Effect of Dietary Amaranth (Amaranthus hybridus chlorostachys) Supplemented with Enzyme Blend on Egg Quality, Serum Biochemistry and Antioxidant Status in Laying Hens

A feeding trial was performed to assess the effects of dietary raw amaranth (Amaranthus hybridus chlorostachys) grain (RAG), with or without an enzyme blend, on the productive performance, blood biochemistry, and antioxidant status in laying hens. The trial was conducted following a completely randomized design by factorial method, including five levels of RAG (0, 10, 20, 30, and 40%, respectively) and two levels of enzyme blend (0 −E and 0.025 +E %). A total of 960 White Leghorn (Hy-line W-36) laying hens (56 weeks of age) were divided into 10 groups with eight repetitions, including 12 birds. The trial period was ten weeks. Results showed that RAG levels in feed (>10%) led to a significant decrease in blood total cholesterol (TC), but they also significantly decreased feed conversion ratio (FCR) (p ˂ 0.05) as measured by feed intake (FI), hen daily production (HDP), egg weight (EW), and mass (EM), leading to overall worse productivity compared to the control group. On the contrary, the addition of the enzyme blend led to an improvement in the investigated production traits (p ˂ 0.05), with the exception of HDP. The enzyme blend was also capable of recovering productive performance when combined with low concentrations of RAG (10%) (p ˂ 0.05), and RAG × enzyme blend groups showed the lowest values of TC (p ˂ 0.05). Moreover, the interaction effects for atherogenic index (LDL/HDL) indicated a significant and promising reduction in response to the addition of RAG both in the presence and absence of the enzyme blend (p ˂ 0.05), and this additive also significantly reduced levels of egg yolk cholesterol (p ˂ 0.05). In summary, the evidence gathered in this trial showed that dietary RAG had positive effects on egg quality characteristics, leading to the production of low-cholesterol eggs, and, at the same time, it may improve the health status of laying hens. Furthermore, the addition of an enzyme blend allowed feeding up to 10% RAG in the diet, leading to an optimal balance between animal productivity and the beneficial effects of RAG.


Introduction
Food safety is a crucial component of the national security of countries, and the increasing demand for healthy nutrition requires innovative solutions encompassing the the Tabriz University. A total of 960 Hy-Line W-36 White Leghorns (56 weeks of age) were assigned to ten dietary treatments with eight replicates having twelve hens in three separate cages of (41 × 23 × 43 cm) with four birds per cage, in a 5 × 2 factorial arrangement by completely randomized design. The dietary treatments consisted of five levels of raw amaranth grain (RAG; 0, 10, 20, 30, and 40%, respectively) with or without enzyme blend addition (0 and 0.025%, respectively), as reported in Table 1. Before starting the feeding trial, egg production of birds was recorded for each individual, and laying hens with equal egg production were allotted in each replication. Hen body weight (BW) and mortality rate were recorded during the 10 weeks of trial duration. Diets were formulated on the basis of linear programming using the UFFDA (User-friendly Feed Formulation, Done Again) software and according to the catalog of Leghorn hens' requirements ( Table 2). Feed and water were offered ad libitum, and lighting program and temperature rearing conditions followed the recommendations of the Hy-line W-36 White Leghorn laying hens.  7 10 + 10 × 0.025 10 RAG +E T 8 20 + 20 × 0.025 20 RAG +E T 9 30 + 30 × 0.025 30 RAG +E T 10 40 + 40 × 0.025 40 RAG +E

Sampling and Analyses
During the feeding trial, daily feed intake (FI), egg weight (EW), egg mass (EM), henday production (HDP), feed conversion ratio (FCR), and Haugh unit were measured. The FI was assessed daily during the trial period, and weekly feed consumption was calculated by subtracting left-over feed from the quantity supplied to laying hens. Separately, the produced eggs were collected and counted daily from each replicate, and EW was recorded. HDP was calculated as total number of eggs collected divided by total number of live hens per day in each group. EM was calculated as per hen per day by multiplying HDP by EW. At the end of the feeding trial, the FCR was calculated as FI/EM.
In order to evaluate the egg quality traits (shell thickness, shell strength, shape index), nine eggs from each replicate were collected for a total of 720 eggs and assessed 24 h after egg collection. Shell strength was measured by a specific instrument (Digital Egg shell force Gauge; Wagner Instruments, Bridgeport, CT, USA). Shell thickness was measured at three locations (air cell, equator, and sharp end) with a digital micrometer instrument (Mitutoyo, Japan). Yolk diameter (D) of all eggs was measured using a compass (Model: Swordfish, Tokyo, Japan), and height of yolk (HY) was measured using a tripod digital micrometer instrument (Model: Mituoyo, Kawasaki, Japan). Yolk index was calculated as [HY/D] ×100, and Haugh unit was calculated as 100 log HA + 7.57 − 1.7 EW 0.37 , where the HA, HY, and EW were albumen height, yolk height, and egg weight, respectively. Moreover, to determine yolk cholesterol content, at the end of the trial period, six eggs from each replicate, for a total of 480 eggs, were transferred to lab and stored at −80 • C for further analysis, as described by Baghban-Kanani et al. [34], after separating the yolk from the albumen. At the end of the trial, six hens from each replicate were randomly selected to collect blood samples from the wing vena. Blood samples were stored in additive-free blood tubes, transferred to lab, and then centrifuged for 10 min at 3000 rpm at 20 • C to obtain plasma for analysis. Plasma samples were used to determine triglycerides (TGs), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and total antioxidant capacity (TAC) using commercial diagnostic kits (enzymatic method) [44]. All samples were subsequently stored at −85 • C in lab for further analysis. TGs were measured using colorimetric enzyme procedure, and HDL and TC were measured using enzymatic photometric method [45,46]. The atherogenic index was calculated as the LDL/HDL ratio. MDA and TAC activities were determined in plasma samples using RANDOX kits (Germany), according to manufacturer instructions. Plasma lipid peroxidation (LP) was determined according to Kei et al. [47] method using 1,1,3,3-tetraethoxypropane standard and based method of the reaction between thiobarbituric acid (TBA) and MDA. The absorbance rate of produced solution was determined spectrophotometrically (V-1201, Shimadzu, Japan) at 532 nm. The plasma lipid peroxidation (LP) values in terms of MDA were expressed as nmol/mL plasma [44,48].

Statistical Analysis
Data were analyzed using a completely randomized design as the factorial arrangement with R Software [49]. In order to assess normal and independent distribution, all random errors were homogenized of variance using the Shapiro-Wilk test and Bartlett's test. Mean differences were compared using Duncan's multiple range test [50] (Duncan, 1955), and values were expressed as means ± standard error of the means (SEM).

Results and Discussion
The characterization of bioactive phytochemicals in amaranth grains, shown in Table 3, shows the abundance of different types of phytosterols and tocopherols and, subsequently, the outstanding presence of squalene, which makes amaranth unique compared to other edible plants. The occurrence of such compounds in food sources indicates the value of superior nutrients, and at the same time, consumers can benefit from the potential benefits of this source to improve health and reduce oxidative stress. The presence of these bioactive compounds and their effects has been reported in other studies by Ogrodowska et al. [51], Tang and Tsao [12], Iftikhar and Khan [52], and Waisundara [53], demonstrating the beneficial effects and the high value of amaranth grain as food or feed.
The findings related to the productive performance of laying hens (Table 4) showed that the main effect of including a high level of RAG in diet determined a decrease in FI and an increase in FCR (p < 0.05). Moreover, increasing RAG levels determined a significant decrease in HDP, EW, and EM (p < 0.05), but the main effect of enzyme addition led to improved productive performance parameters (p < 0.05). The interaction effects indicated that with increasing levels of RAG, the decrease in egg production was significant, but an improvement was observed with the addition of the enzyme blend (p < 0.05). Feeding RAG up to 10% with enzyme blend supplementation led to similar performance to the control group. These results are consistent with the findings of Tillman and Waldroup [54], who reported that the use of amaranth grain at 10, 20, and 30% of the diet of laying hens led to a decrease in FI, but despite the refusal of feed in amaranth diets, no significant variations were observed in daily weight gain or the health status of hens; moreover, feed efficiency was also improved. The same researchers attributed the decrease in FI in birds fed more than 20% of amaranth to the presence of phenolic compounds and saponins in raw amaranth grain [55]. In addition, our results were in line with those of Popiela et al. [23], who reported that the consumption of amaranth grain at 5 and 10% of the diet did not significantly influence the FI of laying hens. It was observed by Pedersen et al. [56] that a high level of lignin in the amaranth cortex was negatively correlated with energy and digestibility, and decreased the bioavailability of nutrients. Decreased FI in laying hens fed high levels of amaranth in their diet could be attributed to increased dietary fiber and non-nutrients, decreased palatability, bitter matter, and less tendency of birds to consume diets [57]. This will increase the availability of important nutrients such as starch, protein, and minerals within the cell walls rich in crude fiber. The observed result of HDP reduction at high levels of amaranth was in contrast to the previous results of Tillman and Waldroup [54], who reported that levels of 10 and 20% of dietary amaranth led to increased HDP. Moreover, at a 30% inclusion level, the same researchers did not observe a decrease in egg production. Perhaps one of the reasons for this difference was the use of extruded processed amaranth grain. Popiela et al. [23] reported that a 5% level consumption of amaranth grain could improve HDP, which was inconsistent with the results of this study, but at 10%, the same researchers reported a decrease in HDP that was in line with our study. Additionally, the increase in EW and HDP reported by Tillman and Waldroup [54] could be attributed to the use of processed amaranth grain and to the presence of linoleic acid, which differed from the results of the present study. The decrease in egg production when fed more than 20% of processed amaranth with or without enzymes was observed recently by Hosseintabar-Ghasemabad et al. [40], who similarly used raw amaranth with or without enzymes. Many studies demonstrated that the efficacy of enzyme blends on laying-type poultry performance, as well as the improvement in EW and EM, was due to the fact that enzymes can lead to reduced concentrations of antinutritional factors and NSP [34,58], and viscosity [59] in edible plants such as amaranth. The use of enzyme blends also improved the activity of lipase and chymotrypsin enzymes in the gut, enhancing the digestibility of nutrients [60] and metabolizable energy [41]. It seems that this set of factors led to improved poultry performance [34,61].
The results shown in Table 5 indicate that at the end of the feeding period, egg quality traits, including shell thickness and strength, shape index, and Haugh unit, were not significantly affected by diets, considering both the main and interaction effects; however, with increasing consumption of RAG in diet, the main and interaction effects showed a significant decrease of 7 to 12% in yolk cholesterol compared to control (p < 0.05). This was in line with the results of Punita and Chaturvedi [32] reporting that the use of raw or processed amaranth at 25% in hens' diet can reduce yolk cholesterol. These researchers assessed that high amounts of fiber, as well as the presence of squalene in amaranth, led to increased hepatic 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase activity, which helps prevent cholesterol deposition in egg yolk. Furthermore, the presence of a high level of linoleic acid in amaranth grains led to the excretion of bile acids, lowering cholesterol levels. Many edible plants and even their by-products, including amaranth, due to the presence of fiber, phenolic compounds, as well as bioactive compounds, can also increase cholesterol-lowering effects without adverse effects on the performance and health status of poultry [36,40,44]. Reklewska et al. [62] reported a decrease in yolk cholesterol using amaranth in laying hen diets, which is in line with the present study. Conversely, Escudero et al. [63] argued that the protein fraction of amaranth grain contains a relatively high amount of fiber, which can lead to anti-cholesterol effects. Several mechanisms have been reported for the effect of amaranth on the reduction in yolk cholesterol. In general, the liver and ovary are the primary sites of cholesterol biosynthesis in laying hens, and the liver is the main site of fat synthesis in the egg yolk. Given that amaranth grains have been proven to be a rich source of squalene, tocopherols, and tocotrienols, they can play an important role in cholesterol biosynthesis. Squalene is an unsaturated triterpene containing 30 polyprenyl carbons; it is structurally similar to beta-carotene, and it is well known as an intermediate of cholesterol synthesis. Squalene and its precursors, including oxidosqualene and bis-oxidosqualene, can be the precursors of 200 triterpenes, which can play many important roles in the body. Squalene in feed can reach the liver with the help of chylomicrons and is used to make steroids and bile acids. Bile acids (colic and deoxycholic) are made from squalene-synthesized cholesterol in liver cells and can combine with glycine and taurine to produce bile salts which have hypocholesterolemic effects in the body. Lower egg cholesterol has a positive correlation with lower cholesterol in the blood and liver [64]. Therefore, any factor that lowers blood cholesterol can lead to a decrease in yolk cholesterol, seeing how a five percent inhibition of HMC-CoA reductase activity leads to a two percent decrease in blood serum cholesterol. Due to the rate of bile secretion in laying hens, which is about 1 mL per kg of body weight per hour, the process of lowering blood cholesterol due to increased turnover of bile acids led to a decrease in egg yolk cholesterol. Feeding of amaranth can also bind cholesterol to bile acids and increase the fermentation effect on the production of short-chain fatty acids (SCFA), preventing the formation of micelles [40]. The cholesterol-lowering potential of amaranth, reviewed by Peiretti [19], supported the hypothesis that this plant may be successfully used in livestock nutrition to produce high-quality products.  The effects of feeding RAG and enzyme blend on blood parameters of laying hens are reported in Table 6. The findings of the main effects of RAG showed no significant differences in hens' blood triglyceride (TG) and LDL (p > 0.05); however, blood total cholesterol (TC) and atherogenic index in all groups fed RAG decreased significantly compared to the control diet. Moreover, blood HDL showed an increase with increasing levels of RAG in diet (p < 0.05). The main effect of enzyme supplementation was effective on blood TC (p < 0.05). The interaction effects showed that RAG without enzyme blend had determined a significant reduction in blood cholesterol at concentrations higher than 10% compared to the control group, whereas including the enzyme blend in the RAG diets led to the lowest blood cholesterol levels (p < 0.05). In addition, regarding blood LDL, only the diet with 40% RAG without enzyme showed the lowest value (p < 0.05). The results of interaction effects for blood HDL indicated that consuming RAG with or without enzyme blend led to higher HDL levels than the control group (p < 0.05). The output of results of the mutual effects on atherogenic index showed that this index was able to show a significant and promising reduction in response to the addition of RAG with or without enzymes (p < 0.05). Previous studies showed that amaranth can play a key role in reducing blood cholesterol in both animals and humans due to the amounts of unsaturated fatty acids, the abundance of squalene, and the high amount of phytosterols and tocopherols it contains [65,66]. In a study, Qureshi et al. [27] reported that supplementing the diet of broilers with grains from two Amaranthus species (Amaranthus hypochondriacus and Amaranthus cruentus) led to a 10-30% of reduction in blood cholesterol. The LDL levels showed up to 40% of reduction, which was consistent with the results in blood cholesterol and LDL in the present study. The aforementioned researchers also demonstrated that the activation of 7-alpha hydroxylase enzyme led to the formation of bile acids from cholesterol and increased the catabolism and excretion of cholesterol. The authors assessed that the dietary feed source containing isoprenoids, while having squalene, tocotrienols, tocopherols, crude fiber, and saponin, can effectively reduce the activity of HMG-CoA enzyme, leading to a significant reduction in blood cholesterol in chickens fed amaranth grains. Lehmann et al. [67] confirmed that feeding of tocopherol and tocotrienols, gamma, and zeta tocotrienols identified in amaranth grain played a role in reducing blood cholesterol. Mendonça et al. [28] and Soares et al. [29] reported that consumption of amaranth grain can reduce cholesterol in hamster blood by 48%. They also stated that when squalene is provided through feed consumption, a certain amount is converted into cholesterol, but such synthesis does not lead to an increase in cholesterol levels. These researchers attributed the decrease in blood cholesterol of hamsters fed amaranth grain to the presence of peptides that limited the production of the HMG-CoA reductase enzyme. However, there are several reports assessing the role of squalene in reducing blood cholesterol [40]. In a study, Janevski et al. [68] observed that total and LDL cholesterol in mice consuming cholesterol-rich food decreased significantly after injection with 3.5 mg of squalene, due to changes in gene expression. Farvin et al. [69] demonstrated that dietary squalene can have beneficial effects on blood pressure and hypercholesterolemia by keeping cholesterol and lipoprotein levels at normal levels. Recently, Leukebandara et al. [70] observed a decrease in blood cholesterol, LDL, and triglycerides by feeding raw and heat-treated amaranth grain in lactating goats.
The presence of sufficient amounts of methionine in amaranth may increase the production of lecithin in the liver, also leading to a decrease in cholesterol and liver fat [71]. Moreover, it was demonstrated that the tocopherols in amaranth grain may potentially reduce the synthesis of cholesterol, LDL, and lipoprotein lipase enzyme, and finally regulate and reduce cholesterol [12,27,[72][73][74]. In addition, the presence of specific phytochemical compounds in amaranth grain, such as 20-hydroxyacidisone (20HE), can lead to antiobesity effects in terms of reduction in triglycerides, cholesterol, and LDL in mice [12,75]. An investigation conducted by Króliczewska et al. [76] on blood parameters in laying hens showed that supplementing up to 10% of diets with amaranth (A. cruentus) led to the significant reduction in blood lipid parameters, which was in agreement with the present study. Conversely, Popiela et al. [23] did not observe any reduction in cholesterol and other blood lipid parameters in laying hens fed with amaranth, and the reason was probably due to the use of low amaranth grain levels (5 and 10%). Furthermore, several reports have also demonstrated that the presence and role of carbohydrate enzymes (such as pectinase, cellulase, furyl-esterase, and glucanase) led to the release of phenolic compounds in amaranth grain, which can indirectly play an effective role in reducing cholesterol and in improving blood lipid parameters [12,30,77].
Investigation of laying hens' blood antioxidant status, in terms of total antioxidant capacity (TAC), malondialdehyde (MDA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), indicated that the main effect of RAG and enzyme as well as their interaction exhibited no differences without any negative effect (p > 0.05). Considering the breeding of laying hens in a cage system and the possible stress conditions in this type of rearing system, there was a fluctuation in blood antioxidant status; therefore, the use of feed sources such as amaranth may improve the antioxidant capacity of birds. In a previous study, Longato et al. [78] reported a significant decrease in serum lipid peroxidation level by feeding Amaranthus caudatus grain to broiler chickens at 0, 5, and 10% of amaranth, which was in disagreement with the findings of the present study. Alvarez et al. [79] found that the presence of phytosterol bioactive compounds, tocopherols, and squalene had a significant role in anti-inflammatory and antioxidant effects, reducing the risks of oxidative stress and also improving the consumer's health. Moreover, vitamin E homologs, having an antioxidant effect, are particularly concentrated in amaranth grains compared to other grains, which can positively regulate physiological and metabolic processes [12,[80][81][82][83]. The experimental diets in the present study did not show any negative effects on the blood antioxidant status of laying hens.

Conclusions
According to the findings of the present feeding trial, the consumption of raw amaranth grain had no negative effect on egg production and quality, mainly due to the abundance of phytosterol and tocopherol bioactive compounds on raw amaranth grains, along with the presence of squalene, leading to an improvement in the health parameters of birds, in terms of their blood lipid profile and antioxidant status. Moreover, a significant reduction in egg yolk cholesterol by feeding amaranth may be considered a valuable option for consumers' choices. Thus, the use of RAG up to 10% along with enzyme blend addition can be recommended as an acceptable practical proposal for the poultry industry.