Determination of Chemical Composition and Apparent Metabolizable Energy Corrected for Nitrogen (AMEn) Content of Amaranth Grain with and without Enzyme in Adult Leghorn Roosters by Regression Method

Document Type: Research Articles


Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran


To determine chemical composition as well as apparent metabolizable energy corrected to zero nitrogen balance (AMEn) estimates of Amaranth grain in adult roosters by regression method, firstly, a specie of Amaranthus hybridus chorostachys were cultivated in a farm pilot at agriculture research station (Khalat Poushan-Tabriz- East Azarbaijan- Iran). After harvesting, amaranth grain was sifted and milled. Finally, the chemical composition of amaranth grain produced in the Advanced Animal Nutrition Laboratory was analyzed. Adult roosters 96-week-old Leghorn in individual metabolic cages for nine daysused to determine AMEn content by the regression method. The basal diet was based on corn-soybean with 3200 kcal/kg metabolizable energy and 12% protein. Two experiments with raw amaranth grain (RAG) and processing amaranth grain (PAG), started separately using 40 leghorn roosters in the form of completely random by factorial method (2´5), that each experiment consisted oftwo levels of Rovabio multi-enzyme (0 and 0.055%) and five amaranth grain levels (0, 15, 30, 45, 60% of replacement or embedment of energizing part of the diet). Each experiment included four replicates that included one bird, it was subjected to biological experiments in metabolic cages by total excreta collection method. The obtained results of regression equations in adult roosters showed that AMEn content for raw amaranth without enzyme (RAG-e), raw amaranth with an enzyme (RAG+e), processed amaranth without enzyme (PAG-e), and processed amaranth with an enzyme (PAG+e) were 3250.24, 3433.18, 3242.18 and 3438.09 kcal/kg, respectively. The total results of this study showed that according to amaranth's adaptable and nutritious agricultural features, it can have a good potential in supplementation of poultry diets with enzymes for improving the energy and nutrition quality.



According to the Food Agriculture Organization (FAO) and the World Health Organization (WHO), Amaranth after the egg and upper than cow's milk, is the second most valuable substance in the world in the protein value chart (Cai et al. 2003). Amaranth is the most important plant from Pseudocereals (FAO/WHO/UNU, 1985). Pseudocereals are plants that produce fruits or seeds which are used and consumed as grains, though botanically pseudocereals are neither grasses nor true cereal grains. Pseudocereals are typically high in protein and other nutrients, gluten-free, and are considered whole grains. Many so-called "ancient grains" are pseudocereals (Gordon, 2006). Today, the introduction and development of forgotten plants such as pseudocereals have been able to help agricultural problems and improve human’s nutritional along with genetically modified plants under the name of new crops (Rastogi and Shukla, 2013). Estimates indicate that the need for cereals, which is one of the most important consumables in poultry nutrition, on a global scale to meet the demand for cereals by 2025 would need an increase in wheat production of 44%, rice production of 43%, and corn production of 56% compared to those of in 2000 (Khush, 1999; Rosegrant et al. 2001). Due to the high protein quality and amino acid balance of Amaranth, which is similar to animal protein sources, as well as the high levels of other nutrients in Amaranth, it has been able to play a valuable role in complementing and replacing nutritional programs in the fight against malnutrition and possible deficiencies of nutrients, especially from cereal sources in dietary foods. According to reports, lysine, methionine and arginine content in Amaranth are 2 to 3 times higher than common legumes (peas, beans, soya, etc.) (Cai et al. 2003). Today Amaranth has become a world-class product of high potential and multipurpose use (Rastogi and Shukla, 2013). The respiratory system in the category of C4 plants belongs to the genus amaranthus (which includes 75 species) and the family amaranthcea (Mozaffarian, 2013). In Iran, 11 species of amaranth are grown and other common names in Persian besides "Taj Khoroos" are "Baroothak" and "Avisi" (Mozaffarian, 1996; Mozaffarian, 2013). Considering the nutritional profile of this research on Amaranth and the classification of the Amaranth according to nutritional division, we can report that there are two major types of Amaranth (Cai et al. 2003). They are the Grain-like Amaranth producers: Amaranthus cruentus, Amaranthus caudatus, Amaranthus hybridus, Amaranthus hypochondriacus, and the Vegetable-like Amaranth: Amaranthus tricolor, Amaranthus dubius, Amaranthus lividus (Cai et al. 2003). Amaranth hybridus is known as "Bari" in the world. At several points in the world, there are South America, Africa, India, China and the United States (He et al. 2002). Amaranth grains are small and sticky. Squalene values in the hybrid species (7.3%) were reported more than other species (Williams and Brenner, 1995; Budin et al. 1996). Amaranth is a world-class herbaceous plant with a higher vegetative and resilient strength than any grain that can be obtained cheaper, and there is a potential for the entry of this plant and its products to nutritional programs (Rastogi and Shukla, 2013). Considering that energy is an important part of the poultry diet and accounts for about 40% of the cost of production, it is important to determine the nutritional energy of the food to properly regulate the poultry diet (Shivazad and Seidavi, 2006). The purpose of this study was to focus on the nutritional potential of Amaranth by determining the chemical composition and numerous nutritional and biological tests on poultry nutrition (metabolic and adult rooster) to provide an accurate assessment of the nutritional value of amaranth.



Planting and production of amaranth

To provide the raw material in this research, which is an herbicide and is an amaranth seed, amaranth farming was carried out in Khalat-Poushan research center (Tabriz, Azarbayjan). The species being studied was A. hybridus. Herbarium specimens of cultivated plants were sent to Iran Botanical Garden (National Botanical Garden of Iran) and approved. In the year 2016, amaranth cultivation was carried out in June and harvesting was in September. After harvest, the prepared samples were milled. Fifty percent of the product was crude (raw amaranth grain (RAG)) and fifty percent of the product was processed under autoclave heat treatment (humidity of 120 ˚C for 5 minutes) (processed amaranth grain (PAG)), that was the raw amaranth processed (RAP). The products were packaged before transferring them for biological and nutritional tests. In addition to thermal processing, in 50 % of treatments, both in the case of grain and in processing enzymatic treatment was carried out of experimental diets (e+). For regression testing, a multi-enzyme was used for enzyme-containing treatments. Rovabio is a multienzyme complex (commercial) and that it is used to increase the digestibility of cereals. Each gram of the multi-enzyme, Rovabio, contains 22000 viscose units of Xylanase enzymes, 2000 units of the enzyme beta-glucanase, as well as other enzymes such as cellulase, pectinase, protease, beta-mannosidase. The reason for using this multi-enzyme Rovabio was to reduce the potential antinutrient effects of Amaranth grain, which is recommended in poultry nutrition as an additive.All samples were transferred to the Advanced Animal Nutrition Laboratory in Tabriz University for analysis and determination composition.


Proximate analysis

In the first stage of the experiments, the chemical and mineral composition of amaranth grain were analyzed according to the Institute of Standards and Industrial Research of Iran (ISIRI, 2003) (Table 1). The nutritional value analysis includes the determination of dry matter according to AOAC (1990) crude protein by Kjeldahl analysis (Kjeltec Analysis Foss 2300 Tecator) according to the reference method of ISIRI 19052, raw fats by Suk according to the method of ISIRI 415, Velp device, ash in electric furnace at 550 ˚C according to the reference method of ISIRI 103 and crude fiber with the Foss Tecator device according to the reference method of ISIRI 3961. Calcium was analyzed using Atomic absorption Shematzo spectroscopy according to the reference method of ISIRI 9266 and phosphorus by the spectrophotometric method (Apel, AA-6300) according to the reference method of ISIRI 513. The gross energy was calculated using a calorimetric bomber Par in the Advanced Animal Nutrition Laboratory in the Faculty of Animal Science, Faculty of the Agriculture University of Tabriz-Iran. Determination of the fatty acids profiles using HPLC QP2010SE GC-MS model based on Institute of Standards and Industrial Research of Iran (ISIRI) 13126-2 was implemented in the ARL (Afshin Rahimi Lab) located in Rasht, Guilan, Iran.


Birds and management

A total of 40 adult roosters (98 wk) were randomly assigned to each treatment. According to the protocol Bourdillon et al. (1990), the adult roosters were studied for 9 days (four days of adaptation to assay diets, one day of fasting, three days of assay diet, and one day of fasting). Due to the thermal processing of the sample as well as the use of the enzyme, the determination method (AMEn) at different levels of the substitution was done by a single regression method on adult Leghorn roosters. According to McNab and Boorman (2002), the reason for using adult roosters was used due to their constant feed intake, low growth rate, higher feed intake than other birds, rapid adjustment to dietary changes, and long maintenance period, results are reported with less variance. Additional reasons for using this bird are consistency, the strength of the digestive system, and the low maintenance requirements. In the regression method, we obtain equations that can be used to predict the amount of energy at each level of the use of the test sample in the experimental diet. The location of these studies was in the adult metabolism laboratory hall at Khalat-Poushan Research Station, Faculty of Agriculture, Tabriz University. In this laboratory, individual cages of 40 × 45 × 40 cm in size were provided with a separate dish and a container for drinking water. The temperature of the metabolism salon of adult roosters was 26 ˚C and ad libitum water consumption, but an experimental diet of 100 g daily was provided for each bird to provide maintenance requirements. Each experiment had 20 birds (five treatments, four replicates of one bird). Two separate assays, once on RAG and once again on PAG to determine AMEn content by regression assay were conducted in a completely randomized design with a factorial arrangement of (2×5) containing two levels of Rovabio enzymes (0 (-e)and 0.055 (+e)%) and in five different levels of experimental (0, 15, 30, 45 and 60% amaranth) and the first treatment was the basal diet and the other treatments included amaranth. Each treatment four replicates for adult Leghorn roosters contains a bird in each of the metabolic cages that were performed by the total excreta collection method. The order of using amaranth and enzyme in experimental diets was as follows in Tables 2 and 3. A basal diet was formulated based on linear programming using UFFDA software (Table 4).


Table 1 Composition of amaranth grain (A. hybridus) and comparison of some corn and wheat composition in NRC (1994)



Table 2 Design of firstexperiment treatments



Table 3 Design of second experiment treatments



Table 4 Ingredient composition basal diet for maintenance requirements for adult roosters Leghorn


1 Vitamin and mineral premix include per kilogram of diet: vitamin A: 7.2 g; vitamin D3: 0.6 g; vitamin E: 14.4 g; Thiamin: 0.72 g; Riboflavin: 3.3 g; Nicotinic acid: 4 g; Pyridoxine: 1.2 g; Ciano Cobalamin: 0.6 g; Menadione: 1.6 g; Folic acid: 0.5 g; Choline chloride: 400 mg; Manganese oxide: 64 g; Iron: 44 g; Sulphate copper: 16 g; Iodine: 0.64 g and Selenium: 80 mg.


The basal diet was formulated only on the of the maintenance requirements (no growth and no production) of the adult rooster. As mentioned, according to protocol Bourdillon et al. (1990), the experimental diet and each of the prepared diets was first prepared for four days for adaptation ad libitum, followed by 24 hours of starvation, and then by placing special trays for collecting feces in a 3-day dietary basis with a daily intake of 100 grams. Upon completion of this course, the animals were hungry for 24 hours to empty the digestive system so that the gastrointestinal tract was completely evacuated, but the accumulation of feces continued during these 24 hours.


Test ingredients and experimental diets

Chemical analysis of experimental diets and excreta samples of biological assay

Experimental rations and excreta collected at the end of the experiment and samples taken in biological experiments were transferred to the Advanced Animal Nutrition Laboratory. The specimens were kept at an oven temperature of 70 ˚C for 72 hours until the wastes dried. After evacuating from the oven and cooling in the refrigerator, samples were transferred to the lab for 24 hours to exchange moisture and then they were milled after weighing. The samples were stored in plastic containers until the chemical decomposition. Chemical analysis of experimental diets and waste samples included determination of dry matter according to AOAC (1990), crude protein by Kjeldahl analysis (Kjeltec Analysis Foss 2300 Tecator) according to the reference method of ISIRI 19052, raw fats by Suk according to the reference method of ISIRI 415, Velp, raw ash in an electric furnace at 550 ˚C according to the reference method of ISIRI 103, crude fiber using atomic absorption spectrometry (Foss Tecator) according to the reference method of ISIRI 3961, atomic absorption spectrometry for calcium according to the method of ISIRI 9266 and phosphorus was also analyzed using (Apel, AA-6300) spectrometry according to the reference method of ISIRI 513. The amount of gross energy was measured using the spectrophotometric method using the calorimeter bomber par.


Calculations and statistics

In order to calculate the AME and AMEn in experimental diets, the formula presented by Sibbald and Wolynetz (1989) and Sibbald (1989) was used.

AME= {](Fi×GEf) – (E×GEe)[ ÷ Fi}

AMEn= {](Fi×GEf) – (E×GEe) – (NR×K) [÷ Fi}


AME: apparent metabolizable energy (kcal/kg).

AMEn: apparent metabolizable energy corrected for nitrogen (kcal/kg).

Fi: feed intake.

E: excreta (g).

GEf: gross energy feed intake (kcal/kg).

GEe: gross energy excreta (kcal/kg).

NR= (Fi×Nf) – (E×Ne).

NR: nitrogen retention.

Ne: percent of nitrogen excreta.

Nf: percent of nitrogen feed intake (feed nitrogen) (%).

K: 8.22 kcal/g nitrogen retained (nitrogen retention corrected coefficient).

Then, to calculate the apparent metabolizable energy of each amaranth sample in experimental treatments and to determine the prediction equations at different levels of replacement in the diet using the regression method, the formulas, and reports proposed in Cao and Adeola (2016) were used as follows:

Cti={Cbd + [(Cad–Cbd) ÷ Pti]}


Cti: AMEn of test ingredient.

Cbd:AMEn of basal diet.

Cad:AMEn of assay diet.

Pti:amaranth replacement percentage.

All data related to the parameters were collected and sorted by Excel 2013 software. Before analyzing data, residual normality test and homogeneity of variance were performed. Data analysis was performed using "R" statistical software. Data were analyzed in a completely randomized design with factorial experiment. To compare the mean of treatments, the Duncan test was used. Regression analysis was used to analyze the apparent metabolizable energy content of the adult roosters.

Yijk= µ + Ai + Sj + (A×S)ij + eijk


Yijk: continuous adjective.

μ: total mean.

Ai: effect of enzymes (i contains 2 levels).

Sj: effect of amaranth in the diet (k contains 4 levels in the regression method).

(A×S)ij: interactive effect of enzymes and amaranth.

eijk: error term of the experiment.



Feed intake (FI) is one of most significant factors in production cost and in supplying poultry requirements, and nowadays it is the main focus of attention of poultry nutritionists and poultry feed industry manufacturers (McNab and Boorman, 2002). Also, FI is one of the effective parameters for metabolizable energy that can affect energy balance through making reductions in endogenous energy loss (EEL) (McNab and Boorman, 2002). The results of Table 5 reveal that, for the main effects, the level of 60 % RAG had the least feed intake (P£0.05). The results of interactions between RAG and enzyme in the parameter of FI show that, with increasing the amaranth level, FI parameter decreased (P£0.05). However, only in level, 60% of RAG with and without enzyme were their significant difference and this level had the least quantity compared to other test groups (P£0.05). Meanwhile, the reported results in Table 6 show that using PAGfor the main effects, at levels of 45% and 60%, the FI reduced significantly (P£0.05). The results of interactions between PAG and enzyme for FI parameter showed that these interactions for levels of 45% and 60% PAG without enzyme had the least amounts (P£0.05). Although in other levels, the FI had decreasing trends compared to the control group, there wasn't a significant difference between test groups (P ≥0.05). Decreasing FI for high levels of amaranth in the diet of trial birds could be due to an increase of fiber in the diet, an increase of non-nutrients, alteration of the balance of nutrients, decrease of palatability, and decreased tendency of the birds to eat the diets (Acar et al. 1988). Overall, the results of feed intake showed that the use of amaranth (raw and processed) without enzyme (-e), could only be used up to 30% for the birds to still eat similar amounts to the control group, and using higher levels of amaranth in the diet resulted in a decrease of FI. If the enzyme was used in diets with RAG, the level of amaranth intake improved up to the level of 45% and was similar to the control group. The impact of the enzyme on the diets with PAG was higher, so intake was similar to the control group up to 60%. Whereas the use of PAG without enzyme (-e) was acceptable only up to 30%. It seems that using an enzyme (+e) was more effective in encouraging FI in diets with amaranth because the use of enzymes leads to an effect on the decomposition of non-nutrients, increasing availability of important nutrients like starch, protein, and minerals in cell walls rich in crude fiber and improved diet palatability. Finally, it helped the endogenous enzymes of the adult birds and improved FI when high levels of amaranth were offered. The results of this study about the decrease of FI with an increased level of amaranth agreed with reports of Waldroup et al. (1985) that had observed the decrease in FI in birds fed high levels of raw amaranth. These researchers, using two species of amaranth (cruentus) and (hypochondriacus( as raw and autoclaved in broiler birds at levels of 20% and 40% in the diet, believed that one of the reasons for the decrease of FI and performance can be due to phenolic compounds and saponins in raw amaranth. This was consistent with the results of the investigations of Connor et al. (1980) that reported that high levels of raw amaranth lead to a decrease in FI and performance. The negligible differences between the results of this study and other reports were due to different levels in intake, bird model, the difference in amaranth variety, and a difference in the processing method. But overall, the results of this study and other researches showed that adding high levels of amaranth without thermal and enzyme processing certainly results in feed intake reduction, and any methods of processing and also using enzyme can help in improving feed intake on a diet with high levels of amaranth. That agreed with other researches on amaranth. In general, reliable energy estimation and availability to it is the basis of the correct formulation of the diet in poultry nutrition (McNab and Boorman, 2002). If the value of GE is closer to the value of metabolizable energy (ME), this illustrates the high nutritional value and high- availability of nutrients (Rochell et al. 2011). The results of Table 5 showed that gross energy (GE) for RAG's main effects, in levels 15% and 60%, had the least and the highest values respectively (P£0.05). Results of Table 6 showed that GE values for the main effects had the least value in levels (without PAG) (P£0.05). Whereas, the results of the main effects of enzyme showed that the level containing enzyme had more GE compared to the groups without enzyme (P£0.05). The interactions of PAG and enzyme had the least value for GE parameter in control group (P£0.05). The values of GE in PAG with enzyme (e+) had significant an incremental trend, so that levels 45% and 60% had the highest values of GE (P£0.05). It seems that thermal and enzyme processing led to an increase of GE in the assay diets. In energy partition discussion, the AMEn has been accepted for poultry. If a feed is not edible by itself or is processed, apparent metabolizable energy is measured by replacing some part of the basal diet with the test ingredient. Based on different percentages of test ingredient replacement, it is possible to write regression equations in any level of use of the test diet for predicting energy values. As apparent metabolizable energy for basal diet is always constant, any energy change in test diets can be attributed to the test ingredient (McNab and Boorman, 2002). Determination of AMEn using the regression method based on replacement method was suggested by Matterson et al. (1965) for the first time, and later many studies and researches were developed by other researchers for determining the metabolizable energy of ingredients in poultry by different protocols (e.g poultry model, time, feeding number, different ingredients, etc.) (Pesti et al. 2005). Sibbald and Walynets (1989) reported that AMEn estimates a more exact estimation of test ingredients of energy. Results of this study showed that increasing RAG level with and without enzyme could increase the AMEn values but there were no significant differences in received content of AMEn by birds for the main effects and also for interactions (P>0.05).


Table 5 Effect of different levels of raw amaranth grain (RAG) with (+e) and without (-e) enzyme replacement on the energy balance of Leghorn adult rooster in the first experiment


AMEn: apparent metabolizable energy corrected for nitrogen and GE: gross energy.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.


AMEn values for the main effects of PAG were the least value for level 0% (without PAG). Levels 30%, 45% and 60% had the highest values (P£0.05). Also, for the main effects of the enzyme, the level containing enzyme had more values of AMEn compared to the group without enzyme (P£0.05). Results of interactions between PAG and enzyme (+e)showed that the AMEn of the control group had the least value (P£0.05). In levels containing PAG with and without enzyme (-e), AMEn values in PAG level 60% without enzyme (-e), and PAG levels 60%, 45%, and 60% with an enzyme (+e), had significant differences and the most values (P£0.05). Use of RAG with an enzyme (+e) led to AMEn values similar to the control group. Also, thermal processing and use of enzyme resulted in an increase of AMEn and a significant difference with the control group and improved energy and gross energy availability (GE) for the birds. The value of AME in the diet, in addition to nutrients and non-nutrients, depends on factors such as age and kind of processing of test ingredient (Larbier and Leclercq, 1994). Also, Bedford and Partridge (2001) believed that increasing FI increased AMEn, because when FI is high, endogenous effects decline. Janmohammadi et al. (2005) reported that factors such as calcium and fat in the ingredient diet can lead to interactions and can affect metabolizable energy values. Choct and Annison (1992) and Annison et al. (1996) and suggested that using multi-enzymes in most cereal grains in poultry nutrition can help in cell wall decomposition, nutrient digestion, and AMEn improvement. They agreed with the present research in enzyme effect on PAG that increased AMEn. However, Kalmendal and Tauson (2012) using multi-enzyme in poultry diet didn’t find any significant effect on AMEn, and this result was consistent with the enzyme results on RAG in assay diets. However, multi-enzyme intake is dependent on factors like diet ingredients, age of poultry and food processing (Bedford and Apajalahtind, 2001).


Table 6 Effect of different levels of processing amaranth grain (PAG) with (+e) and without enzyme (-e) replacement on energy balance of Leghorn adult rooster in second experiment


AMEn: apparent metabolizable energy corrected for nitrogen and GE: gross energy.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.


In the present experiment, equations of prediction were formulated based on different levels of replacement in the diet from 0% to 60% by the regression method, and in Figures 2, 3, 4, and 5 equations and confidence limits (R2), raw amaranth without enzyme (RAG-e), raw amaranth with an enzyme (RAG+e), processed amaranth without enzyme (PAG-e) and processed amaranth with an enzyme (PAG+e) respectively. If (x) is replaced by amaranth substitution percent, we can obtain (y) that represents AMEn. If in all introduced equations, x is replaced by the value 100, AMEnvalues are calculated that results show that AMEnvalues for RAG-e, RAG+e, PAG-e, PAG+e based on regression equations, were respectively 3250.24, 3433.18, 3242.10, 3438.09 and confidence limits (R2) for these parameters were 87, 95, 71, 85 percent respectively. Connor et al. (1980) using amaranth grain species edulis reported that the AMEn of raw amaranth grain is based on dry matter 88.4%, the values of AMEnfor raw grain and autoclaved grain were reported 3145 and 3775 kcal/kg, respectively. In summary, the AMEn values for RAG by Connor et al. (1980), Acar et al. (1988), and Ravindran et al. (1996) were 3145, 3210, and 2832 kcal/kg respectively. Also, the AMEn values for PAG by Connor et al. (1980), Tillman and Waldroup (1986), Laovoravit et al. (1986), Acar et al. (1988), Tillman and Waldroup (1988) and Ravindran et al. (1996) were 3745, 2859, 3475, 2860, 3650, 3040, 3522 and 3133 kcal/kg respectively. In detailing the reports, Laovoravit et al. (1986) determined the AME and TME of amaranth species cruentus using adult broiler cockerels by method of Vohra et al. (1982), the value of AME were in range of 2762 to 3038 kcal/kg. Tillman and Waldroup (1988), in a study named assessment of extruded grain amaranth as a feed ingredient for broilers, estimated the values of AMEnin grain amaranth species cruentus using two methods. These two methods were: 1) total collection method of excreta (TCM) and use of acid-insoluble ash (AIA) as a marker using regression analysis and also the difference method.


Figure 1 AMEn content in assay diet



Figure 2 AMEn content of experimental diets assayed of raw aamaranth grain without enzyme (RAG-e)

Note: when x= 100%, the AMEn value is 3250.24 kcal/kg



Figure 3 AMEn content of experimental diets assayed of raw aamaranth grain with enzyme (RAG+e)

Note: when x= 100%, the AMEn value is 3433.18 kcal/kg



Figure 4 AMEn content of experimental diets assayed of processing aamaranth grain without enzyme (PAG-e)

Note: when x= 100%, the AMEn value is 3242.10 kcal/kg



Figure 5 AMEn content of experimental diets assayed of