Blood Profile of Quails (Coturnix Coturnix Japonica) Fed Ration Containing Silkworm Pupae (Bombyx Mori) Powder Extract

The aim of this research was to assess the use of silkworm pupae (Bombyx mori) powder extract on the blood cells and hematological variables of quails (Coturnix coturnix japonica). The treatments consisted of R0= isoenergy and isoprotein ration without pupae powder extract, R1= isoenergy and isoprotein ration + 1% of pupae powder extract, R2= isoenergy and isoprotein ration + 10% of pupae powder extract, R3= isoenergy and isoprotein ration + 1% of residue of pupae powder extract, R4= isoenergy and isoprotein ration + 10% of residue of pupae powder extract, R5= isoenergy and isoprotein ration + 1% of pupae powder, and R6= isoenergy and isoprotein ration + 10% of pupae powder. The variables measured were blood cell and hematological profiles including the number of leukocytes, erythrocytes, and leukocytes differentiation. Immune response was observed at 12, 24, and 48 h by counting the white blood cells type. The results showed that feed consumption ranged 79.19-154.70 g/quail/wk and the body weight was 71.45-149 g/quail. The addition of pupae extract at a dose of 10% in the diet significantly (P<0.05) increased the number of quail white blood cells. The addition of pupae extract, pupae powder residue, and pupae powder in the ration did not significantly affect the number of quail erythrocytes, hemoglobin, and PCV (packed cell volume). Mean number of white blood cells increased during 24 h after treatment of 10% pupae powder extract (R2) and decreased at the 48th h. It is concluded that the value of leukocytes, lymphocytes, monocytes, and heterophile increased in quails supplemented with 10% pupae powder extract (R2).


INTRODUCTION
Insects have great potential as sources of food and feed as well as a source of nutrients that is comparable to meat and fish and also can be a source of animal protein as the main stay protein in the future for the purposes of poultry farming and fish farming (Usub et al., 2008;Oh et al., 2012;Rumpold & Schluter, 2013). The utilization of some insects such as silkworms and bees for large-scale production with the use of technology was done to anticipate the problem of global food and feed supply (Vantomme et al., 2010;Huis et al., 2013).
Another potential owned by insects is the discovery of a various bioactive substances with beneficial characteristics (Hirose et al., 2013). These substances included compounds with diverse pharmacological properties, including anti-cancer, anti-tumor, anti-viral, and antimicrobial as well as the capacity to boost the immune system against various diseases in human, livestock, poultry, and fish (Chernysh et al., 2002). Insects are not only useful as a source of animal protein in food production as well as feed, but they provide benefits in the pharmacological and medical areas which are able to protect animals and livestock against various risks of many diseases.
In the life cycle of insects, the eggs will hatch into larvae, pupae, and adult metamorphosis. In the stage of insect metamorphosis, pupae under fasting conditions to grow in the cocoon (protective hard). Silkworm cocoon thread is used for spinning into yarn silk, while the pupae are used as animal feed cultivation. Cocoon production for silk in Indonesia since the 2005-2009 period reached 166 tons, which produced about 23 tons of yarn. The wastes produced from the process were around 143 tons.
In some countries such as Korea, Japan, and China pupae are used to produce snacks, canned food, alternative foods for diabetics, candles and beauty products such as soap and hair tonic (Yi et al., 2010). Pupae silkworms can be made into flour pupae that can be used as an ingredient in the manufacture of complementary cookies (crackers and flakes), sponge cake, and nuggets (Astuti & Kusharto, 2009). In addition, the pupae silkworm can be utilized as a bioreactor in the development of antiviral agents and as an immunostimulant in vertebrates (Yao et al., 2006;Swevers et al., 2013;Yeo et al., 2013).
The addition of insect pupae powder on red sea bream feed could boost immune activity against pathogenic bacteria (Ido et al., 2015). Powder extract of Bactrocera cucurbitae pupae was known to activate the innate immunity on rat macrophage cells (Ohta et al., 2014). This immunostimulatory effect was identified and it was derived from the active ingredient in the form of polysaccharides contained in the body of the pupae (Ohta et al., 2014). Silkworm pupae contained polysaccharides (27.9%) (Long et al., 2007) and high protein (55.6%) with a balanced amino acid (Zhou & Han, 2006;Tomotake et al., 2010).
Immunostimulatory effect of silkworm pupae was identified from the polysaccharide contents. High molecular weight polysaccharide can activate the innate immune system. The polysaccharide is the site identifier (marker). Dipterose polysaccharide is synthesized by the Diptera insect pupae such as melons fly and has a molecular weight of 1.01x10 6 mol%. Similarly, it occurs also in insect Lepidoptera such as silkworm, polysaccharides molecular components, namely silk rose, that is synthesized in the pupae and has a molecular weight of 3.15x10 5 mol%. The composition of silkrose and dipterose consist of nine monosaccharides i.e., D-glucose, D-mannose, D-galactose, D-ribose, L-rhamnose, L-fucose, D-glucoronic acid, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine (Ohta et al., 2014).
Silkrose is ethanol-soluble polysaccharide group, so that silkrose bioactive substances can be obtained from the extraction of silkworm pupae powder with ethanol solvent.
L-rhamnose is one component of dipterose with the highest molar ratio as compared to the other monosaccharides. At silkrose, the highest monosaccharide component is D-Galactose with molar ratio reached 48.9%. L-Rhamnose can be found in natural organic components such as flavonoids, terpenoids and saponins (Ohta et al., 2014). Saponins in a particular dosage are secondary compounds that can play a role as immunostimulant. Bomford (1980) reported that saponins can function resemble a membrane antigen of polysaccharide cell at the outside of the cell membrane and it is used as the site identifier (marker) in the immune system of vertebrates. Dipterose and Silkrose were obtained from Musca domestica, melon fly, Hermetia illucens, Antheraea yamamai and Bombyx mori and were able to activate the innate immune response against pathogenic microorganisms and viral infections (Ohta et al., 2014;Ido et al., 2015). Conversely the waste of silkworm pupae in unrefined pupae powder will make some rat macrophage cells die (Ido et al., 2015). The ability of bioactive substances owned by the insects in activating the immune response against pathogens and viral infections in fish and mammals, become the reason to conduct a research to assess the use of silkworm pupa powder extract (B. mori) on the blood profile of quail (Coturnix coturnix japonica).

MATERIALS AND METHODS
A total of 70 laying quails (Coturnix coturnix japonica) with the age of 4 weeks and average body weight of 76.36 g were used in this study and half of them (35 laying quails) were used for blood profile analysis. Raw materials used to formulate the experimental ration were corn, rice bran, soybean meal, fish meal, palm oil, DCP, CaCO 3 , NaCl, Premix, DL-Methionine, and silkworm pupae powder. Additional feed was given in the form of silkworm pupae powder, residue of silkworm pupae powder extraction and pupae powder extraction on the prescribed dose. Pupae powder extraction refers to the maceration extraction method with 95% ethanol as a solvent (Liu & Zhang, 2006). Feed rations were prepared with isoenergy and isoprotein i.e. the energy content of 2950 kcal/kg and protein content of 18% (Table  1) (Leeson & Summers, 2005). The experimental quails were maintained for 8 weeks, with details of treatment were first week for environmental adaptation, second week for control ration adaptation (R0), third week for additional treatment rations, and the last five weeks without ration treatments (R0). Rations and water were given ad libitum.
This study used a completely randomized design with 7 treatments and 5 replications. The treatments were R0= isoenergy and isoprotein ration without pupae powder extract (control), R1= isoenergy and isoprotein ration + 1% pupae powder extract, R2= isoenergy and isoprotein ration + 10% pupae powder extract, R3= isoenergy and isoprotein ration + 1% residue of pupae powder extraction, R4= isoenergy and isoprotein ration + 10% residue of pupae powder extraction, R5= isoenergy and isoprotein ration + 1% pupae powder, and R6= isoenergy and isoprotein ration + 10% pupae powder. Pupae powder was used as a feed supplement with doses of 0% (control), 1% and 10% (Ohta et al., 2014). Parameters measured were blood profile of the experimental quails consisted of number of erythrocytes, hemoglobin concentration, hematocrit percentage, leukocyte count and leukocyte differentiation (Jalees et al., 2011). Blood samples of quail were taken on the 15 th day at the time of observation 12, 24, and 48 h. It was based on the estimation that time of circulating white blood cells in the blood may be brief (Guyton & Hall, 1997). Blood sampling at different times was conducted in order to look at the treatment that had significantly impacted on the number of white blood cells. One milliliters of blood sample was taken and put into EDTA tubes. Preparations of the blood smear with Giemsa staining were made to calculate the number of leukocyte. Total white blood cells and total red blood cells were calculated by using a Neubauer hemocytometer, with a modified diluent solution of Rees & Ecker. Data were tested by using Analysis of Variance (ANOVA) and the differences among treatments′ means were examined by Duncan Multiple Range Test (Mattjik & Sumertajaya, 2002). Data were processed by using SPSS 16.0 statistical software.

Quail Feed Consumption
Feed consumption during the research ranged from 79.19 g/quail/wk to 154.70 g/quail/wk ( Table 2).
The results showed that the supplementation of pupae extract, residue of pupae extraction and pupae powder in R1, R2, R3, R4, R5, and R6 did not affect the average feed consumption. In poultry, there is a limiting factor of feed intake that is the gizzards capacity and energy requirements (North & Bell, 1990). The limiting factors cause quail stop eating when energy needs are fulfilled.
Feed given during the study was in accordance with the needs of quail that have been specified in SNI (1995) with energy content of 2900 kcal/kg. Quail used in this study had the same age, namely four weeks so that the gizzards size was not much different. This caused the feed intake of all treatments was not different, except in R2 treatment (extract pupae powder with dose of 10%).

Quail Body Weight
The observation during the experiment found that the body weight gain of quails from the beginning to the end of experiment increased in quails fed with R2 treatment (extract of pupae powder at a dose of 10%) ( Table   3). The provision of pupae powder in extract form as a force feeding enable all of the ingredients entered the body of quails through gastrointestinal tract without being wasted. Silkworm pupae powder in the form of polysaccharide extract contains active ingredients that can boost the immune system of the experimental quails. These results are consistent with the results reported by Ido et al. (2015), that the administration of extract of house fly pupae powder in red sea bream fish improved growth rate and immune system.

Total Number of Erythrocytes
The results showed that the number of erythrocyte in the group R1, R2, R3, R4, R5, and R6 were similar (P>0.05) ( Table 4). Erythrocytes are blood cells that have a nucleus and play a role in carrying hemoglobin to bind oxygen throughout the body. The ranges of average erythrocytes number of laying quail studied were 2.97-3.46 x 10 6 /mm 3 . This value was still in the normal range. According to Sturkie & Griminger (1976), the normal number of erythrocytes is 2.30-3.86 x 10 6 /mm 3 . This indicates that the active ingredient contained in pupae powder extract, pupae powder residue and pupae powder in the form of polysaccharides silkrose did not Note: *) Based on calculation using the formula of Leeson & Summers (2005). inhibit the formation of erythrocytes, thus laying quail conditions were healthy.

Level of Hemoglobin
The results showed that the hemoglobin concentrations in the quails fed with ration R1, R2, R3, R4, R5, and R6 were not different (P>0.05) ( Table 4). Hemoglobin closely related to the erythrocyte and hematocrit. Hemoglobin is a simple protein, red color poster on erythrocytes, and function in the binding of oxygen. Hemoglobin concentrations of the experimental quails ranged from 7.57% to 12.23 g%. These hemoglobin values are still in the normal range. According to Sturkie & Griminger (1976), the normal hemoglobin level is 12.30 g%.
35.18%. The hematocrit values were in the normal range. According to Sturkie & Griminger (1976), the normal hematocrit value in quail is 37%.
Hematocrit values indicate the total number of erythrocytes in the blood, thus becoming one of the indicators of the determination of the blood ability to carry oxygen (O2) which are commonly known as Oxygen Carrying Capacity (Maheswaran et al., 2008).
Decrease in hematocrit value can be caused by several other factors, namely the level of stress by environmental influences and nutrition, dehydration and parasites in the blood (Challenger et al., 2001). During the maintenance of experimental laying quail, ambient temperature tended to be high, ranging from 31-33°C. Heat stress can occur when the ambient temperature exceeds 32°C (Hemid et al., 2010). The condition causes laying quail consume more water. The high consumption of drinking water causes the water concentration in the blood increases, thereby decreasing the percentage of blood (hematocrit) (Tamzil et al., 2013). It can also be caused by the high levels of yolk precursors (William et al., 2004), and due to the increased concentrations of estrogen in the blood (Wagner et al., 2008), an assumption that illustrates the decline in quail's hematocrit concentrations with higher egg production.

Number of Leukocytes
Further test results indicated that averages numbers of leukocytes of the control group were similar to those of R1, R3, R4, R5, and R6 groups (Table 4). However, the R2 group quails had significantly (P<0.05) higher leukocyte numbers as compared to the other treatments (14.22 x 10 3 /mm 3 ). The increased number of leukocyte occurred at the 24 h after observation and decreased at 48 h (9.86 x 10 3 /mm 3 ) (Figure 1). The ranges of average number of leukocytes in the control (R0), R1, R2, R3, R4, R5, and R6 were 3.55 to 14.22 x 10 3 /mm 3 and this range was lower but still under the normal range. According to Sturkie & Griminger (1976), the normal leukocyte count in quail ranges from 20 to 40 x 10 3 /mm 3 .
The number of leukocytes in this study tended to be lower compared to a standard leukocyte count in quail. This lower number of leukocyte was due to the state of heat stress because the ambient temperature during the experiment was in the range of 31-33 o C. The decrease in the leukocytes number in the experimental laying quail in this study was approximately 50% when compared to standard quail leukocytes that were not in stress conditions (Table 3). Tamzil et al. (2014) reported that the levels of leukocytes in chickens decreased by 40% at the time of heat stress. Heat stress decreased the number of leukocytes so that the immune system became lower (Kusnadi, 2009;Usama et al., 2013). Muhammad (2013) stated that during stress the number of leukocytes and lymphocytes decreased as well as improving the ratio of H/L, which is one important indicator of stress in laying quail, especially during the laying period. Blood sampling in this research was performed at 7 weeks of age at the beginning of laying eggs. Female quails start laying eggs at the age of 6 weeks (Sujana et al., 2012). Blood sampling was conducted at 12, 24, and 48 h after giving the treatment.
Leukocyte is an active unit to provide fast and powerful defense against any material that causes infection (Guyton & Hall, 1997). Leukocytes play a role in responding the immune. The use of silkworm pupa powder was able to maintain the number of leukocytes in laying quails for functioning the immune system, because the silkworm pupae powder contained polysaccharides silkrose which could boost immune function. Polysaccharides silkrose of silkworm pupae could significantly increase macrophage phagocytosis and increase hemolysin antibody and lymphocyte transformation. This indicates that polysaccharide of silkworm pupae clearly indicate the presence of nonspecific immunity (innate immunity) (Sun et al., 2007).
Ethanol extraction could improve the content of polysaccharides found in insect pupae (Liu & Zhang, 2006). Polysaccharides silkrose contained in the 10% of pupae powder extract was assumed can increase the number of laying quail leukocytes which were experiencing stress on the production period. According to Hrabcakova et al. (2014), a lot of factors that affect the levels of leukocytes in poultry i.e., genetic (species and strains), exposure to stress (stress may be temperature, environment uncomfortable cage, handling), system maintenance, feed, production period (starter, grower, layer) and the lighting program.

Leukocyte Differentiation
In this study, the number of heterophile, lymphocytes, and monocytes were observed at 12, 24, and 48 h. This was done to document the effect of treatment on the immune responses by counting white blood cell types. The results of the variance analysis showed that R1, R2, R3, R4, R5, and R6 groups did not have significant different number of heterophile in laying quail blood (Table 5). The average number of heterophile R6. R0= ration without pupae powder extract (control), R1= ration + 1% pupae powder extract, R2= ration + 10% pupae powder extract, R3= ration + 1% residue of pupae powder extraction, R4= ration + 10% residue of pupae powder extraction, R5= ration + 1% pupae powder, and R6= ration + 10% pupae powder. in quail blood in the study ranged between 22.33% and 41.55%. The number of heterophile in this study was quite high, indicating quail treated R1, R2, R3, R4, R5, and R6 had good immune responses. Sturkie & Griminger (1976) stated that the percentage of normal quail heterophile ranged between 20%-30%. In this study, the number of heterophile increased at the 12 th hour and then decreased until the 48 h of observation in the groups R2, R3, R4, and R5 (Figure 2).
The results of variance analysis showed that groups R1, R2, R3, R4, R5, and R6 did not have significantly different in number of lymphocytes in the quail blood studied. The range of lymphocytes in this study was 46.89% to 75.55% (Table 5). Lymphocyte values in this study were quite high, indicating that quail in the groups of R1, R2, R3, R4, R5, and R6 had a good immune desist. Sturkie & Griminger (1976) stated that the percentage of quail lymphocytes normally ranged between 30%-66%. In this study, the number of lymphocytes increased until the 24 h, although decreased in the 48 h (Figure 2). Lymphocytes have very important roles in the immune system (Melvin et al., 1993).
as a foreign body, so it will attract monocyte through its phagositic capabilities. Monocytes are an essential component of the innate immunity (Parihar et al., 2010).

CONCLUSION
Total leukocyte of laying quail in this study significantly increased in quail treated with the addition of extract pupae powder with dose of 10% in the ration. The increase of leukocytes number occurred at the 24 h, and then decreased at 48 h after observation. The addition of silkworm pupae powder extracts containing polysaccharides' silkrose in the ration could increase the number of leukocytes in laying quail at heat stress conditions.