
ZnO‐80	88.88bc	182.14e	290.54	
ZnS‐40	71.00f	145.57h	329.26	
ZnS‐60	72.87ef	170.18f	337.01	
ZnS‐80	84.75c	178.71e	325.67	
ZnG‐40	72.08ef	211.89b	265.97	
ZnG‐60	78.56d	179.61e	285.39	
ZnG‐80	93.36ab	203.31c	285.65	
ZnOA‐40	69.29gf	156.31g	289.37	
ZnOA‐60	77.32ed	191.54d	281.76	
ZnOA‐80	95.46a	235.17a	302.77	
SEM	3.06	3.13	47.78	
p value	0.0001	0.001	0.05	
Note: C: control, ZnO‐40: 40 mg/kg zinc oxide, ZnO‐60: 60 mg/kg zinc oxide, ZnO‐80: 80 mg/kg zinc oxide, ZnS‐40: 40 mg/kg zinc sulphate, ZnS‐60: 60 mg/kg zinc sulphate, ZnS‐80: 80 mg/kg zinc sulphate, ZnG‐40: 40 mg/kg zinc glycine, ZnG‐60: 60 mg/kg zinc glycine, ZnG‐80: 80 mg/kg zinc glycine, ZnOA‐40: 40 mg/kg zinc organic acid, ZnOA‐60: 60 mg/kg zinc organic acid, ZnOA‐80: 80 mg/kg zinc organic acid. Means with different superscript letters (a–h) in each effect are statistically significant (p ˂ 0.05).

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4 DISCUSSION

In recent years, the problem of broken eggs represents a significant financial loss for the poultry industry. Shell quality is crucial for maintaining the shelf life and safety of eggs. Good shell quality can prevent bacterial contamination, essential for maintaining egg laying and hatching performance (Sokolowicz et al., 2018).

The supplementation of organic zinc sources, such as zinc methionine or zinc proteinate, is expected to lead to increased bioavailability and more notable positive effects on laying hen performance, egg quality and immune system function compared to inorganic zinc sources (Qiao et al., 2008). A dose of more than 40 mg Zn/kg of feed is anticipated to generate the most favourable outcomes concerning these parameters. The results of this study showed that the addition of different zinc sources to the diets of laying hens increased their weights compared to controls. The optimal dose of zinc was found to be 80 mg/kg, with a highly significant increase in weight observed 2 months after feeding. This weight increase was attributed to the intake of zinc from all sources.

As the zinc dose increased, so did the weight gain of the laying hens and other parameters, such as egg weight, albumen, yolk and shell. This finding aligns with Torki et al. (2015), who reported that the addition of 40 mg Zn/kg to the basal diet of laying hens increased egg production and weight, improving feed conversion. Korenekova et al. (2007) also indicated that dietary zinc supplementation increased egg mass in quail. However, Cufadar et al. (2020) reported differing results, highlighting the variability in outcomes based on zinc sources and levels, basal diet content, experimental periods and genetic differences among hens.

Organic forms of zinc offer protection against the creation of indigestible complexes with certain anti‐nutrient compounds in the gut and have higher bioavailability than inorganic sources (Huang et al., 2020). Zinc is essential for growth, mineral deposition, enzyme function and protein metabolism in poultry. In laying hens, zinc deficiency affects the quality of the epithelium and epithelial secretions during eggshell membrane synthesis, impacting shell quality and reducing the proportion of cracked eggs, which leads to significant economic losses (Huang et al., 2020).

The results showed significant variations in IgG and IgM immunoglobulins across all zinc concentrations, indicating that zinc supplementation positively influences immune system function. The findings suggest that zinc supplementation improves egg production performance, egg quality and bioavailability and reduces the negative effects of age.

The results of this study demonstrate that zinc supplementation in the diet of laying hens at the end of their production period positively affects body weight, egg production, egg quality and immune function. The optimal zinc dose was found to be 80 mg/kg, with significant improvements observed in all parameters tested. These findings support the need for well‐balanced and effective zinc supplementation strategies to enhance the productivity and health of ageing laying hens.

5 CONCLUSION

In conclusion, this study demonstrates that zinc supplementation in the diet of laying hens at the end of their production period has a significant positive impact on body weight, egg production and egg quality. The optimal zinc dose was determined to be 80 mg/kg, which resulted in notable improvements in egg weight, albumen weight, yolk weight and shell weight. These findings indicate that zinc plays a crucial role in enhancing the overall performance and egg quality of laying hens, likely through its involvement in protein synthesis and albumin deposition.

Additionally, the study found that different zinc sources, whether organic or inorganic, did not have a significant impact on the immunoglobulin levels (IgG and IgM) of the hens. This suggests that although zinc is essential for improving performance and egg quality, its effect on immune system parameters may not vary significantly with the source of zinc.

These insights contribute valuable knowledge to poultry nutrition strategies, underscoring the importance of optimizing zinc supplementation to improve productivity and egg quality in laying hens, particularly as they approach the end of their production period. Tailoring zinc supplementation strategies to the specific needs of the flock can help mitigate the decline in performance associated with ageing, ultimately enhancing the economic viability of poultry farming.

AUTHOR CONTRIBUTIONS

All authors contributed to the study conception and design, commented on previous versions of the manuscript, read and approved the final manuscript as well as discussed the results and contributed to the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

FUNDING INFORMATION

This research received no specific grant from any funding agency in the public, commercial or not‐for‐profit sectors.

CONSENT TO PUBLISH

All authors had confirmed the final version of the manuscript for publication.

ETHICS STATEMENT

This study was approved by the Research Ethics Committee. The protocol and ethics of current study were approved by the Ethical Committee of Islamic Azad University. The authors declare that no human participant was involved in current study.

DATA AVAILABILITY STATEMENT

The data used to support the findings of this study are available from the corresponding author upon request.
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