Phytogenic compounds do not interfere physiological parameters and growth performances on two Indonesian local breeds of ducks

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Aim: The present study was to investigate the interaction between duck's breed and phytogenic compounds as feed additives in the diet on blood lipid and hematological profile, welfare, and growth performance. Materials and Methods: A total of 200
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  Veterinary World, EISSN: 2231-0916 1689Veterinary World, EISSN: 2231-0916Available at www.veterinaryworld.org/Vol.12/November-2019/2.pdf  RESEARCH ARTICLEOpen Access Phytogenic compounds do not interfere physiological parameters and growth performances on two Indonesian local breeds of ducks Ismoyowati Ismoyowati 1 , Diana Indrasanti 1 , Sigit Mugiyono 1  and Mulyoto Pangestu 1,2 1.Department of Animal Production, Faculty of Animal Science, Jenderal Soedirman University, Purwokerto, Indonesia;2.Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia. Corresponding author:  Ismoyowati Ismoyowati, e-mail: moy.moyowati@gmail.com Co-authors:  DI: dianaindrasanti@gmail.com, SM: sigitmugiyono@yahoo.com, MP: mulyoto.pangestu@monash.edu   Received:  25-06-2019, Accepted:  18-09-2019, Published online:   0 4 -11-2019 doi:  www.doi.org/10.14202/vetworld.2019.1689-1697 How to cite this article:  Ismoyowati I, Indrasanti D, Mugiyono S, Pangestu M (2019) Phytogenic compounds do not interfere physiological parameters and growth performances on two Indonesian local breeds of ducks, Veterinary World  , 12(11): 1689-1697. Abstract Aim:  The present study was to investigate the interaction between duck’s breed and phytogenic compounds as feed additives in the diet on blood lipid and hematological profile, welfare, and growth performance. Materials and Methods:  A total of 200 male day-old local breed ducks (Tegal and Muscovy ducks) were used in this experiment. The first factor was duck breed and the second factor was different phytogenic compounds supplementation in the diet: Garlic, turmeric, ginger, and kencur, at 3% each. The observed variables were the blood lipid profiles comprise high-density lipoprotein (HDL), low-density lipoprotein, cholesterol total, triglyceride, blood parameters, welfare (heterophil/lymphocyte [H/L] ratio), and growth performances (feed consumption, body weight gain, feed conversion ratio, and carcass  percentage). Results:  The interaction between breed of ducks and phytogenic compounds had a significant effect on blood triglyceride,  but no significant effect on the blood lipid profile, hematological parameters, and growth performances. While, phytogenic compounds in the diet had significant effects on the blood lipid profile, heterophil (H), lymphocyte (L), and H/L ratio of ducks. The breed factors affected HDL and growth performances. Muscovy duck had a higher HDL and growth performance compare to Tegal duck. Among those, garlic most effectively reduced triglyceride level in Tegal duck. Conclusion:  Phytogenic compounds 3% do not have a negative effect on the physiological parameters of ducks increase ducks welfare (H/L ratio), so it does not affect the growth performances of ducks. Muscovy duck had higher growth  performances than Tegal ducks. Keywords:  blood lipid, growth performances, heterophil/lymphocyte ratio, Indonesian ducks, phytogenic compounds. Introduction Duck (  Anas platyrhynchos  javanicus ) and Muscovy duck ( Cairina moschata ) are waterfowl, which typically have twice the body fat content of chicken. At present, duck becomes a popular cui-sine in Indonesia, its own special class in the society competing with chicken. The population and pro-duction of duck meat and egg in Indonesia during 2015 increased by 3.55%, 5.02%, and 3.49%, respectively [1]. Indigenous Indonesian ducks are usually named according to their srcin region; they are more adaptable to the tropical environment with optimum productivity. They know as Alabio duck, Magelang duck, Mojosari duck, Tegal duck, Cihateup duck, and Pengging duck. Tegal duck is one of the indigenous Indonesian duck strains, which is main-tained around the northern coast of Java. The advan-tages of Tegal duck are higher egg production, with average duck day production is 70.89% and mature age of 154.5 days [2]. Tegal duck is well adapted in tropical and coastal regions. Adaptable local ducks have strategic position as a source of germplasm as well as a source as a research material [3]. The physi-cal characteristics of Tegal ducks are small head, slim neck, long and round body, wings attached tightly to the body, and the tip of the feathers close above the tail [2]. Muscovy duck ( C. moschata ) is one type of meat-producing domesticated waterfowl for a long time in Indonesia whose wing is shorter than a Pekin duck [4]. It is srcinated from Central and South America, which was discovered by conquista-dor from Spain in the early 16 th  century. Initially, the ducklings were preserved by Colombian, Peruvian, and Brazilian Indians. Some ducks were brought to Europe, Africa, and Asia, including Indonesia. It has a great opportunity to become the source of meat for livestock development [2] because it has high breast meat with unique taste, low fat, savory flavor, and least calorie content than Pekin ducks. Muscovy ducks adapt well to various rearing conditions. It is relatively resistant to the disease and is able to use low-qual-ity feed [5,6]. The purpose of raising Muscovy duck  by the rural communities is to make it as an alterna-tive food source and as a parent for incubating duck Copyright: Ismoyowati, et al.  Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the srcinal author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.  Veterinary World, EISSN: 2231-0916 1690Available at www.veterinaryworld.org/Vol.12/November-2019/2.pdf  eggs. Phytogenic compounds are a multifunction feed additive for poultry, including ducks, derived from  plant products used in animal feed to improve perfor-mance and it enhances immune responses and intesti-nal quality [7,8]. Phytogenic compounds are primary and secondary plant substances with pharmacolog-ical effects and widely used in traditional medicine. Phytogenic compounds are derived from leaves, roots, flowers, and whole plants [9,10]. Most plants contain  polyphenol compounds which are classified into fla-vonoids and non-flavonoids [11] so that feed additives using of phytogenic compounds may improve physio-logical conditions, immune system, and growth [12]. There are some known phytogenic compounds added in feed such as garlic (  Allium sativum ), turmeric ( Curcuma domestica ), ginger (  Zingiber officinale ), and aromatic ginger (  Kaempferia galanga , Kencur. Loc.). Garlic   is a popular spice for food or medicine against several diseases. It contains an organosulfur thiosulfinates, with allicin as the active component. Allicin (diallyl thiosulfinate) is a key constituent of garlic and has potential health benefit which promotes growth and antioxidant activity including anti-inflam-matory, antihypertensive (allicin decreases cholesterol and blood pressure) activities [13,14], and increases  production performance [15]. Increasing garlic pow-der level (5%) in broiler feed lowers total cholesterol and low-density lipoprotein (LDL) and increases high-density lipoprotein (HDL) [16]. Ginger contains several compounds such as gingerol, gingerdiol, and gingerdione that possess strong antioxidant activ-ity [17]. Supplementing 5 g/kg of feed with ginger increases the total activity of superoxide dismutase and glutathione peroxidase but decreases malondial-dehyde and cholesterol level in 21- and 42-day-old  broilers [18]. Kencur is an important medicinal plant. It has been traditionally used as carminative, cholera, anti-inflammatory, abdominal pain, dyspepsia, and stomach ache as well as in the diseases of coughs, pec-toral affections, and stoppage of the nasal blocks [19]. It contains saponin, flavonoid, polyphenol and vola-tile oil, ethyl-p-methoxycinnamate, and other import-ant compounds of enormous medicinal values and they are very demanding to the traditional health-care  practitioner [20].Bioflavonoid is one of the most varied and vast natural compound groups and tends to be the most sig-nificant natural phenol. This compound has a broad spectrum of chemical and biological activity and han-dles radical [21]. The bioflavonoid modulator effect on cholesterol and lipid has been tested in some animals such as poultry and laboratory animal. Hematological characteristics can be used as an indicator of poultry’s adaptability toward certain environmental conditions. Welfare levels can be measured from the heterophil/lymphocyte (H/L) ratio [22]. Furthermore, H/L ratio has been widely used as a sensitive indicator of long-term stress related to immune function in bird [23]. Active substances in phytogenic compounds have vasorelaxation and antioxidant properties that help animals overcome environmental distress. There are few researches [3,5,6] about Tegal duck and with regard to phytogenic compounds on blood profile and welfare level.This research was aimed to evaluate the effect of phytogenic compounds on local ducks’ blood lipid  profile, welfare level, and growth on two breeds of ducks (Tegal and Muscovy). Materials and Methods Ethical approval The experimental protocols were approved by the Animal Ethics Committee, Jenderal Soedirman University No. 1923/UN.23.14/PN.01/2018. Preparation of phytogenic compounds powder Raw material, fresh bulbs of garlic (  A. sativum ) and rhizomes of turmeric ( C. domestica ), ginger (  Z. officinale ), and kencur (  K. galanga ) were obtained from local market in Purwokerto, Indonesia. The raw materials were cleaned, sliced, and washed with sterile distilled water and then oven-dried (55°C) and milled. Animals Hundred day-old male Tegal ducks (  A. platy-rhynchos ) weighing 38 g were purchased from duck farmers in the Tegal region and 100 male day-old of Muscovy ducks ( C. moschata ) weighing 40 g were  purchased from farmers in the Banyumas area. Each treatment was taken a sample of eight ducks for blood sample collection and slaughter, so the total was 80 ducks. Ducks were kept in brooders from old duck days to 21 days. After the age of 21 days, the ducklings were moved to a colony cage measuring 1×1.5 m, which was filled with five ducklings. Equipment included feeding container, drinking container, digital scale, box, syringe, Vacutainer filled with anticoagu-lant, and cuvette tube. Feeding Dietary for 1-21 days old was broiler complete feed (Broiler Starter BR I crumble, Japfa Comfeed). Starting from the age of 22 until 70 days, the duck was given experimental feed. The composition of ingredi-ents and nutrient content of the feed is presented in Table-1. Phytogenic compounds Phytogenic compounds were mixed in the basal feed as described in previous studies [13-17]. P0 was  basal feed with no phytogenic compounds, P1 (basal feed with 3% garlic), P2 (basal feed with 3% ginger), P3 (basal feed with 3% turmeric) and P4 (basal feed with 3% kencur). Husbandry, feeding, and supplementation trial Up to the age of 21 days, the ducks were kept in a brooder, temperature of 32°C, density of 25-day-old duck (DOD)/m 2  with 24-h lighting and fed with complete broiler feed. At the age of 22 days, the duck was moved into a colony cage with a density of 5 ducks/1.5 m 2  with 24-h lighting. Phytogenic  Veterinary World, EISSN: 2231-0916 1691Available at www.veterinaryworld.org/Vol.12/November-2019/2.pdf  compounds were given to the ducks from day 22 to 70. The feed and drinking water was given ad libitum. Experimental design The completely randomized factorial design with two factors. The first factor was Breed (B)B1 Tegal ducks (  A. platyrhynchos ) and B2 was Muscovy ducks ( C. moschata ). The second factor is Phytogenic compounds (P).P0 = control feed (basal feed only), P1 = basal feed+3% garlic powder, P2 = basal feed+3% curcuma powder, P3 = basal feed+ginger powder 3%, and P4 = basal feed+kencur powder 3%. Observation were made on blood lipid profiles comprises choles-terol level, HDL, LDL, blood triglycerides and H/L ratios, growth, body weight, and carcass percentage. Blood sample collection, analysis, measurement of growth, and carcass percentage Blood sample was collected at day 70 from bra-chial vein of duck using a sterilized syringe and needles then transferred into vacutainer tubes with anticoagu-lant ethylenediaminetetraacetic acid. Blood samples were centrifuged at 2000×  g   for 30 min. Blood plasma was collected and stored at −20°C for further assay of total cholesterol, LDL, HDL, and triglyceride using a spectrophotometer at a wavelength of 500 nm and 340 nm using Hitachi 704 analyzer [24].Hematological profiles were measure according to Hrabčáková  et al  ., 2014 [25]. The number of red  blood cells (RBCs; million/ml) was measured using a hemocytometer with the Hayem dilution method. Hematocrit or packed cell volume was determined by the microcapillary reader by a microhematocrit cen-trifuge with a maximum relative centrifugal force of 10,000×  g  , which should be reached within 30 s; then, the hematocrit value was indicated on the microcapil-lary reader.The number of white blood cells (WBCs) and differential leukocytes performed using blood smears stained with Wright method, using a Wright Stain Hema-Tek Stain (WSHT, Sigma Aldrich, St. Louis, MO). One hundred WBCs per blood sam- ple were examined using a microscope (Eclipse 100,  Nikon, Japan) with 400×, as well as the identification heterophils, lymphocytes, monocytes, eosinophils, and basophils. The total heterophil and eosinophil count were calculated using a Neubauer hemacytom-eter, with phloxine B cell staining using water pro- pylene glycol. The total number of lymphocytes, monocytes, and basophils was determined indirectly  by calculation of the percentage and total heterophils cells and eosinophils.The concentration of hemoglobin (g/100 ml), total plasma protein (TPP), and blood albumin were measured by spectrophotometry [26]. The total cho-lesterol, HDL cholesterol, triglycerides, and LDL cho-lesterol were measured using Hitachi 704. Body weight and food consumption The measurements of body weight and feed con-sumption were carried out every week for 10 weeks, starting from DODs. Feed conversion is calculated  based on the amount of feed consumed for 10 weeks divided by body weight gain during 10 weeks of maintenance of ducks. At the age of 10 weeks, each sample unit was taken from two ducks from 10 treat-ments, to be slaughtered. Each treatment was four rep-lications so that a total of 80 ducks were slaughtered for the measurement of carcass production. The car-cass weight is the weight without blood, feather, head, neck, shank, and internal organs (liver, gizzard, intes-tine, and heart). The carcass percentage is the carcass weight compared to gross body weight after slaughter. Statistical analysis Data were analyzed using Systat 13 (Systat Software, Inc., San Jose, CA). Data were subject to analysis of variance (ANOVA) according to factorial randomized complete design. Significant differences  between treatment means were determined using a Duncan multiple range test. Statements of signifi-cance were based on p<0.05. Results Blood profiles The result of blood lipid profiles consists of total cholesterol, LDL, HDL, and triglyceride is presented in Table-2. Results of ANOVA show interaction between  breed and phytogenic compounds. Triglyceride level in the blood was significantly affected (Table-2). Group of Tegal duck with garlic powder supplement had the lowest blood triglyceride compared to groups of Muscovy ducks. Turmeric powder supplement had a relatively similar effect on both Tegal and Muscovy ducks. Garlic was the most effective phytogenic to lower blood cholesterol levels and LDL while increas-ing HDL (Table-3).Variety of phytogenic compounds has no effect on RBC, hematocrit, hemoglobin, albumin levels, and fibrinogen (Table-4) on both breeds. These results Table-1:  Ingredient and nutrient composition (g/kg) of diet for ducks (air-dried basis). Ingredient (kg)Starter (1-21 days)Grower/finisher (22-70 days) Broiler complete feed (BR I)100-Maize-40Rice bran-39Soybean meal-12Fish meal-8Premix (vitamin+mineral)-1Total100100Nutrient content*Metabolizable energy (kcal/kg)33002905.45Crude protein (%)20.5016.10Crude fat (%)5.004.36Crude fiber (%)5.703.91Calcium (%)0.951.82Phosphorus (%)0.81.32*Proximate analysis  Veterinary World, EISSN: 2231-0916 1692Available at www.veterinaryworld.org/Vol.12/November-2019/2.pdf  indicate that phytogenic compounds do not interfere with the physiological condition. However, ANOVA showed a significant interaction between breeds and various phytogenic compounds on TPP. Animal with ginger supplementation showed higher TPP. Muscovy ducks that were fed supplemented with ginger powder had lower TPP than the control and turmeric supple-mentation. ANOVA results show that supplementing  phytogenic compounds significantly affected the H/L ratio as a stress indicator and body immunity of fowl (Table-5). All phytogenic compounds lowered H/L ratios compared to the control group. Body weight gain, feed conversion ratio (FCR), and carcass percentage The results showed the interaction between ducks breed and phytogenic compounds, as well as phytogenic compounds had no significant effect (p>0.05) on feed consumption, body weight gain, FCR, and carcass percentage of duck for 10 weeks of maintenance. On the other hand, ducks breed alone has a significant effect (p<0.05) on feed con-sumption, body weight gain, FCR, and the carcass  percentage of duck. The average body weight gain, FCR, and carcass percentage of duck due to phy-togenic compounds are presented in Table-6. Feed consumption, body weight gain, and carcass per-centage for Tegal duck were lower than Muscovy ducks. This indicates that Muscovy duck was more efficient in feed, due to its higher FCR than Tegal duck. Discussion Blood profiles Tegal duck is an Indonesian indigenous duck that possesses unique adaptive traits that permit them to survive and reproduce under harsh climatic, nutri-tional, and management conditions. Muscovy duck is a waterfowl that srcinated from Latin America, is now well adapted to the Indonesian climate that adapted for survival under scavenging free-range con-ditions due to their involvement from the same condi-tions and beneficial to rural people because they are available, adaptable [4].Research about herbal plant has been done [13,27], but the research of phytogenic supple-mentation on waterfowl in Indonesia is still limited. The previous research indicated that phytobiotic prop-erties of garlic (  A. sativum ), turmeric ( C. domestica ), red ginger (  Z. officinale ), and kencur (  K. galanga ) were studied using standard in vitro  antibacterial test and in vivo  feeding trial with ducklings. Weight gain, feed intake, and FCR of ducklings were affected by inclusion of garlic, red ginger, and kencur [28] and substituted with antibiotics in the feed of broiler [29]. The highest antibacterial activity against Salmonella  pullorum and  Escherichia coli  was observed with garlic; it was also significantly effective against Clostridium perfringens  [30].Genetic factors influence blood lipid profile on ducks. Both ducks are waterfowl with high subcu-taneous fat. Tegal ducks are laying type ducks, with Table-2:  Effect of interaction between breeds and phytogenics supplement on blood lipid profile. TreatmentsCholesterol (mg/dl)HDL-C (mg/dl)LDL-C (mg/dl)Triglyceride (mg/dl) B1P0250.00±21.2852.80±3.59197.20±23.04187.50±15.96 a BIP1175.00±13.9893.50±7.5181.50±17.58108.33±21.52 d B1P2186.11±10.6485.25±5.50100.86±9.07129.17±34.36 c B1P3222.22±18.1464.35±4.88157.87±22.72187.50±15.96 a B1P4238.89±37.9561.60±9.51177.29±43.09237.50±55.07 a Average B1214.44±36.0771.50±16.17 b 142.94±51.02170.00±55.30B2P0247.22±13.9874.25±6.81172.97±18.32216.67±23.57 a B2P1177.78±18.14100.10±8.3377.68±19.96154.17±25.00 b B2P2177.78±18.1492.40±9.5185.38±26.08145.83±36.96 c B2P3225.00±18.9867.65±7.49157.35±23.64183.33±13.61 a B2P4244.44±18.1462.70±5.54181.74±22.14170.83±28.46 b Average B2214.44±35.3479.42±16.29 a 135.02±49.67174.00±34.82B1P0=Tegal ducks fed with basal feed, B1P1=Tegal ducks fed with basal feed+3% garlic powder, B1P2=Tegal ducks fed with basal feed+3% turmeric powder, B1P3=Tegal ducks fed with basal feed+3% ginger powder, and B1P4=Tegal ducks fed with basal feed+3% kencur powder, B2P0=Muscovy ducks fed with basal feed, B2P1=Muscovy ducks fed with basal feed+3% garlic powder, B2P2=Muscovy ducks fed with basal feed+3% turmeric powder, and B2P4=Muscovy ducks fed with basal feed+3% kencur powder, B1=Tegal duck, B2=Muscovy duck. a,b,c,d Means within a column with uncommon letters differ at p<0.05. HDL-C=High-density lipoprotein cholesterol, LDL-C=Low-density lipoprotein cholesterol Table-3:  Effect of phytogenics supplement on cholesterol level. PhytogenicsCholesterol (mg/dl)HDL-C (mg/dl)LDL-C (mg/dl) Control248.61±16.73 a 63.53±12.52 c 185.09±23.22 a 3% Garlic176.39±15.07 d 96.80±8.15 a 79.59±17.53 d 3% Turmeric181.94±14.47 c 88.83±8.14 b 93.12±19.88 c 3% Ginger223.61±17.25 b 66.00±6.11 c 157.61±21.46 b 3% Kencur241.67±27.70 b 62.15±7.23 c 179.52±31.80 ba,b,c,d Means within the same column with uncommon superscript differ significantly (p<0.05). HDL-C=High-density lipoprotein cholesterol, LDL-C=Low-density lipoprotein cholesterol  Veterinary World, EISSN: 2231-0916 1693Available at www.veterinaryworld.org/Vol.12/November-2019/2.pdf  lower body weight compared to Muscovy ducks. Genetically, both total cholesterol and HDL in the  blood are determined by endogenous factors and the feed consumed by ducks (Table-2).There is a different breed response on phytogenic supplementation. Garlic reduced blood triglycerides in Tegal duck, while turmeric is more effective in lowering blood triglycerides levels in Muscovy (Table-2). Result in Tables-2 and 3 agreed with the  previous research that supplementing 5% garlic pow- der or 3% garlic powder plus α-tocopherol resulted in significantly lower total and LDL cholesterol levels and greater HDL cholesterol levels compared with the control [16]. Triglyceride, the precursor for energy is the most abundant energy forms stored in the body. When the body needs energy, the lipase enzyme in lipid cells will break the triglycerides into glycerol and fatty acid then release them into the arteries. Fatty acid then undergoes hydrolysis in the liver, yielding  by-products such as cholesterol.Garlic was the most effective agent to reduce  blood lipid. About 3% garlic powder in diet have a much higher antioxidant capacity or biological effects in suppressing the formation of free radicals [31]. Administrating garlic powder decreased the blood LDL and non-esterified fatty acids without affect-ing the blood triglyceride, HDL, and beta-hydroxy- butyric acid concentrations [32]. Turmeric powder ( C. domestica  Val.) up to 0.6% increased the number of erythrocytes, hemoglobin concentration, hema-tocrit, and total lymphocyte percentage, while the number of heterophils, monocytes, eosinophils, and  basophils decreased in duck [33].All types of phytogenics showed a relatively similar effect as hematologic profile, stress, and wel-fare level modulators in two Indonesian local breeds of ducks (Table-4). Harper et al  . [34] indicated that  bioactive substances; allin, allicin, curcumin, biofla-vonoid in ginger, and kencur, serve as antioxidants in the immunomodulatory system. Differences in the responses to ginger on Tegal ducks and Muscovy ducks show that physiological levels of TPP will adjust the protein requirements in the tissues. The  plasma levels of total protein are 3.2-5.6 g/dL, albu-min ranges from 52% to 65%, and globulin from 29.5% to 54%. In normal conditions, the albumin and globulin ratio is 1.2:1. TPPs can function as the body’s  protein reserves. Circulating plasma proteins are not static, they are constantly held in exchanges with tis-sue backup labile, where the number is proportional to the circulating protein resulting in a dynamic balance. At a time of protein deficiency, the body takes up a network of proteins and plasma proteins for metabolic needs [34].Welfare levels can be measured from the H/L ratio [22]. Bioactive substances in garlic, ginger, and turmeric have similar effectiveness to promote the immune system (Table-5). The lymphocyte is one type of agranulocyte leukocyte with predominant    T  a   b   l  e  -   4  :    E   f   f  e  c   t  o   f   i  n   t  e  r  a  c   t   i  o  n   b  e   t  w  e  e  n   b  r  e  e   d  s  a  n   d  p   h  y   t  o  g  e  n   i  c  s  s  u  p  p   l  e  m  e  n   t  o  n   h  e  m  a   t  o   l  o  g   i  c  a   l  p  a  r  a  m  e   t  e  r  s .    T  r  e  a   t  m  e  n   t  s   R   B   C   (  m   i   l   l   /  u   l   )   W   B   C   (  c  e   l   l  s   /  u   l   )   H   b   (  m  g   /   d   l   )   P   C   V   (   %   )   T   P   P   (  g   /   d   l   )   A   l   b  u  m   i  n   (  g   /   d   l   )   F   i   b  r   i  n  o  g  e  n   (  g   /   d   l   )   H  e   t  e  r  o  p   h   i   l   (   %   )   L  y  m  p   h  o  c  y   t  e   (   %   )   M  o  n  o  c  y   t  e   (   %   )   E  o  s   i  n  o  p   h   i   l   (   %   )   H   /   L    B   1   P   0   3 .   2   7   ±   0 .   3   1   1   1 ,   2   1   2 .   5   0   ±   2   2   5   9 .   9   3   1   2 .   9   9   ±   1 .   1   0   3   6 .   0   0   ±   5 .   8   9   4 .   4   9   ±   0 .   5   1   6 .   3   4   ±   0 .   9   3   1 .   9   3   ±   0 .   7   0   5   1 .   2   5   ±   6 .   8   5   3   1 .   7   5   ±   9 .   2   2   4 .   5   0   ±   1 .   7   3   1   2 .   5   0   ±   3 .   4   2   1 .   9   3   ±   0 .   7   0   B   I   P   1   3 .   3   1   ±   0 .   2   4   7   8   8   7 .   5   ±   2   9   3   0 .   4   1   1   3 .   5   ±   1 .   0   5   4   2 .   0   0   ±   4 .   0   8   2 .   7   5   ±   0 .   1   9   6 .   9   8   ±   1 .   6   7   0 .   5   7   ±   0 .   1   1   2   9 .   2   5   ±   2 .   9   9   5   5 .   0   0   ±   2 .   8   3   4 .   7   5   ±   1 .   7   4   1   1 .   0   0   ±   2 .   4   5   0 .   5   7   ±   0 .   1   1   B   1   P   2   2 .   8   3   ±   0 .   8   1   1   0 ,   6   8   7 .   5   0   ±   3   3   0   6 .   1   5   1   2 .   1   8   ±   1 .   6   5   3   7 .   5   0   ±   7 .   5   5   4 .   0   5   ±   1 .   1   5   6 .   5   9   ±   1 .   1   5   1 .   0   0   ±   0 .   3   0   3   9 .   0   0   ±   7 .   5   3   4   0 .   0   0   ±   5 .   4   2   4 .   0   0   ±   1 .   4   1   1   7 .   0   0   ±   6 .   2   2   1 .   0   0   ±   0 .   3   0   B   1   P   3   3 .   2   1   ±   0 .   4   0   1   0 ,   7   8   7 .   5   0   ±   2   3   4   2 .   5   0   1   2 .   6   8   ±   1 .   5   9   4   3 .   7   5   ±   4 .   7   9   4 .   8   5   ±   0 .   7   7   8 .   3   6   ±   1 .   5   0   0 .   8   1   ±   0 .   1   8   3   7 .   0   0   ±   5 .   3   5   4   6 .   2   5   ±   4 .   1   9   5 .   0   0   ±   0 .   8   2   1   1 .   7   5   ±   3 .   3   0   0 .   8   1   ±   0 .   1   8   B   1   P   4   3 .   0   4   ±   0 .   3   6   9   5   0   9 .   2   5   ±   2   3   8   3 .   1   7   1   2 .   1   9   ±   0 .   8   6   3   9 .   9   4   ±   5 .   6   0   4 .   4   1   ±   0 .   7   4   7 .   8   0   ±   0 .   9   1   1 .   3   1   ±   0 .   8   1   4   3 .   5   0   ±   1   1 .   0   9   3   8 .   0   0   ±   1   0 .   6   8   5 .   7   5   ±   3 .   5   9   1   2 .   7   5   ±   2 .   9   9   1 .   3   1   ±   0 .   8   1   B   2   P   0   3 .   1   0   ±   0 .   4   4   9   4   0   9 .   3   8   ±   1   7   6   8 .   3   4   1   2 .   5   2   ±   1 .   3   5   4   2 .   4   4   ±   9 .   1   9   4 .   2   0   ±   0 .   3   7   8 .   5   8   ±   1 .   8   7   1 .   6   7   ±   0 .   5   9   5   1 .   5   0   ±   8 .   5   4   3   3 .   0   0   ±   6 .   5   8   4 .   7   5   ±   1 .   5   0   1   0 .   7   5   ±   2 .   6   3   1 .   6   7   ±   0 .   5   9   B   2   P   1   3 .   1   1   ±   0 .   5   7   7   6   0   0 .   0   0   ±   1   9   1   8 .   3   3   1   2 .   2   8   ±   1 .   2   4   4   2 .   5   0   ±   7 .   7   2   2 .   9   0   ±   0 .   1   2   6 .   2   5   ±   0 .   8   7   0 .   5   7   ±   0 .   2   7   3   1 .   7   5   ±   1   0 .   3   4   5   8 .   2   5   ±   6 .   9   9   4 .   5   0   ±   1 .   2   9   8 .   0   0   ±   2 .   8   3   0 .   5   7   ±   0 .   2   7   B   2   P   2   2 .   8   8   ±   0 .   7   0   1   0 ,   5   0   0 .   0   0   ±   2   9   2   0 .   0   5   1   2 .   9   8   ±   1 .   5   8   4   1 .   7   5   ±   9 .   1   8   4 .   8   5   ±   0 .   6   2   8 .   6   6   ±   1 .   2   8   1 .   1   2   ±   0 .   6   2   3   9 .   5   0   ±   1   1 .   5   0   4   0 .   5   0   ±   1   2 .   7   7   4 .   7   5   ±   1 .   5   0   1   5 .   2   5   ±   3 .   7   7   1 .   1   2   ±   0 .   6   2   B   2   P   3   3 .   1   4   ±   0 .   5   7   8   5   0   0 .   0   0   ±   2   1   7   1 .   0   2   1   2 .   7   5   ±   0 .   8   5   4   2 .   5   0   ±   7 .   7   2   3 .   5   0   ±   0 .   8   1   7 .   9   3   ±   1 .   6   7   0 .   6   9   ±   0 .   1   8   3   4 .   7   5   ±   4 .   9   9   5   2 .   0   0   ±   7 .   7   0   5 .   0   0   ±   1 .   4   1   8 .   2   5   ±   2 .   6   3   0 .   6   9   ±   0 .   1   8   B   2   P   4   3 .   0   2   ±   0 .   4   3   8   8   2   5 .   0   0   ±   2   9   1   5 .   9   0   1   1 .   8   3   ±   1 .   0   0   4   1 .   0   0   ±   7 .   3   9   3 .   0   0   ±   0 .   9   1   6 .   9   8   ±   1 .   1   4   1 .   1   2   ±   0 .   6   4   4   1 .   0   0   ±   1   0 .   5   5   4   2 .   7   5   ±   1   4 .   1   0   4 .   0   0   ±   1 .   4   1   1   2 .   2   5   ±   3 .   8   6   1 .   1   2   ±   0 .   6   4   B   1   P   0  =   T  e  g  a   l   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d ,   B   1   P   1  =   T  e  g  a   l   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %   g  a  r   l   i  c  p  o  w   d  e  r ,   B   1   P   2  =   T  e  g  a   l   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %    t  u  r  m  e  r   i  c  p  o  w   d  e  r ,   B   1   P   3  =   T  e  g  a   l   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %   g   i  n  g  e  r  p  o  w   d  e  r ,  a  n   d   B   1   P   4  =   T  e  g  a   l   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %    k  e  n  c  u  r  p  o  w   d  e  r ,   B   2   P   0  =   M  u  s  c  o  v  y   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d ,   B   2   P   1  =   M  u  s  c  o  v  y   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %   g  a  r   l   i  c  p  o  w   d  e  r ,   B   2   P   2  =   M  u  s  c  o  v  y   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %    t  u  r  m  e  r   i  c  p  o  w   d  e  r ,  a  n   d   B   2   P   4  =   M  u  s  c  o  v  y   d  u  c   k  s   f  e   d  w   i   t   h   b  a  s  a   l   f  e  e   d  +   3   %    k  e  n  c  u  r  p  o  w   d  e  r .   a ,   b ,  c ,   d    M  e  a  n  s  w   i   t   h   i  n  a  c  o   l  u  m  n  w   i   t   h  u  n  c  o  m  m  o  n   l  e   t   t  e  r  s   d   i   f   f  e  r  a   t  p  <   0 .   0   5 .   R   B   C  =   R  e   d   b   l  o  o   d  c  e   l   l ,   W   B   C  =   W   h   i   t  e   b   l  o  o   d  c  e   l   l ,   P   C   V  =   P  a  c   k  e   d  c  e   l   l  v  o   l  u  m  e ,   T   P   P  =   T  o   t  a   l  p   l  a  s  m  a  p  r  o   t  e   i  n ,   H   /   L  =   H  e   t  e  r  o  p   h   i   l   /   l  y  m  p   h  o  c  y   t  e
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