THE EFFECT OF BEE BREAD ON THE EGG QUALITY OF JAPANESE QUAILS DURING LAYING PERIOD

The aim of the study was to detect effect of supplementation bee bread in diet on Japanese quail’s egg quality during laying. The quails were divided into four groups as follows: E1 with addition of 2 g, E2 with 4 g, E3 with 6 g bee bread per 1 kg of feed mixture and the control without additives (C). The groups were kept under the same conditions. The supplementation of BB into diets affected the laying parameters in favor of the experimental groups. The average egg weight was significantly (P≤0.05) affected by diet and by age. E2 had significantly lower egg weight than C and E3. The animals had significantly lower egg weight regardless of the experimental group at age of 16 weeks. The most significant differences between the C and E groups were in eggshell thickness. All experimental groups had a thinner eggshell than C. The average egg weight was significantly (P≤0.05) affected by diet and by age. The weight of the egg, yolk and albumen were significantly lower at the beginning of laying (P<0.01). We recorded significant differences between the control group and some experimental groups in the weight and width of the albumen; in the percentage of albumen and yolk between the control group and E2 (P<0.05). The control group had darker eggs (9.58oHLR) than experimental groups (from 8.89 to 9.34oHLR). We can conclude that the BB supplementation in diet had significant effect on selected parameters of egg quality of Japanese quails, mainly on the eggshell thickness.


INTRODUCTION
In the field of animal production, the most growing sector include poultry farming. Japanese quail (Coturnix japonica) is the alternative poultry species, which can be used for egg production. Japanese quail is the smallest poultry species, which provides several advantages such as its resistance to numerous poultry diseases that affect chickens, its greater capacity to benefit from food, high reproduction proportions, and also low feed intake (Santos et al., 2011). Recently, the Japanese quail is used in the world not only to produce eggs and meat, but also as a model animal for experiments (genetic, physiological, nutritional, ethological and technological), the conclusions of which also apply to other economically important avian species, especially hens and turkeys (Genchev et al., 2005, Hanusová et al., 2018. The egg quality is influenced by numerous factors such as the genotype of birds Al-Obaidi and Al-Shadeedi (2017) found no significant differences between chicken and quail egg in albumin content (11.76 vs. 11.80%) and egg yolk content (17.59 vs. 15.58%), respectively. They also found that quail egg cholesterol and LDL (mg.g -1 ) levels were significantly lower than in the chicken egg. These findings imply that the quail egg is relatively healthier than the chicken egg especially for people suffering from cardiac problems. The antibiotics growth promotors for poultry diets have been banned in the European Union in 2006. Therefore, studies on alternative products that could result in promotion of growth, improved feed utilization and maintains of gut health are taking place (Zhang et al. 2005). For this reason, natural bee products are being widely investigated (Babaei et al., 2016;Haščík et al., 2017Haščík et al., , 2017Pavelková et al., 2020a, b). Honey and other bee products are also used in poultry diet for their anti-stress effects (Tatli et al., , 2009Ipek et al., 2007). The fashion for a healthy lifestyle leads to a situation where a number of people start taking care of their health. They search for the highest quality products, preferably with health benefits, rich in vitamins, valuable bioelements and nutrients. Biologically active substances of natural origin always arouse a great interest. This also applies to bee products because of their powerful healing properties. Bee products are multicomponent natural substances necessary for the proper course of basic life reactions (Bobis et al., 2010). These include the following: honey, pollen, bee bread, propolis, royal jelly, and bee venom. Bee products demonstrate a wide range of healing effects. They increase the level of ATP, thus neutralize an effect of many toxic agents, increase immunity of an organism, and improve the energy balance of tissues. They participate in many stages of protein metabolism. Bee bread (ambrosia) is a unique product, which is very important not only for humans, but also for the bees. Bee bread (BB) is in fact fermented and naturally preserved pollen. Pollen is gathered by bees and mixed with its own digestive enzymes, carried in the hive and preserved with a tiny layer of honey and bee wax. It is a fermented bee product made from plant pollen, honey and bee saliva, which undergoes different chemical processes due to the action of specific enzymes, micro-organisms, moisture and temperature (35-36 °C) for 2 weeks ( Bee bread is composed of balanced proteins containing all essential amino acids, the full spectrum of vitamins (C, B1, B2, E, H, P, nicotinic acid, folic acid), pantothenic acid, pigments and other biologically active compounds, like enzymes as saccharase, amylase, phosphatases, flavanoids, carotenoids, hormones (Nagai et al., 2005). According to the study by Aliyazicioglu et al. (2005), bee bread is a product with high potential for use as a food supplement. Bee bread composition and bioactive proprieties have not been studied thoroughly until now and there are only few publications which present detailed studies of its chemical composition and proprieties. However, worldwide interest increases with highlighting chemical and therapeutic properties of bee bread. Some studies indicate that fermentation process is necessary to modify the outer layer of the pollen known as exine, made of sporopollenin, a compound that provides chemical resistance to pollen and preserves the compounds which are within it, and is responsible for the limited capacity for absorbing nutrients and bioactive substances that are inside the pollen grain (Atkin et al., 2011 cited by Zuluaga et al., 2015). The bees can transform the bee pollen in bee bread by an The aim of the study was to detect effect of supplementation bee bread in diet on Japanese quail's egg quality during laying. The quails were divided into four groups as follows: E1 with addition of 2 g, E2 with 4 g, E3 with 6 g bee bread per 1 kg of feed mixture and the control without additives (C). The groups were kept under the same conditions. The supplementation of BB into diets affected the laying parameters in favor of the experimental groups. The average egg weight was significantly (P≤0.05) affected by diet and by age. E2 had significantly lower egg weight than C and E3. The animals had significantly lower egg weight regardless of the experimental group at age of 16 weeks. The most significant differences between the C and E groups were in eggshell thickness. All experimental groups had a thinner eggshell than C. The average egg weight was significantly (P≤0.05) affected by diet and by age. The weight of the egg, yolk and albumen were significantly lower at the beginning of laying (P<0.01). We recorded significant differences between the control group and some experimental groups in the weight and width of the albumen; in the percentage of albumen and yolk between the control group and E2 (P<0.05). The control group had darker eggs (9.58 o HLR) than experimental groups (from 8.89 to 9.34 o HLR). We can conclude that the BB supplementation in diet had significant effect on selected parameters of egg quality of Japanese quails, mainly on the eggshell thickness.

ARTICLE INFO
anaerobic fermentation process. Pollen transformation into bee bread occurs as a result of successive interventions of different enzymes, some species of microorganisms such as Pseudomonas, Lactobacillus, Saccharomyces that are naturally present in pollen, moisture and temperature (35-36º C) ( Barene et al., 2015). During this fermentation, the wall of the pollen is disrupted and in this way the bee bread has a better bioavailability. Comparatively with bee pollen, beebread is better tolerated by the human organism and has a lower pH (3.8-4.3) duo to its content of lactic acid which offers a good stability during the storage at room temperature (Berene et al., 2015). Until now, there are only a few studies regarding the effects of bee bread in human health. The benefits of bee bread on the hepatic function and blood parameters in alcohol abuse patients suffering from chronic hepatitis was demonstrated in the study of Čeksterytė et al. (2012). The results showed that bee bread helps to regulate the lipid metabolism and exerts a positive effect on the immune system of patients suffering from chronic arthritis and cardiovascular diseases and type 2 diabetes. The conclusion of this study was that bee bread used together with medicaments and Livosan supplement exerted a hepatoprotective effect and improved liver function. Egg quality is factor which contributes for better economy price of fertile and table eggs (Kocevski et al., 2011). Egg quality is usually commented in connection with consumers' requirements and is performed by groups of methods, which give a general characteristic of egg with intact eggshell (freshness, weight, size and shape, eggshell appearance) and the quality of egg parts (albumen, yolk and eggshell) (Genchev, 2012). It has also been indicated that laying hen genotype (breed) and age have a significant impact on egg quality traits. It was demonstrated that egg examined the effect of using BB as a natural supplement on productive and physiological performance of hens. They found that egg number and egg mass per hen and laying rate were significantly improved for the groups fed diets supplemented with different BB levels as compared to the control group during the overall experimental period (24-47 wks.), whereas the group fed with 0.5 g BB.kg -1 had insignificantly higher values of these traits as compared to control group. All studied egg quality parameters were not significantly affected by used BB supplementation compared to the control. The aim of study was to investigate the effect of supplementing the diets with different concentration of bee bread on the internal and external egg quality during laying period of Japanese quails.

Animals and diet
The experiment was carried out on the Department of Small Farm Animals of the National Agricultural and Food Centre -Research Institute for Animal Production in Nitra. Japanese quails of meat line were included in the experiment. The quails were divided into four groups as follows: the control group received no additives (C), the experimental group E1 received bee bread at a dose of 2 g per 1 kg of feed mixture, experimental group E2 received 4 g of bee bread per 1 kg of feed mixture and E3 group 6 g bee bread per 1 kg of feed mixture. Bee bread was of Slovak origin (Medula Ltd., Bratislava). The groups were kept under the same conditions. The quails were reared using a cage technology, each cage was equipped with a feed disperser and water intake was ensured ad libitum through a self-feed pump up to whole experiment. Table 1 shows the list of the ingredients and nutrient content of the basal diets formulated to provide the nutrient requirements of quails according to the recommended reference levels. The feed mixture was produced without any antibiotics and coccidiostats.

Egg quality
Japanese quails began to lay from the age of 7 weeks. The laying was recorded daily. We monitored the effect of the bee bread addition on the laying parameters of Japanese quail during the three months of laying (from 15 to 27 weeks of age). At the age of 16 (age 1), 20 (age 2) and 24 (age3) weeks of females, we performed an analysis of eggs. Total 360 eggs were analysed: 30 eggs from each group once a month for three consecutive months. Egg weight was individually determined to 0.001 g accuracy using a laboratory scale Kern 572-32 (KERN, Balingen, Germany). Egg length (along the longitudinal axis) and egg width (along the equatorial axis) were measured with a micrometer. Egg shape index was calculated as the ratio of egg width to egg length (%) by method of Anderson et al. (2004). After the eggs were broken, egg shells were washed with water and dried to clean the remaining albumen. Following Andreson´s procedure, shell weight (with membrane) was measured using a laboratory scale Kern 572-32 and the percentage proportion of the shell in the egg was calculated. Shell thickness (with membrane) was measured at the sharp poles, blunt poles and equatorial parts of each egg. Shell thickness was obtained from the average values of these three parts. The albumen weight was calculated as the difference between the egg weight, and the yolk and shell weight. The percentage proportion of the albumen in the egg was calculated. Albumen index (%) was determined according to Alkan et al. (2010) based on the ratio of the albumen height (mm) measurement taken with a micrometer to the average of width (mm) and length (mm) of this albumen with 0.01mm accuracy × 100. Individual Haugh Unit score (Haugh, 1937) was calculated using the egg weight and albumen height as follows (Monira et al., 2003): HU = 100 log (H -1.7w 0.37 + 7.6) Hobserved height of albumen in mm wweight of egg in g Yolk weight with 0.001 g accuracy was determined using a laboratory scale Kern 572-32 and its percentage proportion was calculated. Yolk index (%) calculated based on the ratio of the yolk height (mm) to the yolk width (mm) was measured according to Funk (1948) using micrometer with 0.01mm accuracy. Yolk colour was determined with La Roche scale (Hoffman-La Roche, Switzerland). Egg length (along the longitudinal axis) and egg width (along the equatorial axis) were measured with a micrometer. Egg shape index was calculated as the ratio of egg width to egg length (%) by method of

Statistical analysis
Statistical analysis was done using the SAS 9.2 statistical software (2009). The GLM model was applied to study the influence of effects causing variation of individual egg quality parameter and age in Japanese quail. The following model was applied: where: Yijindividual egg quality parameters μintercept Di -fixed effect of diet (C, E1, E2, E3); Σi E = 0 Aj -fixed effect of age (1, 2, 3); Σj A = 0 eijrandom error; eij = N (0, Iσ 2e) i=1 to 4 j=1 to 3 Differences among means were obtained using Tukey´s test for the same number of observations in each class of individual effects.

RESULTS AND DISCUSSION
The result of laying performance of control group (C) and group with addition of different concentration of bee bread in Japanese quails' diet are given in Table 2.
As we can see from the table, we found the best laying indicators during three months laying period in the experimental group with the addition of 6 g per kg of feed. The control group had the lowest average laying performance both in the number of eggs per laying hen and in the laying intensity. Similar results were found by other authors with application of bee bread. Arafa et al. (2016) were investigated the effect of dietary bee bread (B), bee pollen (P) and their mixtures (BP) as antioxidants on productive performance and functional properties of table eggs in Hy-Line hens strain. They indicated that all the experimental diets produced greater egg number than control diet, but a clear improvement was observed in the hens fed with diet contained supplemented with 0.15% and 0.2% P and 0.1% B + 0.1%P. Egg mass per hen was significantly improved by the diet contained 0.2% P comparing with the control diet. The diets supplemented with 0.2% B, 0.15% P and 0.1% B recorded significantly lower values of triglycerides in fresh eggs yolk than control diet. The total cholesterol in stored yolks ranged from 18.02 to 45.99 mg.dl -1 in the hens fed diets with BHT, 0.2% P, 0.1% B + 0.1% P, 0.15% B, 0.15% P and 0.2% B respectively while it was 63.79 mg.dl -1 in control diet. The digestibility coefficient of ether extracts was significantly improved by the diet with 0.15% B and 0.15% P compared to the control diet. Awad et al. (2013) investigated the effect of supplementing bee bread (BB) as a growth promoter and antioxidant material at levels of 0, 0.5, 1.0 and 1.5 g.kg -1 diet as well as a group supplemented with 250 mg ascorbic acid.kg -1 as a positive control (PC) on laying performance and egg quality traits. Egg number and egg mass per hen and laying rate were significantly improved for the groups fed diets supplemented with different BB levels as compared to the control group during the overall experimental period (24-47 weeks), whereas the group fed 0.5 g BB.kg -1 had insignificantly higher values of these traits compared to PC group. Least squares means and standards errors for external and internal egg quality are given in Table 3. Egg weight is among the most important parameters not only for consumers, but for egg producers as well (Genchev, 2012). In our experiment, average egg weight was significantly (P≤0.05) affected by diet and by age of quails. The quails of experimental group 2 had significantly lower egg weight than control and E3. At the beginning of the laying, the weight of the eggs (age 1) was significantly lower (12.84 g, versus 13.51 and 13.40 g). Similarly, the weight of the egg yolk and albumen were significantly lower at the beginning of laying. Gradually, the weight of the egg, yolk and albumen increased (P<0.01). The animal had significantly lower egg weight regardless of the experimental group at age of 16 week (age 1). We also found significant differences between the control group and experimental group 2 in some morphometric parameters of the egg (egg length and egg index). The eggs of experimental group 2 were shorter compared to the control group. Legend: Ccontrol group, E1-experimental group1 received bee bread at a dose of 2 g, E2experimental group 2 received bee bread at a dose of 4 g, E3experimental group3 received bee bread at a dose of 6 g per 1 kg of feed mixture The most important quality traits of the egg shell are its thickness. We found in the thickness of the shell the most significant differences between the control group and the experimental groups. All experimental groups had significantly thinner shell in all measured parameters compared to the control one. In opposite to our work Wang et al. (2007) found that the laying hens supplemented with 1.5% bee pollen significantly increased shell thickness, yolk weight by about 9.22 and 6.89%, respectively than that of the control group. Similarly, Mehaisen et al.  Fletcher et al., 1983; Zita et al., 2013). From yolk characteristics of eggs, we found significant differences only in yolk percentage and yolk colour between control group and E2 with addition of 4 g of bee bread. The control group had darker eggs (9.58 o HLR) than experimental groups (from 8.89 to 9.34 o HLR). The average yolk weight was insignificantly higher in experimental group than in control one. The highest relative yolk weight values may be due to the effects of antioxidants which may be attributed to release of vitellogenin from liver to improve those measurements (Whitehead et al., 1998;Bowry and Ingold, 1999). From the indicators of the internal egg quality, we recorded significant differences between the control group and some experimental groups in the weight and width of the albumen. We also found a significant difference in the percentage of albumen and yolk between the control group and E2 (P<0.05). Awad et al. (2013) investigated the effect of supplementing BB on egg quality of chicken. All studied egg quality parameters were not significantly affected by feeding BB diets as compared to the control. In our experiment the albumen weight as well as the percentage of albumen was lower in all experimental groups compared to the control group. Similar results were observed by Awad et al. (2013). Relative egg albumin weight was significantly decreased by feeding 1.0 (58.59±0.28% albumen weight) and 1.5 g BB.kg -1 diets (58.35±0.49 %) than those fed PC diet.
(59.97±0.54). They found significant differences between the groups in the width of the albumen. We did not find any significant differences in the Haugh Unit. In our work, we found a lower weight of the yolk and its percentage in the control group compared to the experimental groups. Similar results regarding the yolk content in hens feeding with bee bread were found in Award et al. (2013).
Evaluating the impact of age on the quality of eggs, we recorded significant evidence in several indicators between the quality of eggs at the beginning of laying and the quality of eggs in the later laying period. From external indicators of egg quality, we recorded significant differences between individual age categories in egg weight and egg width (Table 3). The weight and width of the egg have been shown to increase with age. Genchev (2012) also found similar results in the quails. During the experiment, the shape of eggs became more elongated (34.34 mm to 34.88 mm). Similar results confirmed Genchev (2012) in Japanese quails of Pharaoh and Manchurian golden lines. From the internal egg quality, we found significant differences in the weight of the albumin, where at the beginning of the laying the average weight was lower than at the end of the laying (from 7.60 g to 8.01 g). Similarly, the weight of the yolk increased with age (from 4.12 g to 4.40 g 1:2 + . 2:3 ++ Legend: µ -least squares mean, -standard error, + -P<0.05, ++ -P<0.01

CONCLUSION
In our experiment, we evaluated the effect of the addition of bee bread in diet on Japanese quail's egg quality during laying. We recorded significant differences between the control group and some experimental groups in the weight and width of the albumen; in the percentage of albumen and yolk between the control group and E2. The control group had darker eggs than experimental groups. The most significant differences between the C and E groups were in eggshell thickness. All experimental groups had a thinner eggshell than C. We can conclude that the adding bee bread into Japanese quails' diets affected the laying parameters in favor of the experimental groups. The age of the quail had a more significant effect on the weight of both the whole egg and its individual components (albumen, yolk).