Comparative effects of zinc hydroxy chloride, zinc sulfate, and zinc-methionine on egg quality and quantity traits in laying hens
Abstract
The aim of this experiment was to evaluate the effect of different levels of zinc supplements on egg quality and quantity traits as well as egg enrichment with zinc in laying hens from 40 to 50 weeks of age. A total of 240 Hy-line laying hens were distributed among eight treatments and five replications (six birds per replication). The control group received no zinc diet, while the other treatments were supplemented with varying levels of zinc sulfate (80, 120, and 160 mg/kg) or zinc hydroxy chloride (50, 75, and 100 mg/kg). An additional group of zinc-methionine supplement at 124 mg/kg was also included. Results showed that different levels of zinc supplementation caused a significant improvement in eggshell resistance, eggshell percentage, feed conversion ratio, and Haugh unit compared to the control group. Adding organic and hydroxy sources of zinc significantly increased zinc contents in egg yolk, tibia bone, and blood. In addition, the treatments containing zinc supplements caused an increase in the antibody level against the Newcastle disease compared to the control (P < 0.05). Different levels and sources of zinc had no significant effect on eggshell thickness, specific gravity, and egg mass. Results showed that adding zinc in hydroxy chloride form at 100 mg/kg could improve performance indices, safety, and egg enrichment with zinc.
1 INTRODUCTION
As an indispensable element, zinc plays significant roles in biochemical and physiological processes in the body. This element plays a unique role in a healthy life, especially in old age, by preventing the neoplasia process in cells (Silvia Cr, 2021). Zinc deficiency can cause a disorder in the function of digestive enzymes (Ibrahim et al., 2022)—also a severe disorder in taste dysfunction and dysgeusia (Han et al., 2015; Henkin, 1984). This essential metal plays a role in developing and maintaining the innate and acquired immune systems. Zinc affects the maturation of dendritic cells (DCs), which is influential in shaping the response of T lymphocytes to pathogens (George et al., 2016). Zinc controls diabetes by increasing insulin hexamers and their storage in the pancreas (Gembillo et al., 2022). In many Asian countries, the problem of zinc deficiency is due to the calcareous nature of the soil, high pH, lack of organic matter, salinity stress, long-term drought, high bicarbonate of water, and unbalanced application of nitrogen (N), phosphorus (P) and potassium (K), collectively known as the NPK fertilizer (Narimani et al., 2010). According to a new report, over a half of the world's population suffer from zinc deficiency (Cakmak, 2008). Approximately 80% of Iran's agricultural lands are deficient in zinc (Ghasemi et al., 2015). By influencing the process of cell apoptosis, zinc prevents the replication of damaged DNA. Zinc plays a regulatory role in programmed cell death. Hence, the lack of zinc in the body of people disrupts programmed death, causes transcription from the damaged cell, and increases the risk of contracting or aggravating all types of cancer (Walker & Black, 2004). In an experiment to enrich eggs vitamins, it was observed that dietary increments led to incremental accumulation in the egg. However, the storage of other vitamins of the same group tended to decrease. For example, by increasing the amount of retinol as a precursor of vitamin A in the feed of laying hens, an increase in its amount in the egg yolk was observed. However, the amount of tocopherol (E) stored in the egg was decreased (Singh et al., 2012). Nowadays, selenium-enriched eggs are available in more than 25 countries. Selenium-enriched eggs, which are slightly more expensive than regular eggs, are produced by adding selenium to the feed of laying hens (Hassanien, 2011). Zinc enrichment is done in various food items, such as wheat flour, as a staple in some countries (Wang et al., 2023). Although animal sources such as beef, pork, and oysters are rich in zinc, these sources are expensive. Therefore, zinc deficiency is widely felt in the human population. The present experiment looked into enriching eggs with zinc from different zinc sources in a comparative study.
2 MATERIALS AND METHODS
This experiment was conducted using 240 Hy-line laying hens (36-W) from 40 to 50 weeks of age in a completely randomized design with eight treatments, five replications, and six birds per replication. Experimental treatments to compare organic (zinc methionine chelate) and inorganic (sulfate and hydroxy chloride) sources of zinc included a control (without zinc supplement), zinc sulfate at 80, 120, and 160 mg/kg (designated as treatment 2, 3, and 4 respectively), and zinc hydroxychloride at 50, 75, and 100 mg/kg (designated as treatment 5, 6, and 7 respectively). Treatment 8 included 124 mg/kg of the zinc-methionine (with a purity of 22%). Zinc sulfate and zinc hydroxychloride had a purity of 34 and 55%, respectively. Zinc hydroxychloride was provided by Ayman Nano Fam Zanjan Knowledge Base Company. All dietary treatments provided an equal amount of zinc (25 mg/kg). Laying hens with similar weights (1590 ± 25 g) were randomly distributed among 40 battery cages (six birds in each cage). The repetitions related to each treatment were placed in different classes to comply with the uniformity principle. To acclimatize the birds to the cages and deplete the zinc reserves, an acclimatization period was carried out 2 weeks before the beginning of the experimental period, during which they were fed a zinc-free basal diet (control). The composition of the basal diet (based on the guide of the strain and according to the bird's age) and its approximate analysis were shown in Table 1. Experimental diets were provided to laying hens once a day. The house temperature was 24 ± 2°C, and a lighting schedule of 16 h of light and 8 h of darkness was used throughout the experiment. The concentrations of zinc element in organic and inorganic sources and hydroxy used in this experiment were determined by atomic absorption spectrometry (AOAC, 1990). The Animal Care and Use Committee of Shahrekord University approved all of the animal welfare and experimental protocols used in the study (SKU-1402R).
| Ingredient | % |
|---|---|
| Corn grain | 54.44 |
| Soybean meal | 22.58 |
| Wheat grain | 4 |
| Limestone | 10.16 |
| Soybean oil | 3.47 |
| Wheat bran | 2.7 |
| Dicalcium phosphate | 1.54 |
| Common salt (NaCl) | 0.39 |
| Vitamin premixa | 0.25 |
| Mineral premixb | 0.25 |
| DL-methionine | 0.14 |
| Lysine | 0.08 |
| Calculated analysis | |
| ME (kcal/kg) | 2835 |
| CP (%) | 15.90 |
| Calcium (%) | 4.35 |
| Available phosphorus (%) | 0.45 |
| Na (%) | 0.18 |
| Methionine (%) | 0.38 |
| Met + cys (%) | 0.65 |
| Lysine (%) | 0.80 |
| Arginine (%) | 0.90 |
| Threonine (%) | 0.58 |
- a Vitamin supplement composition for each kilogram of ration includes 3,000,000 international units of vitamin A, 1,300,000 international units of vitamin D3, 750 international units of vitamin E, 980 mg of B12, 5 mg of vitamin K, 2000 mg Gram riboflavin, 3000 mg niacin, 4 g choline chloride, 250 mg biotin, 3 mg thiamine, and 4 mg pyridoxine.
- b Mineral supplement composition for each kilogram of basic ration includes 75 mg of manganese, 75 mg of iron, 5 mg of copper, 0.76 mg of iodine, and 0.1 mg of selenium. Zinc supplement in different forms (organic and inorganic) was added to the diet of the control group in the amounts tested. Treatment 1 without zinc supplement, treatments 2, 3, and 4 containing, respectively, 80, 120, and 160 mg/kg zinc sulfate supplement, treatments 5, 6, and 7, respectively, containing 50, 75, and 100 mg/kg zinc hydroxy chloride supplement, and treatment 8 containing 124 mg/kg is zinc-methionine chelate supplement.
2.1 Performance parameters
During the experiment, egg mass, feed conversion ratio, and eggshell thickness were calculated biweekly. At the end of the second, fourth, sixth, and eighth weeks of the experiment, three eggs were randomly selected from each replicate, and the specific gravity of the eggs was determined by the method of preparing salt solutions with a range of specific gravity from 1.058 to 1.102 with 0.004 increments (Keshavarz, 2003). The eggs were then broken, and some qualitative parameters were measured, including Haugh unit, shell thickness, and shell strength. After measuring the height of the egg white, the Haugh unit was calculated using the equation Haugh unit = 100 log (albumen height + 7.57 − 1.7 egg weight0.37), where albumen height was in millimeters and egg weight in grams. After drying and separating the shell's inner membrane, the shells were weighed. Then, the thickness of the eggshell was measured using a thickness gauge (OSK 13469) with an accuracy of 0.01 (average of three points, two ends, and the center in millimeters). The resistance of the shell against breaking was determined by means of a digital eggshell force gauge (model-II) in the form of kilograms of force required to break the shell in a cross-section of one square centimeter (Mohiti Asli et al., 2007).
2.2 Blood biochemical parameters and antibody response
On 47th day of the experiment, a dual oral vaccine against Newcastle + Bronchitis was administered to the birds after 2 h of water restriction.
The live dual vaccine was administrated via drinking water on 47th days of experiment and blood samples were taken after 23 days (70th of experiment) to assay antibody titer. The experiment lasted from 40 to 50 weeks of age (70 days).
The Pars test kit was used to measure blood zinc and albumin levels (Abd El-Hack et al., 2018). Also, the antibody titer against the Newcastle virus was measured by HI test for each sample as the logarithm of the second base of the last dilution (Marquardt et al., 1985).
2.3 Bioavailability (zinc storage in the tibia)
At the end of the experiment, one bird was randomly selected and killed from each repetition to measure the amount of bone. The tibia of the right leg was separated. After separating the moisture and fat in the furnace, the amount of zinc was evaluated by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES Analysis). The ICP device uses the emission spectrometry technique and relies on the fact that excited electrons emit energy at the irradiated wavelength. This excitation is done in the ICP device by hot argon plasma. The intensity of the radiated energy in a specific wavelength is directly proportional to the present concentration of that element in the analyzed sample. Therefore, the value in the sample was measured by determining which element was emitting at which wavelength and by measuring its intensity. To prepare the samples, the samples were first digested in acid, and then the zinc level was set at 206.200 nm wavelength to read. ICP-OES Analyzer SPECTRO ARCOS, made in Germany, did data reading and recording.
2.4 Stored zinc in egg yolks and eggshells
To measure zinc in eggs at the end of the fourth and eighth week of the main experiment, three eggs were randomly selected from each repetition, and for this purpose, the white and yolk were separated. Then, after drying, the sample was digested with sultan water and hydrochloric acid. The concentration of zinc was measured by ICP-OES analysis according to the usual instructions. Also, on the last day of the experiment, one sample was selected from each replication to measure the amount of zinc in the eggshell (Kumaravel & Alagusundaram, 2014).
2.5 Statistical analysis
Data were analyzed in a completely random design. For this purpose, the GLM procedure of SAS software was used, and the Tukey's test was used at a significant level of 0.05 to compare the means (SAS Institute (2003) SAS/STAT® User's Guide: Statistics Version 6.12.; SAS Institute Inc., Cary, NC, USA).
3 RESULTS
3.1 The egg weight and mass
The findings pertaining to the performance indices are presented in Table 2. Various levels and sources of zinc had no significant effect on egg weight. Moreover, the treatments did not influence egg mass (P > 0.05). These results indicate that the addition of zinc at different levels and from various sources did not significantly impact egg production or egg weight.
| Source of zinc | Egg weight (g) | Egg mass (g/hen/day) | ||||||
|---|---|---|---|---|---|---|---|---|
| 2th week | 4th week | 6th week | 8th week | 2th week | 4th week | 6th week | 8th week | |
| Control | 63 ± 1.10 | 63 ± 2.80 | 62 ± 1.97 | 62 ± 2.27 | 57.33 ± 1.01 | 58.89 ± 2.55 | 56.50 ± 1.79 | 56.23 ± 2.07 |
| Sulfate (80 mg/kg) | 63 ± 2.24 | 63 ± 3.39 | 63 ± 2.92 | 65 ± 2.53 | 57.31 ± 2.03 | 57.76 ± 3.08 | 57.66 ± 2.65 | 59.07 ± 2.30 |
| Sulfate (120 mg/kg) | 62 ± 2.91 | 64 ± 2.97 | 63 ± 3.12 | 64 ± 2.98 | 56.95 ± 2.64 | 57.96 ± 2.70 | 57.50 ± 2.84 | 58.71 ± 2.71 |
| Sulfate (160 mg/kg) | 63 ± 1.95 | 64 ± 2.77 | 64 ± 2.55 | 65 ± 2.71 | 57.50 ± 1.77 | 58.06 ± 2.51 | 57.92 ± 2.32 | 58.90 ± 2.46 |
| Hydroxy chloride (50 mg/kg) | 63 ± 3.07 | 63 ± 2.23 | 65 ± 3.14 | 65 ± 3.01 | 57.61 ± 2.80 | 57.63 ± 2.03 | 59.39 ± 2.86 | 59.44 ± 2.74 |
| Hydroxy chloride (75 mg/kg) | 62 ± 3.63 | 64 ± 1.95 | 64 ± 3.61 | 66 ± 4.79 | 57.17 ± 3.30 | 58.18 ± 1.77 | 58.69 ± 3.29 | 60.46 ± 4.37 |
| Hydroxy chloride (100 mg/kg) | 63 ± 2.37 | 63 ± 3.09 | 65 ± 2.26 | 65 ± 7.50 | 57.35 ± 2.15 | 57.39 ± 2.81 | 59.27 ± 2.06 | 59.37 ± 6.84 |
| Zinc methionine chelate (124 mg/kg) | 63 ± 2.58 | 64 ± 2.05 | 65 ± 3.19 | 66 ± 2.20 | 57.39 ± 2.34 | 58.01 ± 1.87 | 59.44 ± 2.90 | 60.32 ± 2.01 |
| P-value | 0.9999 | 0.9997 | 0.5766 | 0.6897 | 0.9999 | 0.9997 | 0.5765 | 0.6892 |
| Source of zinc | Feed conversion ratio | Eggshell (%) | ||||||
|---|---|---|---|---|---|---|---|---|
| 2th week | 4th week | 6th week | 8th week | 2th week | 4th week | 6th week | 8th week | |
| Control | 2.18a ± 0.08 | 2.27 ± 0.11 | 2.27 ± 0.08 | 2.31a ± 0.08 | 8.04 ± 1.93 | 7.16b ± 1.31 | 7.46 ± 1.63 | 7.22c ± 1.23 |
| Sulfate (80 mg/kg) | 2.00b ± 0.06 | 2.08 ± 0.07 | 2.09 ± 0.06 | 2.19b ± 0.13 | 8.67 ± 0.69 | 8.42ab ± 0.61 | 8.15 ± 0.51 | 8.26bc ± 0.66 |
| Sulfate (120 mg/kg) | 1.99b ± 0.07 | 2.15 ± 0.16 | 2.20 ± 0.09 | 2.24ab ± 0.09 | 8.65 ± 0.65 | 8.52ab ± 0.59 | 8.19 ± 0.62 | 8.45ab ± 0.73 |
| Sulfate (160 mg/kg) | 2.00b ± 0.05 | 2.14 ± 0.21 | 2.18 ± 0.16 | 2.23ab ± 0.15 | 8.61 ± 0.84 | 8.50ab ± 0.58 | 8.14 ± 0.35 | 8.39ab ± 0.21 |
| Hydroxy chloride (50 mg/kg) | 2.01b ± 0.06 | 2.05 ± 0.20 | 2.12 ± 0.15 | 2.17b ± 0.06 | 8.51 ± 0.71 | 8.22ab ± 0.28 | 8.12 ± 0.64 | 8.15bc ± 0.90 |
| Hydroxy chloride (75 mg/kg) | 1.96b ± 0.05 | 2.09 ± 0.07 | 2.10 ± 0.07 | 2.13b ± 0.07 | 8.60 ± 0.20 | 8.60ab ± 0.27 | 8.37 ± 0.25 | 8.42ab ± 0.67 |
| Hydroxy chloride (100 mg/kg) | 1.95b ± 0.06 | 1.99 ± 0.04 | 2.07 ± 0.14 | 2.12b ± 0.06 | 8.98 ± 0.42 | 9.06a ± 0.62 | 8.97 ± 0.95 | 9.42a ± 1.29 |
| Zinc methionine chelate (124 mg/kg) | 1.98b ± 0.07 | 2.03 ± 0.05 | 2.06 ± 0.13 | 2.15b ± 0.10 | 9.13 ± 0.22 | 8.85a ± 0.98 | 8.51 ± 0.59 | 9.20ab ± 0.62 |
| P-value | 0.0001 | 0.0579 | 0.0777 | 0.0321 | 0.6807 | 0.0142 | 0.2304 | 0.0135 |
- Note: a, b, c: Within columns, mean values with common superscript(s) are not different (P > 0.05).
3.2 Feed conversion ratio
As presented in Table 2, the dietary treatments exhibited a significant influence on the feed conversion ratio (P < 0.05), with the lowest value observed in the hydroxy and chelate forms of zinc methionine. It is worth noting that zinc hydroxy chloride at doses of 50 and 75 mg/kg produced similar outcomes to the organic chelate and mineral sulfate forms.
3.3 Eggshell percentage
The proportion of eggshell weight to egg weight was significantly differed in all of the dietary treatments supplemented with zinc sources in comparison with the control treatment (P < 0.05) at the end of 4th weeks of experiment. There were significant differences among dietary treatments with control except treatments fed diets supplemented with zinc sulfate and zinc hydroxy chloride at the level of 80 and 50 mg/kg, respectively (P < 0.05) when measured at the end of 8th weeks of experiment. No significant difference was observed in eggshell weight percentage between organic (zinc-methionine chelate) and inorganic (zinc sulfate and zinc hydroxy chloride) sources (P > 0.05).
3.4 Specific gravity
As evident from the data presented in Table 3, the specific gravity of eggs remained unaffected by the treatments throughout the experiment (P > 0.05).
| Source of zinc | Specific gravity (g/cm3) | Haugh unit | ||||||
|---|---|---|---|---|---|---|---|---|
| 2th week | 4th week | 6th week | 8th week | 2th week | 4th week | 6th week | 8th week | |
| Control | 1.070 ± 0.041 | 1.066 ± 0.038 | 1.08 ± 0.023 | 1.066 ± 0.034 | 77.26 ± 2.516 | 76.05 ± 3.215 | 75.42 ± 3.619 | 72.69c ± 4.928 |
| Sulfate (80 mg/kg) | 1.066 ± 0.040 | 1.062 ± 0.039 | 1.062 ± 0.036 | 1.064 ± 0.022 | 79.93 ± 2.160 | 79.49 ± 1.949 | 79.27 ± 2.80 | 78.70ab ± 3.449 |
| Sulfate (120 mg/kg) | 1.064 ± 0.039 | 1.061 ± 0.037 | 1.064 ± 0.635 | 1.064 ± 0.021 | 80.05 ± 2.120 | 79.36 ± 1.840 | 79.32 ± 2.852 | 79.68ab ± 5.469 |
| Sulfate (160 mg/kg) | 1.062 ± 0.048 | 1.064 ± 0.040 | 1.064 ± 0.034 | 1.064 ± 0.020 | 79.18 ± 3.018 | 79.50 ± 1.962 | 79.07 ± 2.638 | 76.72ab ± 3.505 |
| Hydroxy chloride (50 mg/kg) | 1.063 ± 0.027 | 1.060 ± 0.032 | 1.058 ± 0.036 | 1.054 ± 0.030 | 78.76 ± 1.865 | 79.41 ± 2.624 | 79.32 ± 2.464 | 80.31ab ± 1.952 |
| Hydroxy chloride (75 mg/kg) | 1.064 ± 0.034 | 1.063 ± 0.033 | 1.062 ± 0.033 | 1.060 ± 0.031 | 79.44 ± 2.813 | 79.46 ± 2.547 | 80.08 ± 2.575 | 80.50ab ± 3.062 |
| Hydroxy chloride (100 mg/kg) | 1.068 ± 0.026 | 1.064 ± 0.023 | 1.064 ± 0.025 | 1.064 ± 0.030 | 79.03 ± 2.150 | 79.73 ± 2.926 | 79.56 ± 2.671 | 82.24a ± 4.426 |
| Zinc methionine chelate (124 mg/kg) | 1.066 ± 0.045 | 1.064 ± 0.031 | 1.066 ± 0.034 | 1.064 ± 0.033 | 79.52 ± 3.165 | 79.58 ± 3.196 | 79.53 ± 3.038 | 80.65ab ± 4.099 |
| P-value | 0.9999 | 0.9998 | 0.9850 | 0.9985 | 0.7471 | 0.3735 | 0.2804 | 0.0217 |
| Source of zinc | Eggshell thickness (mm) | Eggshell strength (kg/cm2) | ||||||
|---|---|---|---|---|---|---|---|---|
| 2th week | 4th week | 6th week | 8th week | 2th week | 4th week | 6th week | 8th week | |
| Control | 0.40 ± 0.012 | 0.39 ± 0.014 | 0.39 ± 0.010 | 0.38 ± 0.019 | 2.20 ± 0.18 | 2.15 ± 0.23 | 2.11 ± 0.22 | 1.77c ± 0.07 |
| Sulfate (80 mg/kg) | 0.41 ± 0.016 | 0.41 ± 0.012 | 0.41 ± 0.016 | 0.41 ± 0.015 | 2.45 ± 0.28 | 2.33 ± 0.35 | 2.33 ± 0.30 | 2.31ab ± 0.28 |
| Sulfate (120 mg/kg) | 0.41 ± 0.018 | 0.41 ± 0.015 | 0.41 ± 0.018 | 0.41 ± 0.018 | 2.41 ± 0.19 | 2.37 ± 0.15 | 2.35 ± 0.18 | 2.24b ± 0.30 |
| Sulfate (160 mg/kg) | 0.41 ± 0.017 | 0.41 ± 0.019 | 0.41 ± 0.017 | 0.41 ± 0.015 | 2.40 ± 0.17 | 2.36 ± 0.14 | 2.31 ± 0.49 | 2.20b ± 0.48 |
| Hydroxy chloride (50 mg/kg) | 0.41 ± 0.011 | 0.41 ± 0.010 | 0.41 ± 0.009 | 0.41 ± 0.013 | 2.44 ± 0.13 | 2.34 ± 0.19 | 2.32 ± 0.11 | 2.18b ± 0.13 |
| Hydroxy chloride (75 mg/kg) | 0.42 ± 0.010 | 041 ± 0.011 | 0.41 ± 0.012 | 0.41 ± 0.017 | 2.46 ± 0.16 | 2.41 ± 0.16 | 2.34 ± 0.20 | 2.30ab ± 0.17 |
| Hydroxy chloride (100 mg/kg) | 0.42 ± 0.015 | 0.42 ± 0.015 | 0.42 ± 0.013 | 0.42 ± 0.014 | 2.53 ± 0.23 | 2.44 ± 0.25 | 2.40 ± 0.16 | 2.49ab ± 0.20 |
| Zinc methionine chelate (124 mg/kg) | 0.41 ± 0.017 | 0.41 ± 0.016 | 0.42 ± 0.020 | 0.41 ± 0.022 | 2.49 ± 0.15 | 2.39 ± 0.22 | 2.37 ± 0.23 | 2.63a ± 0.76 |
| P-value | 0.8175 | 0.3740 | 0.2962 | 0.1470 | 0.2696 | 0.5975 | 0.4635 | 0.0019 |
- Note: a, b, c: Within columns, mean values with common superscript(s) are not different (P > 0.05).
3.5 Haugh unit
According to Table 3, the Haugh unit was significantly influenced by the dietary treatments at the end of the experiment (P < 0.05). The highest value was associated with zinc hydroxy chloride at 100 mg/kg, while the lowest value was observed in the control treatment. The sources of zinc did not significantly affect the Haugh unit (P > 0.05).
3.6 Shell thickness and resistance
Data presented in Table 3 demonstrates that zinc supplementation did not have a significant effect on shell thickness and resistance (P > 0.05). However, significant improvements in eggshell resistance were observed at the end of the experiment under the influence of different sources and zinc levels. The hydroxy and organic chelate treatments exhibited the highest resistance to external force applied to the egg surface. Additionally, the inclusion of zinc supplements at various levels and in both organic and inorganic forms led to enhanced shell resistance compared to the control treatment (P < 0.05).
3.7 Blood biochemical parameters and bioavailability
Table 4 illustrates that zinc levels in blood samples and tibia ash were influenced by the amount of zinc in the diet. Different sources and levels of zinc supplementation resulted in significant effects on blood albumin and antibody levels against the Newcastle vaccine (P < 0.05). In comparison with control, all of dietary treatments supplemented with zinc significantly (P < 0.05) increased the level of zinc in blood. There were also significant differences among the levels of zinc in blood between the birds received zinc sulfate and birds fed diets supplemented with zinc hydroxy chloride or zinc-methionine chelate, except for 50 mg/kg of with zinc hydroxy chloride (P < 0.05). The level of albumin and antibody titers against the Newcastle vaccine was significantly affected by the level and source of the zinc element (P < 0.05). The highest value of blood albumin was associated with the organic form of zinc. A significant difference was observed between the control treatment and the 80 mg/kg sulfate treatment (P < 0.05). However, there were no significant differences compared to other treatments (P > 0.05). The antibody response against Newcastle disease vaccine was influenced by the treatments, with the highest antibody levels associated with different levels of zinc hydroxy chloride and the organic source of zinc-methionine chelate (P < 0.05). Moreover, adding zinc supplements in the form of sulfate at doses of 80 and 120 mg/kg did not result in significant changes compared to the control treatment (P > 0.05). There were significant differences among dietary treatments with respect to the stored-levels of zinc in tibia bone (P < 0.05). The highest amount of zinc stored in the bone was observed in 75 and 100 mg/kg of zinc hydroxy chloride, as well as the organic form of zinc. These treatments exhibited significant differences compared to other treatments (P < 0.05).
| Source of zinc | Zinc level in blood mg/dL | Albumin level in blood mg/dL | Antibody titers against NDV (log2) | Zinc on tibia mg/kg |
|---|---|---|---|---|
| Control | 1.52d ± 0.08 | 1.57c ± 0.20 | 2.60c ± 0.52 | 125c ± 26 |
| Sulfate (80 mg/kg) | 1.95c ± 0.09 | 2.30ab ± 0.45 | 3.07c ± 0.30 | 136bc ± 19 |
| Sulfate (120 mg/kg) | 1.96c ± 0.16 | 2.21b ± 0.36 | 3.03c ± 0.58 | 150bc ± 27 |
| Sulfate (160 mg/kg) | 1.90c ± 0.08 | 2.27ab ± 0.34 | 3.41ab ± 0.26 | 163bc ± 39 |
| Hydroxy chloride (50 mg/kg) | 1.99bc ± 0.11 | 2.51ab ± 0.44 | 4.25ab ± 0.44 | 186b ± 35 |
| Hydroxy chloride (75 mg/kg) | 2.31ab ± 0.14 | 2.56a ± 0.30 | 4.61a ± 0.55 | 277a ± 25 |
| Hydroxy chloride (100 mg/kg) | 2.34a ± 0.36 | 2.61a ± 0.25 | 4.79a ± 0.56 | 299a ± 27 |
| Zinc methionine chelate (124 mg/kg) | 2.24ab ± 0.18 | 2.70a ± 0.35 | 4.60a ± 0.45 | 275a ± 40 |
| P-value | 0.0001 | 0.0004 | 0.0001 | 0.0001 |
- Note: a, b, c: Within columns, mean values with common superscript(s) are not different (P > 0.05).
3.8 Zinc storage in eggshell and yolk
Table 5 confirms that the storage of zinc in the eggshell was not affected by the level and source of this element in the diet of laying hens (P > 0.05). The storage of zinc in egg yolk was significantly influenced by the supplementation of feed with zinc, as depicted in Table 5 (P < 0.05). These effects were observed in the fourth and eighth weeks of the experiment. The significance of the effect varied depending on the level and type of the zinc source used, taking into account the purity of zinc sources. The highest storage rate of zinc in the yolk was associated with zinc hydroxy chloride at a rate of 100 mg/kg. Considering this amount was twice the recommended requirement, based on the purity percentage of the source, such a response was expected. Additionally, the performance of the organic form of zinc showed a significant difference compared to the mineral forms at the same dosage (P < 0.05).
| Source of zinc |
Stored in egg yolk 4th week (mg/kg) |
Stored in egg yolk 8th week (mg/kg) |
Stored in eggshell 8th week (mg/kg) |
|---|---|---|---|
| Control | 29.93c ± 3.79 | 27.93c ± 4.81 | 2.56 ± 1.99 |
| Sulfate (80 mg/kg) | 31.40c ± 2.50 | 31.53c ± 5.65 | 3.24 ± 1.62 |
| Sulfate (120 mg/kg) | 33.00c ± 2.23 | 30.00c ± 7.37 | 3.03 ± 2.77 |
| Sulfate (160 mg/kg) | 31.73c ± 4.96 | 29.60c ± 4.91 | 3.14 ± 1.02 |
| Hydroxy chloride (50 mg/kg) | 33.33c ± 4.55 | 30.73c ± 2.99 | 3.17 ± 3.13 |
| Hydroxy chloride (75 mg/kg) | 41.46ab ± 3.35 | 41.47b ± 1.88 | 3.56 ± 2.19 |
| Hydroxy chloride (100 mg/kg) | 44.66a ± 9.69 | 49.33a ± 9.11 | 4.36 ± 2.99 |
| Zinc methionine chelate (124 mg/kg) | 40.60b ± 5.85 | 41.20b ± 5.57 | 3.28 ± 1.61 |
| P-value | 0.0001 | 0.0001 | 0.9665 |
- Note: a, b, c: Within columns, mean values with common superscript(s) are not different (P > 0.05).
4 DISCUSSION
Regarding the lack of any significant difference between dietary treatments in terms of egg weight and egg mass, it appears that the levels of other nutrients, such as amino acids and carbohydrates, may play a prominent role in determining the weight of the eggs. On the other hand, biological limitations of laying hens allow for an increase in egg weight, especially during the peak period. These findings are consistent with those reported in studies where the replacement of various levels of organic chelates of copper, zinc, and manganese instead of their sulfate salts did not affect the productive performance of laying hens (Bai et al., 2017). The results of this research contradicted the findings of other studies in which the addition of different sources of zinc across various zinc levels significantly affected egg weight (Bahakaim et al., 2014). The results regarding the feed conversion ratio opposed a previously published report (Swiatkiewicz & Koreleski, 2008). A study reported that different levels of zinc did not significantly affect feed consumption in broilers, but it improved the feed conversion ratio (Sahraei et al., 2013). Zinc supplementation in broiler diets has been shown to improve the feed conversion ratio (Dibaiee-nia et al., 2017). An improvement in the feed conversion ratio can be attributed to the role of zinc in dipeptidyl peptidase 3 (DPP3), a zinc-dependent peptidase enzyme. This enzyme is involved in the hydrolysis of amide bonds to separate dipeptide fragments from oligopeptides (Younas et al., 2023).
In relation to the proportion of eggshell weight, the enzymatic activity of carbonic anhydrase is responsible for the decomposition of carbonic acid into carbon dioxide and water. This particular enzyme is a metalloenzyme consisting of zinc (Mabe et al., 2003; McDowell, 1992). A deficiency in zinc levels can detrimentally impact the function of this enzyme, subsequently resulting in a reduction in eggshell weight (Nys et al., 2001). This information explains how the introduction of zinc supplements could have yielded a significant effect on the percentage of shell weight. The present findings regarding specific gravity conformed to earlier investigations (Behjatian Esfahani et al., 2021). Comparative analysis of organic and inorganic zinc sources on the egg quality of laying hens did not yield a statistically significant impact on the specific gravity of the eggs (Londero et al., 2020). Given that the eggshell tends to gradually thin over time, including varying levels of zinc helps restore this situation and thereby maintains the specific weight of the egg. The results obtained from the measurement of zinc reserves in the tibia corroborate the fact that birds receiving the control treatment utilized zinc from their bone reserves, thereby rendering their specific weights comparable to the control group. The Haugh unit serves as a quality indicator of eggs and has a strong correlation with other quality indicators, such as resistance to breakage (International, 2007). Zinc has been reported to enhance the Haugh unit in laying hens. Notably, in the eighth week of the experiment, this quality index showed a significant improvement in treatments containing organic zinc when compared to the control treatment. As the shell thickness diminishes with advancing age, it is expected that notable differences will arise among various sources during the final phase of laying hen production. Zinc supplementation increased eggshell thickness in experiment (Yang et al., 2012). However, a study has demonstrated that diverse sources of zinc and manganese do not yield a significant effect on shell thickness (Swiatkiewicz & Koreleski, 2008). Incorporating 100 mg/kg of zinc in the form of organic chelate as well as the mineral form has proven to enhance eggshell resistance, particularly during the later stages of laying hen development (Swiatkiewicz & Koreleski, 2008).
Zinc supplementation has been found to increase the resistance of the eggshell. This improvement can be attributed to enhanced calcification within the eggshell matrix. Filamentous proteins attach the matrix tissue to the external shell during the eggshell formation process. As the eggshell forms, calcite crystals descend haphazardly into the matrix, giving rise to an adhesive mass. It is believed that low-consumption minerals, such as zinc, manganese, and copper, influence shell quality primarily through their role as cofactors for enzymes involved in eggshell formation and their reciprocal influence on calcite crystals during the shell development. Empirical evidence supports that zinc and manganese enhance calcium utilization in laying hens and improve the qualitative components of shell resistance (Mabe et al., 2003). Although shell thickness significantly contributes to shell strength, it does not singularly dictate the overall durability of the shell (Afshar Bakeshlou et al., 2020). In line with these findings, researchers have indicated that dietary zinc supplementation administered to laying ducks towards the end of their production cycle could enhance production performance and shell quality by influencing HCO3− secretion (Zhang et al., 2022). This observation might explain the heightened strength of the eggshell despite similar shell thickness.
The predictable increase in circulatory levels of albumin can be attributed to the fact that 70% of zinc in the blood is transported through protein chelation, primarily with albumin. This finding was consisted with previous studies reported by Vallee and Falchuk (1993) and Vikbladh (1950).
Zinc serves as an essential cofactor for the thymus. Thymulin, a thymic hormone exclusively synthesized by the thymic epithelial cells, consists of a nonapeptide component coupled with the zinc ion, thus conferring biological activity to the molecule (Reggiani et al., 2009). Accordingly, the enhanced immune response can be attributed to the increased maturation of T lymphocytes and the activation of B lymphocytes facilitated by helper cells (Fraker et al., 1977).
One method for assessing the bioavailability of mineral elements is to examine the reserves of these elements in tibia bone. Zinc methionine chelate likely exhibits greater bioavailability due to enhanced absorption. The composition of zinc hydroxy chloride remains protected from phytate interference owing to its remarkable stability in the upper regions of the digestive tract. As the quantities of hydroxy reaching levels of 75 and 100 mg, based on the purity of its source, exceed the recommended amounts indicated in the breeding guideline by 1.5 and 2 times, respectively, a decrease in bone reserve utilization during peak production and retention of zinc within the bird's bone structure was observed (P < 0.05).
Zinc hydroxy chloride is less reactive than ionic forms of zinc (Cao et al., 2000), suggesting a higher bioavailability value compared to zinc sulfate. The absorption of zinc from zinc sulfate is more affected by the presence of other minerals and phytic acid. Treatments containing varying levels of zinc sulfate and 50 mg/kg hydroxy exhibited a similar performance. The control treatment had the lowest zinc content in the bone. Consistent with these findings, literature has reported that the addition of zinc sulfate led to increased zinc deposition in the tibia bone (Kumar et al., 2021). Zinc content in the broiler tibia depends upon the source type and its dietary level. Notably, nano-particles exhibit a greater zinc retention in the tibia compared to its sulfate form (Mohammadi et al., 2015).
The main objective of this research was to explore the feasibility of enriching eggs with zinc through dietary zinc supplements. In this regard, the results were consistent with a study that compared the effects of zinc oxide and organic zinc-methionine chelate on performance, egg quality, and bioavailability in laying hens. The researchers concluded that supplementing zinc in the diet of laying hens yielded a significant impact on the zinc storage in eggs (Behjatian Esfahani et al., 2021). In a report, it was stated that high levels of zinc cause an increase in vitilin protein as a carrier of minerals. It seems that this protein, as a part of the egg yolk protein, increases the amount of zinc in the yolk (Park et al., 2004). The findings revealed that different levels of dietary zinc had a significant effect on zinc storage in eggs, and the highest level of storage was associated with an organic zinc form at a concentration of 150 mg/kg (Bahakaim et al., 2014).
The process of adding one or more essential nutrients to food at levels higher than their natural content is known as fortification. The purpose of fortification is to prevent and address deficiencies caused by inadequate levels of certain nutrients present in the food. To ensure the optimal intake of vitamins and minerals while avoiding excessive absorption and unwanted side effects, recommended daily intake reference values for individuals are determined based on the Recommended Nutrient Intake (RNI) guidelines provided by the World Health Organization. In this particular experiment, the inclusion of zinc in the hydroxy chloride form at various levels resulted in a remarkable increase of over 35% in the element's reserve within the yolk compared to the sulfate form. This indicates that by incorporating 100 mg/kg of zinc hydroxy chloride, not only the reserve of zinc could be augmented, but also caused a reduction in overall cost.
Fortification plays a crucial role in addressing nutrient deficiencies and improving the nutritional quality of food. By strategically supplementing specific nutrients, such as zinc, we can enhance the health benefits associated with consuming fortified products. However, it is essential to carefully consider the appropriate levels of fortification to ensure optimal nutrient absorption, minimize any potential adverse effects, and adhere to established guidelines recommended by organizations like the World Health Organization.
ACKNOWLEDGMENTS
This research was funded by Shahrekord University, Shahrekord, Iran.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest for this article.
