Effect of organic and inorganic manganese supplementation on performance and eggshell quality in aged laying hens

Abstract Background Manganese (Mn) is an important trace element for laying hen's nutrition, which is required in small amounts in the diet. Its deficiency results in lowered production performance and eggshell quality. Objectives This experiment was conducted to investigate the influence of different sources and levels of Mn on egg production performance and eggshell quality in aged laying hens. Methods A total of 720, 83‐week‐old Hy‐Line W‐36 laying hens were fed a non‐Mn supplemented basal diet for 4‐week (to ime Mn‐exhaustion of body) and then were allocated to a completely randomized design with 10 treatments, six replicates and 12 birds each. Concentration of Mn in the non‐Mn supplemented basal diet was 10.34 mg/kg (treatment 1), the added doses of dietary Mn were included 30, 60, and 90 mg/kg of three different sources (Mn‐oxide, Mn‐sulphate, and Mn‐organic) for treatments 2‐10, respectively. The experiment lasted for 12 week. Results Dietary supplementation with either organic or inorganic Mn sources significantly enhanced egg production (EP), egg mass (EM), feed conversion ratio (FCR), and relative eggshell weight (RESW) compared with the non‐Mn supplemented diet. However, the experimental diets did not influence feed intake (FI), egg weight, and other eggshell quality traits. Based on the broken line regression models, the performance traits were optimized at 30–40 mg/kg Mn concentration when supplemented by Mn‐sulphate or Mn‐organic. Although, it was 80–90 mg/kg when supplemented by Mn‐oxide. The relative bio‐efficacy of inorganic Mn sources include Mn‐oxide and Mn‐sulphate in compare with Mn‐organic were estimated 45% and 87% (for EP trait), 30% and 94% (for EM trait), 36% and 99% (for FCR trait), and 37% and 78% (for RESW trait), respectively. Conclusions In the aged laying hens, Mn requirement is higher than the NRC's recommendation. Sulphate and organic sources of Mn are more effective than Mn‐oxide.


INTRODUCTION
Since the thin eggshell and broken and cracked eggs are the main problem in the egg production industry, the eggshell quality improvement can be one of the main issues in laying hen farms (Manangi et al., 2015).
The percentage of broken or cracked eggs increases with increasing laying hens age and egg size (Khatibi et al., 2021). Mineral nutrition is a remarkable side for optimum egg production and quality in commercial layers. Deficiency of minerals is one of the influential factors responsible for eggshell disorders in laying hens. The saving of optimum mineral concentration in the diets is crucial for getting high-quality eggs for sale (Saleh et al., 2020). Trace minerals are substantial in poultry nutrition due to their role in various biochemical processes needed for normal growth , maturation, and eggshell deposition (Saleh et al., 2020).
Manganese (Mn) is an important trace element for laying hen's nutrition, which is required in small amounts in the diet (Olgun, 2017).
Mn is a cofactor for a wide range of metalloenzymes. It is utilized by almost all forms of life (Culotta et al., 2005). It is also involved in skeletal system development, energy metabolism, nervous system function, immune system function, and reproductive hormone function (Santamaria, 2008). Mn functions as essential accessory factors to enzymes involved in the synthesis of cholesterol, the precursor for the production of steroid hormones and essential for proper reproductive function (Saleh et al., 2020). It is also a component of the principal mitochondrial antioxidant enzyme (Abreu & Cabelli, 2010;Johnson & Giulivi, 2005). Mn deficiency results in lowered egg production and reduced eggshell strength (Lu et al., 2007;Suttle, 2010).
Further, the eggshell ultrastructure becomes abnormal, especially in the morphology of mammillary knobs (Xiao et al., 2015).
However, the National Research Council had recommended a dietary Mn concentration of 20-25 mg/kg for laying hens (NRC, 1994).
This suggested concentration may no longer be sufficient to maintain the optimal performance and eggshell quality of laying hens due to the considerable improvement in their egg production (Olgun, 2017).
Additionally, the reported Mn requirement may vary with the type of diet and supplement used. In the recent diets, the Mn requirement is considered higher than these values (mHy-Line, 2020; Olgun, 2017).
Minerals are added to the poultry diets in inorganic or organic forms.
In organic form, minerals linked with amino acids and become stable and more bioavailable . Commercial diets are typically supplemented with inorganic trace minerals at a proportion greater than the recommendation (Saleh et al., 2020). Meanwhile, use of inorganic trace minerals holds several risks including the formation of insoluble complexes, low digestibility, and environmental pollution via their excretion in urine and faeces . The bioavailability of Mn is very low in most practical feedstuffs, and there is evidence that practical ingredients reduce the bioavailability of inorganic dietary Mn (Halpin & Baker, 1986). Previous results showed that the absorption rate of organic minerals in the gut was greater than it was in inorganic form (Saleh & El-Magd, 2018). Some studies reported that organic Mn sources have more positive effects on performance (Klecker et al., 2002) and eggshell quality (Klecker et al., 2002;Xiao et al., 2015) than the inorganic sources. In contrast, other studies indicate no difference between organic and inorganic Mn sources (Lim & Paik, 2003;Mabe et al., 2003). Given this background, this study was undertaken to evaluate the effects of different levels of dietary Mn supplementation of organic (Mn-amino acid) and inorganic (Mn-oxide and Mn-sulphate) sources on egg production performance and eggshell quality of aged laying hens. In addition, it was hypothesized that it is possible to find an optimal dietary Mn concentration and evaluate the relative bio-efficacy of inorganic to organic Mn sources in aged laying hens.

Feedstuffs analysis
Before the experiment, the basal ingredients such as corn (

Egg production performance
Egg production (number and weight) and mortality were recorded daily, egg weight was recorded weekly, and feed intake and egg quality traits were recorded monthly. Feed intake was calculated by weighing the residual feed in feeders for the respective period. Feed consumption was adjusted for mortality. The feed conversion ratio (FCR) was adjusted for mortality and calculated as follows: total feed intake divided by total egg mass.

Egg quality
For evaluating egg quality traits, six eggs/replicate (36 eggs/treatment) from eggs laid throughout the 3 consecutive days by the end of every

Bone sampling and analytical methods
At the end of 98 weeks of age, after random selection of one bird from each replicate, the birds were first weighed and then killed by cervical dislocation to measure tibia characteristics. Each bird's left tibia was removed, transferred to sealed plastic bags, and maintained at −20 • C for further analysis. Based on the method proposed by the Association of Official Analytical Chemists (Latimer, 2016), the left tibias were first subjected to 5 min boiling to loosen the muscle tissue; then, the meat, connective tissue, and fibula bone were completely removed using scissors and forceps. After cleaning the tibiae, they were placed in an ethanol container (to remove water and polar lipids) for 48 h. The bones were then extracted in anhydrous ether for 24 h (to remove nonpolar lipids). The tibias were dried at 105 • C for 24 h and then weighed.
The weight of the tibia was recorded. To determine tibia ash content, the ash of the bone was used in a muffle furnace for 18 h at 600 • C.

Chemical analysis
The oven drying and Kjeldahl methods were performed according to methods 926.12 and 981.10 of the AOAC International, respectively, for determining DM and CP contents in feed samples (Latimer, 2016).
To analyze the dietary Mn content, a sample from each treatment (three replicates/treatment) was ashed at 550 • C for 14 h; it was then dissolved in 10 mL of 6 N HCl and 30 mL demineralized water at high temperature using a sand heater (300 • C for 15 min). After filtration, the volume was increased to 100 mL using demineralized water (Mabe et al., 2003). The Mn content in ash sample was measured using flame atomic absorption spectrophotometer (Perkin Elmer A Analyst 100, Wellesley, MA, USA).

Statistical analysis
All data were analyzed for normality using SAS 9.1 software through the Univariate plot normal procedure; then, the data were analyzed by using the General Linear Model procedure of SAS Institute (SAS, 2003).
According to analysis of variance (ANOVA), differences among treatments mean were compared using Tukey test, and the values obtained were considered different at a statistical level of 5%. Orthogonal polynomials for linear and quadratic responses to diet Mn concentration were calculated to explore the relationships between dietary Mn level as independent variables and the respective traits as dependent variables. The dietary Mn concentration for maximum response in performance variables, that is R 2 was significant, was predicted using the broken-line regression models, using the nonlinear modelling option in SAS, with the dietary nutrient's density as the independent variable (Robbins et al., 2006). The iterative procedure makes repeated estimates for coefficients and minimizes residual error before the best-fit line is achieved. To assist in choosing a suitable model, coefficient of determination (R 2 ), adjusted R 2 (adj. R 2 ), root means square error (RMSE), and Akaike's information criterion (AIC) values were calculated using the following formulas (Kazemi et al., 2022): (corrected total sum of squares − sums of squares for error) corrected total sum of squares , , U is the rate constant, yt is the observed values,ŷt is the predicted values, N is the number of observations, P = k + 1, and k is the number of parameters.
A multi-linear regression model was applied to estimate the relative bio-efficacy (RBE) value of inorganic Mn (Mn-oxide and Mn-sulphate) to Mn-organic. The nonlinear models procedure (PROC NLIN) of the SAS system was applied, and the models were fit using the following equation: where Y is the dependent variable, a is the intercept (parameters with the basal diet), b is the asymptote response, b 1 is the slope ratio for Mnoxide, b 2 is the slope ratio for inorganic Mn-sulphate, b 3 is the slope ratio for organic Mn, and X 1 , X 2, and X 3 are dietary supplemented levels of Mn-oxide, Mn-sulphate, and organic Mn, respectively.
The RBE value is the ratio between the standard amount and testing source that is required to generate equivalent responses in which a nutrient source at different levels is used and then it is compared to a reference standard based on a biological response such as growth and bone mineralization (Littell et al., 1997). The RBE values for inorganic Mn sources relative to organic Mn were provided by comparing the slope ratios:

Egg production performance
The effect of different treatments on egg production performance indices is shown in Table 3 An improvement was noticed in egg production performance traits (EP, EM, and FCR) by using diet supplementation with Mn-organic and Mnsulphate up to 60 mg/kg Mn, but by increasing dietary supplementation to higher level (90 mg/kg Mn), birds showed undesirable performance (quadratic effect, p < 0.05).

Eggshell quality
The effects of dietary supplementation with Mn on egg quality characteristics are presented in

Tibia bone characteristic
The influence of dietary sources and levels of Mn on the tibia bone characteristic of laying hens at 98 weeks of age is shown in

Estimated Mn requirement
In Mn-organic for egg production performance traits (EP, EM, and FCR).

Relative bio-efficacy of Mn sources
The slope ratio method for the variables of performance and eggshell quality response was employed to measure the RBE of inorganic Mn (Mn-oxide and Mn-sulphate) compared with Mn-organic ( Abbreviation: Non-Sup., non-supplemented. a Every mean is the average of six replicates and six eggs for each replicate (36 eggs for each treatment were measured).   the current study, we observed a few eggshell quality parameters, such as eggshell relative weight, that exhibited a significant response to the Mn dietary supplement levels and sources (p < 0.05), and it is reasonable to apply these sensitive parameters to evaluate the RBE of organic

Egg production performance
This study indicates that dietary Mn supplementation levels and sources affect egg production and feed efficiency, which is consistent with previous studies. It has been shown that fed diet supplemented with Mn can improve egg production performance in laying hens (Fassani et al., 2000;Hossain & Bertechini, 1998;Mabe et al., 2003;Xiao et al., 2015Xiao et al., , 2014. The effects of graded levels of Mn (25, 50, and 75 mg/kg) on performance of commercial layers were evaluated during the period from 42 to 52 weeks of age (Hossain & Bertechini, 1998).
When egg production and egg weight data were regressed against the level of Mn in the diet, a quadratic response for egg production and a linear response for egg weight were obtained. The highest egg production was observed with supplementation of 50 mg/kg Mn, whereas the highest egg weight was observed with 75 mg/kg of Mn in the diet. Xiao et al. (2015) in an experiment that was conducted for bioefficacy comparison of organic Mn with inorganic Mn (0, 25, 50, 100, and 200 mg/kg) in Hy-Line Brown laying hens reported that dietary Mn supplementation with organic Mn at 50 mg/kg level had the highest feed efficiency and the lowest feed intake, when the data were extrapolated from the 12-week feeding trial (55-62 weeks of age). In contrast with the current study and above reports, many studies (Faria et al., 1999;Mabe et al., 2003;Sazzad et al., 1994;Venglovska et al., 2014;Xiao et al., 2015Xiao et al., , 2014Yıldız et al., 2011) Faria et al. (1999) showed that dietary Mn levels (70, 140, or 210 mg/kg) did not affect the performance of laying hens as did Venglovska et al. (2014), who stated that feeding 120 mg/kg did not affect the performance of 20-week-old hen layers. In an experiment conducted by Yildiz et al. (2011), dietary addition of Mn (15-70 mg/kg), regardless of its form, did not affect performance, but egg weight was increased by supplementation with organic Mn (Mn-Bioplex) in laying hens at 49 weeks of age. In the current study, the basal diet Mn content was 10 mg/kg, and the age of the birds was 87-98 weeks; it is reasonable that the effect of adding Mn to aged layers was more apparent than young layers. On the other hand, lowering the Mn content in the basal diet should reinforce the dose-response to Mn dietary supplementation. Mn is one of the enzyme cofactors involved in the synthesis of cholesterol, which is the main structure of ovarian steroids (Olgun, 2017). Dietary deficiency of Mn caused changes in circulating ovarian steroids in laying hens, and consequently reduced reproduction in hens (Feng & Feng, 1998).

Eggshell quality
As shown in Table 4, eggshell relative weight was significantly improved by the dietary supplementation of Mn in aged laying hens. These results are in correspondence with other reports that have shown that fed diet supplemented with Mn can improve eggshell strength in laying hens (Fassani et al., 2000;Mabe et al., 2003;Xiao et al., 2014). Xiao et al. (2015) showed that as an organic source, Mn-amino acid complexes improved eggshell quality parameters in aged laying hens compared to inorganic Mn-sulphate (Xiao et al., 2015). Conversely, other studies comparing the availability of inorganic with organic sources of Mn have reported no significant difference in either performance or eggshell quality in laying hens (Swiatkiewicz & Koreleski, 2008;Yildiz et al., 2011). The positive impacts of organic microelements in laying hens' nutrition and egg quality characteristics were documented by Gheisari et al. (2011) who demonstrated that inclusion of organic Zn, Mn, and Cu in corn-soybean diets was able to improve eggshell and egg albumen qualities (Gheisari et al., 2011). In addition, Favero et al. (2013) concluded that the replacement of inorganic sulphates of Zn, Mn, and Cu with organic amino acid complexes of these microelements in broiler breeders enhanced eggshell quality (i.e., eggshell weight, and thickness) and reduced early embryo mortality (Favero et al., 2013). It is well known that Mn from organic or inorganic sources could affect the mechanical properties of eggshell by affecting the calcite crystal formation and adjusting crystal holographic structure of the eggshell (Mabe et al., 2003;Swiatkiewicz & Koreleski, 2008). Mn is an activator of enzymes that participated in the synthesis of mucopolysaccharides and glycoproteins, which participate in shell organic matrix formation (Saleh et al., 2020).

Tibia bone characteristic
The Mn supplementation levels and sources in the present study did not affect the tibia bone quality traits (Table 5) and composition such as phosphorus, iron, zinc, Mn, and magnesium contents of the tibia bone (data not shown). However, it has been proved that Mn plays an important role in bone formation (Olgun, 2017) and in many biochemical processes by activating enzymes, such as pyruvate carboxylase, superoxide dismutase, and glycosyltransferase (Suttle, 2010). The current experiment's results indicate that bone properties in older hens are not sensitive to the dietary Mn source and level. There is a lack of literature data on the effect of dietary Mn on the bone quality in laying hens (Wang et al., 2002).

Estimated Mn requirement
The current experiment's data indicated that a certain level of dietary Mn requirement for maximal egg production performance traits in laying hens, as a result of improvement in genetics, management, and nutrition over the past 50-60 years, is estimated to be higher than NRC levels. For instance, the Mn requirement of laying hens was recommended as 50 mg/kg (Ochrimenko et al., 1992), 60 mg/kg (Leeson & Summers, 2009), 105 mg/kg (Sazzad et al., 1994), and 120 mg/kg for laying hens (Fassani et al., 2000). In contrast to the results of the current experiment, dietary Mn addition was not necessary for the basal diet (14 mg/kg Mn) of 22-to 42-week-old laying hens (Yildiz et al., 2011). The dietary requirements for Mn depend on the criteria used in studies. For example, a total of 25 mg/kg Mn is considered sufficient to support maximum egg production, egg weight, and FCR, but for maximal eggshell quality, the minimal requirement for laying hens is between 50 and 100 mg/kg (Yang et al., 2012). Additionally, reported the Mn requirement may vary with the type of diet and supplement used (NRC, 1994). The bioavailability of Mn is very low in most practical feedstuffs, and there is evidence that practical ingredients reduce the bioavailability of inorganic dietary Mn (Halpin & Baker, 1986). For example, the Mn requirement 14 mg/kg reported (Southern & Baker, 1983) for chicks fed a semi-purified dextrose-casein diet is much lower than 50-70 mg/kg for growth and bone development in chicks (Caskey et al., 1939), pekin ducks (Wu & Shen, 1978), turkeys (Kealy & Sullivan, 1966), and pheasants (Scott et al., 1959) fed with diet containing practical ingredients. The same levels (60 mg/kg) were reported as the requirement for adult turkeys (Atkinson et al., 1976), pheasants, and quails (NRC, 1994). Overall, the current study's result confirms that the Mn requirement (25 mg/kg) suggested by NRC (1994) is inadequate for aged layers.

Relative bio-efficacy of Mn sources
In the current experiment, the RBE of Mn-oxide and Mn-sulphate was estimated to be 30%-45% and 76%-99%, respectively, as efficacious as Mn-organic at equimolar Mn levels. However, limited reports are available on the RBE of different Mn sources for laying hens, and certain published results are inconsistent (Attia et al., 2010;Fernandes et al., 2008;Xiao et al., 2015). The RBE of organic Mn source depends on its absorption, and inorganic Mn source depends on its solubility in the intestine (Olgun, 2017). It is generally accepted that the organic trace element is advantageous for absorption and, thus, causes less environmental contamination (Ao et al., 2006;Li et al., 2005;mHy-Line, 2020;Xiao et al., 2015;Yan & Waldroup, 2006). As an Mn source, Mn-sulphate is reported to be more available than Mn-oxide and Mncarbonate (Black et al., 1984;Korol et al., 1996). Similarly, it was noted that the dietary addition of Mn-sulphate instead of Mn-oxide improved performance parameters such as feed intake and feed conversion ratio of laying hens and decreased the percentage of broken eggs (Gheisari et al., 2011).

CONCLUSION
The Mn-oxide and Mn-sulphate in comparison with organic source (Mnorganic) were estimated 45% and 87% (for EP trait), 30% and 94% (for EM trait), 36% and 99% (for FCR trait), and 37% and 78% (for RESW trait). Overall, in the aged laying hens, Mn requirement is higher than the NRC's (1994) recommendation, and feed supplementation with organic source is more effective than Mn-oxide.

AUTHOR CONTRIBUTIONS
Heydar Zarghi and Nafise Barzegar designed and carried out the experimental trail. Nafise Barzegar performed lab analysis. Heydar Zarghi performed the statistics, tabulated the data, and wrote the draft paper.
Ahmad Hassanabadi revised and reviewed the manuscript.