Turmeric powder in the diet of Japanese quails improves the quality of stored eggs

This study evaluated the effect of turmeric powder (TP) on the productivity and egg quality of quails and on the quality of eggs stored at different temperatures for 7 or 14 d. Quails were distributed in three treatments that consisted of sorghum-based diets with 1.5% and 3% TP or zero TP inclusion, with five replicates for 84 d. Eggs were stored at ambient temperature or refrigerated for 7 or 14 d in a 3 × 2 × 2 factorial arrangement with three TP levels × two storage temperatures (ST) × two storage periods (SP). Inclusion of TP did not affect the performance of the quails or egg quality at 84 d. Interaction SP × ST influenced the height and diameter of yolk and albumen, and the Haugh unit value. Eggs of quails with a diet supplemented with 3% TP and stored for 14 d showed specific gravity similar to the eggs stored for 7 d, despite the TP supplementation. It was concluded that diets with 3% TP did not affect the performance and quality of fresh eggs but improved the quality of eggs stored for 14 d at ambient temperature.


INTRODUCTION
Corn is the main energetic ingredient used in animal diets. Several feeds may sometimes be used as a corn substitute to reduce the production costs. Sorghum is the most commonly used feed in these situations. However, sorghum has very low β-carotene (CHE et al., 2016) and when used in the diet of laying hens, a pigment must be added to the diet to avoid light yolks. Yolk color is one of the main factors that influences the buying decision of consumers because it is associated with the nutritional value of the egg (MOURA et al., 2010). Another consumer demand is the consumption of healthy food with no synthetic additives (ATTIA et al., 2018). Hence, turmeric powder may be used as a natural pigment. Turmeric rhizomes (Curcuma longa) are widely used as condiments and pigments in food. Curcumin is the main pigment in the turmeric rhizome (ATTIA et al., 2017). According to Bartov & Bornstein (1966), birds do not synthesize pigments, but 20%-60% of the ingested pigment is deposited in the yolk. Egg quality reflects the physical and chemical status of the eggs. Nutrition affects egg characteristics such as egg weight, egg yolk, and egg white proportion (RÉHAULT-GODBERT et al., 2019). Turmeric is a food that may improve egg quality by increasing the functions of the liver, where nutrient metabolism occurs and vitellogenin is produced, and reproductive organs, such as the magnum and uterus, where the albumen and the eggshell are produced, respectively (SARASWATI et al., 2013a). Curcuminoids found in turmeric rhizomes (2.5%-6.0%) consisted of curcumin (curcumin I), demethoxycurcumin (curcumin II), bisdemethoxycurcumin (curcumin III), and a cyclocurcumin. In commercial turmeric, curcumin I represents 70%, curcumin II, 17%, and curcumin III, 3% (LEE et al., 2013). Turmeric contains antioxidant (SUN et al., 2019), anti-inflammatory (HEWLINGS & KALMAN, 2017), antiviral (ALAGAWANY et al., 2015), antitumor, and antimicrobial (MOSELHY et al., 2018) substances that improve body function (SARASWATI et al., 2013a), and consequently, productive performance, as shown by Attia et al. (2017), Nuraini & Djulardi (2019), Kennedy et al. (2020), and Liu et al. (2020). Most of the studies have focused only on the quality of fresh eggs; thus, this study was carried out to evaluate the effect of dietary supplementation with turmeric powder on the productivity and egg quality of Japanese quails as well as on the quality of eggs stored at ambient temperature or under refrigeration for 7 or 14 d.

MATERIAL AND METHODS
This study was approved by the Ethics Committee on Animal Use of the Universidade de Rio Verde (protocol n. 01/14, approved on March 18, 2014). In total, 105 Japanese quails, 50 d old, were distributed in a completely randomized design to three treatments and five replicates. The experimental period lasted 84 d. Treatments consisted of sorghum-based diets containing turmeric powder (0%, 1.5%, and 3%). The evaluated levels were chosen according to preliminary studies published in the literature to elucidate contradictory results. Turmeric powder (TP) was obtained after washing, slicing, drying, and grinding of the rhizomes. Experimental diets were formulated according to the nutritional requirements for quails as recommended by Rostagno et al. (2011) (Table 1). Water and diets were provided ad libitum throughout the experiment. Birds were housed in galvanized wire cages (25 L × 15 H × 33 W, cm) equipped with gutter-type feeders and drinkers. The temperatures in the production facilities, from August to November, were 18 o C (min) and 31 o C (max). The light program was initiated when the birds were 40 d old with an initial supply of 14 h of light/day and increased per week of 30 min until it reached 17 h of light/day, which was retained until the end of the experimental period. The evaluated parameters were productive performance (laying rate, egg mass, daily feed consumption, and feed conversion) and egg quality (weight, specific gravity, Haugh unit, and pH of the egg; weight, height, and diameter of the yolk and albumen and yolk color; and weight and thickness of the eggshell). The specific gravity of eggs was determined by immersing them in saline solutions of different densities (1.05-1.10 g/cm 3 ). The Haugh unit was calculated using the following formula: HU = 100 × log (H -1.7 × W 0.37 + 7.6), where H is albumen height (mm), and W is egg weight (g). Albumen weight (g) was calculated as the difference between the weight of the entire egg and the combined weight of the yolk and eggshell (g). Eggshell thickness was measured as reported by Attia et al. (2020). During the last 3 d, all the eggs produced in each cage were weighed, and three were used to determine the weight, height, and diameter of the yolk and albumen. Eggshells were weighed. The albumen weight was obtained by subtracting the weight of the yolk and eggshell from the egg weight. The height and diameter of the yolk and albumen were measured using a formal caliper. Based on the data, the percentage of each component was calculated, along with the Haugh unit following the formula: UH = 100 × log (H −1.7 × W 0.37 + 7.6), where H is the albumen height (mm), and W is the egg weight (g). The remaining eggs were used to determine the specific gravity by immersion of the eggs in containers of saline solution (NaCl), whose densities ranged from 1.050 to 1.100, with intervals of 0.005. Eggshells were washed and air-dried to obtain the weight and thickness that was measured at three different points (at both the poles and lateral region of the shell) with a digital caliper with a precision of 0.0 mm. On the 84 th day, birds continued to receive the experimental diets. In total, 72 eggs were collected on the 85 th day and were stored at ambient temperature (30 °C ± 2.1 °C) for 7 d (36 eggs) and 14 d (36 eggs). Further, 72 eggs were collected on the 86 th day and stored under refrigeration (4 °C ± 0.6 °C) for 7 d (36 eggs) and 14 d (36 eggs). The experimental design was completely randomized in a 3 × 2 × 2 factorial arrangement, with three TP levels (0%, 1.5%, and 3%) in the diets, two storage temperatures (Tp), and two storage periods (SP), with four replicates as the experimental unit with three eggs each, totaling 12 eggs per treatment. The same egg quality parameters were evaluated. The results of the productive performance and egg quality obtained with the sorghum-based diets, with or without TP, were analyzed by ANOVA using the software SISVAR ® (FERREIRA, 2011). Results of the quality of the stored eggs were also analyzed by ANOVA, and when necessary, Tukey's test was used to compare the means at 5% probability.

RESULTS AND DISCUSSION
Inclusion of TP did not affect (P > 0.05) the productive performance of the quails ( Table 2). Inclusion of TP did not alter the productive performance, which means that it did not alter the diet palatability and nutrient use, and consequently, did not change the feed conversion, laying rate, or egg mass. Feed intake may be affected by feed palatability and glucose levels in the blood. Curcumin has an effect similar to insulin in the blood glucose control (AL-SAUD, 2020;HARTOGH et al., 2020) and stimulates bile secretion (WANG et al., 2016), which is important for digestion. However, in studies with quails using TP doses of 0.5-2.0% (SILVA et al., 2018) or 0.1%, 0.2%, and 0.3% (LAGANÁ et al., 2019), no change was observed in the feed intake and nutrient use. Working with laying hens, Laganá et al. (2011) evaluated a 2% turmeric inclusion in the diet but did not note its effects on the productive performance of the birds. Similarly, Malekizadeh et al. (2012) studied the inclusion of 1% and 3% turmeric in the diet and did not report differences in egg production or egg mass. Egg quality was not affected (P > 0.05) by treatment at the 84 th day of rearing (Table 3) (2019) noted a significant change in the yolk color using 5 to 20 ppm turmeric powder in the diet of Japanese quails; the values varied from 8.32 (control) to 10.55 (20 ppm) as per the colorimetric fan. Klassing (1998) elucidated that pigment deposition in tissues depends on the amount present in the diet and the ability of the bird to digest, absorb, and metabolize the pigment. Regarding eggshell quality, curcumin could improve the uterine microenvironment and thus, increase calcium deposition and consequently, the weight and thickness of the eggshell (Radwan et al. 2008). However, this effect was not observed in our study. Saraswati et al. (2013b) reported no effect of TP on egg and eggshell weight, but eggs had a thinner eggshell and a higher diameter and height of yolk and albumen, with a higher Haugh unit value. Later, Saraswati et al. (2016) evaluated turmeric at doses of 0, 54, and 108 mg/bird/day and did not verify the effect on the egg weight, weight, thickness of the eggshell, height and diameter of the yolk and albumen, and Haugh unit. There was no effect (P > 0.05) of TP × storage temperature × storage period on egg quality. However, albumen weight was lower (P < 0.05) in eggs stored at ambient temperature and increasing the storage period from 7 d to 14 d resulted in lower (P < 0.05) weight of the egg and albumen (Table 4). The decline in the quality of stored eggs occurred because of the degradation of the egg protein, ovalbumin. This degradation results in water formation, which migrates to the yolk, increases its weight, and causes dissociation of carbonic acid, one of the buffer system components in albumen, which dissociates in water and CO2 (FEDDERN et al., 2017). Refrigeration delays these reactions, and at ambient temperature, the albumen loses water more easily to the environment and to the yolk, which contributed to the 0.3 g reduction in weight. Al-Sagan et al. (2020) demonstrated that pH is related to water-holding capacity in broiler meat. Independent of the storage temperature, increasing the storage period negatively influenced albumen weight, and consequently, the egg weight was reduced by 0.43 g. Similar results were found by Nepomuceno et al. (2014), who also did not note the changes in the weight, percentage of yolk, and eggshell thickness of the eggs of quails due to the storage period (5 and   With respect to yolk color of the eggs, the results concur with those of Laganá et al. (2012), who did not observe changes in the yolk color of eggs from hens fed a diet containing 2% curcumin and stored at ambient temperature for up to 28 d, compared with the control treatment (with no curcumin). The inclusion of 3% turmeric powder in the diet caused an increase in albumen diameter (Table 5). Hens supplemented with curcumin at 200 mg/kg showed higher estrogen levels, according to Liu et al. (2020). Albumen is mainly synthesized in the tubular gland cells in the magnum and comprises ovalbumin, conalbumin, ovomucoid, and lysozyme. The synthesis of these molecules has been associated with the hormone estrogen (MISHRA et al., 2020). The interaction of temperature × period of storage affected (P < 0.05) the height and diameter of the yolk and albumen as well as the Haugh unit value of the eggs. Increasing the storage period from 7 to 14 d, independent of the storage temperature, resulted in a reduction (P < 0.05) in yolk height, particularly when the eggs were stored at ambient temperature. Eggs stored at ambient temperatures exhibited yolk with lower diameter (P < 0.05), albumen with a lower height, and consequently, lower Haugh unit values (Table 5). Storing the eggs for 14 days, independent of the storage temperature, resulted in reduction of the yolk height, particularly when the eggs were stored at ambient temperatures, compared to eggs stored for 7 days. Eggs stored at ambient temperatures had lower yolk diameter, albumen height and, consequently, lower Haugh unit values. Further, as the egg ages, the yolk absorbs water from the albumen and becomes wider with a fragile vitelline membrane, which causes a reduction in its height. The water loss is influenced by the temperature and storage period. In eggs stored for 7 d, independent of the temperature, the yolk height did not change; however, in eggs stored for 14 d under refrigeration, the yolk height was higher (6.00 mm) than in the eggs stored at ambient temperature (4.14 mm).
The reduction in the Haugh unit value occurred due to the dissociation of the carbonic acid-producing water and CO2. The CO2 is lost to the environment, and the pH of the egg becomes more alkaline. Mucin fibers provide the gel structure to the egg. In an alkaline environment, the fibers become more resistant and the albumen becomes more watery, decreasing its height and increasing its diameter, which will reduce the Haugh unit value (EKE et al., 2013).
According to Rocha et al. (2013), the ovalbumin also undergoes hydrolysis with a destruction of its protein structure, weakening of its vitelline membrane and liquefaction, loss of viscosity, and subsequent reduction of the albumen height. Similar results were found by Park et al. (2012), who did not verify an effect of turmeric on the eggs but increased the storage period from 7 to 14 d, resulted in hens' eggs with a lower Haugh unit value and by Lee et al. (2016), who studied laying hens' eggs stored for 30 d. Turmeric supplementation increased albumen diameter without decreasing its height. It is possible that active substances in the turmeric stimulate the growth of epithelial cells and tubular glands in the magnum, responsible for synthesizing and secreting albumen (SARASWATI et al. 2013b). The interaction of turmeric powder supplementation × storage period was significant (P < 0.05) for eggs from quails with diets supplemented with 3% turmeric powder and stored for 14 d, showing specific gravity similar to the eggs stored for 7 d, independent of the turmeric supplementation (Table 6). Specific gravity may be positively correlated to the resistance of the eggshell and negatively correlated with the air chamber size (ABDALLAH et al., 1993). The reduction in specific gravity occurs due to water loss from the egg, which leads to a progressive increase in the air chamber (SANTOS et al., 2009). This loss is higher in eggs stored at ambient temperatures or for long time periods. As mentioned before, curcumin could improve the uterine microenvironment where the eggshell is formed and increase calcium deposition and the eggshell weight. No effect was observed on the shell, but it is possible that a higher calcium deposition occurred in eggs obtained from the birds fed with diets containing 3% turmeric powder and stored for 14 d. Curcumin is a weak acid with three labile protons (LEE et al., 2013;PRIYADARSINI, 2014), and intestinal acidification, even mild, results in higher solubilization and absorption of minerals, including calcium. In addition, turmeric also contains flavonoids that act like estrogen, improving calcium deposition in the eggshell (RAHARDJA et al., 2015). Laganá et al. (2012) evaluated the quality of eggs from hens fed with diets containing 2% curcumin and stored at ambient temperatures for up to 21 d. The authors did not report effect of the pigment; however, there was a linear decline in the specific gravity due the increase in the storage period.
Egg pH was influenced by the interaction between turmeric levels and storage temperature (P < 0.05). Refrigerated eggs showed similar pH values, independent of dietary turmeric level. Eggs stored at ambient temperatures had a more alkaline pH (8.08) when they were from non-supplemented quails ( Table 7). During ovalbumin degradation, water and CO2 are formed. The CO2 is lost to the environment through the eggshell pores, which results in an increase (alkalinization) of the egg pH (FEDDERN et al., 2017). Refrigerated eggs had similar pH values, independent of dietary turmeric level. However, eggs from non-supplemented quails and stored at ambient temperature had pH values superior to those of the eggs from birds fed diets supplemented with 1.5% and 3% turmeric, demonstrating the antioxidant effect of curcumin. Egg pH increases as storage time and storage temperature also increase (AYOOLA et al., 2016;LEE et al., 2016;HAGAN and EICHIE, 2019), and antioxidants may delay the degradation of ovalbumin and inhibit lipid peroxidation in the yolk and, thus, retain the pH and quality of the eggs for longer, as demonstrated by Attia et al. (2017) in broiler meat.