https://orcid.org/0000-0002-1903-2753
Abstract
This experiment was conducted in order to investigate the effect of the chelated form of trace elements in diet on the production traits, immunity, and blood parameters of Ross 308 broiler breeder chickens. Treatments included Treatment 1 (Negative control): Mn, Se, Cu, Fe and Zn provided as non-chelated form; Treatment 2 (Positive control): Mn, Se, Cu, Fe and Zn provided as chelated form; Treatment 3: Mn, Se, Cu, Fe and Zn provided as 50% chelated form and 50% non-chelated form; Treatment 4: Zn provided as chelated form; Treatment 5: Cu provided as chelated form; Treatment 6: Fe provided as chelated form; Treatment 7: Mn provided as chelated form; Treatment 8: Se provided as chelated form. The results showed that over the entire production period egg production and egg mass were affected by the experimental treatments (p < 0.05) and that the highest levels were observed in treatments 2 and 3. The chelated form of trace elements had a significant effect, increasing the number of settable eggs and decreasing the unsettable eggs over the entire production period (p < 0.05). Average egg weight over the entire period was not significantly changed (p > 0.05). The highest percentage of 2-yolk, thin shell, broken and small eggs was observed in 61st week of age significantly (p < 0.05). The experimental treatments had a significant effect on the immunity and the antibody titre against Newcastle disease showed a significant increase in treatments 2 and 8 (p < 0.05). The concentration of glucose, triglyceride, progesterone, AST and ALT were affected by the experimental treatments (p < 0.05). In conclusion, the use of a basic diet containing Zn, Fe, Cu, Se and Mn in the chelated form or the use of a basic diet containing mineral elements of Zn, Fe, Cu, Se and Mn with 50% non-chelated form and 50% in the chelated form improves the performance of production traits, immunity and blood parameters of Ross 308 broiler breeder chickens.
Chelated form of trace elements in the diet led to improvement in productive traits, immune response and blood parameters compared to the mineral form
Basic diet containing mineral elements of Zn, Fe, Cu, Se, and Mn, 50% in the non-chelated form and 50% in the chelated form led to a significant improvement in egg production, egg mass, egg weight and egg specific gravity
Chelated form of trace elements in the diet led to a decrease in the number of abnormal eggs including double-yolk, thin shell, broken, deformed, and small eggs
Highlights
Introduction
Broiler breeders are a main component of the poultry industry, playing a key role in supplying the protein needs of humans. They are increasingly produced all over the world (Hosseini Asl et al. Citation2013). It is very important to provide diets with the highest nutritional value for these birds. Among the most important nutrients in the diet are trace elements Zn, Fe, Cu, Se, and Mn. These minerals are often not provided in sufficient quantities in commercial maize and soy based diets. The presence of antinutritional substances such as phytate (Aksu et al. Citation2012) further complicates the matter. To meet this challenge, breeders use mineral salts in the form of oxides, sulphates, and carbonates in the diet. A large proportion of these mineral elements are excreted in the environment due to low bio-absorption resulting in environmental pollution (Vieira Citation2008). Chelated forms of trace elements can increase bioavailability and so reduce waste and soil phytotoxicity (Nys et al. Citation2018). There is a great interest in using trace elements in organic forms (chelated) due to the high rate of intestinal absorption and bioavailability compared to mineral forms (Wang et al. Citation2019; Muszy’nski et al. Citation2018a, 2018b).
The chelated form of trace elements have other benefits, including improving the immune system (Nagalakshmi et al. Citation2015; Meshreky et al. Citation2015; Vieira et al. Citation2013; Feng et al. Citation2010), increasing the level of antibodies IgA, IgM, and IgG (Feng et al. Citation2010), reducing lipid peroxidation (Bun et al. Citation2011) and improving superoxide dismutase and glutathione peroxidase activity (Oviedo-Rondon et al. Citation2013; Zhang et al. Citation2018, Citation2021). In addition, the chelated form of trace elements has led to an improvement of cellular immune responses of broilers infected with Eimeria tenella (Bun et al. Citation2011). Some studies have introduced the chelated form of trace elements as an alternative to antibiotics, and have reported that this has led to a decrease in the population of E. coli in the intestine (Kim et al. Citation2011; Oviedo-Rondon et al. Citation2013).
It is very important to prepare broiler diets that lead to maximum production and profitability. Studies have shown that the chelated form of minerals in the diet of birds can lead to an increase in the weight of eggs (Kita et al. Citation1997; Klecker et al. Citation2002; Yildiz et al. Citation2011; Ghasemi et al. Citation2020, Citation2022) and offspring productivity (Noetzold et al. Citation2022). Zhang et al. (Citation2017). Niknia et al. (Citation2023) showed that the chelated form of trace elements led to a greater increase in hen-housed egg production compared to the mineral form. Studies on mineral forms of the elements have identified antagonism between the mineral form of zinc and copper (Ao and Pierce Citation2013). Study of the chelated form of trace elements such as Zn, Fe, Cu, Se, and Mn is of great practical importance. The purpose of this study was to investigate the effect of the chelated form of trace elements (Zn, Fe, Cu, Se, Mn) on production traits, immune system, and blood parameters in Ross 308 broiler breeder chickens.
Materials and methods
Experimental birds
The experiment was carried out at Navid Morgh Guilan Company (Rasht, Iran). Five hundred and sixty Ross 308 broiler breeder chickens (520 females and 40 males) with an average age of 52 weeks were used. There was an adaptation for 2 weeks (weeks 52 and 53) and the beginning of the experiment was in week 54. The experimental area was designed with forty 1.5 × 2.5 m pens, separated by a fences. Each pen had a drinker, a feeder and a two-story nest. The roosters were fed in separate pens.
Lighting and feeding during the study were provided according to the recommendations for female Ross 308 broiler breeders from the latest handbook (2021). Birds were exposed to 14 h of light and 10 h of darkness. The roosters were collected from the experimental units before darkness, and the next day, after lighting and feeding, one rooster was randomly added to each experimental unit. Other environmental parameters including temperature (22 °C), drinkers (4 nipple drinkers/13 birds), feeder space (15 cm/bird), bird density (3.47 bird/m2), humidity (606–5%), light (60 lux), and ventilation were the same for all treatments. All experimental dietary groups were provided with the same amount of feed and water per bird. The birds were monitored daily and any abnormalities were recorded and treated if necessary.
Experimental treatments
A randomised design was used in the form of 8 treatments, and 5 replicates. Each replicate comprised 13 broiler breeder hens with one rooster for 8 weeks (from week 54 to week 61). The basic diet was formulated according to the recommendations of the Ross 308 broiler breeder’s handbook (2021). In addition, experimental diets received two mineral (non-chelated) and organic (chelated with amino acid) elements of selenium, zinc, iron, copper, and manganese prepared by ZINPRO (Eden Prairie, MN, USA® Zinpro). The mineral form of the trace elements under study includes iron sulphate (FeSO4,6H2O, copper sulphate (CuSO4,5H2O, zinc sulphate (ZnSO4, H2O, manganese sulphate (MnSO4,H2O, and selenium premix (SeSO4, + Carrier. All these were formulated by GivanChemi, Tehran, Iran. The organic forms of trace elements were in the form of iron-methionine (Fe-Met- Availa®-Fe 100 USA), manganese-methionine (Mn-Met-Availa®-Mn 80 USA), copper-methionine (Cu-Met- Availa ®-CU 100 USA), zinc-methionine (Zn-Met- Availa®-Zn 1000 USA) and selenium-methionine (Se-Met- Availa®-Se 1000 USA).
Eight experimental diets were prepared:
Treatment 1 (Negative control): Mn, Se, Cu, Fe and Zn provided as non-chelated form;
Treatment 2 (Positive control): Mn, Se, Cu, Fe and Zn provided as chelated form;
Treatment 3: Mn, Se, Cu, Fe and Zn provided as 50% chelated form and 50% non-chelated form;
Treatment 4: Zn provided as chelated form;
Treatment 5: Cu provided as chelated form;
Treatment 6: Fe provided as chelated form;
Treatment 7: Mn provided as chelated form;
Treatment 8: Se provided as chelated form.
Table 1. Supplemented amounts of trace minerals in experimental treatments (mg/kg).
Table 2. Ingredients and chemical composition of experimental diet (negative control) for broiler breeder chickens.
Production traits
During the experiment (from week 54 to week 61), the number of eggs was recorded six times per day. The birds were weighed to an accuracy of ±0.1 grams (GF-300 balance, A&D Weighing, San Jose, California). The percentage of egg production (eggs/bird/day), egg mass (g/bird/day), average egg weight (g/egg), and egg specific gravity for each experimental unit was calculated for each week and for the entire period (from week 54 to week 61).
To calculate the percentage of egg production, the number of eggs laid each day in each experimental unit was divided by the number of live broiler breeder chickens. The egg mass-produced was determined from the product of egg weight and production percentage. Hen day production data was calculated every week from the total number of eggs divided by hen days in that period. Since there was no mortality, hen day production was equal to a percentage of egg production and so is not present as a separate parameter.
The egg weight of each experimental unit was weighed with an accuracy of ± 0.1 g at the end of each day using a GF-300 balance. The average weekly egg weight for each experimental unit was calculated and recorded at the end of the week.
Every two weeks, all the eggs collected from two consecutive days were weighed in order to check the specific gravity. This was determined by immersion in defined saltwater solutions. Nine salt water solutions were prepared with densities from 1.060 to 1.100 g/cm3 in steps of 0.05 (Hamilton Citation1982). To ensure that eggs were at room temperature and the pores of the shell were closed, this was done 12 h after the eggs were placed in the refrigerator of the farm. The prepared solutions were was kept at about 18 °C. To perform this test, the eggs were placed in a basket for immersion in the salt solutions. When the specific gravity of the egg was equal to the solution, the eggs sank just to the surface of the solution.
The number of settable eggs suitable for hatching was recorded daily. These were stored in a cool room (12 °C, RH 70%). Unsettable eggs were divided into two-yolk eggs, deformed eggs, thin-shell eggs, broken eggs, and small eggs. These were recorded weekly.
Immune responses
Anti-Newcastle antibodies (NDVs; Live LaSota strain; Vetrina, Zagreb, Croatia) and phytohemagglutinin injections were used in order to evaluate the immune system of the tested birds. Antibodies of Newcastle virus were taken on the 44th day from two birds in each replicate. The hemagglutination inhibition (HI) test was performed on the samples according to the OIE standard in two stages on the 30th day of rearing. 96-well microplates were used for the experiment. First, 25 μL of PBS was added to each well. Then 25 microliters of bird serum were pipetted into the first well of a 96-well plate and its dilution was performed until the last well. Then 25 microliters of Newcastle antigen were added to the wells. The microplate was placed on a mechanical shaker for 1 min and then incubated at 25 °C for 30 min. Next, 25 microliters of 1% red blood cells were added to all wells and the microplate was again placed on a mechanical shaker for 15 s. Then the microplate was held at 25 °C for 30 min and the results were recorded. A 4-unit antigen (Pasouk, Iran) was used to perform the hemagglutination inhibition (HI) test (Seidavi et al. Citation2014). Titres were expressed as log2 of the highest dilution indicating agglutination. 1% red blood cells were obtained from specific pathogen-free broilers.
Cell-mediated immunity
Cell-mediated immunity was assessed through injection of phytohaemglutinine (Sigma Aldrich, St. Louis, MO, USA) as reported earlier with the following slight modification. Two birds per replicate (n = 8 per group) were randomly selected at 30 days and the phytohaemglutinine, prepared in sterile phosphate buffered saline (PBS) was injected intradermally (100 μg/100 μL/chicken) between the 3rd and 4th digits of the right foot. The left foot served as control and was injected with 100 μL of PBS. The increase in thickness of the injected sites was evaluated 24 h post-injection using pressure sensitive micrometer. The immune response (foot web index) to phytohaemglutinine was measured by subtracting the left foot thickness from that of the right foot.
Blood biochemistry parameters
To measure blood biochemistry parameters, blood samples were collected from the vein under the wing of two chickens from each experimental unit in test tubes without EDTA anticoagulant. Then the blood samples were divided into two parts to measure serum and plasma. To separate the plasma, the samples were centrifuged at 3500 rpm for 20 min (Sorvall super T21, Du Point Co, USA). The serum and plasma samples were transferred into 2 ml microtubes and the blood samples after separating the serum were kept in a freezer (Emersun freezer, NRF3292D Emersun Co, Tehran, Iran) at −20 C. Levels of glucose, triglyceride, progesterone and liver enzymes (ALT, AST) in serum samples was determined using commercial kits of Pars Azmoun and by autoanalyzer (Hitachi, Ltd. Tokyo, Japan), according to the instructions of commercial kits (RANDOX Laboratories Ltd. Ardmore, Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY).
Statistical analysis
Statistical analysis was performed using SPSS 24.0 statistical software (SPSS Inc., Chicago, IL, USA). The comparison between groups was obtained in the form of a completely randomised design using one-way analysis of variance (ANOVA) and then Duncan’s multi-range test was used for mean comparison. p < 0.05 was considered as statistically significant. For Tables and , the data was analysed based on a factorial arrangment based on completely randomised design using a two-way analysis of variance (ANOVA) with the following statistical model: Yijk=μ+Ai+Bj+ABij+eijk.
Table 3. Effect of chelated/non-chelated minerals on egg quantity characteristics (±SEM) at 54th – 61st weeks of ages.
Table 4. Effect of chelated/non-chelated minerals, age (week) and their interaction on egg specific gravity.
Table 5. Effect of chelated/non-chelated minerals, age (week) and their interaction on percentage of 2-yolk, thin shell, deformed, broken and small eggs.
Results and discussion
Production traits
The results of the effect of the chelated form of trace elements on the egg production of Ross 308 broiler breeder chickens are presented in Table . This shows that for the entire period, the percentage of egg production was affected by the experimental treatments significantly (p < 0.05), and the highest levels were observed in treatments 2 and 3. In weeks 54, 59, 60, and 61, a significant difference was observed between the experimental treatments (p < 0.05) (supplementary data) with the highest egg production being in treatments 2 and 3. The effect of the chelated form of trace elements on the egg mass of broiler breeder chickens of the Ross 308 is presented in Table . The egg mass produced per bird per day in production weeks 54, 57, 59, 60, and 61 was been affected by experimental treatments (p < 0.05) (supplementary data). The greatest egg mass was in Treatment 3 and the smallest in Treatment 7 (containing 100% manganese in chelated form). The average egg weight was affected by the experimental treatments only in the 54th week of the production period (p < 0.05) (supplementary data). The lowest egg weight was in the negative control treatment and the highest was in the treatment 6. No significant difference was observed between experimental treatments over the entire period (p > 0.05). The results of the chelated form feeding of trace elements on the egg specific gravity show a significant difference between the treatments in weeks 55 and 61 (p < 0.05) with the treatments containing the chelated form of trace elements showing a higher specific gravity than treatment 1 (basic diet containing a non-chelated form of the mineral elements of Zn, Fe, Cu, Se, Mn) (p < 0.05) (supplementary data).
The economic efficiency of a broiler breeder flock has a high correlation with factors such as egg production, so improving the quantitative and qualitative traits of eggs is important. Special attention should therefore be paid to the vital role of minerals in the body’s metabolic processes and egg production (Fakler et al. Citation2002). Many factors including genotype (Fathi et al. Citation2022), age of broiler breeders chickens (Santos et al. Citation2022), environmental conditions including breeding systems (þuk’c- Stojci’c et al. 2009; Faghih-Mohammadi et al. Citation2022), as well as additives (Safaa et al. Citation2008; Bai et al. Citation2022), change the quantitative and qualitative characteristics of eggs. The absorption of trace minerals presented in the chelated form is significantly higher than that for minerals presented in the non-chelated form (Muszy’nski et al. Citation2018a, 2018b).
There are many other published reports showing the benefits of chelated minerals in the diet. These include Kita et al. (Citation1997), Klecker et al. (Citation2002) and Yildiz et al. (Citation2011) who showed that the chelated form of manganese in the diet of broiler breeder chickens increased egg weight. Zhang et al. (Citation2017) and Yang et al. (Citation2022) showed that the chelated trace elements led to improved production performance in broiler breeder chickens. Maciel et al. (Citation2010), Favero et al. (Citation2013a, Citation2013b) and Paton et al. (Citation2002) compared the chelated and mineral forms of Zn, Mn and Cu in the diet of broiler breeder chickens and showed that the chelated forms resulted in increased egg weight, specific gravity, shell thickness, improved of egg conversion ratio and egg quality without having any effect on egg production. Kim et al. (Citation2022) and Khoshbin et al. (Citation2023) showed that manganese-methionine chelate improved antioxidant activity, immune system and egg manganese enrichment in old laying hens. However, some studies do not report a difference in egg production performance or egg quality between the chelated and non-chelated forms (Tabatabaie et al. Citation2007; Liao et al. Citation2018). Pekel and Alp (Citation2011) using a copper-chelated form instead of a mineral form showed no beneficial effect on egg production and egg mass and Yenice et al. Citation2015 failed to show that the use of the chelated form of Mn, Zn,Cu, and Cr had significant effect on the egg production traits or egg weight in broiler breeder chickens.
In general, the results of the present study showed an increase in egg production and egg mass in Ross 308 broiler breeder chickens with the consumption of a basic diet containing mineral elements of Zn, Fe, Cu, Se, Mn as 50% non-chelated form and 50% chelated form (treatment 3) compared to other treatments. This may be due to the interaction of the complex of minerals in the chelated form with each other without the effects of antagonism, leading to an increase in the production of the desired traits. The differences between the various published studies may be due to differences in the sources of mineral elements, experimental periods and genetic differences, age, and physiological condition of the birds.
In the trial, the number of unsettable eggs in the entire production period and in week 60 was affected by the experimental treatments (p < 0.05). Treatment 2 had the lowest number of unsettable eggs (p < 0.05) (supplementary data). The experimental treatments also resulted in a significant difference in the production of settable eggs in the entire production period and all the production weeks, except for weeks 57 and 58 (p < 0.05) (supplementary data). The highest number of settable eggs was observed in treatments 2, 3 4 and 8 (p < 0.05) (Table ). The diets for treatments 4 and 8 contained chelated zinc and selenium respectively.
The results of the chelated form feeding of trace elements and age (week) on egg-specific gravity shows a significant difference between the treatments in weeks 55 and 61 (p < 0.05) and the egg-specific gravity reduced with the layer’s age in all experimental treatments (Table ). The highest egg-specific gravity was at 55, 57 and 59 weeks of age in Ross 308 broiler breeder hens in comparison with 60 weeks (p < 0.05). Also, Table shows no statistically significant effects of the interaction between chelated/non-chelated minerals and age (week) on egg-specific gravity.
Another result of this study is the effect of the chelated form of trace elements on the types of unsettable eggs. The effect on the production of double-yolk eggs is presented in supplementary data. No significant differences in the production of double yolk eggs could be found in any one week's data however the whole production period, was affected by experimental treatments (p < 0.05) (supplementary data). Treatment 1 showed the lowest production of double-yolk eggs and treatments containing chelated manganese and iron (4 and 7) resulted in the highest production of two-yolk eggs (p < 0.05). Obtained results also show that chelated trace elements did not result in a statistically significant difference in the percentage of thin-shell eggs (p > 0.05). but numerically, the base diet containing the no chelated mineral elements showed the highest percentage of thin shells eggs. Results show no statistically significant effects of the treatments on malformed egg production over the entire production period in the trial results except for the percentage of deformed eggs resulting from treatment 7 being higher than for treatment (p < 0.05).
Obtained results presented in supplementary data show that no statistically significant effects of the treatments on the percentage of small or broken eggs were found. With increasing bird age the percentage of small and broken eggs increased for all treatments. Duman et al. Citation2016, Bi et al. Citation2018, Sirri et al. Citation2018 have also observed this change in performance which results in a large economic loss in the egg production industry and affects egg marketing programs (Sirri et al. Citation2018). The strength of the egg shell is crucial in reducing the proportion of broken eggs to prevent economic losses (Zhang et al. Citation2019). Eggshell is formed by the interaction of an organic matrix and calcium carbonate (Nys et al. Citation2004). The physical and mechanical properties, including the elasticity of eggshells, strongly depend on their microstructure (Rodriguez-Navarro et al. Citation2002). Eggshell ultrastructure consists of two eggshell membranes, a mammary layer, a palisade layer, a vertical crystalline layer and a cuticle layer (Lammie et al. Citation2006). The mamillary knob is important for eggshell calcification, and the distance between its nucleus locations may affect the morphology of eggshell crystals. Studies have shown that decreasing mammary thickness and mamillary knob width significantly increases eggshell-breaking strength. This is also strongly related to effective thickness (Zhang et al. Citation2019). Zamani et al. (Citation2005) reported that the addition of trace elements to the diet may change the ultrastructure of eggshells and thus change their mechanical properties. Trace elements such as zinc, copper, and manganese also play an important role as part of the enzymes involved in the process of eggshell or membrane formation or by directly interfering in the formation of eggshell structural components. Such processes include the formation of mucopolysaccharides (Leach et al. Citation1981), desmosine, and isodesmosine or as part of the enzyme carbonic anhydrase (Nys et al. Citation1999). They have catalytic properties as key enzymes and interact with calcite minerals that increase eggshell strength and thickness by improving eggshell ultrastructure (Zhang et al. Citation2017, Nys et al. Citation2018, Qiu et al. Citation2020a).
Due to its presence in the structure of carbonic anhydrase enzyme and cofactor for a keratinase enzyme, zinc plays an effective role in the formation of eggshells and their base membrane. Zinc deficiency also indirectly affects the structure of the epithelium and epithelial secretions during the synthesis of the eggshell membrane (Tabatabaie et al. Citation2007). Therefore, zinc plays an important role in the excretion of albumin in the magnum and the production of egg membranes (Nys et al. Citation2004).
Eggshell membranes in the isthmus are produced from a derivative of lysine and the amino acids of proline, histidine, and cystine under the influence of the enzyme lysine oxidase. Copper is used as an activator in the composition of the lysyl oxidase enzyme and causes the production of elastin. Elastin in the ovary plays an important role in preventing the production of blood-stained eggs and also in creating eggshell membranes (Fakler et al. Citation2002). Copper also leads to an increase in shell strength (Pekel et al. Citation2012). Manganese, with its metabolic and enzyme activities in the isthmus and uterus, plays also an important role in the production of eggshell membranes and eggshell lime making eggshells hard and strong by creating a compact mould of minerals (Fakler et al. Citation2002).
Fakler et al. (Citation2002) showed that the combined feeding of chelated zinc and manganese to laying hens improves the eggshell thickness and strength. Swiatkiewicz and Koreleski (Citation2008), replacing the mineral form of zinc (30 mg/kg) and manganese (50 mg/kg) with the chelated form and found this improved the quality of the eggshell in the last phase of the laying cycle. Hudson et al. (Citation2004a, Citation2004b) reported that broiler breeder chickens fed with a chelated form of zinc produced eggs with better shell quality. Therefore, the mineral elements of manganese, zinc, and copper in the form of chelated and inorganic forms increase the effective thickness, breaking the strength of the eggshell and the ratio of the shell, and reducing mamillary density. Most studies reported that the use of trace elements in chelated form compared to mineral form led to a reduction in broken, cracked, porous, or thin-shelled eggs in broiler breeder chickens (Zamani et al. Citation2005). In another study, the ratio of cracked eggs in hens receiving minerals in the chelated form was consistently lower (Olukosi et al. Citation2018).
The chelated form of manganese (protein-Mn) led to a decrease in the percentage of deformed eggs compared to its mineral form (Yildiz et al. Citation2011). These beneficial effects are probably related to the change in the excretion of mineral elements in chickens and especially the accumulation of manganese in bones, which is promoted by the use of chelated forms of manganese (Yildiz et al. Citation2011). Therefore, these effects are due to the increase in the breaking strength of eggs receiving the chelated form of zinc, manganese, and copper (Gheisari et al. Citation2011; Ramos-Vidales et al. Citation2019). Recently, the findings of Ghasemi et al. (Citation2022), when examining the quality of eggs under the influence of 33, 66, and 100% replacement of mineral supplements with chelated form, found that the breaking strength of the eggshell increases with the increase in the amount of replacement of minerals into chelated form. On the contrary, no changes in eggshell quality parameters were observed in hens fed with chelated form compared to mineral form (Manangi et al. Citation2015). However, factors such as the age of the hens, the race of the hens and the duration of the experiment, and the structure of the chelated form compared to the mineral form, may influence the observed effects (Mabe et al. Citation2003; Maciel et al. Citation2010).
Table also shows that effect of chelated/non-chelated minerals, age (week) and their interaction on percentage of 2-yolk, thin shell, deformed, broken and small eggs. These findings shows that chelated trace elements did not result in a statistically significant difference in the percentage of 2-yolk, thin shell, deformed, broken and small eggs (p > 0.05). But the results showed that percentage of 2-yolk, thin shell, deformed, broken and small eggs increases with the age of hens (Table ), and the highest percentage of 2-yolk, thin shell, broken and small eggs was observed in 61st week of age in Ross 308 broiler breeder hens in comparison with 54th week of age (p < 0.05). Also, Table shows no statistically significant effects of the interaction between chelated/non-chelated minerals and age (week) on the percentage of 2-yolk, thin shell, deformed, broken and small eggs.
Immune system
The results of the effect of the chelated form of trace elements instead of the mineral form on the cellular and humoral immunity of Ross 308 broiler breeder chickens are presented in Table . The results show that the antibody against Newcastle at the age of 44 days has been affected by the experimental treatments (p < 0.05). Treatments 2 and 8 showed the highest amount of antibody production against Newcastle. The results of cellular immune response with phytohemagglutinin solution on day 30 in experiments 1 and 2 were affected by the experimental treatments, and treatments 2 and 8 showed the highest levels compared to other experimental treatments (p < 0.05).
Table 6. Effect of chelated/non-chelated minerals on immunity and blood constitutes and sexual hormone (±SEM) at 61st week of ages.
The resistance of broiler breeder chickens against the Newcastle virus depends on the body’s immune system, which in turn, trace elements play a key role in maintaining the normal function of the immune system. The mechanism of action is that trace elements in the chelated form lead to an increase in the content of immunoglobulin and anti-inflammatory cytokines in the body’s inflammatory response and reduce the mRNA expression level of pro-inflammatory cytokines in the tissue and play an important role in stabilising the immune defense system (Pan et al. Citation2007; Manangi et al. Citation2015; Jarosz et al. Citation2017).
Other authors report results consistent with the result of this study. Oviedo-Rondon et al. (Citation2013) report that replacement (30%) of the mineral form of Zn, Mn, and Cu with the chelated form in the diet of broiler breeder chickens has a positive effect on the humoral immune response and the level of antibodies against the Newcastle disease virus after vaccination. Manangi et al. (Citation2015) found that hens consuming diets containing 25-50% zinc, copper, and manganese in the chelated form compared to the mineral form led to an increase in blood antibody levels. The chelated form of manganese can significantly reduce the number of Salmonella in the caecum and the mRNA expression levels of IL-1b and IL-6 in the intestinal tonsils, which indicates the immunity of the chickens infected with Salmonella is improved by diet (Pan et al. Citation2007). In addition, Jarosz et al. (Citation2017) reported that the chelated form of zinc significantly increased blood interleukin-2 (IL-2) and IL-10 contents compared to zinc sulphate, suggesting the role of minerals in the chelated form increases the body’s resistance to infection compared to the mineral form.
Blood parameters, liver enzymes, and sexual hormones
The results of the effect of the chelated form of trace elements instead of the mineral form on blood parameters, liver enzymes, and sex hormones are presented in Table showing a significant effect of the chelated form of trace elements on blood glucose, triglyceride, and progesterone levels (p < 0.05). The amount of glucose is the highest in the negative control treatment and the lowest in treatment 4.
Blood triglyceride and progesterone levels increased with increasing the composition of minerals in chelated form (p < 0.05) and the negative control treatment showed the lowest levels of blood triglycerides and progesterone. In a study by Hajilari et al. (Citation2019), broilers fed with chelated zinc and copper did not show a significant effect on serum glucose and triglyceride concentrations. The study of Pekel and Alp (Citation2011) showed that the consumption of the chelated form of copper by replacing the mineral form of copper with the chelated form does not have any beneficial effect on total cholesterol, plasma cholesterol, and triglycerides (Pekel and Alp, Citation2011).
The activity of liver enzymes AST and ALT has been affected by the effect of the chelated form of trace elements, and the highest levels of ALT were observed in treatments 2 and 8 (p < 0.05), but the level of AST was the highest in treatment 2 and the lowest in treatment 5 (supplementary data). Treatment 8 which contained selenium in chelated form) showed the highest ALT level. But the AST in treatments 1, 2, and 3 showed the highest levels (p < 0.05).
Since copper, manganese, zinc, and iron are important components of some oxidoreductases as well as important antioxidant substances in broiler breeder chickens and can remove excessive oxidative substances produced by the body to maintain the homeostasis of the body’s antioxidant system (Liu et al. Citation2019; Wang et al. Citation2019), trace elements can significantly increase the activities of liver enzymes (Li et al. Citation2019; Wang et al. Citation2019; Sarlak et al. Citation2021). Zinc is a cofactor for liver enzymes such as AST, and ALT (Bennett et al. Citation2001) and is involved in many metabolic and enzymatic functions (Prasad et al. Citation2009). Das et al. (Citation2014a) reported that the activity of these liver enzymes is influenced by the form of chelated zinc in diets (Das et al. Citation2014b), which is consistent with the results of the present study. On the other hand, it has been reported that the chelated form of trace elements does not affect the activity of AST and ALT compared to the mineral form (Idowu et al. Citation2011; Das et al. Citation2014b).
Conclusions
In conclusion, the chelated form of trace elements led to improved performance, productive traits, immune response, and blood parameters compared to the mineral form. Basic diet containing mineral elements of Zn, Fe, Cu, Se, and Mn as much as 50% non-chelated form and 50% chelated form (treatment 3) leads to a significant improvement in the amount of egg production, egg mass, and egg specific gravity. In addition, the chelated form of trace elements led to a decrease in the percentage of abnormal (double-yolk, thin shell, broken, deformed, and small) eggs.
Ethical approval
The study was approved by the research committee of the authors’ institutions (11750103971005; 13971218).
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Author contributions
FFM, AS and MB participated in preparing the manuscript equally, and all authors approved of the manuscript.
Supplemental Material
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Raw data is available from corresponding author upon resonable request.
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References
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