Multimineral Fortification of Chicken Egg by Supplementing a Combination of Sodium Selenite, Ferrous Sulphate and Zinc Sulphate in Layer Diet

Since the bioavailability of organic minerals are highly effective in supplying minerals to the body, enriching of eggs with multiminerals could be an effective way to supply minerals to human beings. This study was conducted to investigate the effect of Sodium selenite (Na 2 SeO 3 ), Ferrous sulphate (FeSO 4 ) and Zinc sulphate (ZnSO 4 ) incorporated diet on Selenium (Se), Iron (Fe) and Zinc (Zn) concentrations of eggs, egg quality and productivity traits of layers. A feeding trial was conducted with Shaver Brown laying hens (65–week of age) for eight weeks. The dietary groups were basal diet and basal diet plus Na 2 SeO 3 , FeSO 4 and ZnSO 4 with the concentrations of Se 0.3 mg/kg, Fe 120 mg/ kg and Zn 150 mg/kg. Feed intake and egg production were recorded daily. The feed conversion ratio was calculated for the whole period. Concentrations of Se, Fe and Zn in egg samples were determined by using inductively coupled plasma mass spectrometer after microwave digestion in acidic media. Egg quality parameters including egg weight, shape index, eggshell strength, shell thickness, Haugh unit, yolk index, and yolk color were measured weekly and a sensory evaluation of hard boiled eggs was carried out at the end of the experimental period. Supplementation of mineral incorporated diet significantly increased Se and Zn concentrations in egg white by 28.6% and 50.9% (P<0.05) respectively. Fe concentration in egg yolk was increased by 15.7% (P<0.05). Feed intake/hen/day, egg production/hen/week and feed conversion ratio/kg eggs were not significantly different among the treatment and control (P>0.05). Mineral supplemented diet markedly increased (P<0.05) egg weight and egg white weight than the control. The other egg quality characteristics showed a positive improvement, but with no statistically significant difference compared to the control group (P>0.05). The results of sensory evaluation showed higher consumer preference to the yolk color (P>0.05) of mineral fortified eggs than the control. Mineral supplemented diet with Na 2 SeO 3 (0.3 mg/kg), FeSO 4 (120 mg/kg) and ZnSO 4 (150 mg/kg) can be recommended to fortification of eggs with Se, Fe and Zn successfully and, it results positive improvements in egg quality especially the yolk color. The egg production parameters are not affected by the fortification of mineral concentrations and combination used in the current study. by Supplementing a Combination Selenite, Ferrous Sulphate and Zinc Sulphate Layer Diet.


Introduction
Egg fortification with trace minerals is considered as a route towards the prevention of Micro Nutrient Malnutrition (MNM), which has increased greatly over the last few years (Food and Agriculture Organization/World Health Organization FAO/WHO [1]. MNM is one of the health problems widely found in both industrialized and developing countries in the world which contributes greatly to the global burden of diseases FAO/WHO [2]. MNM adversely affects all age groups, but risk of micronutrient deficiency is somewhat high DOI: 10.26717/BJSTR.2021. 35.005683 27559 among young children and women in reproductive age. According to the FAO/WHO report 2004, iodine, iron, vitamin A and zinc deficiencies are the world's most serious health risk factors FAO/ WHO [1]. Role of Se in human health and disease has been discussed in detail along with the deficiency problems in worldwide. As an essential trace element Se plays an important role in antioxidant selenoproteins for protection against oxidative stress originated by surplus reactive oxygen species and reactive nitrogen species in human body. A clear relationship has been observed between low levels of Se and increased risk of diseases, such as cancer and heart diseases Tinggi [3]. The low selenium concentration in the blood is associated with male subfertility Oguntibeju [4]. Recent studies on Se have been directed towards kidney protective efficiency of it. Randjelovic, et al. [5] have shown that the ability of selenium to reduce cardiovascular disease by reducing oxygen free radicals and lipid peroxidation. According to Wiesen, et al. [6] plasma selenium level has been decreased in patients with acute renal injury. But all over the world human populations in many countries are not provided with adequate level of selenium from their food WHO [7].
The level of selenium in human food varies among regions since the content of this element in plant-based food depends on its availability in soil Fisinin, et al. [8].
Iron is an integral part of many proteins that required for maintaining good health. In humans, iron serves as an essential component of proteins involved in oxygen transport Dallman [9]. It is vital for the regulation of cell growth and differentiation Andrews [10]. Iron deficiency limits oxygen delivery to cells, resulting in fatigue, poor work performances and decreased immunity Haas, et al. [11]. Iron is a part of haemoglobins and cytochromes and Zinc functions as essential accessory factor to enzymes Paul [12,13].
Around two billion people in the developing world are deficient with zinc Prasad [14]. It causes infections and diarrhoea which accounts for about 800,000 death per year among children worldwide Hambidge [15]. It is also responsible for the immune deficiency resulting susceptibility to infection especially in elderly population Meydani, et al. [16]. In poultry, Se increases egg production, fertility, antioxidant status, growth rate and reduces feather loss in young laying hens. It positively influences on storage capacity of eggs Edens [17]. Further, Se supplementation has shown to increase the settable eggs, fertility, hatch of fertile eggs, hatchability, and A-grade chicks and reduced embryonic mortality Khan, et al. [18].
Egg fortification is an effective way to deliver these important trace minerals to human body. It may be a good solution for the people who are at risk of trace mineral deficiency. In Sri Lanka chronic kidney disease of unknown etiology (CKDu) has become a significant burden health issue. In some districts the prevalence of CKDu is 15.1-22.9% Rajapakse [19]. As deficiency of Se has a association, Se enriched egg consumption may have a significant effect on alleviation of this situation. However, in Sri Lanka fortified eggs are not common and the information on multimineral fortification in tropical climates are limited. In a successful mineral supplementation, the levels and combination of minerals used should not negatively affect to the egg production parameters and the quality of eggs while enriching eggs. Therefore, the objectives of this study were to investigate the effect of incorporation of combination of Sodium selenite, Ferrous sulphate and Zinc sulphate to layer diet on Se, Fe and Zn concentrations of eggs and to compare the quality characteristics of egg along with the hen's performance such as feed intake, feed conversion rate and egg production under tropical climatic conditions.

Experimental Hens and Feeding Regimen
The feeding trial was conducted at the Livestock Field Station, University of Peradeniya, Peradeniya, Sri Lanka. Shaver brown laying hens (65 weeks old), were randomly allocated into two groups as the treatment and the control. Each group consisted of three replicates of ten laying hens and they were reared according the deep litter system. Commercially manufactured layer ration was ground and used as the basal diet. For the preparation of experimental diet, the basal diet was mixed with Na 2 SeO 3 , FeSO 4 and

Productive Performances of Laying Hens
Feed intake and egg production were recorded daily. Feed conversion rate was calculated as kilograms of feed consumed per kilogram of eggs.

External Quality of Eggs
Five eggs from each replicate were collected every week for analyses of egg weight, shape index, shell strength and shell thickness. Eggs were weighted individually, the width and the DOI: 10.26717/BJSTR.2021.35.005683 27560 length of individual egg were measured. After breaking eggs, two halves of eggshell were wiped with tissue and kept upside down and allowed to remove albumen properly. Dried out shell was used to take the shell thickness. Shape index was calculated using the formulation (breadth/length x100) which described by Reddy, et al. [24].

Internal Quality of Eggs
The albumin weight, yolk weight, yolk index, Haugh unit, yolk color of five eggs from each replicate were used to determined weekly. Eggs were broken and yolk and albumin contents were separated and weights were recorded. Yolk height and albumin height were determined using a Haugh tester (ORKA digital Haugh tester, Lakeview Dr, Bountiful UT, USA). Yolk color was determined using Roche yolk color fan (ROCHE, Yolk color fan, Switzerland).
Yolk index was obtained by the formulation described by Funk (1948). Individual Haugh unit was calculated using according to Haugh [25] (Table 1).

Evaluation of Se, Fe and Zn Content in Egg
Eggs (

Sensory Evaluation
The yolk color, appearance, aroma, flavor and overall acceptability of two hard-boiled egg samples from two groups were evaluated in sensory evaluation with 30 untrained panelists using nine point hedonic scale system (1 -Dislike extremely, 9like extremely). The sensory profiles were conducted on three digit-coded samples.

Statistical Analyses
The experimental design was Completely Randomized Design and data were analyzed using Minitab software package.
Differences at P<0.05 were considered as significant. Sensory data were analyzed using Friedman non-parametric test using Minitab software package.

Productive Performances of Laying Hens
The feed intake of the control and the treatment groups were 110.8±2.9 and 110.2±3.8 g/hen/day respectively and not significantly different (P>0.05). Though hens supplemented with minerals incorporated diet has resulted higher egg production       (Table 2).

Productive Performances of Laying Hens
The feed intake, egg production and feed conversion rates were not significantly difference between the two groups of the present study. Skrivan, et al. [20] have also reported similar productive performance results when supplementing Sodium selenite, Se-  [20,21]. They have used organic and inorganic sources of Se as the same level as the current study. Feed conversion rate/kg of eggs also were not significantly different. Bahakaim, et al. [22], have found that the improvement in feed conversion, but was not significant at the level of 150 mg zinc/kg compared to the control diet. The feed conversion ratio of poultry is heritable and also can be affected by gastrointestinal tract traits De Verdal [27].

External Quality of Eggs
Egg Weight: The current study showed significant increase of egg weights compared to the control (Figure 1). Similarly Ramadan, et al. [26] have found a remarkable increase of egg weights when layers fed with supplemental iron at the level of 100 mg/kg of diet in combination with Zn. The increment was 20% as compared to the control diet. Mean egg weight reported by Skrivan, et al. [20] was in comparable with the current study, for the hens fed with organic and inorganic selenium sources supplemented diet. Bahakaim, et al. [22] have observed significant difference in egg weight by supplementing zinc sources to the laying hen's diet. Saldanha [28] reported that this could be related to the positive effect of organic selenium on the absorption and/or protection of fat-soluble vitamins. Further, the combination of the trace minerals used in this study might have facilitated better utilization of nutrients resulting higher egg weights than the control even though, the feed conversion ratio was not significantly different among the two groups.

Egg Width, Length, Shell Thickness and Shape Index:
Arpasova, et al. [29] have found similar results compared to the current study with regard to the egg width and length when supplementing organic and inorganic sources to the laying hens. and 57.89% respectively than the control group. Skrivan, et al. [23] have observed that the FeSO 4 incorporated diet has increased the Fe concentration in egg yolk and white by 6.3 and 2.2%, respectively.
Moreover, the same concentration of Fe in the diet was used in the present study [35].