DOI: 10.1111/jpn.12116
ORIGINAL ARTICLE
Effect of dietary supplementation with an ethanolic extract of propolis on broiler intestinal morphology and digestive enzyme activity C. Eyng, A. E. Murakami, C. R. A. Duarte and T. C. Santos Departamento de Zootecnia, Universidade Estadual de Maring a, Maringa, Brazil
Summary The present study aimed to evaluate the effect of different levels of an ethanolic extract of propolis (EEP) on broiler performance, carcass characteristics, weight of gastrointestinal organs, intestinal morphometry and digestive enzyme activity. 1020 male broiler chicks were assigned in a completely randomised experimental design to six treatments (EEP supplement levels of 0, 1000, 2000, 3000, 4000 and 5000 ppm) and five replications, and 34 birds per experimental unit. The experimental diets were administered from 1 to 21 days of age, and the birds were subsequently provided a ration based on corn and soybean meal. EEP supplementation from 1 to 7 days negatively affected (p < 0.05) the weight gain and feed intake. The proventriculus weight at 7 days exhibited a quadratic response (p < 0.05), which predicted a lower weight at a dose of 2865 ppm of the EEP. For the duodenum at 21 days of age, the response pattern (p < 0.05) predicted that birds that were fed 2943 and 3047 ppm of the EEP would exhibit an improved crypt depth and villus-to-crypt ratio respectively. The villus height, crypt depth and villus-to-crypt ratio in the jejunum and the ileum were not affected (p > 0.05). With increased EEP doses, the duodenal sucrase activity linearly decreased at 7 days of age and linearly increased in the jejunum at 21 days of age (p < 0.05), while pancreatic enzyme activity was unaffected (p > 0.05). Although the carcass and cut yields did not improve, the percentage of abdominal fat decreased (p < 0.05). The supplementation of the broiler pre-starter diet with 1000–5000 ppm of the EEP impaired performance at this stage, most likely due to the decreased sucrase activity. However, the EEP supplementation from 3000 ppm improved intestinal morphophysiology at 21 days of age and did not affect the performance or carcass yield at 42 days of age. Keywords disaccharidases, growth promoter, natural additive, pancreatic enzymes ^ncias Agrarias, Universidade Estadual de Maringa, Av. Correspondence Profa. Dra. A. E. Murakami, Departamento de Zootecnia, Centro de Cie Colombo, 5790, Bloco J45, Maring a, PR 87020-900, Brazil. Tel: +55 44 3011 8942; Fax: +55 44 3011 8977; E-mail: [email protected] Received: 16 October 2012; accepted: 7 July 2013
Introduction For decades, the poultry production chain has benefited from the use of antibiotics at subtherapeutic doses for improving performance indices. However, possible microbial resistance and the requirements of certain consumer markets for healthy products without health risks have led to the search for alternative additives, which consist mainly of natural products with important therapeutic functions that can replace growth-promoting antibiotics. Propolis is a resinous and balsamic substance that is produced by bees when they combine plant exudates, wax and pollen with their salivary secretions, which has been described because of antibacterial, antioxidant, antiviral, anti-inflammatory, antifungal and
immunostimulatory properties (Marcucci, 1995), among others. These properties are attributed to more than 300 compounds, including phenolic acids, flavonoids, esters, aromatic aldehydes, fatty acids, amino acids, vitamins and minerals (Bankova et al., 2000; Lofty, 2006). Among them, the flavonoids are the most studied as potent antioxidants, although the flavonoids also interfere with many physiological processes, such as carbohydrate metabolism, showing an antihyperglycaemic property in rats (Matsui et al., 2004). Due to the presence of these important compounds and their effects on animal physiology, the use of propolis in broiler feed, whether in the form of crude propolis or an ethanolic extract of propolis (EEP), promotes improved gut health by controlling the growth
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of pathogenic micro-organisms in the gastrointestinal tract, which consequently improves digestive and absorptive functions. Additional advantages derive from the use of propolis in poultry diets, such as the absence in carcasses of toxic residues that might compromise human health (Denli et al., 2005) and improvements in the immune system (Freitas et al., 2011) and in the performance of laying hens and broilers (Shalmany and Shivazad, 2006; Tatli Seven et al., 2008; C ß etin et al., 2010; Tekeli et al., 2011). In broiler chickens, Tatli Seven et al. (2008) showed that ethanolic extract of propolis supplementation is more effective than vitamin C on performance and carcass qualities under heat stress that can be attributed to beneficial effects of propolis on reduction in the adverse effects of oxidative stress (Tatli Seven et al., 2009; Seven et al., 2010). Moreover, broiler chickens fed with 1000 ppm of propolis presented a similar performance compared with chickens fed antibiotic growth promoter (Tekeli et al., 2011). Shalmany and Shivazad (2006) have shown better performance and low mortality rate in birds fed with propolis. Although many authors showed beneficial effects of propolis on animal performance and antioxidant and immune status, there are scarce studies that correlate these effects with changes in intestinal morphophysiology. Some evidences show that propolis has protective effect on liver morphology of broiler chickens (Babi nska et al., 2013) and it can modulate the intestinal microbiota (Kac aniov a et al., 2012). Thus, the present study evaluated the effect of different ethanolic extract of propolis levels in the feed of broilers on their performance, carcass characteristics, organ weights, intestinal morphometry and digestive enzyme activity.
extract of propolis (0, 1000, 2000, 3000, 4000 and 5000 ppm, each fed to 34 birds per replicate). The broilers received the experimental diets until 21 days of age followed by a conventional diet until 42 days of age. The experimental diets (Table 1) were based on corn and soybean meal and were formulated using the feed chemical composition values and the nutritional requirements for the average performance of male broilers according to Rostagno et al. (2005). The ethanolic extract of propolis was prepared using equal amounts of the propolis and the alcohol. The Table 1 Ingredients and nutrient composition of the experimental diets
Ingredient (%) Corn Soybean meal 45% Soybean oil Limestone Dicalcium phosphate NaCl Inert* DL- Met 98% L- Lys HCl, 78.5% L- Thr 98% Supplement minerals and vitamins† Calculated composition CP (%) ME (kcal/kg) Calcium (%) Available phosphorus (%) Digestible Met + Cys (%) Digestible Lys (%) Digestible Thr (%) Digestible Trp (%)
1–7 days
8–21 days
22–42 days
55.75 37.04 2.20 0.92 1.94 0.40 0.50 0.36 0.35 0.15 0.40
58.63 34.39 2.51 0.88 1.80 0.40 0.50 0.24 0.19 0.05 0.40
64.45 28.68 3.15 0.82 1.58 0.40 0.23 0.23 0.06 0.40
22.04 2.950 0.939 0.470 0.944 1.330 0.865 0.213
20.79 3.000 0.884 0.442 0.814 1.146 0.745 0.183
18.72 3.125 0.793 0.395 0.752 1.045 0.679 0.177
The protocol for this experiment was approved by the Ethics Committee on Animal Use in Experimentation (CEAE/UEM) of the Universidade Estadual de Maringa (UEM, Maring a, Paran a, Brazil), and birds were cared according to the Ethical Principles for Animal Experimentation established by the Brazilian Society of Laboratory Animal Science (SBCAL) and National Council for the Control of Animal Experimentation (CONCEA). In all, 1020 1-day-old male Cobb-Vantressâ chicks from 40-week-old breeders were distributed in a completely randomised experimental design among five replicates of six treatments, which consisted of six levels of supplementation of the diet with an ethanolic
*The ethanolic extract of propolis was added in replacement for inert (kaolin). †(1–21 days) Vitamin mixture (content per kg of diet): vitamin A (retinyl acetate), 2917 IU; vitamin D3 (cholecalciferol), 583 IU; vitamin E (DL-a-tocopheryl acetate), 8750 IU; vitamin K3 (menadione dimethylpyrimidinol), 433 IU; vitamin B1 (thiamine mononitrate), 408 mg; vitamin B12 (cyanocobalamin), 4166 lg; niacin (niacinamide), 8983 mg; calcium pantothenate, 3167 mg; folic acid, 200 mg; D-biotin, 25 mg. Mineral mix (content per kg of diet): Fe (iron sulphate monohydrate), 12.6 g; Cu (copper sulphate pentahydrate), 3072 mg; I (calcium iodate), 248 mg; Zn (zinc oxide), 12.6 g; Mn (manganous oxide), 60 mg; Se (sodium selenite), 61 mg; Co (cobalt sulphate), 50 mg. (22–42 days) Vitamin mixture (content per kg of diet): vitamin A (retinyl acetate), 2250 IU; vitamin D3 (cholecalciferol), 450 IU; vitamin E (DL-a-tocopheryl acetate), 7000 IU; vitamin K3 (menadione dimethylpyrimidinol), 418 IU; vitamin B1 (thiamine mononitrate), 300 mg; vitamin B12 (cyanocobalamin), 2300 lg; niacin (niacinamide), 7000 mg; calcium pantothenate, 2500 mg; folic acid, 140 mg; D-biotin, 14 mg. Mineral mix (content per kg of diet): Fe (iron sulphate monohydrate), 12.5 g; Cu (copper sulphate pentahydrate), 3000 mg; I (calcium iodate), 250 mg; Zn (zinc oxide), 12.5 g; Mn (manganous oxide), 15 mg; Se (sodium selenite), 75 mg; Co (cobalt sulphate), 50 mg.
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Materials and methods
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composition of the extract was determined according to Singleton and Rossi (1965) and Pierpoint (2004) for the total polyphenols and using the aluminium chloride colorimetric methods for the total flavonoid content. The broilers and the feed were weighed at 1, 7, 21 and 42 days of age to evaluate performance, which was measured as the feed intake, weight gain and feed conversion rate. Every bird that was sampled or died was weighed, and the pen feed intake at that time was recorded; these data were used to correct the feed conversion rate. At 7 and 21 days of age, 5 birds per treatment were sacrificed to collect the gastrointestinal organs (proventriculus, gizzard, small and large intestines, pancreas and liver). The organs were cleaned with physiological saline solution, dried with filter paper and weighed. The weight of each organ was expressed relative to the total body weight (g/100 g BW). The length of the intestine was also measured (cm). Fragments of approximately 5 cm were obtained from each of the following segments of the small intestine: the duodenum (from the pylorus to the distal portion of the duodenal loop), the jejunum (from the distal portion of the duodenal loop to Meckel’s diverticulum) and the ileum (the anterior portion of the ileocecal junction) to evaluate the intestinal morphology. The fragments from each segment were then placed on polystyrene sheets, opened longitudinally, washed in saline solution, fixed in 10% formaldehyde solution, dehydrated and embedded in paraffin. Thin sections from each segment were cut at a thickness of 5 lm and stained with haematoxylin and eosin according to Luna (1968). The measurements of villus height and crypt depth were taken using a light microscope and a system that analyses computerised images (Motic Image Plus 2.0; Motic China Group, Hong Kong, China). The height of 30 villi and the depth of 30 crypts were measured from each segment and replicate. The mean was obtained for each EEP treatment and intestinal segment from these values. Portions of each segment of the small intestine were freed of residual food, frozen in liquid nitrogen and stored in a freezer at 80 °C until they were assayed to determine the activity of intestinal disaccharidases by the Dahlqvist (1964). Each segment was opened longitudinally, and the mucosa was scraped off with a glass microscope coverslip. The collected mucosa was homogenised after the addition of 4 parts of ice-cold deionised water. Maltase and sucrase activities were assayed by incubating aliquots of the homogenates with the appropriate substrate in malate buffer at pH 6.4. Released glucose was determined by the glucose
The concentration of the total polyphenols and flavonoids in the EEP was 357.71 mg/l and 112.72 mg/l respectively. At 7 days of age, the weight gain and feed intake deteriorated linearly with increasing EEP levels (Table 2, p < 0.05), and the feed intake was lower in the animals fed 5000 ppm of the EEP than it was in the control animals (Dunnett’s test, p < 0.05). However, the feed conversion at this age did not differ among the experimental groups (p > 0.05). At 21 and 42 days of age, the performance parameters were similar among the experimental groups (Table 2, p > 0.05).
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oxidase method (Laborlab, Guarulhos, Brazil). The enzyme activity is expressed as units per gram of protein, which was determined by the method of Bradford (1976). The activity of the pancreatic enzymes was determined after the whole pancreas was homogenised (1:20 wt/vol) in 50 mM Tris–HCl buffer (pH 8) containing 50 mM CaCl2. The trypsin activity was determined according to methodology proposed by Kakade et al. (1974). A similar method was used for the determination of chymotrypsin (Erlanger et al., 1966). Amylase activity was determined by the iodometric method modified by Caraway (1959) (Gold Analisa, Belo Horizonte, Minas Gerais, Brazil). Lipase activity was obtained using the BALB-DNTP method (Gold Analisa). At the end of the experimental period, the carcass yield was determined in 2 birds at random per experimental unit (12 birds per treatment). The birds were identified, starved for 6 h and killed by electric stunning and bleeding. The carcass yield was calculated as the ratio of the hot eviscerated carcass to the BW before euthanasia. The prime cut yield (the whole breast, thigh and legs with their skin and bones) was calculated in relation to the weight of the eviscerated carcass. Abdominal fat associated with the cloaca, the bursa of Fabricius, the gizzard, the proventriculus and the adjacent abdominal muscles was removed as described by Smith (1993). The data were examined in relation to the EEP levels using analysis of variance followed by Dunnett’s test and regression analysis by polynomial decomposition of the degrees of freedom. SAEG (System for Statistical and Genetic Analysis, Universidade Federal de Vicßosa, 1997) software was utilised for the analyses, and a probability (p) < 0.05 was considered significant. Results
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Table 2 The performance of broiler chickens fed with diets containing different levels of ethanolic extract of propolis EEP levels 1–7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 1–21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 1–42 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Weight gain (g)
Feed intake (g)
F:G
134.28 136.07 130.08 132.54 126.70 126.07 1.37 L† (p = 0.04) 0.195
164.35 170.16 160.47 158.29 161.19 153.85* 1.40 L‡ (p = 0.002) 0.010
1.224 1.254 1.235 1.196 1.274 1.220 0.01 ns 0.167
792.87 826.30 822.13 841.27 809.55 789.36 7.24 ns 0.286
1183.73 1231.45 1234.16 1242.14 1221.08 1162.02 10.17 ns 0.163
1.496 1.492 1.502 1.476 1.509 1.472 0.01 ns 0.868
2596.80 2701.43 2651.15 2678.31 2639.62 2591.42 22.00 ns 0.689
4617.63 4782.09 4664.83 4801.16 4647.34 4696.99 31.84 ns 0.471
1.780 1.770 1.760 1.795 1.761 1.814 0.01 ns 0.425
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Y = 137.310 0.00233937x (R2 = 0.79). ‡Y = 170.366 0.00319139x (R2 = 0.71).
The weight of the proventriculus varied quadratically with the concentration of the EEP supplement at 7 days, and the quadratic equation predicted a lower weight for animals fed at an EEP level of 2865 ppm (Table 3, p < 0.05). The weights of the other organs evaluated did not vary with the EEP level at either 7 or 21 days of age (p > 0.05). At 7 days of age, the intestinal morphology did not differ among the experimental groups (p > 0.05). For the duodenum at 21 days, the crypt depth and the ratio of the villus height to crypt depth varied quadratically with the level of the EEP supplementation; the quadratic equation predicted a lower crypt depth in broilers fed diets supplemented with 2943 ppm of the EEP and a higher villus-to-crypt ratio when diets were supplemented with 3047 ppm of the EEP (Table 4, p < 0.05). The treatments had no effect (p > 0.05) on 396
the crypt depth or the villus height-to-crypt ratio in the jejunum or the ileum or on the villus height in any of the intestinal segments. At 7 days of age, the sucrase activity in the duodenum decreased linearly with increasing EEP supplement levels (Table 5, p < 0.05) and displayed lower values in the group fed 3000 ppm of the supplement than in the control group (Dunnett’s test, p < 0.05). Although not statistically significant in the regression tests (p > 0.05), the sucrase activity in the jejunum was higher in the animals fed 5000 ppm of the EEP supplement than it was in the control animals (Dunnett’s test, p < 0.05). At 21 days of age, the sucrase activity in the jejunum increased linearly with increasing EEP levels (p < 0.05) and exhibited lower values in the group fed 1000 ppm than in the control group (Dunnett’s test, p < 0.05). The maltase activity in the jejunum was higher in the animals fed 4000 ppm of the EEP than in the control animals (Table 5, Dunnett’s test, p < 0.05). The EEP treatments did not affect (p > 0.05) the sucrase activity in the ileum at 7 or 21 days of age or in the duodenum at 21 days of age nor the maltase activity in any of the intestinal segments at 7 or 21 day of age. The activities of the lipase, amylase, trypsin and chymotrypsin did not vary in the pancreas at 7 or 21 days of age with the level of the EEP supplement in the diet (Table 6, p > 0.05). The carcass and cut yields were similar among the EEP supplement levels (Table 7, p > 0.05). The abdominal fat percentage was lower in the animals fed 1000 and 3000 ppm of the EEP than in the control animals (Dunnett’s test, p < 0.05). Discussion The polyphenol and flavonoid levels can present differences between samples according to variations in the extraction process, such as the area of the contact surface relative to weight of the raw propolis, the type of solvent used for extraction and the duration of the extraction process, all of which may influence the final concentrations of these therapeutic substances. Moreover, it is very difficult to compare the results showed by another authors due the differences in the propolis composition, which depends on geographical origin and seasonal effect (Bankova et al., 2000). Among the polyphenols present in the EEP, the flavonoid group has important therapeutic properties, such as antimicrobial, antioxidant and anti-inflammatory qualities. The high concentration of these compounds in propolis may be responsible for its Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Table 3 Relative weight (g/100 g live weight) of proventriculus, gizzard, small (SI) and large (LI) intestines, liver and pancreas and length of small intestine (cm) of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels 7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Proventriculus
Gizzard
SI
LI
Liver
Pancreas
SI Length
1.009 1.076 0.973 0.976 1.007 1.102 0.01 Q* (p = 0.003) 0.060
4.689 4.361 4.538 4.264 4.694 4.855 0.08 ns 0.325
9.625 9.817 10.180 9.759 10.189 10.171 0.11 ns 0.534
1.637 1.715 1.609 1.517 1.744 1.578 0.05 ns 0.991
3.481 3.755 3.381 3.534 3.419 3.648 0.06 ns 0.890
0.534 0.538 0.498 0.506 0.525 0.481 0.01 ns 0.581
97.35 98.12 98.85 98.25 97.67 98.00 0.61 ns 0.657
0.596 0.611 0.647 0.614 0.569 0.585 0.01 ns 0.418
2.460 2.487 2.293 2.424 2.413 2.553 0.04 ns 0.662
7.444 7.276 7.445 7.069 6.900 7.508 0.13 ns 0.727
1.041 1.172 1.114 1.256 1.372 1.338 0.04 ns 0.242
2.543 2.658 2.753 2.649 2.610 2.839 0.04 ns 0.301
0.352 0.412 0.353 0.399 0.379 0.351 0.01 ns 0.090
156.30 158.80 168.20 157.60 161.70 156.00 1.89 ns 0.442
ns, not significant; SEM, standard error of means. *Y = 1.21395 0.000172607x + 0.00000003012x2 (R2 = 0.98); maximum point: 2865 ppm.
therapeutic activity (Fu et al., 2005). Because these substances can interfere with adhesion by pathogenic bacteria (Jamroz et al., 2006) and modulate intestinal microbiota, mainly qualitatively, by favouring the growth of beneficial bacteria, such as lactobacilli and bifidobacteria (Guo et al., 2004), supplementing the diet with these compounds would influence many important physiological processes, such as nutrient uptake and immunity (Romier et al., 2009; Hanhineva et al., 2010). Moreover, the flavonoids have an aglycone hydroxyl grouping positioned similarly to that of oestrogen, predicting their possible growth hormone activity (Ziaran et al., 2005). Thus, the use of propolis in animal feed could promote better nutrient use by animals by improving their intestinal health. In fact, Kac aniov a et al. (2012) showed the microbiota modulation by ethanolic extract of propolis in broiler chickens, with a reduction in pathogenic bacteria. The EEP supplements impaired performance in 1to 7-day-old animals by reducing their weight gain and feed intake as the EEP levels increased, although propolis contains substances, such as resins, waxes and honey, that are considered palatable (Shalmany and Shivazad, 2006; Tatli Seven, 2008). According to Acßikg€ oz et al. (2005), adding a high level of propolis (4000 ppm) during early broiler development causes decreased protein digestion and poor growth.
However, according to the performance and carcass yield results reported here, the EEP dietary supplements had no negative effects at 21 days of age or at the end of the rearing period, and thus, the carcass and cut yields were not impaired at 42 days of age. This result indicates that the EEP supplementation was only detrimental within the first week of life at the levels studied because the animal performance parameters recovered at 21 and 42 days of age. Moreover, the EEP supplementation caused an abdominal fat reduction, which was attributable to the lipolytic action of the propolis. Regarding this suggested mechanism, compounds in the propolis are capable of inhibiting phosphodiesterase, which is responsible for degrading cyclic AMP, and increasing intracellular phosphodiesterase levels, which consequently increases lipolysis (Kuppusamy and Das, 1992; Jin et al., 1998). The effects of EEP dietary supplements on birds are still unclear, given that some studies detected no effects on animal performance (Acßikg€ oz et al., 2005; Ziaran et al., 2005), whereas others have demonstrated positive effects (Denli et al., 2005; Shalmany and Shivazad, 2006; Galal et al., 2008; Tatli Seven et al., 2008; Tekeli et al., 2011). Tatli Seven et al. (2008) showed that supplementing the diet with 500– 3000 ppm of propolis extract improved broiler performance under heat stress, with the mean weights of
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Table 4 Villus height (lm), crypt depth (lm) and villus/crypt ratio of segments of the small intestine of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels Control 7 days Villus height Duodenum 1033.99 Jejunum 448.15 Ileum 366.76 Crypt depth Duodenum 166.43 Jejunum 126.57 Ileum 95.24 Villus/crypt depth Duodenum 6.44 Jejunum 3.54 Ileum 3.91 21 days Villus height Duodenum 1652.58 Jejunum 880.02 Ileum 486.72 Crypt depth Duodenum 193.40 Jejunum 138.17 Ileum 106.80 Villus/crypt depth Duodenum 8.80 Jejunum 6.42 Ileum 4.57
1000 ppm
2000 pm
3000 pm
4000 ppm
5000 ppm
SEM
Reg
p-values
981.83 468.07 330.53
1000.47 452.41 369.67
929.89 465.19 354.53
904.11 477.77 363.40
977.15 479.63 351.06
20.20 13.05 8.18
ns ns ns
0.501 0.982 0.787
178.66 130.71 85.19
182.91 129.82 91.19
191.39 131.69 101.12
204.66 142.93 97.88
194.07 118.90 88.52
4.98 2.87 2.86
ns ns ns
0.303 0.275 0.633
5.69 3.60 3.89
5.54 3.50 4.07
4.85 3.55 3.56
4.50 3.35 3.76
5.06 4.02 4.15
0.22 0.09 0.11
ns ns ns
0.133 0.449 0.733
1510.36 885.44 490.30
1581.30 756.38 549.24
1610.86 688.36 480.33
1533.33 823.57 505.71
1598.09 750.15 535.85
28.92 27.59 10.85
ns ns ns
0.765 0.222 0.353
212.22 141.91 107.73
180.72 121.22 112.71
192.07 123.15 103.82
187.34 130.26 104.60
213.83 130.33 118.05
4.18 2.77 1.96
Q*(p = 0.003) ns ns
0.119 0.208 0.273
7.15 6.30 4.60
8.77 6.19 4.89
8.45 5.57 4.66
8.24 6.40 4.87
7.52 5.89 4.61
0.24 0.21 0.12
Q† (p = 0.02) ns ns
0.264 0.853 0.956
ns, not significant; SEM, standard error of means. *Y = 244.232 0.0412346x + 0.00000700631x2 (R2 = 0.81); minimum point: 2943 ppm. †Y = 5.676 + 0.00194855x – 0.000000319772x2 (R2 = 0.84); maximum point: 3047 ppm.
the supplemented, stressed animals similar to those of animals reared under thermoneutral conditions. These inconsistencies are most likely caused by the difficulty of standardising the EEP treatments due to considerable variability in the composition of propolis from various sources and differences in propolis extraction methods. Furthermore, there is wide variation in the EEP levels used in the different studies. The impaired performance at 7 days of age cannot be explained by effects of the EEP supplements on the relative size of the gastrointestinal organs because there were no differences related to the digestive or absorptive capacities of these organs. Only the weight of the proventriculus was influenced by the supplements, and it was influenced in a quadratic manner; this influence may have been related to the reduced feed intake that was observed simultaneously. Although the EEP supplements did not affect the relative weight of the small intestine, this organ underwent important morphophysiological alterations. At 21 days of age, the duodenal crypt depth
varied quadratically with the EEP supplementation level; the shallowest crypt depth was predicted to occur at a level of 2943 ppm, and there was a consequent effect on the villus-to-crypt ratio, which was predicted to increase at the 3047 ppm level of supplementation. A shallower crypt depth is correlated with a reduced need for cell renewal (Oliveira et al., 2008), reducing energy and protein expenditures for this process and, thus, enabling growth of other tissues (Miles et al., 2006). In addition, higher villus-to-crypt ratios suggest higher digestive and absorptive capacities (Silva et al., 2009). Thus, the increased ratios obtained here can be attributed to the beneficial effect of the propolis compounds in controlling proliferation of pathogenic bacteria and avoiding possible damage to the intestinal mucosa, which could result in reduced dimensions of the villi (Sayrafi et al., 2011). These beneficial changes in the intestinal morphometry occurred when the sucrase activity increased in the jejunum. Thus, the recovery of the performance indices at 21 day of age could be attributed to these changes.
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Table 5 Maltase and sucrase activities (U/mg of protein) of segments of the small intestine of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels
7 days Sucrase activity Duodenum Jejunum Ileum Maltase activity Duodenum Jejunum Ileum 21 days Sucrase activity Duodenum Jejunum Ileum Maltase activity Duodenum Jejunum Ileum
Control
1000 ppm
2000 pm
3000 pm
12.03 6.40 7.43
12.70 9.10 5.82
13.41 11.26 8.48
14.07* 6.75 4.75
27.08 29.39 25.47
26.89 30.40 28.00
24.81 34.75 28.19
4.22 8.91 6.93 22.58 27.00 24.48
4.53 5.61* 7.20 23.71 24.44 25.40
4000 ppm
5000 ppm
SEM
Reg
p-values
9.58 8.88 7.12
11.17 12.79* 8.94
0.40 0.47 0.76
L† (p = 0.035) ns ns
0.007 0.028 0.647
21.02 20.94 25.25
27.44 34.02 25.18
25.95 33.57 30.50
0.87 1.82 1.71
7.10 10.36 9.02
9.81 7.66 7.05
6.25 9.43 8.63
7.13 10.36 7.80
0.71 0.49 0.45
26.25 37.34 30.55
34.31 25.08 31.36
35.03 38.59* 32.74
24.02 26.99 26.76
2.37 1.59 1.31
ns ns ns
ns L‡ (p = 0.012) ns ns ns ns
0.282 0.327 0.946
0.234 0.018 0.705 0.491 0.008 0.351
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Y = 14.2523 0.000689074x (R2 = 0.36). ‡Y = 6.15381 + 0.000858245x (R2 = 0.45).
Table 6 Pancreatic enzyme activities of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP)
EEP levels 7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Amylase (UA/mg of protein)
Trypsin (nmol/mg of protein
Chymotrypsin (nmol/mg of protein)
Lipase (IU/mg of protein)
5.89 7.83 9.13 5.43 11.88 6.64 0.94 ns 0.425
25.42 22.37 26.95 22.71 27.08 28.70 2.59 ns 0.985
3.36 3.34 3.13 3.28 5.52 3.58 0.31 ns 0.285
3.82 4.31 7.09 4.24 4.90 6.67 0.63 ns 0.579
5.27 6.55 3.97 4.52 3.80 4.95 0.47 ns 0.642
12.50 23.53 20.74 13.88 19.13 17.96 2.09 ns 0.738
3.60 4.43 5.29 4.33 4.97 4.21 0.32 ns 0.759
14.31 8.70 11.78 13.33 15.25 13.77 1.51 ns 0.642
ns, not significant; SEM, standard error of means.
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The EEP dietary supplement decreased the duodenal sucrase activity at 7 day of age, which may have caused the decreased weight gain in the animals at this age. Several authors have shown that an EEP has an antihyperglycaemic effect in rats (Skopec et al., 2010). The propolis acts as an a-glycosidase inhibitor in the small intestine, inhibiting carbohydrate digestion into absorbable monosaccharides and thus decreasing or delaying their absorption (Matsui et al., 2004). These authors further suggest that caffeoylquinic acid compounds may be responsible for this effect. Moreover, the large amounts of phenolic compounds present in propolis affect glucose metabolism by inhibiting intestinal absorption and stimulating insulin secretion, among other mechanisms (Hanhineva et al., 2010). In fact, some authors have shown that flavonoids decrease glucose uptake in enterocytes by decreasing SGLT1 and GLUT2 transport activity (Johnston et al., 2002; Kwon et al., 2007). Although the EEP dietary supplement altered the intestinal disaccharidase activities, the supplement had no observable effect on the pancreatic enzyme activities at the EEP levels tested in the present study. There are no studies in the literature reporting the effects of polyphenols on the digestive enzyme activity of broilers. Several articles describe for mammals an 399
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Table 7 Carcass yield (%) of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP)
EEP levels
Carcass†
Breast
Thigh and leg
Wings
Abdominal Fat
Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
71.36 72.77 71.55 72.53 72.63 72.67 0.23 ns 0.326
36.12 37.08 36.18 37.29 37.70 36.75 0.18 ns 0.071
31.04 30.60 30.72 30.12 29.84 30.22 0.15 ns 0.230
11.15 11.03 10.87 10.98 10.97 11.17 0.07 ns 0.802
2.40 1.75* 2.52 2.01* 2.34 2.16 0.06 ns 0.001
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Carcass without neck, giblets and abdominal fat and expressed on a relative basis to the full-fed live weight carcass yield of broilers.
inhibitory effect of polyphenols on pancreatic enzyme activity. Hanhineva et al. (2010) described several studies that show inhibition by polyphenols of aamylase in vitro. Similar inhibition is described for lipase activity, and this inhibition may mediate favourReferences Acßikg€ oz, Z.; Y€ ucel, B.; Altan, O., 2005: The effects of propolis supplementation on broiler performance and feed digestibility. Archiv F€ ur Gefl€ ugelkunde 69, 117–122. Babi nska, I.; Kleczek, K.; Makowski, W.; Szarek, J., 2013: Effect of feed supplementation with propolis on liver and kidney morphology in broiler chickens. Pakistan Veterinary Journal 33, 1–4. Bankova, V. S.; Castro, S. L.; Marcucci, M. C., 2000: Propolis: recent advances in chemistry and plant origin. Apidologie 31, 3–15. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Caraway, W. T., 1959: A stable starch substrate for the determination of amylase in serum and other body fluids. American Journal of Clinical Pathology 32, 97– 99. C ucßl€ u, B. K., ß etin, E.; Silici, S.; C ßetin, N.; G€ 2010: Effects of diets containing different concentrations of propolis on hematological and immunological variables in laying hens. Poultry Science 89, 1703– 1708.
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able effects on lipid metabolism and control of weight gain, with the polyphenol anthocyanin considered the most potent inhibitor (You et al., 2011). As for literature data concerning the proteolytic enzymes trypsin and chymotrypsin, only trypsin appears to be inhibited by dietary polyphenols (Goncßalves et al., 2007). According to the results of the present study, adding 1000–5000 ppm of the EEP to the pre-starter broiler diet compromised performance during this stage, which was most likely due to the altered sucrase activity. The EEP promoted improved intestinal morphometry and sucrase enzyme activity at 21 days of age, with subsequent recovery of the performance parameters and carcass yield at 42 days of age; thus, the supplementation of the broiler diet with the EEP from 3000 ppm is indicated after the animals are 7 days old. Acknowledgements This work was supported by grants from National Counsel of Technological and Scientific Development (CNPq) and Financing Agency for Studies and Projects (FINEP), Brazil.
Dahlqvist, A., 1964: Method for assay of intestinal disaccharidases. Analytical Biochemistry 7, 18–25. Denli, M.; Cankaya, S.; Silici, S.; Okan, F.; Uluocak, A. N., 2005: Effect of dietary addition of Turkish propolis on the growth performance, carcass characteristics and serum variables of quail (Coturnix coturnix japonica). AsianAustralasian Journal of Animal Sciences 18, 848–854. Erlanger, B. F.; Edel, F.; Cooper, A. G., 1966: The action of chymotrypsin on two new chromogenic substrates. Archives of Biochemistry Biophysics 115, 206–210. Freitas, J. A.; Vanat, N.; Pinheiro, J. W.; Balarin, M. R.; Sforcin, J. M.; Venancio, E. J., 2011: The effects of propolis on antibody production by laying hens. Poultry Science 90, 1227–1233. Fu, S.; Yang, M.; Wen, H.; Chern, J., 2005: Analysis of flavonoids in propolis by capillary electrophoresis. Journal of Food and Drug Analysis 13, 43–50. Galal, A.; Abd El-Motaal, A. M.; Ahmed, A. M. H.; Zaki, T. G., 2008: Productive performance and immune response of laying hens as affected by dietary propolis supplementation. International Journal of Poultry Science 7, 272–278.
Goncßalves, R.; Soares, S.; Mateus, N.; de Freitas, V., 2007: Inhibition of trypsin by condensed tannins and wine. Journal of Agricultural and Food Chemistry 55, 7596–7601. Guo, F. C.; Williams, B. A.; Kwakkel, R. P.; Li, H. S.; Li, X. P.; Luo, J. Y.; Li, W. K.; Verstegen, M. W., 2004: Effects of mushroom and herb polysaccharides, as alternatives for an antibiotic, on the cecal microbial ecosystem in broiler chicken. Poultry Science 83, 175–182. Hanhineva, K.; T€ orr€ onen, R.; BondiaPons, I.; Pekkinen, J.; Kolehmainen, M.; Mykk€ anen, H.; Poutanen, K., 2010: Impact of dietary polyphenols on carbohydrate metabolism. International Journal of Molecular Sciences 11, 1365–1402. Jamroz, D.; Werteleki, T.; Houszka, M.; Kamel, C., 2006: Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90, 255–268. Jin, L. Z.; Ho, Y. W.; Abdullah, N.; Jalaludin, S., 1998: Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets
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containing Lactobacillus cultures. Poultry Science 77, 1259–1265. Johnston, K. L.; Clifford, M. N.; Morgan, L. M., 2002: Possible role for apple juice phenolic compounds in the acute modification of glucose tolerance and gastrointestinal hormone secretion in humans. Journal of the Science of Food and Agriculture 82, 1800–1805. Kac aniov a, M.; Rovn a, K.; Arp asov a, H.; Cubo n, J.; Hleba, L.; Pochop, J.; Kunov a, S.; Hascık, P., 2012: In vitro and in vivo antimicrobial activity of propolis on the microbiota from gastrointestinal tract of chicken. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances & Environmental Engineering 47, 1665–1671. Kakade, M. L.; Rackis, J. J.; McGhee, J. E.; Puski, G., 1974: Determination of trypsin inhibitor activity of soy products. A collaborative analysis of an improved procedure. Cereal Chemistry 51, 376–382. Kuppusamy, U. R.; Das, N. P., 1992: Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes. Biochemical Pharmacology 44, 1307–1315. Kwon, O.; Eck, P.; Chen, S.; Corpe, C. P.; Lee, J. H.; Kruhlak, M.; Levine, M., 2007: Inhibition of the intestinal glucose transporter GLUT2 by flavonoids. FASEB Journal 21, 366–377. Lofty, M., 2006: Biological activity of bee propolis in health and disease. Asian Pacific Journal of Cancer Prevention 7, 22– 31. Luna, L. G., 1968: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edn. McGraw-Hill Book, New York, NY. Marcucci, M. C., 1995: Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26, 83–99. Matsui, T.; Ebuchi, S.; Fujise, T.; Abesundara, K. J.; Doi, S.; Yamada, H.; Matsumoto, K., 2004: Strong antihyperglycemic effects of water-soluble fraction of Brazilian propolis and its bioactive constituent, 3,4,5-tri-O-caffeoylquinic acid. Biological and Pharmaceutical Bulletin 27, 1797–1803. Miles, R. D.; Butcher, G. D.; Henry, P. R.; Littell, R. C., 2006: Effect of antibiotic
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growth promoters on broiler performance, intestinal growth parameters, and quantitative morphology. Poultry Science 85, 476–485. Oliveira, M. C.; Rodrigues, E. A.; Marques, R. H.; Gravena, R. A.; Guandolini, G. C.; Moraes, V. M. B., 2008: Performance and morphology of intestinal mucosa of broilers fed mannan-oligosaccharides and enzymes. Arquivo Brasileiro de Medicina Veterin aria e Zootecnia 60, 442–448. Pierpoint, W. S., 2004: The extraction of enzymes from plant tissues rich in phenolic compounds. Methods in Molecular Biology 244, 65–74. Romier, B.; Schneider, Y. J.; Larondelle, Y.; During, A., 2009: Dietary polyphenols can modulate the intestinal inflammatory response. Nutrition Reviews 67, 363–378. Rostagno, H. S.; Albino, L. F. T.; Donzele, J. L.; Gomes, P. C.; Oliveira, R. F.; Lopes, D. C.; Ferreira, A. S.; Barreto, S. L. T., 2005: Tabelas brasileiras para aves e suınos: composicß~ao de alimentos e exig^encias nutricionais, 2nd edn. Universidade Federal de Vicßosa, Vicßosa, Minas Gerais. Saeg, 1997: Statistical Analysis System and Genetics – SAEG. Stat. version 8.0. Universidade Federal de Vicßosa, Vicßosa, Minas Gerais. Sayrafi, R.; Shahrooz, R.; Soltanalinejad, F.; Rahimi, S., 2011: Histomorphometrical study of the prebiotic effects on intestine morphology and growth performance of broiler chickens. Veterinary Research Forum 2, 45–51. Seven, I.; Aksu, T.; Tatli Seven, P., 2010: The effects of propolis on biochemical parameters and activity of antioxidant enzymes in broilers exposed to leadinduced oxidative stress. Asian-Australasian Journal of Animal Science 23, 1482– 1489. Shalmany, S. K.; Shivazad, M., 2006: The effect of diet propolis supplementation on Ross broiler chicks performance. International Journal of Poultry Science 5, 84–88. Silva, M. A.; Pessotti, B. M. S.; Zanini, S. F.; Colnago, G. L.; Rodrigues, M. R. A.; Nunes, L. C.; Zanini, M. S.; Martins, I. V. F., 2009: Intestinal mucosa structure of broiler chickens infected experimen-
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tally with Eimeria tenella and treated with essential oil of oregano. Ci^encia Rural 39, 1471–1477. Singleton, V. L.; Rossi, J. A. Jr, 1965: Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture 16, 144–158. Skopec, M. M.; Green, A. K.; Karasov, W. H., 2010: Flavonoids have differential effects on glucose absorption in rats (Rattus norvegicus) and American robins (Turdis migratorius). Journal of Chemical Ecology 36, 236–243. Smith, M. O., 1993: Parts yield of broilers reared under cycling high temperatures. Poultry Science 72, 1146–1150. Tatli Seven, P., 2008: The effects of dietary Turkish propolis and vitamin C on performance, digestibility, egg production and egg quality in laying hens under different environmental temperatures. Asian-Australasian Journal of Animal Sciences 21, 1164–1170. Tatli Seven, P.; Seven, I.; Yilmza, M.; € G., 2008: The effects of TurkSimsek, U. ish propolis on growth and carcass characteristics in broilers under heat stress. Animal Feed Science and Technology 146, 137–148. Tatli Seven, P.; Yilmaz, S.; Seven, I.; Cerci, I. H.; Azman, M. A.; Yilmaz, M., 2009: Effects of propolis on selected blood indicators and antioxidant enzyme activities in broilers under heat stress. Acta Veterinary Brno 78, 75–83. Tekeli, A.; Kutlu, H. R.; C ß elik, L., 2011: Effects of Z. officinale and propolis extracts on the performance, carcass and some blood parameters of broiler chicks. Current Research in Poultry Science 1, 12–23. You, Q.; Chen, F.; Wang, X.; Luo, P. G.; Jiang, Y., 2011: Inhibitory effects of muscadine anthocyanins on a-glucosidase and pancreatic lipase activities. Journal of Agricultural and Food Chemistry 59, 9506–9511. Ziaran, H. R.; Rahmani, H. R.; Pourreza, J., 2005: Effect of dietary oil extract of propolis on immune response and broiler performance. Pakistan Journal of Biological Sciences 8, 1485–1490.
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ORIGINAL ARTICLE
Effect of dietary supplementation with an ethanolic extract of propolis on broiler intestinal morphology and digestive enzyme activity C. Eyng, A. E. Murakami, C. R. A. Duarte and T. C. Santos Departamento de Zootecnia, Universidade Estadual de Maring a, Maringa, Brazil
Summary The present study aimed to evaluate the effect of different levels of an ethanolic extract of propolis (EEP) on broiler performance, carcass characteristics, weight of gastrointestinal organs, intestinal morphometry and digestive enzyme activity. 1020 male broiler chicks were assigned in a completely randomised experimental design to six treatments (EEP supplement levels of 0, 1000, 2000, 3000, 4000 and 5000 ppm) and five replications, and 34 birds per experimental unit. The experimental diets were administered from 1 to 21 days of age, and the birds were subsequently provided a ration based on corn and soybean meal. EEP supplementation from 1 to 7 days negatively affected (p < 0.05) the weight gain and feed intake. The proventriculus weight at 7 days exhibited a quadratic response (p < 0.05), which predicted a lower weight at a dose of 2865 ppm of the EEP. For the duodenum at 21 days of age, the response pattern (p < 0.05) predicted that birds that were fed 2943 and 3047 ppm of the EEP would exhibit an improved crypt depth and villus-to-crypt ratio respectively. The villus height, crypt depth and villus-to-crypt ratio in the jejunum and the ileum were not affected (p > 0.05). With increased EEP doses, the duodenal sucrase activity linearly decreased at 7 days of age and linearly increased in the jejunum at 21 days of age (p < 0.05), while pancreatic enzyme activity was unaffected (p > 0.05). Although the carcass and cut yields did not improve, the percentage of abdominal fat decreased (p < 0.05). The supplementation of the broiler pre-starter diet with 1000–5000 ppm of the EEP impaired performance at this stage, most likely due to the decreased sucrase activity. However, the EEP supplementation from 3000 ppm improved intestinal morphophysiology at 21 days of age and did not affect the performance or carcass yield at 42 days of age. Keywords disaccharidases, growth promoter, natural additive, pancreatic enzymes ^ncias Agrarias, Universidade Estadual de Maringa, Av. Correspondence Profa. Dra. A. E. Murakami, Departamento de Zootecnia, Centro de Cie Colombo, 5790, Bloco J45, Maring a, PR 87020-900, Brazil. Tel: +55 44 3011 8942; Fax: +55 44 3011 8977; E-mail: [email protected] Received: 16 October 2012; accepted: 7 July 2013
Introduction For decades, the poultry production chain has benefited from the use of antibiotics at subtherapeutic doses for improving performance indices. However, possible microbial resistance and the requirements of certain consumer markets for healthy products without health risks have led to the search for alternative additives, which consist mainly of natural products with important therapeutic functions that can replace growth-promoting antibiotics. Propolis is a resinous and balsamic substance that is produced by bees when they combine plant exudates, wax and pollen with their salivary secretions, which has been described because of antibacterial, antioxidant, antiviral, anti-inflammatory, antifungal and
immunostimulatory properties (Marcucci, 1995), among others. These properties are attributed to more than 300 compounds, including phenolic acids, flavonoids, esters, aromatic aldehydes, fatty acids, amino acids, vitamins and minerals (Bankova et al., 2000; Lofty, 2006). Among them, the flavonoids are the most studied as potent antioxidants, although the flavonoids also interfere with many physiological processes, such as carbohydrate metabolism, showing an antihyperglycaemic property in rats (Matsui et al., 2004). Due to the presence of these important compounds and their effects on animal physiology, the use of propolis in broiler feed, whether in the form of crude propolis or an ethanolic extract of propolis (EEP), promotes improved gut health by controlling the growth
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of pathogenic micro-organisms in the gastrointestinal tract, which consequently improves digestive and absorptive functions. Additional advantages derive from the use of propolis in poultry diets, such as the absence in carcasses of toxic residues that might compromise human health (Denli et al., 2005) and improvements in the immune system (Freitas et al., 2011) and in the performance of laying hens and broilers (Shalmany and Shivazad, 2006; Tatli Seven et al., 2008; C ß etin et al., 2010; Tekeli et al., 2011). In broiler chickens, Tatli Seven et al. (2008) showed that ethanolic extract of propolis supplementation is more effective than vitamin C on performance and carcass qualities under heat stress that can be attributed to beneficial effects of propolis on reduction in the adverse effects of oxidative stress (Tatli Seven et al., 2009; Seven et al., 2010). Moreover, broiler chickens fed with 1000 ppm of propolis presented a similar performance compared with chickens fed antibiotic growth promoter (Tekeli et al., 2011). Shalmany and Shivazad (2006) have shown better performance and low mortality rate in birds fed with propolis. Although many authors showed beneficial effects of propolis on animal performance and antioxidant and immune status, there are scarce studies that correlate these effects with changes in intestinal morphophysiology. Some evidences show that propolis has protective effect on liver morphology of broiler chickens (Babi nska et al., 2013) and it can modulate the intestinal microbiota (Kac aniov a et al., 2012). Thus, the present study evaluated the effect of different ethanolic extract of propolis levels in the feed of broilers on their performance, carcass characteristics, organ weights, intestinal morphometry and digestive enzyme activity.
extract of propolis (0, 1000, 2000, 3000, 4000 and 5000 ppm, each fed to 34 birds per replicate). The broilers received the experimental diets until 21 days of age followed by a conventional diet until 42 days of age. The experimental diets (Table 1) were based on corn and soybean meal and were formulated using the feed chemical composition values and the nutritional requirements for the average performance of male broilers according to Rostagno et al. (2005). The ethanolic extract of propolis was prepared using equal amounts of the propolis and the alcohol. The Table 1 Ingredients and nutrient composition of the experimental diets
Ingredient (%) Corn Soybean meal 45% Soybean oil Limestone Dicalcium phosphate NaCl Inert* DL- Met 98% L- Lys HCl, 78.5% L- Thr 98% Supplement minerals and vitamins† Calculated composition CP (%) ME (kcal/kg) Calcium (%) Available phosphorus (%) Digestible Met + Cys (%) Digestible Lys (%) Digestible Thr (%) Digestible Trp (%)
1–7 days
8–21 days
22–42 days
55.75 37.04 2.20 0.92 1.94 0.40 0.50 0.36 0.35 0.15 0.40
58.63 34.39 2.51 0.88 1.80 0.40 0.50 0.24 0.19 0.05 0.40
64.45 28.68 3.15 0.82 1.58 0.40 0.23 0.23 0.06 0.40
22.04 2.950 0.939 0.470 0.944 1.330 0.865 0.213
20.79 3.000 0.884 0.442 0.814 1.146 0.745 0.183
18.72 3.125 0.793 0.395 0.752 1.045 0.679 0.177
The protocol for this experiment was approved by the Ethics Committee on Animal Use in Experimentation (CEAE/UEM) of the Universidade Estadual de Maringa (UEM, Maring a, Paran a, Brazil), and birds were cared according to the Ethical Principles for Animal Experimentation established by the Brazilian Society of Laboratory Animal Science (SBCAL) and National Council for the Control of Animal Experimentation (CONCEA). In all, 1020 1-day-old male Cobb-Vantressâ chicks from 40-week-old breeders were distributed in a completely randomised experimental design among five replicates of six treatments, which consisted of six levels of supplementation of the diet with an ethanolic
*The ethanolic extract of propolis was added in replacement for inert (kaolin). †(1–21 days) Vitamin mixture (content per kg of diet): vitamin A (retinyl acetate), 2917 IU; vitamin D3 (cholecalciferol), 583 IU; vitamin E (DL-a-tocopheryl acetate), 8750 IU; vitamin K3 (menadione dimethylpyrimidinol), 433 IU; vitamin B1 (thiamine mononitrate), 408 mg; vitamin B12 (cyanocobalamin), 4166 lg; niacin (niacinamide), 8983 mg; calcium pantothenate, 3167 mg; folic acid, 200 mg; D-biotin, 25 mg. Mineral mix (content per kg of diet): Fe (iron sulphate monohydrate), 12.6 g; Cu (copper sulphate pentahydrate), 3072 mg; I (calcium iodate), 248 mg; Zn (zinc oxide), 12.6 g; Mn (manganous oxide), 60 mg; Se (sodium selenite), 61 mg; Co (cobalt sulphate), 50 mg. (22–42 days) Vitamin mixture (content per kg of diet): vitamin A (retinyl acetate), 2250 IU; vitamin D3 (cholecalciferol), 450 IU; vitamin E (DL-a-tocopheryl acetate), 7000 IU; vitamin K3 (menadione dimethylpyrimidinol), 418 IU; vitamin B1 (thiamine mononitrate), 300 mg; vitamin B12 (cyanocobalamin), 2300 lg; niacin (niacinamide), 7000 mg; calcium pantothenate, 2500 mg; folic acid, 140 mg; D-biotin, 14 mg. Mineral mix (content per kg of diet): Fe (iron sulphate monohydrate), 12.5 g; Cu (copper sulphate pentahydrate), 3000 mg; I (calcium iodate), 250 mg; Zn (zinc oxide), 12.5 g; Mn (manganous oxide), 15 mg; Se (sodium selenite), 75 mg; Co (cobalt sulphate), 50 mg.
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Materials and methods
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composition of the extract was determined according to Singleton and Rossi (1965) and Pierpoint (2004) for the total polyphenols and using the aluminium chloride colorimetric methods for the total flavonoid content. The broilers and the feed were weighed at 1, 7, 21 and 42 days of age to evaluate performance, which was measured as the feed intake, weight gain and feed conversion rate. Every bird that was sampled or died was weighed, and the pen feed intake at that time was recorded; these data were used to correct the feed conversion rate. At 7 and 21 days of age, 5 birds per treatment were sacrificed to collect the gastrointestinal organs (proventriculus, gizzard, small and large intestines, pancreas and liver). The organs were cleaned with physiological saline solution, dried with filter paper and weighed. The weight of each organ was expressed relative to the total body weight (g/100 g BW). The length of the intestine was also measured (cm). Fragments of approximately 5 cm were obtained from each of the following segments of the small intestine: the duodenum (from the pylorus to the distal portion of the duodenal loop), the jejunum (from the distal portion of the duodenal loop to Meckel’s diverticulum) and the ileum (the anterior portion of the ileocecal junction) to evaluate the intestinal morphology. The fragments from each segment were then placed on polystyrene sheets, opened longitudinally, washed in saline solution, fixed in 10% formaldehyde solution, dehydrated and embedded in paraffin. Thin sections from each segment were cut at a thickness of 5 lm and stained with haematoxylin and eosin according to Luna (1968). The measurements of villus height and crypt depth were taken using a light microscope and a system that analyses computerised images (Motic Image Plus 2.0; Motic China Group, Hong Kong, China). The height of 30 villi and the depth of 30 crypts were measured from each segment and replicate. The mean was obtained for each EEP treatment and intestinal segment from these values. Portions of each segment of the small intestine were freed of residual food, frozen in liquid nitrogen and stored in a freezer at 80 °C until they were assayed to determine the activity of intestinal disaccharidases by the Dahlqvist (1964). Each segment was opened longitudinally, and the mucosa was scraped off with a glass microscope coverslip. The collected mucosa was homogenised after the addition of 4 parts of ice-cold deionised water. Maltase and sucrase activities were assayed by incubating aliquots of the homogenates with the appropriate substrate in malate buffer at pH 6.4. Released glucose was determined by the glucose
The concentration of the total polyphenols and flavonoids in the EEP was 357.71 mg/l and 112.72 mg/l respectively. At 7 days of age, the weight gain and feed intake deteriorated linearly with increasing EEP levels (Table 2, p < 0.05), and the feed intake was lower in the animals fed 5000 ppm of the EEP than it was in the control animals (Dunnett’s test, p < 0.05). However, the feed conversion at this age did not differ among the experimental groups (p > 0.05). At 21 and 42 days of age, the performance parameters were similar among the experimental groups (Table 2, p > 0.05).
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oxidase method (Laborlab, Guarulhos, Brazil). The enzyme activity is expressed as units per gram of protein, which was determined by the method of Bradford (1976). The activity of the pancreatic enzymes was determined after the whole pancreas was homogenised (1:20 wt/vol) in 50 mM Tris–HCl buffer (pH 8) containing 50 mM CaCl2. The trypsin activity was determined according to methodology proposed by Kakade et al. (1974). A similar method was used for the determination of chymotrypsin (Erlanger et al., 1966). Amylase activity was determined by the iodometric method modified by Caraway (1959) (Gold Analisa, Belo Horizonte, Minas Gerais, Brazil). Lipase activity was obtained using the BALB-DNTP method (Gold Analisa). At the end of the experimental period, the carcass yield was determined in 2 birds at random per experimental unit (12 birds per treatment). The birds were identified, starved for 6 h and killed by electric stunning and bleeding. The carcass yield was calculated as the ratio of the hot eviscerated carcass to the BW before euthanasia. The prime cut yield (the whole breast, thigh and legs with their skin and bones) was calculated in relation to the weight of the eviscerated carcass. Abdominal fat associated with the cloaca, the bursa of Fabricius, the gizzard, the proventriculus and the adjacent abdominal muscles was removed as described by Smith (1993). The data were examined in relation to the EEP levels using analysis of variance followed by Dunnett’s test and regression analysis by polynomial decomposition of the degrees of freedom. SAEG (System for Statistical and Genetic Analysis, Universidade Federal de Vicßosa, 1997) software was utilised for the analyses, and a probability (p) < 0.05 was considered significant. Results
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Table 2 The performance of broiler chickens fed with diets containing different levels of ethanolic extract of propolis EEP levels 1–7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 1–21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 1–42 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Weight gain (g)
Feed intake (g)
F:G
134.28 136.07 130.08 132.54 126.70 126.07 1.37 L† (p = 0.04) 0.195
164.35 170.16 160.47 158.29 161.19 153.85* 1.40 L‡ (p = 0.002) 0.010
1.224 1.254 1.235 1.196 1.274 1.220 0.01 ns 0.167
792.87 826.30 822.13 841.27 809.55 789.36 7.24 ns 0.286
1183.73 1231.45 1234.16 1242.14 1221.08 1162.02 10.17 ns 0.163
1.496 1.492 1.502 1.476 1.509 1.472 0.01 ns 0.868
2596.80 2701.43 2651.15 2678.31 2639.62 2591.42 22.00 ns 0.689
4617.63 4782.09 4664.83 4801.16 4647.34 4696.99 31.84 ns 0.471
1.780 1.770 1.760 1.795 1.761 1.814 0.01 ns 0.425
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Y = 137.310 0.00233937x (R2 = 0.79). ‡Y = 170.366 0.00319139x (R2 = 0.71).
The weight of the proventriculus varied quadratically with the concentration of the EEP supplement at 7 days, and the quadratic equation predicted a lower weight for animals fed at an EEP level of 2865 ppm (Table 3, p < 0.05). The weights of the other organs evaluated did not vary with the EEP level at either 7 or 21 days of age (p > 0.05). At 7 days of age, the intestinal morphology did not differ among the experimental groups (p > 0.05). For the duodenum at 21 days, the crypt depth and the ratio of the villus height to crypt depth varied quadratically with the level of the EEP supplementation; the quadratic equation predicted a lower crypt depth in broilers fed diets supplemented with 2943 ppm of the EEP and a higher villus-to-crypt ratio when diets were supplemented with 3047 ppm of the EEP (Table 4, p < 0.05). The treatments had no effect (p > 0.05) on 396
the crypt depth or the villus height-to-crypt ratio in the jejunum or the ileum or on the villus height in any of the intestinal segments. At 7 days of age, the sucrase activity in the duodenum decreased linearly with increasing EEP supplement levels (Table 5, p < 0.05) and displayed lower values in the group fed 3000 ppm of the supplement than in the control group (Dunnett’s test, p < 0.05). Although not statistically significant in the regression tests (p > 0.05), the sucrase activity in the jejunum was higher in the animals fed 5000 ppm of the EEP supplement than it was in the control animals (Dunnett’s test, p < 0.05). At 21 days of age, the sucrase activity in the jejunum increased linearly with increasing EEP levels (p < 0.05) and exhibited lower values in the group fed 1000 ppm than in the control group (Dunnett’s test, p < 0.05). The maltase activity in the jejunum was higher in the animals fed 4000 ppm of the EEP than in the control animals (Table 5, Dunnett’s test, p < 0.05). The EEP treatments did not affect (p > 0.05) the sucrase activity in the ileum at 7 or 21 days of age or in the duodenum at 21 days of age nor the maltase activity in any of the intestinal segments at 7 or 21 day of age. The activities of the lipase, amylase, trypsin and chymotrypsin did not vary in the pancreas at 7 or 21 days of age with the level of the EEP supplement in the diet (Table 6, p > 0.05). The carcass and cut yields were similar among the EEP supplement levels (Table 7, p > 0.05). The abdominal fat percentage was lower in the animals fed 1000 and 3000 ppm of the EEP than in the control animals (Dunnett’s test, p < 0.05). Discussion The polyphenol and flavonoid levels can present differences between samples according to variations in the extraction process, such as the area of the contact surface relative to weight of the raw propolis, the type of solvent used for extraction and the duration of the extraction process, all of which may influence the final concentrations of these therapeutic substances. Moreover, it is very difficult to compare the results showed by another authors due the differences in the propolis composition, which depends on geographical origin and seasonal effect (Bankova et al., 2000). Among the polyphenols present in the EEP, the flavonoid group has important therapeutic properties, such as antimicrobial, antioxidant and anti-inflammatory qualities. The high concentration of these compounds in propolis may be responsible for its Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Table 3 Relative weight (g/100 g live weight) of proventriculus, gizzard, small (SI) and large (LI) intestines, liver and pancreas and length of small intestine (cm) of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels 7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Proventriculus
Gizzard
SI
LI
Liver
Pancreas
SI Length
1.009 1.076 0.973 0.976 1.007 1.102 0.01 Q* (p = 0.003) 0.060
4.689 4.361 4.538 4.264 4.694 4.855 0.08 ns 0.325
9.625 9.817 10.180 9.759 10.189 10.171 0.11 ns 0.534
1.637 1.715 1.609 1.517 1.744 1.578 0.05 ns 0.991
3.481 3.755 3.381 3.534 3.419 3.648 0.06 ns 0.890
0.534 0.538 0.498 0.506 0.525 0.481 0.01 ns 0.581
97.35 98.12 98.85 98.25 97.67 98.00 0.61 ns 0.657
0.596 0.611 0.647 0.614 0.569 0.585 0.01 ns 0.418
2.460 2.487 2.293 2.424 2.413 2.553 0.04 ns 0.662
7.444 7.276 7.445 7.069 6.900 7.508 0.13 ns 0.727
1.041 1.172 1.114 1.256 1.372 1.338 0.04 ns 0.242
2.543 2.658 2.753 2.649 2.610 2.839 0.04 ns 0.301
0.352 0.412 0.353 0.399 0.379 0.351 0.01 ns 0.090
156.30 158.80 168.20 157.60 161.70 156.00 1.89 ns 0.442
ns, not significant; SEM, standard error of means. *Y = 1.21395 0.000172607x + 0.00000003012x2 (R2 = 0.98); maximum point: 2865 ppm.
therapeutic activity (Fu et al., 2005). Because these substances can interfere with adhesion by pathogenic bacteria (Jamroz et al., 2006) and modulate intestinal microbiota, mainly qualitatively, by favouring the growth of beneficial bacteria, such as lactobacilli and bifidobacteria (Guo et al., 2004), supplementing the diet with these compounds would influence many important physiological processes, such as nutrient uptake and immunity (Romier et al., 2009; Hanhineva et al., 2010). Moreover, the flavonoids have an aglycone hydroxyl grouping positioned similarly to that of oestrogen, predicting their possible growth hormone activity (Ziaran et al., 2005). Thus, the use of propolis in animal feed could promote better nutrient use by animals by improving their intestinal health. In fact, Kac aniov a et al. (2012) showed the microbiota modulation by ethanolic extract of propolis in broiler chickens, with a reduction in pathogenic bacteria. The EEP supplements impaired performance in 1to 7-day-old animals by reducing their weight gain and feed intake as the EEP levels increased, although propolis contains substances, such as resins, waxes and honey, that are considered palatable (Shalmany and Shivazad, 2006; Tatli Seven, 2008). According to Acßikg€ oz et al. (2005), adding a high level of propolis (4000 ppm) during early broiler development causes decreased protein digestion and poor growth.
However, according to the performance and carcass yield results reported here, the EEP dietary supplements had no negative effects at 21 days of age or at the end of the rearing period, and thus, the carcass and cut yields were not impaired at 42 days of age. This result indicates that the EEP supplementation was only detrimental within the first week of life at the levels studied because the animal performance parameters recovered at 21 and 42 days of age. Moreover, the EEP supplementation caused an abdominal fat reduction, which was attributable to the lipolytic action of the propolis. Regarding this suggested mechanism, compounds in the propolis are capable of inhibiting phosphodiesterase, which is responsible for degrading cyclic AMP, and increasing intracellular phosphodiesterase levels, which consequently increases lipolysis (Kuppusamy and Das, 1992; Jin et al., 1998). The effects of EEP dietary supplements on birds are still unclear, given that some studies detected no effects on animal performance (Acßikg€ oz et al., 2005; Ziaran et al., 2005), whereas others have demonstrated positive effects (Denli et al., 2005; Shalmany and Shivazad, 2006; Galal et al., 2008; Tatli Seven et al., 2008; Tekeli et al., 2011). Tatli Seven et al. (2008) showed that supplementing the diet with 500– 3000 ppm of propolis extract improved broiler performance under heat stress, with the mean weights of
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Table 4 Villus height (lm), crypt depth (lm) and villus/crypt ratio of segments of the small intestine of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels Control 7 days Villus height Duodenum 1033.99 Jejunum 448.15 Ileum 366.76 Crypt depth Duodenum 166.43 Jejunum 126.57 Ileum 95.24 Villus/crypt depth Duodenum 6.44 Jejunum 3.54 Ileum 3.91 21 days Villus height Duodenum 1652.58 Jejunum 880.02 Ileum 486.72 Crypt depth Duodenum 193.40 Jejunum 138.17 Ileum 106.80 Villus/crypt depth Duodenum 8.80 Jejunum 6.42 Ileum 4.57
1000 ppm
2000 pm
3000 pm
4000 ppm
5000 ppm
SEM
Reg
p-values
981.83 468.07 330.53
1000.47 452.41 369.67
929.89 465.19 354.53
904.11 477.77 363.40
977.15 479.63 351.06
20.20 13.05 8.18
ns ns ns
0.501 0.982 0.787
178.66 130.71 85.19
182.91 129.82 91.19
191.39 131.69 101.12
204.66 142.93 97.88
194.07 118.90 88.52
4.98 2.87 2.86
ns ns ns
0.303 0.275 0.633
5.69 3.60 3.89
5.54 3.50 4.07
4.85 3.55 3.56
4.50 3.35 3.76
5.06 4.02 4.15
0.22 0.09 0.11
ns ns ns
0.133 0.449 0.733
1510.36 885.44 490.30
1581.30 756.38 549.24
1610.86 688.36 480.33
1533.33 823.57 505.71
1598.09 750.15 535.85
28.92 27.59 10.85
ns ns ns
0.765 0.222 0.353
212.22 141.91 107.73
180.72 121.22 112.71
192.07 123.15 103.82
187.34 130.26 104.60
213.83 130.33 118.05
4.18 2.77 1.96
Q*(p = 0.003) ns ns
0.119 0.208 0.273
7.15 6.30 4.60
8.77 6.19 4.89
8.45 5.57 4.66
8.24 6.40 4.87
7.52 5.89 4.61
0.24 0.21 0.12
Q† (p = 0.02) ns ns
0.264 0.853 0.956
ns, not significant; SEM, standard error of means. *Y = 244.232 0.0412346x + 0.00000700631x2 (R2 = 0.81); minimum point: 2943 ppm. †Y = 5.676 + 0.00194855x – 0.000000319772x2 (R2 = 0.84); maximum point: 3047 ppm.
the supplemented, stressed animals similar to those of animals reared under thermoneutral conditions. These inconsistencies are most likely caused by the difficulty of standardising the EEP treatments due to considerable variability in the composition of propolis from various sources and differences in propolis extraction methods. Furthermore, there is wide variation in the EEP levels used in the different studies. The impaired performance at 7 days of age cannot be explained by effects of the EEP supplements on the relative size of the gastrointestinal organs because there were no differences related to the digestive or absorptive capacities of these organs. Only the weight of the proventriculus was influenced by the supplements, and it was influenced in a quadratic manner; this influence may have been related to the reduced feed intake that was observed simultaneously. Although the EEP supplements did not affect the relative weight of the small intestine, this organ underwent important morphophysiological alterations. At 21 days of age, the duodenal crypt depth
varied quadratically with the EEP supplementation level; the shallowest crypt depth was predicted to occur at a level of 2943 ppm, and there was a consequent effect on the villus-to-crypt ratio, which was predicted to increase at the 3047 ppm level of supplementation. A shallower crypt depth is correlated with a reduced need for cell renewal (Oliveira et al., 2008), reducing energy and protein expenditures for this process and, thus, enabling growth of other tissues (Miles et al., 2006). In addition, higher villus-to-crypt ratios suggest higher digestive and absorptive capacities (Silva et al., 2009). Thus, the increased ratios obtained here can be attributed to the beneficial effect of the propolis compounds in controlling proliferation of pathogenic bacteria and avoiding possible damage to the intestinal mucosa, which could result in reduced dimensions of the villi (Sayrafi et al., 2011). These beneficial changes in the intestinal morphometry occurred when the sucrase activity increased in the jejunum. Thus, the recovery of the performance indices at 21 day of age could be attributed to these changes.
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Table 5 Maltase and sucrase activities (U/mg of protein) of segments of the small intestine of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP) EEP levels
7 days Sucrase activity Duodenum Jejunum Ileum Maltase activity Duodenum Jejunum Ileum 21 days Sucrase activity Duodenum Jejunum Ileum Maltase activity Duodenum Jejunum Ileum
Control
1000 ppm
2000 pm
3000 pm
12.03 6.40 7.43
12.70 9.10 5.82
13.41 11.26 8.48
14.07* 6.75 4.75
27.08 29.39 25.47
26.89 30.40 28.00
24.81 34.75 28.19
4.22 8.91 6.93 22.58 27.00 24.48
4.53 5.61* 7.20 23.71 24.44 25.40
4000 ppm
5000 ppm
SEM
Reg
p-values
9.58 8.88 7.12
11.17 12.79* 8.94
0.40 0.47 0.76
L† (p = 0.035) ns ns
0.007 0.028 0.647
21.02 20.94 25.25
27.44 34.02 25.18
25.95 33.57 30.50
0.87 1.82 1.71
7.10 10.36 9.02
9.81 7.66 7.05
6.25 9.43 8.63
7.13 10.36 7.80
0.71 0.49 0.45
26.25 37.34 30.55
34.31 25.08 31.36
35.03 38.59* 32.74
24.02 26.99 26.76
2.37 1.59 1.31
ns ns ns
ns L‡ (p = 0.012) ns ns ns ns
0.282 0.327 0.946
0.234 0.018 0.705 0.491 0.008 0.351
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Y = 14.2523 0.000689074x (R2 = 0.36). ‡Y = 6.15381 + 0.000858245x (R2 = 0.45).
Table 6 Pancreatic enzyme activities of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP)
EEP levels 7 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values 21 days Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
Amylase (UA/mg of protein)
Trypsin (nmol/mg of protein
Chymotrypsin (nmol/mg of protein)
Lipase (IU/mg of protein)
5.89 7.83 9.13 5.43 11.88 6.64 0.94 ns 0.425
25.42 22.37 26.95 22.71 27.08 28.70 2.59 ns 0.985
3.36 3.34 3.13 3.28 5.52 3.58 0.31 ns 0.285
3.82 4.31 7.09 4.24 4.90 6.67 0.63 ns 0.579
5.27 6.55 3.97 4.52 3.80 4.95 0.47 ns 0.642
12.50 23.53 20.74 13.88 19.13 17.96 2.09 ns 0.738
3.60 4.43 5.29 4.33 4.97 4.21 0.32 ns 0.759
14.31 8.70 11.78 13.33 15.25 13.77 1.51 ns 0.642
ns, not significant; SEM, standard error of means.
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The EEP dietary supplement decreased the duodenal sucrase activity at 7 day of age, which may have caused the decreased weight gain in the animals at this age. Several authors have shown that an EEP has an antihyperglycaemic effect in rats (Skopec et al., 2010). The propolis acts as an a-glycosidase inhibitor in the small intestine, inhibiting carbohydrate digestion into absorbable monosaccharides and thus decreasing or delaying their absorption (Matsui et al., 2004). These authors further suggest that caffeoylquinic acid compounds may be responsible for this effect. Moreover, the large amounts of phenolic compounds present in propolis affect glucose metabolism by inhibiting intestinal absorption and stimulating insulin secretion, among other mechanisms (Hanhineva et al., 2010). In fact, some authors have shown that flavonoids decrease glucose uptake in enterocytes by decreasing SGLT1 and GLUT2 transport activity (Johnston et al., 2002; Kwon et al., 2007). Although the EEP dietary supplement altered the intestinal disaccharidase activities, the supplement had no observable effect on the pancreatic enzyme activities at the EEP levels tested in the present study. There are no studies in the literature reporting the effects of polyphenols on the digestive enzyme activity of broilers. Several articles describe for mammals an 399
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Table 7 Carcass yield (%) of broiler chickens fed with diets containing different levels of ethanolic extract of propolis (EEP)
EEP levels
Carcass†
Breast
Thigh and leg
Wings
Abdominal Fat
Control 1000 ppm 2000 ppm 3000 ppm 4000 ppm 5000 ppm SEM Regression p-values
71.36 72.77 71.55 72.53 72.63 72.67 0.23 ns 0.326
36.12 37.08 36.18 37.29 37.70 36.75 0.18 ns 0.071
31.04 30.60 30.72 30.12 29.84 30.22 0.15 ns 0.230
11.15 11.03 10.87 10.98 10.97 11.17 0.07 ns 0.802
2.40 1.75* 2.52 2.01* 2.34 2.16 0.06 ns 0.001
ns, not significant; SEM, standard error of means. *Indicates a significant difference between control group and experimental group (Dunnett’s test, p < 0.05). †Carcass without neck, giblets and abdominal fat and expressed on a relative basis to the full-fed live weight carcass yield of broilers.
inhibitory effect of polyphenols on pancreatic enzyme activity. Hanhineva et al. (2010) described several studies that show inhibition by polyphenols of aamylase in vitro. Similar inhibition is described for lipase activity, and this inhibition may mediate favourReferences Acßikg€ oz, Z.; Y€ ucel, B.; Altan, O., 2005: The effects of propolis supplementation on broiler performance and feed digestibility. Archiv F€ ur Gefl€ ugelkunde 69, 117–122. Babi nska, I.; Kleczek, K.; Makowski, W.; Szarek, J., 2013: Effect of feed supplementation with propolis on liver and kidney morphology in broiler chickens. Pakistan Veterinary Journal 33, 1–4. Bankova, V. S.; Castro, S. L.; Marcucci, M. C., 2000: Propolis: recent advances in chemistry and plant origin. Apidologie 31, 3–15. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Caraway, W. T., 1959: A stable starch substrate for the determination of amylase in serum and other body fluids. American Journal of Clinical Pathology 32, 97– 99. C ucßl€ u, B. K., ß etin, E.; Silici, S.; C ßetin, N.; G€ 2010: Effects of diets containing different concentrations of propolis on hematological and immunological variables in laying hens. Poultry Science 89, 1703– 1708.
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able effects on lipid metabolism and control of weight gain, with the polyphenol anthocyanin considered the most potent inhibitor (You et al., 2011). As for literature data concerning the proteolytic enzymes trypsin and chymotrypsin, only trypsin appears to be inhibited by dietary polyphenols (Goncßalves et al., 2007). According to the results of the present study, adding 1000–5000 ppm of the EEP to the pre-starter broiler diet compromised performance during this stage, which was most likely due to the altered sucrase activity. The EEP promoted improved intestinal morphometry and sucrase enzyme activity at 21 days of age, with subsequent recovery of the performance parameters and carcass yield at 42 days of age; thus, the supplementation of the broiler diet with the EEP from 3000 ppm is indicated after the animals are 7 days old. Acknowledgements This work was supported by grants from National Counsel of Technological and Scientific Development (CNPq) and Financing Agency for Studies and Projects (FINEP), Brazil.
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