The effect of dried white mulberry (Morus alba) pulp supplementation in diets of laying quail

Objective. This study was conducted to research the effects of different levels of dried white mulberry (Morus alba) pulp supplementation in diets of laying quail on yield performances, egg quality, blood parameters, yolk fatty acid profiles and cholesterol concentrations. Materials and Methods. A completely randomized experimental design, with four treatments and four replicates, was applied. The experimental treatments were M0: control diet; M4: dietary inclusion of 4% mulberry pulp; M8: dietary inclusion of 8% mulberry pulp; M12: dietary inclusion of 12% mulberry pulp. This experiment was carried out for 4 weeks, and 128 7-week-old female quail were used. Results. Addition of dried mulberry pulp to the diet significantly affected weekly feed intake, egg yield, albumin index, yolk weight, triglyceride, LDL, serum cholesterol and yolk cholesterol levels (p<0.05, p<0.01). The feed conversion ratio, egg weight, and egg yolk fatty acid profile were not significantly affected by the dried mulberry pulp in the diet. Conclusions. As a result, it may be stated that adding dried mulberry pulp up to 8% of the diets of laying quail does not cause any adverse effects and may be used without any problems.


INTRODUCTION
Mulberry is a kind of fruit grown in many parts of the world due to its high adaptability to different climatic and soil conditions (1). Mulberry originates from four main species, the white mulberry (Morus alba), M. multicaulis, M. bombycis and M. atropurpurea. The varieties of mulberry which are adapted to different climatic conditions can be grown in areas up to 4,000 m above sea level and in damp and semi-arid areas. Although mulberry (Morus sp.) cultivation is generally carried out for silkworm breeding in the world, in some countries, the plant is cultivated for its leaves and fruits which are used as animal feed (2).
Mulberry is a fruit that has been produced in Turkey for thousands of years and is consumed in different ways including fresh and dried. Turkey's total annual production of mulberry is 69,334 tons (3), and 97% of this production is white mulberry (4). While the mulberry fruit is consumed as fresh and dried, it is also used for production of goods such as molasses, jelly, dried rollup, mulberry paste, ice cream, churchkhela, vinegar, concentrated fruit juice and methylated spirit (5). Pekmez is a Turkish food, produced in Turkey's rural areas traditionally and enjoyed in the winter season. Mulberries harvested in the summer season are boiled for a few hours in large cauldrons while they are fresh, and then juices and pulp are separated by pressing. (6). The remaining pulp after pressing the boiled mulberry to make molasses is dried by spreading under the sun. The dried mulberry pulp is generally milled and fed to ruminants. When Turkey's annual production of mulberry is considered, it may be guessed how high the pulp volume released after processing will be. The chemical composition of sun-dried mulberry pulp is reported as 855.9 g/kg (fresh sample) of dry matter, 876.0 g/kg DM of organic matter, 218.6 g/kg DM of crude protein, 208.5 g/kg DM of WSC, 490.6 g/kg DM of NDF and 379.6 g/kg DM of ADF (7).
It is not common practice to feed mulberry pulp to poultry. The high price of feed raw materials is one of the most important problems in animal production which increase costs and reduce profitability. This raises the use of waste products of the agricultural production and food processing industry in animal nutrition. Mulberry pulp is one of the pulps that can be used in animal breeding. It can be used for feeding animals, and it is usually preserved by sun-drying or storage in silos.
Supplementation of mulberry meal has a potential for reduction of blood cholesterol, it could modulate the antioxidative status of laying hens and improve their production performance and egg quality, and without adverse effects, it could used in the diets of laying hens to a certain level (8,9,10). Liu et al (11) reported that dried and ground mulberry promotes beneficial cholesterol and helps regulate carbohydrate digestion. Some researchers suggested that mulberry leaf meal addition may be utilized by up to 10% without any adverse effects (8,12).
In particular, there is a need for studies on the effects of mulberry pulp use in poultry diets. Therefore, a study was planned for the first time on poultry, and the effects of mulberry pulp in the diets of quail during laying were investigated. Diets. The mulberry pulps that were used in the study were obtained by collecting the remaining wet pulps after molasses production. The collected mulberry pulps were dried under the sun for 4 days and then ground in the laboratory. Table 1 shows the chemical composition analysis results of the dried mulberry. Nutrient analyses of all raw materials to be used in the diets were conducted, and the experimental diets were prepared according to the results of these analyses. In the experiment, 4 different diets were used, one as control (without mulberry pulp) and 3 as treatment groups (containing 4%, 8% and 12% mulberry pulp). The diets of quail belonging to the experimental groups were prepared as isocaloric and isonitrogenic. All groups were fed ad libitum with feed containing 20% HP and 3000 kcal/kg ME during the experiment (  Feed conversion ratio (FCR) is calculated using the following formula (14): FCR = (FI (g of feed/bird/period))/(EM (g of egg/ bird/period)) FI: Feed intake EM: Egg mass Parameters. In this study, the feed intake, feed efficiency, egg yield, egg weight, egg quality characteristics, some blood parameters, yolk fatty acid profile and cholesterol levels of quail were determined. The feed consumption, egg yield and egg weight of the groups were measured daily. Egg quality characteristics were measured twice, in the 2 nd and 4 th weeks. Blood parameters, yolk fatty acid profiles and yolk cholesterol levels were determined by the samples taken at 4 weeks.

MATERIALS AND METHODS
Egg quality measurement. An electronic scale was used for weighing the eggs with 0.01 g accuracy, a digital caliper was used for egg width and length measurements, a tripod micrometer was used for yellow and white heights, and a digital micrometer was used for measuring shell thickness. The eggs being examined were collected for 3 days and stored at room temperature for 24 hours before the measurements. The eggs were first numbered then weighed. The width and length of the eggs were then measured. After these procedures, 10 minutes after the eggs were broken on a glass on the prepared table, measurements related to internal quality were made. The weights of the egg yolks were determined by separating the yolks from the egg whites. On the other hand, the eggshells were washed in water and dried at room temperature for 24 hours. The dried shells were first weighed together with the shell membranes, and their weights were determined. Then, the thickness of the shell was measured from three different places, namely the pointed, blunt and middle parts of the egg, and the shell thickness was determined by averaging these three measurements. The yolk color of the eggs was determined using the DSM Yolk Color Fan.
The shape index is calculated using the following formula: Shape Index=(Egg width (mm))/(Egg length (mm)) x 100 Albumen index. Albumen height of the broken egg was measured with a three-legged micrometer, and albumen length and width were measured with a digital caliper. The albumen index is calculated using the following formula.
Albumen Index=(Albumen height (mm))/ ([Albumen length (mm) + Albumen width (mm)]/2) x 100 Yolk index. Egg yolk was measured with a three-leg micrometer, yolk diameter was measured with a digital caliper. The yolk index is calculated using the following formula.
Yolk Index=( Yolk height (mm))/(Yolk diameter (mm)) x 100 Haugh Unit. Haugh unit was calculated using the formula. HU = 100 log (H + 7.57 -1.7 * W0.37) H = Albumen height (mm) W = Egg weight (g) Blood analysis. At the end of the experiment, 3 animals from each group (1 from each repetition group) were slaughtered, and their samples were placed into yellow-cap blood collection tubes with BD Vacutainer gel. After the tubes were centrifuged at 3000 rpm for 10 minutes, the samples obtained were stored frozen at -80°C until analysis. Glucose (Beckman Coulter Glucose OSR 6121), triglyceride (TG), LDL-cholesterol, total cholesterol, Ca, P, Mg, K and Na analyses of the serum samples were carried out with an autoanalyzer (Olympus AU400 Chemistry Analyzer-OLY-AU400), using commercial-use kits (Beckman Coulter OSR) and by the photometric method.
Lipid extraction. The method described by Folch (15)  Statistical analysis. The data obtained from the study were analyzed using the SAS 9.1.3 program. Analysis of variance was performed by using the PROC GLM command, and Duncan's test was used to determine the differences between the means which were considered significant.

Feed consumption and feed efficiency.
There were no significant differences between the control and treatment groups in the first 2 weeks of the experiment in terms of their daily feed consumption values, while the differences in the 3 rd and 4 th weeks were statistically significant (p<0.05, p<0.01). Table 3 shows the daily feed consumption values of the groups. Daily feed consumption of the groups started to show differences starting from the 3 rd week.
In the 3 rd week, the control group, 4% and 8% mulberry-supplemented groups consumed more feed than the 12% group. A similar situation was observed in the 4 th week, and the 12% supplemented group consumed less feed than the other groups (except the 4% group). The results showed that addition of 12% mulberry pulp to the diet caused a significant (p<0.05 and p<0.01) reduction in daily feed consumption in the 3 rd and 4 th weeks (Table 4). Although the feed conversion ratio of the control group was partially better than the treatment groups in the third week of the experiment, the difference was not statistically significant. The same situation was observed in the 4 th week of the experiment, but the mean values of the feed conversion ratios of the groups were found to be closer to each other. It may be stated that addition of dried mulberry pulp up to 12% in the diets of laying quail does not cause a significant reduction in feed efficiency.
Egg production and egg weight. When the control and treatment groups were compared in terms of egg production, no significant differences were found between the groups in the 1 st , 2 nd and 4 th weeks. In the third week, the differences between the mean egg production values of the groups were statistically significant (p<0.01). Table 5 shows the weekly egg production values of the groups during the 4-week period. In the third week of the experiment, the 12% mulberry-supplemented group yielded significantly (p<0.01) fewer eggs than the control and other treatment groups. The egg yields of the other 2 treatment groups (4%, 8% supplemented groups) were also partially reduced, but this difference was not significant. In the 4 th week, the differences between the egg yields of the groups disappeared, and all groups provided similar values. It may be stated that the egg production difference between the groups in the 3 rd week and the disappearance of this difference in the 4 th week was because it was taking time for the quail to get used to the mulberry pulp.
When the control and treatment groups were compared in terms of egg weight, no significant differences were found between the groups. Table 6 shows the values of the egg weights of the groups for the 4-week period. In the 3 rd and 4 th weeks, the quail belonging to the 12% supplemented group had lower egg weights in comparison to the other groups. However, these differences were not statistically significant. It was determined that mulberry pulp added to the diet was not effective on egg weight for the first 2 weeks and caused a partial decrease in the 12% supplemented group in the 3 rd and 4 th weeks.
Egg Quality. In the 2 nd and 4 th weeks of the experiment, some quality characteristics of the eggs collected from the control and treatment groups were measured. Table 7 shows the obtained results. Significant differences were observed between the control and treatment groups in terms of the albumen index, yolk index and Haugh unit (p<0.05). The differences between the mean values in terms of egg weight, yolk weight, albumen weight, shell weight, shell thickness, yolk color and shape index were not significant. The values of the treatment groups in terms of the albumen index and yolk index were generally higher than the control group. However, the highest values for these two properties were obtained from the 8% supplemented group. The differences between the groups in terms of yolk weight were found to be significant (p<0.05), while the differences between other quality characteristics were not significant. Addition of mulberry pulp (in the 4 th week) significantly affected yolk weight (p<0.05).  in the 4 th week of the experiment. Addition of mulberry pulp to the diet significantly affected blood triglyceride, total cholesterol and LDL (Low-density lipoprotein) levels (p<0.05, p<0.01). Other blood parameters (Glucose, Ca, P, Mg, Na and K) of the groups were not affected by mulberry pulp addition. The triglyceride levels were lower in the 12% supplemented group than those in the other groups (p<0.05). The triglyceride levels in the 8% supplemented group showed a partial decrease, although not statistically significant, compared to the control group. Addition of mulberry pulp to the diet may be stated to cause a decrease in blood triglyceride levels starting from 8%. When the total cholesterol levels of the groups were examined, it was seen that the differences between the means were significant (p<0.05).
The results on the cholesterol levels were similar in the control group, 8% supplemented group and 12% supplemented group, but higher in the 4% supplemented group. Addition of mulberry pulp to the diet does not have a significant effect on the 8% and 12% groups.
Yolk cholesterol. Table 9 shows the cholesterol analysis results of the egg yolks collected from the control and treatment groups in the 4 th week. The differences between the cholesterol values of the groups were significant (p<0.05). The lowest cholesterol level was obtained from the 8% supplemented group, while the results of the other groups were similar. Addition of 8% mulberry pulp to the diet may be stated to have a positive effect on egg yolk cholesterol levels. Blood parameters. Table 10 shows the results of the analysis performed for some parameters with the blood samples taken from the quail In terms of serum LDL levels, the differences between the means of the control and treatment groups were significant. The LDL levels were higher in the 4% and 12% supplemented groups than the control and 8% supplemented groups (p<0.01). The results on the LDL levels were similar in the control and 8% supplemented groups. It may be stated that the effect of mulberry supplementation on serum LDL levels in quail varies for different levels.

DISCUSSION
While the differences in the daily feed consumptions of the quail in the control and experiment groups on whose diets mulberry pulp was added were not significant at the 1st and 2nd weeks, they were significant at the 3rd and 4th weeks (p<0.05, p<0.01). The daily feed consumptions were found to be the lowest in the group that was fed with feed containing 12% mulberry pulp. While adding mulberry pulp into the diets by up to 8% did not affect the feed consumption of the experiment groups, significant decreases were observed in feed consumption at increased ratios (12%). The quail in the group fed with the feed containing 8% mulberry pulp had the highest feed consumption values.
While the findings obtained regarding feed consumption were in agreement with another study which reported that addition of mulberry flour into the rations of broiler quail increased their feed consumption (16), they were different to the results of another study where mulberry leaf flour was added to the rations of laying quail (17). Values regarding feed consumption obtained in studies where mulberry leaf flour was added to the rations of laying hens were different to the findings in this study (8,9,10,18) and partially similar to Panja's findings (19).
The differences between the feed conversion ratios of the quail in the control and experiment groups were not significant. The results showed that, at all weeks, adding mulberry pulp into the rations by up to 12% did not affect feed efficiency negatively. The results were different to those reported by a study where mulberry flour was added to the diets of broiler quail (16). On the other hand, in comparison to those in other studies where mulberry leaf flour was added to the diets of laying hens, the results were similar to a great extent (8,9,10,18).
When the results on the egg yields of the groups were examined, it was seen that the egg yields were similar at the 1st, 2nd and 4th weeks. However, at the 3rd week, significant reductions were observed in the case of increasing the mulberry pulp level in the diets above 8% (p<0.01). The results obtained on egg yield were similar to those reported by Al-Kirshi et al. (8) and Hermana et al (17) and different to those reported by Lin et al (9), Kamruzzaman et al (10) and Kamruzzaman (18).
Considering the values on egg weight, the results of all groups were similar. Addition of mulberry pulp to the diets of the quail by different ratios did not significantly affect their egg weight values. The results on the egg weights were similar to those reported by a previous study (17) where mulberry leaf flour was added to the rations of laying quail by up to 10% and other studies (10,18) where mulberry leaf flour was added to the rations of laying hens. On the other hand, the results were different to those in another study where mulberry leaf flour was added to the diets of laying hens (8).
When the eggs of the control and experiment groups were evaluated in terms of their internal and external quality characteristics, there were significant differences in the albumen index, yolk index and Haugh unit values at the 2nd week and yolk weight at the 4th week (p<0.05).
The results were similar in terms of the other quality characteristics. It was determined that addition of mulberry pulp to the diets of quail by up to 12% did not have a negative effect on the internal and external quality characteristics of eggs. Panja (19) reported that mulberry leaves do not affect yolk color because diets have enough corn gluten. The yolk finding in this study was similar to the study by Panja (19), but it differred with the results of Olteanu (20).
Other results were in agreement with those of Al-Kirshi et al (8) and Panja (19). Addition of different ratios of mulberry to the rations did not significantly affect the yolk fatty acid profile, and the mean values of the control and experiment groups were similar.
The differences in the mean values of the groups were found significant in terms of the yolk cholesterol levels (p<0.05). Panja (19) reported that inclusion of mulberry leaves decreased blood cholesterol same as the yolk cholesterol content but had no effects on thigh meat cholesterol levels. According to ration content, yolk cholesterol levels may change between 5% and 30% (21). While the cholesterol levels were similar in the control, 4% and 12% groups, they were lower in the 8% group. The findings that were obtained were similar to those reported by Kamruzzaman et al (10).
Considering the results of the groups regarding the blood parameters, it was determined that the differences in the triglyceride, total cholesterol and LDL levels were significant (p<0.05, p<0.01), while those in the other parameters were insignificant. Zhang (12) reported that the blood triglyceride levels in their 15% dietary mulberry leaves group were significantly lower than those in the control group. Their findings were similar to the results of this study. However, they reported that inclusion of 15% mulberry leaf flour also provided significantly lower values than those in the control group. The findings differed for these parameters. The lowest values were found in the 8% group in terms of the blood cholesterol levels and the 12% group in terms of the blood triglyceride levels. The results were similar to those reported by Kamruzzaman et al (10).
In conclusion, in this study, it was found that adding different levels (0, 4%, 8% and 12%) of dried mulberry pulp to the diets of laying quail was effective on daily and weekly feed intake, egg yield, albumen index, yolk index, Haugh unit, yolk weight, yolk cholesterol, triglyceride, total cholesterol and LDL levels. Addition of mulberry pulp to the diet did not affect the egg yolk fatty acid levels significantly. As a result, when the effects of 4%, 8% and 12% dried mulberry pulp added to the diets of laying quail were examined, it was observed that mulberry pulp had no negative effect up to the ratio of 8%. When the mulberry pulp supplement reaches 12%, some negative effects (especially feed intake and egg yield) may be observed. Therefore, it may be stated that it is beneficial to add an 8% ratio of dried mulberry pulp to the diet.