Impact of Storage on Chemical Composition of Wheat and Efﬁciency of Its Utilization in Broilers

: This present study was designed to evaluate the effect of storage duration (1.5 years and 2.5 years storage) on wheat quality and the impact of the inclusion of stored wheat in the diet of broilers on performance, nutrient digestibility, and carcass parameters. A total of 560 one-day-old male broilers were randomly assigned to the 56 pens, and each pen had 10 birds. A corn-soybean-based diet was considered as the control diet and the other six dietary treatments were prepared by replacing corn of the corn-soybean diet with 50% and 100% replacement of fresh, 1.5-year stored, and 2.5-year stored wheat. The data of proximate composition of wheat represents that dry matter was improved ( p < 0.05) and acid detergent ﬁber was decreased in 2.5-year-old wheat ( p < 0.05). Performance parameters data represents that both in the starter phase and ﬁnisher phase, the replacement of corn with 50% and 100% fresh wheat in the diet signiﬁcantly reduced the feed intake, nutrient digestibility, and body weight gain ( p < 0.05), and 100% replacement of corn with fresh wheat resulted in higher FCR ( p < 0.05). Based on the ﬁndings of this current study, it is concluded that the storage of wheat improves the performance of birds.


Introduction
Cereal grains are mostly used in the diet of poultry birds because of their high-energy content and digestibility.A sufficient number of grains are stored every year all over the world to meet the demands of the human population and poultry birds.In Pakistan, PASSCO (Pakistan Agricultural Storage and Services Corporation) stores wheat to meet human needs, especially during the scarcity period.Every year, wheat in excess of the requirements of human consumption is sold to the PPA (Pakistan Poultry Association) for use in poultry diets.
Several studies have reported that long-term storage lowers the anti-nutritional factors of wheat, especially non-starch polysaccharides (NSPs) [1].NSPs are dietary fibers, and water-soluble NSPs of the wheat increase the viscosity of the digesta by the formation of thick viscous solutions that lower the feed intake and passage rate of digesta, while water non-soluble NSPs present in the cell wall reduce the availability of nutrients to the poultry birds by blocking the bird's endogenous enzymes' access to the cell contents, creating a "cage effect".These non-digestible dietary fibers inhibit the enzymes to perform their functions and ultimately result in low digestibility of feed [1,2].Negative effects of NSPs could be reduced by storage of wheat before inclusion in the diet of the broiler birds because it has been reported that storage is responsible for the activation of endogenous seed enzymes that lower the negative effects of anti-nutritional factors, especially NSPs [3].
Ravindran et al. [4] and Perttilä et al. [5] reported that the inclusion of stored ingredients mainly wheat and barley in the diet of broiler chickens increased the growth performance, nutrient digestibility, and improved the feed conversion ratio (FCR) based on the assumption that ingredients' storage durations decrease the total starch content, soluble NSP, acid detergent fiber (ADF), and lignin contents and increase the level of free sugar contents.Gras et al. [6] studied the effect of storage temperature (23,35, or 40 • C) and the oxygen concentration (1, 4.6, or 21%) on the quality of flour milled from stored wheat grain and reported that storage temperatures at or below 23 • C ensured constant flour quality, while the oxygen concentration did not affect it.This may indicate that both the temperature and duration of storage influence the activity of in-seed enzymes.However, the data of 1.5-year or 2.5-year stored wheat supplementation in broiler rations are limited.No previous study has compared the stored wheat and new wheat effects on the performance parameters of the broiler chickens.
Based on the above literature, it was hypothesized that the 1.5-year and 2.5-year stored wheat-based diet may perform better than the fresh wheat-based diet in broilers.This present study was designed to evaluate the effect of storage durations (1.5 years and 2.5 years) on wheat quality and the impact of the inclusion of stored wheat in the diet of broilers on performance, nutrient digestibility, and carcass parameters.

Materials and Methods
Experimental protocols were approved by the Advanced Studies and Research Board, University of Agriculture, Faisalabad, Pakistan (939#12-10-2021).

Cultivation of Wheat Used in Experimental Treatments
Wheat used in feed was harvested from carefully grown crops during mid-June 2019, mid-June 2020, and mid-November 2021 in controlled condition production system (lysimeters in aerated glasshouse) of 6 m × 15 m × 2 m dimensions keeping average temperature of 25 • C ± 5 • C, average humidity of 55% ± 10%, and photosynthetically active radiation (PAR) at 850 ± 50 mmol m −2 .A sum of 400 mm/m cube irrigation was applied in splits along with a basal dose of nitrogen of 1.1 kg and phosphorus, 0.55 kg, mixed in top 0.15 m depth of employed lysimeter.After harvesting of wheat, wheat was stored in home-made steel silos at room temperature.

Bird Management and Experimental Treatments
One month before the arrival of chicks, shed was cleaned thoroughly and fumigated.A total of 560, day-old male broiler chicks (Ross-308) were procured from Arslan Chicks, (Pvt Ltd.) Islamabad.A total of seven dietary treatments were used in this experiment.Experimental diets were formulated as, control diet = corn-soybean based diet, no wheat; 50% new wheat (50% NW) = 50% replacement of control diet corn with new wheat; 100% NW = 100% replacement of control diet corn with new wheat; 50% 1.5-year stored wheat (50% 1.5-YOW) = 50% replacement of control diet corn with 1.5-year-old wheat; 100% 1.5-YOW = 100% replacement of control diet corn with 1.5-year-old wheat; 50% 2.5-YOW = 50% replacement of control diet corn with 2.5-year-old wheat; and 100% 2.5-YOW = 100% replacement of control diet corn with 2.5-year-old wheat.The ingredient data used in the diet formulation was taken from Brazilian Tables for Poultry and Swine [7].Ingredients inclusion level and chemical composition of starter and finisher diets are presented in Tables 1 and 2. Each of the seven dietary treatments was randomly assigned to the 56 pens, each pen had 10 birds.Pens were covered with three-inch wood shavings for chicks' bedding.During the experiment, chicks were reared for 35 days keeping the same environmental conditions for all treatments.Fresh and clean water was offered around the clock.Birds were vaccinated according to local vaccination program.A circular bottom feeder was provided for each pen, and nipple drinking system allowed for continuous water availability.Control diet = no wheat; 50%-NW = replacement of corn with new wheat (50%); 100%-NW = replacement of corn with new wheat (100%); 50%-1.5YOW= replacement of corn with 1.5-year-old wheat (50%); 100%-1.5YOW= replacement of corn with 1.5-year-old wheat (100%); 50%-2.5YOW= replacement of corn with 2.5-year-old wheat (50%); 100%-2.5YOW= replacement of corn with 2.5-year-old wheat (100%); 1 PBM = poultry by-product meal, 2 MCP = mono calcium phosphate. 3Each kg of premix will provide per kg of diet:   Control diet = no wheat; 50%-NW = replacement of corn with new wheat (50%); 100%-NW = replacement of corn with new wheat (100%); 50%-1.5YOW= replacement of corn with 1.5-year-old wheat (50%); 100%-1.5YOW= replacement of corn with 1.5-year-old wheat (100%); 50%-2.5YOW= replacement of corn with 2.5-year-old wheat (50%); 100%-2.5YOW= replacement of corn with 2.5-year-old wheat (100%); 1 PBM = poultry by-product meal, 2 MCP = mono calcium phosphate. 3Each kg of premix will provide per kg of diet:

Data Collection for Growth Performance
Birds were individually weighed and assigned to 56 pens (10 birds per pen).On weekly basis body weight was measured to record body weight gain.Estimation of feed intake was done by subtracting the amount of feed refused from the total feed offered during the course of the week.Feed intake and weight was used to calculate FCR as described in the previous study [8].

Digestibility Assay
A digestibility assay was carried out on day 35.Experimental birds were fed diets mixed with acid-insoluble ash (Celite ® ).Diets were analyzed for proximate composition according to the standard method of the AOAC (2002) [9,10].This feeding plan continued until the end of the trial.Fecal digesta samples were collected for digestibility assay on day 35.The floor of each pen was covered with plastic sheets to prevent contamination from bedding material.Excreta was completely cleaned of feathers and other extraneous objects from each pen.Each pen's excreta was weighed, homogenized, dried in the oven at 55 • C for 72 h, and ground for further chemical analysis.Samples were dried in hot air oven at 105 • C for 2 h for dry matter determination.Ash was estimated by burning the sample in muffle furnace at temperature of 650 • C for 2 h.Ether was used for determination of ether extract.Nutrient digestibility was determined using following formula Digestibility (%) = 100 − (100× % marker in feed % marker in feces × % nutrient in feces % nutrient in feed )

Data Collection for Carcass Characteristics
At the end of trial, two birds from each treatment were selected at random, weighed individually, and slaughtered.In order to get data on carcass characteristics, live body weight of birds was recorded.
After slaughtering, feathers were removed followed by evisceration.

Dressing percentage
The carcass was weighed and recorded live weight data were used to calculate dressing percentage by using the following formula.
Breast weight Slaughtered birds' breast was separated and weighed to obtain breast weight.
Thigh weight Slaughtered birds' thigh was separated and weighed to obtain thigh weight.

Chemical Analysis
Triplicate samples of new, 1.5-year, and 2.5-year-old wheat were collected and dried using the hot air oven at 65 • C. The dried samples were ground and passed through a 0.5 mm sieve.Ground samples were further analyzed for proximate composition [9].Samples were also analyzed for neutral detergent fiber and acid detergent fiber by following [11].Lignin content in the samples was also determined based on AOAC Official Method 973.18.Fecal samples were composited replicate-wise and dried at 65 • C to preserve the samples to determine the nutrient contents of feces [9].Extraction of phytate from wheat samples was done by Digestion and Wade Procedures.A 5 g sample was extracted with 100 mL 2.4% HCl at room temperature and centrifuged, precipitates were discarded.Then, 2.4% HCl extract (1 mL/5 mL) was diluted to 25 mL with distilled water.Afterward, 10 mL of diluted sample was passed through a 200-400 mesh AG1-X8 chloride anion exchange column (0.5 g).Inorganic phosphorus was eluted with 15 mL of 0.1 M NaCl and followed by elution of phytate with 0.7 M NaCl.The 3 mL extract was removed immediately, and 1 mL Wade reagent was added for phytate measurement within 15 min.Then, 5 mL aliquots were removed for digestion and colorimetric measurement of liberated phosphorus (approximately 2-3 h required).Sugar contents were analyzed spectrophotometrically using the color reaction with anthrone in concentrated, purified H 2 SO 4 .Prior to measurements, the samples were deproteinized using zinc acetate Zn(CHCOO) 2 •H 2 O 275.12 g/L −1 water and potassium ferrocyanide K 4 Fe(CN 6 ) 3H 2 O 171.99 g/L −1 water.Extinction was measured at a wavelength of λ = 620 nm.The starch content in the wheat sample was analyzed in triplicate using the "Total Starch (AA/AMG) Assay Kit" (Megazyme) following the standard protocols.

Statistical Analysis
Collected data were analyzed using General Linear Model of Minitab Statistical software 17 [12] under completely randomized design.Significant means were tested using Tukey's test.

Agronomic Traits and Chemical Composition of Wheat
The data of agronomic traits of fresh, 1.5-year-old and 2.5-year-old wheat is presented in Table 3. Results showed that plant height, spikes per plant, seed yield per plant, and 1000-grain weight were not affected by the cultivation period (p > 0.05).The data on the proximate composition of wheat used in the experimental diets are presented in Table 4.The results of dry matter data represent that the dry matter was influenced by storage (p < 0.05).High dry matter (DM) was observed in 2.5-year-old wheat and less dry matter was observed in new wheat (p < 0.05).Similarly, crude protein data represents that the crude protein was influenced by storage (p < 0.05) and higher crude protein was observed in 2.5-year-old wheat (p < 0.05).Results of the chemical composition of wheat and stored wheat explored that there was no significant difference in the CF, ash, phytate, and sugar contents of stored and fresh wheat (p > 0.05).However, ADF and NDF contents were influenced by the storage period (p < 0.05).ADF content was observed higher in new wheat while low ADF was observed in 2.5-year-old wheat (p < 0.05).Moreover, lignin contents were also influenced by the storage periods (p < 0.05).Values which are not followed by common superscript within a column differ significantly.

Growth Performance
Data on the influence of experimental diets on performance parameters are presented in Table 5.Both in the starter phase and finisher phase, the replacement of corn with fresh wheat 50% and 100% in the diet of broilers significantly reduced the feed intake (p < 0.05), while the replacement of corn with 100% 1.5-YOW and 2.5-YOW had no effect on feed intake as compared to the control.Similarly, the replacement of corn with fresh wheat 50% and 100% in the diet of broilers significantly reduced the body weight gain (p < 0.05) both in the starter and finisher phase, while the replacement of corn with 100% 2.5-YOW had no effect on body weight gain as compared to the control.The results of FCR represent that the replacement of corn with fresh wheat, 50% and 100%, in the diet of broilers had a significant effect on FCR in the finisher phase, and numerically higher FCR was observed in a 100% NW-based diet (p < 0.05).Control diet = no wheat; 50%-NW = replacement of corn with new wheat (50%); 100%-NW = replacement of corn with new wheat (100%); 50%-1.5YOW= replacement of corn with 1.5-year-old wheat (50%); 100%-1.5YOW= replacement of corn with 1.5-year-old wheat (100%); 50%-2.5YOW= replacement of corn with 2.5-year-old wheat (50%); 100%-2.5YOW= replacement of corn with 2.5-year-old wheat (100%).Values which are not followed by common superscript within a column differ significantly.

Nutrient Digestibility
Data on the inclusion of stored wheat in the broiler diet on dry matter digestibility is presented in Table 6.Results revealed that the inclusion of fresh or stored wheat in the diet of broilers influenced dry matter digestibility (p > 0.05).Replacement of corn with fresh wheat, 50% and 100%, in the diet of the broiler significantly reduced the DM, CF, EE, and ash digestibility in broilers (p < 0.05), while replacement of corn with 2.5-YOW and 1.5-YOW had no effect on dry matter digestibility as compared to the control.Overall, better DM, CF, EE, and ash digestibility was observed in birds fed the control diet, the 2.5-YOW diet, and the 1.5-YOW diet (p < 0.05).Poor DM, CF, EE, and ash digestibility were observed in the birds fed a 50%-NW diet and a 100%-NW diet (p < 0.05).Control diet = no wheat; 50%-NW = replacement of corn with new wheat (50%); 100%-NW = replacement of corn with new wheat (100%); 50%-1.5YOW= replacement of corn with 1.5-year-old wheat (50%); 100%-1.5YOW= replacement of corn with 1.5-year-old wheat (100%); 50%-2.5YOW= replacement of corn with 2.5-year-old wheat (50%); 100%-2.5YOW= replacement of corn with 2.5-year-old wheat (100%).Values which are not followed by common superscript within a column differ significantly.

Discussion
Grains are a major energy source for commercial poultry production.In poultry feed, mill grains are procured from farmers and used on a fresh basis but most commonly grains are stored to provide feed ingredient reserves during scarcity periods.It has been reported that new season grains are problematic for broiler production due to the high contents of soluble NSPs that are responsible for increasing the digesta viscosity [13].However, storing grains for three to four months improves their nutritive values and has a positive impact on broiler chicken production [5,14].In the present study, special attention was given to the changes in the nutritive value of wheat and the chemical composition of wheat during post-harvest storage.The dry matter contents of the wheat decreased in 1.5-YOW and 2.5-YOW, which could be due to the raising temperature of Faisalabad that ranges from 12 • C to 46.0 • C after harvest in Faisalabad.The other possible reason for the decreased dry matter contents could be a decrease in the ADF, lignin, and starch contents of wheat during storage in the current study.It has been reported previously that four-month storage of wheat grains at room temperature reduces the starch, soluble NSP, ADF, and lignin and increased sugar content [15,16] and these findings are consistent with the findings of this current study.
Results of the chemical analysis of wheat used in the feed explored that starch contents decreased during storage in 1.5-YOW and 2.5-YOW.Our results are in accordance with the study by [17], where they reported decreased starch content in wheat during storage duration.Decreased sugar contents during storage have also been reported previously in barley storage time [18].However, in the current study, no effect on sugar content has been seen but a numerical increase in sugar content has been observed.Previously, it has been documented that the chemical composition of wheat changes from one variety to the other, and varieties do not respond in a uniform manner, even within a variety the chemical composition of nutrients changes based on the year of harvest, harvesting time, and growing location of the wheat [16,[19][20][21].
In this present study, there was no significant difference in the phytate content of new wheat and stored wheat.Our results are like the study of Fuente, De Ayala, Flores, and Villamide [18], who reported that storage conditions are considered important for the activation of endogenous phytase in barley and wheat but storage did not affect the phytate content of stored barley.This agrees with, Kim, Lorenz, and Patterson [3] who did not find a reduction in the phytate content of wheat samples kept for 6 months in dry conditions.It is well-established that after the harvesting of grains, endogenous enzymes such as glycanases activate during storage and speed up the soluble non-starch polysaccharide breakdown which may result in decreased levels of starch and ADF and increased sugar content concentration in the stored grains, as observed in this current study.Similar findings have also been reported in a previous study where the four-month storage of wheat grains at room temperature reduced starch, soluble NSP, ADF, and lignin and increased the sugar content [15,16].Our results of the chemical composition of stored and new wheat represent that wheat harvested and stored at room temperature for 2.5 years decreased the total starch and ADF.Similar findings have also been reported in previous studies that reported that storage of wheat for months at room temperature decreased the total starch and ADF contents in wheat [15,16].A previous study by Kim, Mullan, Simmins, and Pluske [16] reported that CP contents in wheat are inversely correlated to the total starch content.The ether extract of this current study was relatively constant in fresh wheat and wheat stored for 2.5 years.In this current study, a numerical increase in the NDF contents of stored grains was observed.It has been reported previously that the NDF increases and the NSP decreases as the grains are stored for longer periods of time [22].
In this present study, the inclusion of 1.5-year-old wheat and 2.5-year-old wheat in the diet of broilers had almost the same impact as compared to the control on broiler performance in the starter phase as well as in the finisher phase.On days 21 and 35, poor feed intake and weight gain were observed in birds fed the 100% new wheat diet as compared to a corn-based diet and a 1.5-YOW and 2.5-YOW diet.Findings of feed intake and weight gain in the wheat-based diet are consistent with findings of the previous study by Scott and Pierce [23], who recorded that the six-month storage of wheat improved voluntary feed intake and body weight gain of broiler chicks by 16.1 and 22.7%, respectively.Similarly, Yaghobfar and Kalantar [24] also reported that voluntary feed intake in birds and average daily gain decreased in the birds fed new wheat.Scott and Pierce [23] reported that the inclusion of stored wheat in the diet of broilers has a positive impact on voluntary feed intake.Another factor that could influence the feed intake in broilers could be digesta viscosity, and it has been reported [25] that higher digesta viscosity reduces feed passage rate [23], which would be the reason for less feed intake of the new wheat diet in this current study.Previously, it has also been reported that factors that influence water consumption also change feed consumption [23].Hence, in the current study, if there was a palatability "factor" in the new wheat diet that may limit water intake, it would also be expected to result in reduced feed intake.Higher feed intake and higher nutrient digestibility could be the reason for the better weight gain in the birds [26].A better feed conversion ratio was observed in the birds fed the control diet, having no wheat, and birds fed a diet having stored wheat had a poor feed conversion ratio, which was also observed in the birds fed diets with 50%-NW and 100%-NW.The new season wheat as the ingredient in the diet of broilers has a negative effect on the feed efficiency and that could be attributed to the high NSP content in wheat that enhances digesta viscosity [13].Higher digesta viscosity is responsible for lesser feed intake, nutrient digestibility, and growth performance in broilers.Similar results on the feed conversion ratio have been reported by Yaghobfar and Kalantar [24].
Nutrient digestibility is influenced by the inclusion of stored grains in the diet of broilers [27].In this current study, better dry matter and crude fiber digestibility was observed in birds fed a corn-based diet and diet that contained stored wheat.Storage of grains not only improves the quality of grains but results in a breakdown of soluble NSPs by the activation of endogenous enzymes such as glycanases [28,29].Higher crude fiber digestibility in the current study was due to the activation of endogenous seed glycanase and glycosidase enzymes by storage.Activated seed enzymes resulted in the partial cleavage of the NSPs to monosaccharides, which removes their anti-nutritive activities.Similar results have been reported by [30].Higher crude fiber and dry matter digestibility in broiler chickens fed barley, oats, and wheat have also been reported in another study [30].
In this current experiment, CP digestibility was better in broiler birds fed a corn-based diet and stored-wheat-based diet while poor CP digestibility was seen in broilers fed the new wheat-based diet.It has been reported that starch granules are embedded in the protein matrix, and it is well established that starch is rapidly digested as compared to protein [31][32][33].In the current study, crude fiber digestibility results indicated that carbohydrate digestion was high in the corn-based diet and stored wheat-based diet that may release the protein matrix available for broiler's endogenous proteolytic enzymes to digest protein and result in higher protein digestibility.Previous studies have reported that the protein digestibility varies between ingredients used as bird feeds [34] and improved nutritive value, especially protein and carbohydrate in stored wheat may be the reason for enhanced crude protein digestibility coupled with efficient work of gastric and pancreatic juice in the gastrointestinal tract of broilers.Ash represents mainly the mineral content of the diet and higher digestibility was observed in a corn-based diet and birds fed stored wheat, which could be due to the improvement of the nutritional value during storage and the breakdown of complex structures in grains that makes minerals more available for digestion and absorption [30].
No significant differences were observed in the carcass characteristics of birds by feeding all seven dietary treatments.Our results are in accordance with the study by [31].This study only focused on the use of stored wheat in a broiler diet and no exogenous enzymes were added to the diet.It is well established that exogenous enzymes in a wheatbased diet have beneficial effects on broiler production, therefore, additional studies should be conducted to explore the impact of exogenous non-starch polysaccharidases when broilers are fed a stored-wheat-based diet.

Conclusions
Based on the findings of this current study, it is concluded that storage improves the nutritional value of wheat and was successfully replaced with corn and new wheat in the diet of broilers.Furthermore, stored wheat has a positive impact on the performance parameters, and nutrient digestibility as compared to the new wheat.Therefore, based on this study, if in replacement of corn wheat is used, then stored wheat can be used in the diet of broiler birds.

Table 4 .
Chemical composition of fresh and stored wheat.

Table 5 .
Growth performance of broilers fed fresh or stored wheat.

Table 6 .
Nutrient digestibility of broilers fed fresh or stored wheat.

Table 7 .
Carcass characteristics of broilers fed fresh or stored wheat.