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Growth performance, meat quality, and gut microflora of broiler chickens fed with cranberry extract1
PROCESSING, PRODUCTS, AND FOOD SAFETY Growth performance, meat quality, and gut microflora of broiler chickens fed with cranberry extract1 G. Leusink,* H. Rempel,* B. Skura,† M. Berkyto,† W. White,† Y. Yang,† J. Y. Rhee,† S. Y. Xuan,† S. Chiu,† F. Silversides,* S. Fitzpatrick,* and M. S. Diarra*2 *Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, PO Box 1000, 6947 Highway 7, Agassiz, British Columbia, Canada V0M 1A0; and †University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada V6T 1Z4 ABSTRACT Cranberry fruit components have been reported to have antimicrobial activities against a variety of pathogenic bacteria and to be beneficial for human health. Studies on their effects are very limited in animals and especially in chickens. This study investigated the effect of feed supplementation with a commercial cranberry fruit extract (CFE) on the performance, breast meat quality, and intestinal integrity of broiler chickens. Twelve hundred male 1-d-old broiler chicks were allocated randomly to CFE treatments at 0, 40, 80, or 160 mg/kg of feed from d 0 to 35. Cloacal and cecal samples were collected weekly to evaluate the influence of treatments on the intestinal population of generic Escherichia coli, Clostridium perfringens, Enterococcus spp., and Lactobacillus spp. At d 35, BW were 1.62, 1.60, 1.61, and 1.64 kg for the control birds and birds fed 40, 80, and 160 mg of CFE/kg of feed,
respectively. Feed intake ranged from 2.7 to 2.8 kg and feed efficiency from 1.8 to 1.9 g of feed/g of BW. However, the treatment effects on bird performance were not statistically significant (P > 0.05). The mortality rate tended to be lower (P = 0.09) in birds fed 40 mg of CFE/kg of feed. Feed supplementation with CFE did not significantly alter any broiler meat properties evaluated when compared with the control diet (P > 0.05). At d 28, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower (P < 0.05) in birds receiving CFE at 160 mg/kg of feed than the other groups. No significant differences were noted between the control and the treatment groups for general health and intestinal integrity (P > 0.05). These findings suggest that more studies are needed to investigate potential beneficial effects of CFE or its derivatives in broiler production.
Key words: cranberry fruit extract, broiler performance, bacteria, breast meat quality 2010 Poultry Science 89:1514–1523 doi:10.3382/ps.2009-00364
INTRODUCTION Antimicrobial agents are used in broiler chicken feed for growth promotion and to prevent infectious diseases (Butaye et al., 2003; Singer and Hofacre, 2006). These agents improve feed conversion and BW gain presumably by altering the composition and activities of gut microflora (Knarreborg et al., 2002; Collier et al., 2003). Antimicrobial feed supplementation may create a selective pressure in favor of antibiotic-resistant bacteria (Singer and Hofacre, 2006). Recently, we reported the presence of multi-antibiotic-resistant bacteria on commercial broiler chicken farms (Diarrassouba et al., 2007) and that the phenotype and distribution of antibiotic resistance genes in chicken gut bacteria can be
©2010 Poultry Science Association Inc. Received July 17, 2009. Accepted February 10, 2010. 1 Pacific Agri-Food Research Centre contribution number 790. 2 Corresponding author: [email protected]
modulated by feed supplementation with some antimicrobial agents (Diarra et al., 2007). In response to the emergence of antibiotic resistance, several European countries have restricted or banned the use of antibiotics as growth promoters (Anonymous, 2007). Several alternatives to antibiotics in poultry are under investigation (Doyle, 2001; Dahiya et al., 2006). None of these alternatives have yielded an efficient control equivalent to in-feed antibiotic supplementation. Consequently, there is an urgent need to develop effective approaches that would preclude changes to existing poultry production practices. Consumers are becoming increasingly health conscious, and poultry meat quality is receiving considerable attention recently due to the emergence of problems associated with poor water-holding capacity, poor texture, and pale color called pale, soft, and exudative (Fletcher, 1999; Baéza, 2004). The ultimate cause of pale, soft, and exudative meat needs to be identified. However, in commercial broiler operations, it creates value-added processing problems and affects consumer
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EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
satisfaction. Nutritional methods including the use of antioxidants have been investigated (Wood and Enser, 1997; Gladine et al., 2007; Jang et al., 2008) to maximize growth and yield and to maintain or improve breast meat functional properties. Cranberry (Vaccinium macrocarpon Ait.) is an important commercial crop in the United States and Canada. About 500 million tons are produced every year and cranberries are used as ingredients in more than 700 products for human consumption (Puupponen-Pimiä et al., 2005). Cranberry fruit has received considerable attention for its putative human health benefits. Most of the focus is on the flavonoid constituents due to their relatively high antioxidant activity in various assays. In vitro chemical assays have rated cranberries as having the highest antioxidant values of over 21 fruits, and the overall phenolic content appears to correlate with the level of antioxidant activity (Vinson et al., 2001; Sun et al., 2002). The phenolic classes identified in cranberries include phenolic acids, anthocyanins, flavonols, and flavan-3-ols, which consist of both monomers and the polymer classes of procyanidins and proanthocyanidins (Vinson et al., 2001; Sun et al., 2002). The proanthocyanidins in plants are well known to improve nitrogen nutrition in ruminant animals and as powerful antioxidants with beneficial effects on human health and immunity (Lin et al., 2002). Particular interest is being shown for the proanthocyanidins from cranberry (Foo et al., 2000). Cranberry constituents are known to exert antiadhesion and antimicrobial activities against several pathogenic bacteria and have been suggested to prevent urinary tract infections (Foo et al., 2000; Nogueira et al., 2003; Howell, 2007). The cranberry A-type proanthocyanidins have been recognized for their antiadherence activities against uropathogenic P-type Escherichia coli (Foo et al., 2000) and may play a role in urinary tract health. Cranberry juice extracts were found to inhibit the specific adhesion of Helicobacter pylori to immobilized human mucus, erythrocytes, and cultured gastric epithelial cells (Burger et al., 2000; Zhang et al., 2005; Shmuely et al., 2007). In addition, cranberry may affect biofilm formation via inhibition of extracellular polysaccharide synthesis, which promotes Streptococcus sobrinus (Steinberg et al., 2004; Yamanaka et al., 2004; Labrecque et al., 2006). Limited laboratory research has examined the biological effect of cranberry extract using animal models. The purpose of the present work is to evaluate the growth performance, breast meat quality, gut bacterial numbers, and intestinal health in broiler chickens receiving a commercial cranberry extract through feed.
MATERIALS AND METHODS Broiler Chickens and Housing Twelve hundred Ross 308 one-day-old male broiler chicks were obtained from a local commercial hatchery
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and placed in 24 pens (50 birds per pen). Before placement, all chicks were visually examined for health and inferior chicks were not included in the trial. The clean and disinfected concrete floor was covered with approximately 3 in. (7.6 cm) of clean softwood shavings and the bird density was approximately 0.75 ft2 (0.07 m2) per bird. Ventilation was provided by negative pressure with fans. Heat was provided by gas-fired brooders; water and feed were offered ad libitum through nipple drinkers and tube feeders, respectively. The temperature was initially set at 32°C at d 0 and then was progressively reduced by 1.7°C each week to reach 23°C at 35 d of age. Chicks were exposed to light for 24 h for the first day, 23 h for the second and third days, then 18 h thereafter. The composition of the feed used in this study is presented in Table 1. The starter, grower, and finisher diets were formulated in accordance with the broiler diet used in Western Canada with wheat, barley, and corn as the principal cereals and soybean and canola meals as protein concentrates to meet the NRC nutrient requirements for broiler chickens (NRC, 1994). Analysis for DM, total proteins, soluble carbohydrates, fatty acids, and some of the most common minerals was performed at the Centre de Recherche en Sciences Animales de Deschambault (CRESAD, Deschambault, Quebec, Canada). All experimental procedures performed in this study were approved by the Animal Care Committee of the Pacific Agri-Food Research Center (Agassiz, British Columbia, Canada) according to guidelines described by the Canadian Council on Animal Care (CCAC, 1993).
Study Design The chicks were assigned at random to 4 treatments (6 pens per treatment): a control untreated group without antibiotics and 3 groups fed rations containing the following per kilogram of feed: 160, 80, and 40 mg of a commercial cranberry fruit extract (CFE) supplied by Decas Botanical Synergies (Wareham, MA). No additional anticoccidials or antibiotics were administrated to the birds throughout the trial. The concentrations of CFE were chosen to have a comparative range of the concentrations of some approved antimicrobial agents used in broiler feed (i.e., bacitracin, 55 to 110 mg/kg; salinomycin, 60 mg/kg; and virginiamycin, 11 to 22 mg/ kg). As described by the manufacturer, the CFE used in this study contained at least 30% organic acids, 2 to 3.8% total phenolic compounds, 0.3 to 1% anthocyanins, 0.8 to 1.5% proanthocyanidins, 300 to 435 µg/g of quercetin, and were 100% soluble in water.
Data Collection Chicks were weighed at the start of the trial (d 0) and every week thereafter; feed intake was measured at d 14, 28, and 35 from each pen and was used to determine feed efficiency (g of gain per g of feed). During the trial, all mortalities were removed from the pens
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Leusink et al. Table 1. Composition of the feed used in this study Item, % of inclusion in diet Ingredient Wheat Soy Barley Canola Canola oil Corn Corn gluten Limestone Dicalcium phosphate1 Vitamin-mineral mix2 Lysine Methionine Salt Avizyme3 Calculated energy (kJ/kg) Analyzed nutrient4 DM Ash Protein Fat Glucose Fructose Acid detergent fiber Neutral detergent fiber Ca Mg K P Na Fe Zn Mn
Starter (d 0 to 14)
Grower (d 14 to 28)
Finisher (d 28 to 35)
34.96 23.00 10.00 9.00 8.60 7.00 2.30 1.60 1.60 1.00 0.40 0.18 0.31 0.05 13.16
35.03 0.00 0.00 22.00 7.00 25.00 6.00 1.30 1.50 1.00 0.71 0.09 0.32 0.05 13.35
40.79 0.51 0.00 18.00 7.00 25.00 4.00 1.20 1.40 1.00 0.63 0.10 0.32 0.05 13.44
89.01 6.42 24.84 10.58 19.25 21.95 7.42 13.35 0.99 0.22 0.89 0.80 0.22 0.04 0.02 0.02
88.70 5.75 21.34 10.35 17.28 22.68 7.18 13.72 0.99 0.22 0.55 0.81 0.26 0.04 0.02 0.02
88.02 5.81 19.81 10.35 18.58 24.43 6.62 14.31 1.01 0.22 0.53 0.79 0.26 0.04 0.02 0.02
1A
mixture of mono- and dicalcium phosphate containing 18% calcium and 21% phosphate. per kilogram of diet: vitamin A, 9,000 IU; cholecalciferol, 1,500 IU; vitamin E, 10 IU; vitamin K, 0.5 mg; vitamin B12, 0.007 mg; thiamine, 0.4 mg; riboflavin, 6 mg; folic acid, 1 mg; biotin, 0.15 mg; niacin, 135 mg; pyridoxine, 4 mg; choline chloride, 1,000 mg; dl-methionine, 1,184 mg; ethoxyquin, 125 mg; NaCl, 2 g; manganese sulfate, 60 mg; copper sulfate, 5 mg; selenium (sodium selenium), 0.1 mg; iodine, 0.35 mg; and zinc sulfate, 50 mg. 3Multi-Enzyme System for Wheat-Based Poultry Feed (Halchemix Canada Inc., Toronto, Ontario, Canada) containing 5,000 U/g of xylanase and 1,600 U/g of protease. 4Analyzed nutrient contents on a DM basis determined at the Centre de Recherche en Sciences Animales de Deschambault (CRESAD, Deschambault, Quebec, Canada). 2Supplied
and recorded. Necropsies were performed on all mortalities, which were categorized as early mortality (due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration), physiological (due to sudden death syndrome, ascites, heart failure, and unknown reasons), and culls (due to spondylolisthesis, valgus, and varus deviations of tibiotarsi, rotated tibia, and runts). At d 36 (in the Fraser Valley of British Columbia, most of the broilers are marketed between 35 and 40 d), the broilers were fasted for 12 h and 18 birds (3 per pen) from each treatment were randomly selected (a total of 72 birds) and transported to a commercial slaughterhouse situated at a distance of 80 km. Birds were scalded, plucked, and manually eviscerated. The circumference of the breast and the carcass weight before and after evisceration were obtained. The individual carcasses were then vacuum-packaged and stored at −20°C until they were used for quality tests.
Meat Quality Chickens were partially thawed in a 2°C refrigerator overnight and the left and right breast muscles (pectoralis major and pectoralis minor) were removed and weighed. The left breast muscle samples were used for pH, percentage of lipid, and water content analyses according to the official methods of analysis (Association of Official Analytical Chemists, 1995). Raw finely chopped middle sections of the breasts were used for pH measurement. Two-gram samples were homogenized with 10 mL of distilled deionized water in a blender then transferred to a 50-mL beaker. The pH was measured and recorded in triplicate for every sample at room temperature. Fat was analyzed using the BlighDyer lipid extraction method (Bligh and Dyer, 1959). Samples (4 ± 0.001 g) of chicken meat were cut from the lower tip of the breast. Muscle moisture was de-
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
termined using a vacuum oven (model number 1430, VWR Scientific Products, Batavia, IL) at 70°C under pressure (<100 mmHg) for 18 h (Park and Bell, 2004). The fleshiest part near the top of the right breast muscle was excised and analyzed for color and texture. Color (lightness = L*; redness = a*; and yellowness = b*) of each sample was measured in triplicate using a Hunterlab LabScan XE colorimeter (Hunter Associates Laboratory Inc., Reston, VA) using a D65 illuminant. The instrument was calibrated with a white-and-black tile before analysis in agreement with the International Commission on Illumination (Fletcher et al., 2000). After the color measurements, breast muscle samples were then wrapped in aluminum foil and cooked at 95°C for 20 min and cooled at room temperature. Then, 2 × 2 × 1 cm samples were cut for texture (shear force and hardness) analyses using a TA/XT2 texture analyzer (Stable Microsystems, Surrey, UK) as described previously (Fletcher, 1999). A 3-mm blade was used for texture measurement.
Bacteriological Analyses Ten chicks at d 0 and 2 birds per pen at each sampling time (7, 14, 21, 28, and 35 d of age) were killed by cervical dislocation. Cloacae samples and cecal contents from the 2 killed birds were aseptically collected and transferred to peptone buffer in test tubes and sterile Whirl-Pak plastic bags (Nasco, Fort Atkinson, WI), respectively, for bacteriological culture. The samples were placed on ice and transported to the microbiology laboratory for bacteriological analysis that was carried out the same day. Sample weights were estimated by subtracting the weight of the container without sample from the weight with the samples. From d 7 to 35, bacteriological analyses were performed on a total of 120 cloacae and 120 cecal samples (pooled samples from 2 birds of the same pen constituting 1 sample). The generic E. coli population was estimated using E. coli and coliform Petrifilms (3M, St. Paul, MN) as described previously (Diarra et al., 2007). Enterococcus spp. populations were determined by spreading 10-fold dilutions of samples on KF Streptococcal Agar CM0701 (Oxoid, Nepean, Ontario, Canada) and incubating at 37°C for 48 h (Hayes et al., 2003). Clostridium perfringens was enumerated according to Knarreborg et al. (2002). Briefly, samples were spread on tryptose sulfite agar (Oxoid) supplemented with cycloserine (SR088E, Oxoid) and incubated anaerobically for 24 h at 37°C. Lactobacillus spp. populations were quantified using Lactobacilli MRS Agar (Oxoid) according to the manufacturer’s methods.
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coccidiosis and necrotic enteritis because this disease occurs most commonly in broiler chickens of 2 to 5 wk of age. The intestines were longitudinally opened and intestinal mucosa were scored on a scale of 0 to 3 for necrotic enteritis lesions for each of the upper gut, mid gut, lower gut, and ceca according to the method of Collier et al. (2003). Body weights of killed birds were determined.
Statistical Analysis Statistical analyses were conducted according to a randomized complete block design using the GLM procedure of SAS (2000) with treatment groups as sources of variation and the individual pens as experimental units (6 pens per treatment group). Data from bacterial counts were log-transformed before analysis and the sample origins (ceca and cloacae) and bird ages (d) were included as sources of variation. Least significance difference was used to separate treatments means whenever the F-value was significant. The 0.05 P-value was used to declare significance.
RESULTS Broiler Performance The effects of diet supplementation with CFE on BW, feed intake, feed efficiency, and mortality estimated are presented in Table 2. No significant differences were noted between the treatment groups for all parameters evaluated from d 0 to 28 (P > 0.05). At d 35, the highest feed intake and BW values for birds fed 160 mg of CFE were not statistically significant (P > 0.05). All mortalities and culls were necropsied and on d 35, fatal necrotic enteritis was observed in 1 bird from one pen receiving 80 mg/kg. Most of the mortalities were culls (runts or leg disorders especially in CFE, 80 and 160 mg/kg) or routine physiological conditions including acute death syndrome or ascites (Figure 1). The percentage of mortality in birds in the 40 mg of CFE treatment tended to be lower than for chickens from the other treatments or the control (P = 0.09), possibly due to the decrease of early mortality in this treatment. Early mortality was decreased by 50% with diet containing CFE at 40 mg/kg of feed when compared with the control untreated bird (Figure 1). The highest mortality (11.1%) observed in CFE 160 mg/kg treatment may be due to the increased physiological mortality (sudden death syndrome, ascites, heart failure, and unknown reasons) or an increased number of culled birds of this treatment, or both.
Intestinal Health
Carcass and Meat Quality
At each of 21, 28, and 35 d of age, the intestines of killed birds (2 per pen) were examined for evidence of
Chickens fed CFE at 80 and 160 mg/kg of feed had higher breast circumference and carcass weight (Figure
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Leusink et al.
Figure 1. Categories of mortalities: early mortality due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration; physiological due to sudden death syndrome, ascites, heart failure, and unknown; culls due to spondylolisthesis, valgus and varus deviations of tibiotarsi, rotated tibia, and runts.
2 A and B), but these effects were not statistically significant (P > 0.05). The characteristics of the breast meat from the broilers fed different concentrations of CFE are presented in Table 3. The means of pH values, fat content, and moisture were 6.39, 2.11, and 76.25%, respectively.
The lightness (L*) values ranged from 48.7 (control) to 50.38 (40 mg/kg), the yellowness (b*) values ranged from 13.50 (40 mg/kg) to 14.14 (160 mg/kg), and the redness (a*) values ranged from 2.71 (40 mg/kg) to 3.56 (80 mg/kg). Meat from the control birds and birds fed 160 mg of CFE/kg of feed had the highest values
Table 2. Performance of broiler chickens fed diets containing cranberry fruit extract (CFE) at different concentrations (0 to 160 mg/kg of feed) Concentration of CFE (mg/kg of feed) Parameter BW (g) Initial 0 to 14 d 14 to 28 d 28 to 35 d Feed intake (g) 0 to 14 d 14 to 28 d 28 to 35 d 0 to 35 d Feed efficiency3 (feed:gain) 0 to 14 d 14 to 28 d 28 to 35 d 0 to 35 d Total mortality (%) 0 to 35 d
0
40
80
160
SEM1
P-value2
41.5 399.8 1,208.3 1,619.8
41.3 395.9 1,183.1 1,573.8
41.2 402.9 1,199.1 1,611.7
41.3 402.9 1,200.4 1,637.9
0.185 5.466 19.771 32.764
0.65 0.77 0.83 0.56
430.7 1,386.0 951.2 2,767.8 1.2 2.0 2.4 1.8 6.9
425.7 1,369.4 950.5 2,745.8 1.2 2.0 2.8 1.9 5.2
420.5 1,387.4 961.3 2,769.2 1.2 2.0 2.6 1.9 9.5
425.4 1,389.0 1,003.4 2,817.8 1.2 2.0 2.5 1.9 11.1
6.748 23.805 20.463 42.181 0.023 0.026 0.019 0.028 1.803
0.77 0.93 0.23 0.67 0.59 0.55 0.52 0.67 0.09
1Cranberry fruit extract was administrated via feed from 0 to 35 d. Data represent means ± SE of 6 replicates/ treatment (n = 6 pens of at least 30 chickens/pen) arranged in a completely randomized block design. 2P-value obtained by ANOVA. 3Grams of feed per gram of BW gain.
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
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Bacteriological Analyses In general, the populations of generic E. coli, C. perfringens, Enterococcus spp., and Lactobacillus spp. were higher (P < 0.05) in the cecal samples than in the cloacal samples (Table 4). The log C. perfringens numbers per gram of sample in ceca were greater (P < 0.05) at d 7 (4.5), 28 (4.2), and 35 (4.5) than at d 14 (2.1) to 21 (2.4), but no significant treatment effects were noted (P > 0.05). The population of this bacterium remained lower than those of other bacteria (P < 0.05). The populations of E. coli or Enterococcus spp. were highest on d 7 and slowly declined thereafter. There was no significant effect of any treatment on E. coli population in any sample (cloacae or ceca, P > 0.05). At d 28, counts of Enterococcus spp. in cecal and cloacae samples were significantly lower (P < 0.05) in birds receiving 80 mg of CFE/kg of feed than the other groups. The counts of Lactobacillus were not influenced by the treatments and remained relatively stable in ceca and cloacae from d 7 to 35.
Intestinal and General Health
Figure 2. Breast circumference and carcass weight of broiler chicken fed 0 to 160 mg/kg of cranberry fruit extract in feed.
for texture. However, no statistically significant differences in fat content (%), moisture content (%), pH, texture (hardness and shear force), and color (L*, b*, and a*) were observed between the treatment groups (P > 0.05).
Gross observations of the health status and intestinal mucosa were performed weekly, beginning at 21 d of age. In general, health and body condition were good, and bursal size and anatomical features were normal in all birds examined. In most birds, intestinal mucosa was normal on gross observation. However, on d 28 and 35, some birds were seen with rough, pitted mucosa involving the duodenum and the upper part of the jejunum, proximal to Meckel’s diverticulum. In birds examined on d 21, 28, and 35, small foci of necrosis from approximately 1 to 5 mm in diameter (lesions score of 1) in the upper intestine were observed in 7 (4.9%) of the 144 sample birds, none on d 21, 2 (1 in each of 80 and 160 mg/kg treatments) on d 28, and 5 (2, 1, and 2 in the control, 80, and 160 mg/kg treatments, respectively). None of the birds receiving the lower CFE concentration (40 mg/kg) were found to have intestinal lesions.
Table 3. Characteristics of breast meat from broiler chickens fed diets containing cranberry fruit extract (CFE) at different concentrations (0 to 160 mg/kg of feed) Concentration of CFE (mg/kg of feed) Meat characteristic Fat (%) pH Moisture (%) Lightness (L*) Yellowness (b*) Redness (a*) Hardness (N) Shear force (Nm)
0
40
80
160
SEM1
P-value2
2.01 6.39 76.19 48.70 14.03 2.88 35.74 0.29
2.03 6.37 76.26 50.38 13.50 2.71 33.49 0.26
2.2 6.37 76.17 48.65 13.83 3.56 33.40 0.26
2.22 6.41 76.38 49.90 14.14 3.47 35.82 0.30
0.189 0.025 1.110 0.728 0.315 0.314 2.100 0.023
0.35 0.63 0.76 0.14 0.79 0.18 0.41 0.81
1Cranberry fruit extract was administrated via feed from 0 to 35 d. Data represent means ± SE of 6 replicates/ treatment (n = 6 pens of 3 carcasses/pen) arranged in a completely randomized block design. 2P-value obtained by ANOVA.
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Table 4. Log10 bacterial number per gram of sample obtained from ceca and cloacae samples from broiler chickens fed diets containing cranberry fruit extract (CFE) at 0, 40, 80, and 160 mg/kg of feed1 Concentration of CFE (mg/kg) Bacteria2 Escherichia coli d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Enterococcus spp. d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Lactobacillus spp. d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Clostridium perfringens d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL a,bValues
0
40
80
160
SEM3
P-value4
8.9 7.1
8.9 7.2
8.9 7.1
8.8 7.3
0.26
0.989
8.2 6.3
8.2 6.4
8.1 6.8
8.3 6.4
0.244
0.879
7.4 6.4
7.1 6.4
6.6 6.7
7.5 6.3
0.34
0.849
6.3 6.1
6.7 6.5
6.6 6.2
7.2 6.2
0.269
0.288
7.0 6.4
6.8 6.6
7.2 6.2
7.0 6.5
0.302
0.997
8.5 7.6
8.4 7.8
8.4 7.7
8.4 7.6
0.256
0.986
6.8 5.9
6.5 5.6
7.2 5.8
6.7 5.7
0.301
0.44
6.2 5.8
5.6 5.4
5.6 5.4
6 5
0.263
0.175
5.3a 5.4
5.5ab 5.7
5.0a 5.2
5.9b 5.7
0.182
0.001
5.7 5.6
5.0 5.5
5.9 5.3
5.9 5.9
0.256
0.129
8.6 7.9
8.5 7.6
8.6 7.5
8.6 7.5
0.082
0.064
8.9 7.9
8.9 8.1
8.4 7.8
9.1 7.9
0.193
0.159
8.0 7.3
7.8 7.1
8.2 7.1
8.2 7.4
0.217
0.589
8.2 7.0
8.3 8.2
7.9 8.3
8.5 8.4
0.59
0.56
8.4 8.3
8.6 8.5
8.5 7.7
8.6 8.2
0.178
0.17
4.5 1.8
4.7 2.6
4.8 2.1
4.4 1.8
0.512
0.626
2.1 1.1
2.6 0.5
2.7 1.5
2.7 0.6
0.589
0.828
2.4 1.8
1.7 2.2
1.1 1.4
2.9 2.1
0.506
0.1
4.2 3.7
4.4 3.7
4.4 3.1
4.5 3.4
0.626
0.961
4.5 3.1
4.4 3.2
4.9 4.1
4.4 3.1
0.848
0.769
within a row with different superscripts are statistically different (P < 0.05). of colony-forming units per gram of sample. 2CE = ceca sample; CL = cloacae sample. According to ANOVA, statistically significant differences (P < 0.05) were observed between bacterial numbers from different locations (ceca vs. cloacae). 3Standard error of means of 6 replicates/treatment (n = 6 pens of 2 chickens/pen) arranged in a completely randomized block design. 4P-value obtained by ANOVA. 1Mean
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
No mortality due to necrotic enteritis was seen during necropsies. There was no clinical evidence of infectious disease during the feeding trial. No significant effect of treatments on intestinal and general health was noted.
DISCUSSION Because cranberries have received considerable attention for their putative human health benefits (Vinson et al., 2001; Sun et al., 2002), we evaluated the use of cranberry extracts as alternatives to traditional antibiotics in broiler chicken production. We did not find that growth performance of broiler chickens was statistically improved when they were fed diets containing commercial CFE at concentrations of 40, 80, and 160 mg/kg of feed. The lack of significance of our observed effects on growth performance was not surprising because we previously reported no significant effects of traditional approved individual antimicrobial agents including salinomycin and bacitracin on performance using our broiler model (Diarra et al., 2007). This may be due to the high hygienic and biosecurity practices we used before and throughout the experimental protocol. Mortality in research trials is often high, possibly related to handling. The CFE used at 40 mg/kg seemed to decrease early mortality due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration. For animal care concerns, higher mortalities were due to culled birds and acute deaths mainly in treatment with 80 and 160 mg/kg of CFE. The severity of necrotic enteritis within the population was not extensive. Only about 5% of birds examined from d 21 to 35 presented a lesion score of 1. We can speculate that most cases observed during sampling likely would have healed and the affected birds would have survived with some loss of performance. Only 1 case of fatal necrotic enteritis was seen. Further work is needed to confirm and explain the effect of CFE used at different concentrations in diets on the mortality and the overall bird health. We did not estimate the cost (which was beyond the scope of the study) of the use of CFE in broiler diets. However, it would be interesting to investigate the effects on production parameters of similar or greater doses of CFE in commercial farming conditions because the beneficial effects of growth-promoting agents have been reported under stress and poor management conditions often found in commercial poultry production (Sims et al., 2004). In recent years, consumers have become concerned about poultry meat quality. Several factors including bird sex, age at slaughter, strain, method of processing, exposure to chemicals, irradiation, and freezing have been shown to affect poultry meat quality (Froning, 1995; Le Bihan-Duval et al., 1999; Zhou et al., 2009). It has been reported that proximate composition of nonfrozen breast meat from broilers fed diets supplemented with medicinal herb extracts mix was not affected, but the total phenol content of the breast meat from these birds receiving supplemented diets was significantly dif-
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ferent and displayed higher antioxidant activities when compared with those from the control diet (Jang et al., 2008). In our study, we used frozen samples and the CFE in feed was not found to affect the broiler meat quality parameters that were evaluated. Whether freezing might have any adverse effects on tested meat samples is unknown; however, it would be interesting to evaluate fresh meat characteristics. Feed supplementation with oregano extracts resulted in greater a* and b* values in chicken meat than in the meat of control chickens (Young et al., 2003). Larrain et al. (2008) showed in familial hypercholesterolemic pigs that continuous feeding with cranberry juice powder altered the time-course changes of bacon color. These authors reported that lipid oxidation, but not TBA reactive substances, were significantly high in frozen meat samples from cranberry-fed pigs. In our study, lipid peroxidation was not investigated. However, our findings suggest the need for more rigorous studies on the effects of cranberry feeding on overall broiler meat quality including sensory tests after processing and storage. In our study, the cranberry extracts used did not affect intestinal health when compared with the nonsupplemented diet. Escherichia coli, Enterococcus spp., Clostridium spp., and Lactobacillus spp. are normal inhabitants of the gastrointestinal tract of the chicken. Feed supplementation with cranberry extract had no effect on the population of these commensal bacteria species in the ceca and cloacae of the broiler chickens at d 7 to 21 and 35. However, at d 28, in birds receiving CFE at 80 mg/kg of feed, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower, whereas greater numbers of this bacterium were observed in chickens receiving feed containing CFE at 160 mg/kg. The mechanism underlining this observation on the enterococci number is not known. However, because enterococci are lactic acid-producing bacteria, we can speculate that the increase in their population might translate into elevated lactic acid levels that might affect the growth of some other bacteria. Some Enterococcus spp. are used in meat and dairy processing as starter cultures (Giraffa, 2003). Bacteriocin producing Enterococcus was isolated from chickens, suggesting the possible development of such isolates as a probiotic that could reduce the burden of pathogens in poultry (Strompfová and Lauková, 2007). On the other hand, Enterococcus spp. are ubiquitous commensal bacteria, but some strains of enterococci, particularly Enterococcus faecium and Enterococcus faecalis, are important in food safety and public health due to their potential to cause disease and their ability to acquire multiple antibiotic-resistant genes. Much more work is needed to investigate the potential in vivo effects of cranberry extract on the gut microflora of poultry. The consequences of poultry production for the environment, food safety, and animal welfare issues are now part of consumers’ opinions and demands. Replacement strategies based on novel or alternative feeding practices without antibiotics are favored by the industry.
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This study did not find significant effects of 3 levels of cranberry extract on bird performance. We evaluated 1 raw commercial sample. It would be interesting to evaluate samples from different sources as well as subfractions (derivatives) of such samples. Although our studied cranberry extract did not show significant beneficial effects in broilers, more investigation of cranberry fruit compounds could lead to the development of feeding strategies for chicken to improve bird health and on-farm food safety that will reduce the use of antibiotics as growth promoters.
ACKNOWLEDGMENTS This work was financially supported by Agriculture and Agri-Food Canada and the Canadian Cranberry Growers Coalition (Abbotsford, British Columbia, Canada). We thank L. Struthers and M. Fraser (Pacific Agri-Food Research Centre) and William R. Cox (Canadian Animal Health Management Services Ltd., Chilliwack, British Columbia, Canada) for assistance. We also thank Decas Botanicals for providing cranberry extract and acknowledge the help of D. Mah and S. Li.
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sistant phenotype, and distribution of antimicrobial resistance determinants in Escherichia coli. Appl. Environ. Microbiol. 73:6566–6576. Diarrassouba, F., M. S. Diarra, S. Bach, P. Delaquis, J. Pritchard, E. Topp, and B. J. Skura. 2007. Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolated from commercial broiler chicken farms. J. Food Prot. 70:1316–1327. Doyle, M. E. 2001. Alternatives to antibiotic use for growth promotion in animal husbandry. Pages 1–17 in Food Research Institute Briefings. Food Research Institute, University of Wisconsin– Madison. Fletcher, D. L. 1999. Broiler breast meat color variation, pH, and texture. Poult. Sci. 78:1323–1327. Fletcher, D. L., M. Qiao, and D. P. Smith. 2000. The relationship of raw broiler breast meat color and pH to cooked meat color and pH. Poult. Sci. 79:784–788. Foo, L. Y., Y. Lu, A. B. Howell, and N. Vorsa. 2000. A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated Escherichia coli. J. Nat. Prod. 63:1225–1228. Froning, G. W. 1995. Color of poultry meat. Poult. Avian Biol. Rev. 6:83–93. Giraffa, G. 2003. Functionality of enterococci in dairy products. Int. J. Food Microbiol. 88:215–222. Gladine, C., C. Morand, E. Rock, D. Bauchart, and D. Durand. 2007. Plant extracts rich in polyphenols (PERP) are efficient antioxidants to prevent lipoperoxidation in plasma lipids from animals fed n-3 PUFA supplemented diets. Anim. Feed Sci. Technol. 136:281–296. Hayes, J. R., L. L. English, P. J. Carter, T. Proescholdt, K. Y. Lee, D. D. Wagner, and D. D. White. 2003. Prevalence and antimicrobial resistance of Enterococcus species isolated from retail meats. Appl. Environ. Microbiol. 69:7153–7160. Howell, A. B. 2007. Bioactive compounds in cranberries and their role in prevention of urinary tract infections. Mol. Nutr. Food Res. 51:732–737. Jang, A., X.-D. Liu, M.-H. Shin, B.-D. Lee, S.-K. Lee, J.-H. Lee, and C. Jo. 2008. Antioxidative potential of raw breast meat from broiler chicks fed a dietary medicinal herb extract mix. Poult. Sci. 87:2382–2389. Knarreborg, A., M. A. Simon, R. M. Engberg, B. B. Jensen, and G. W. Tannock. 2002. Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Appl. Environ. Microbiol. 68:5918–5924. Labrecque, J., C. Bodet, F. Chandad, and D. Grenier. 2006. Effects of a high-molecular-weight cranberry fraction on growth, biofilm formation and adherence of Porphyromonas gingivalis. J. Antimicrob. Chemother. 58:439–443. Larrain, R. E., C. G. Krueger, M. P. Richards, and J. D. Reed. 2008. Color changes and lipid oxidation in pork products made from pigs fed with cranberry juice powder. J. Muscle Foods 19:17–33. Lin, L. C., Y.-C. Kuo, and C.-J. Chou. 2002. Immunomodulatory proanthocyanidins from Ecdysanthera utilis. J. Nat. Prod. 65:505–508. Nogueira, M. C., O. A. Oyarzabal, and E. D. Gombas. 2003. Inactivation of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella in cranberry, lemon, and lime juice concentrates. J. Food Prot. 66:1637–1641. NRC. 1994. Nutrient Requirements of Poultry. 9th ed. National Academy Press, Washington, DC. Park, Y. W., and L. N. Bell. 2004. Determination of moisture and ash contents in foods. Pages 55–82 in Handbook of Food Analysis: Residues and Other Food Component Analysis. L. M. L. Nollet, ed. CRC Press, New York, NY. Puupponen-Pimiä, R., L. Nohynek, H. L. Alakomi, and K. M. Oksman-Caldentey. 2005. Bioactive berry compounds—Novel tools against human pathogens. Appl. Microbiol. Biotechnol. 67:8–18. SAS. 2000. Release 8.02. SAS Institute Inc., Cary NC. Shmuely, H., J. Yahav, Z. Samra, G. Chodick, R. Koren, Y. Niv, and I. Ofek. 2007. Effect of cranberry juice on eradication of Helico-
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN bacter pylori in patients treated with antibiotics and a proton pump inhibitor. Mol. Nutr. Food Res. 51:746–751. Sims, M. D., K. A. Dawson, K. E. Newman, P. Spring, and D. M. Hoogell. 2004. Effects of dietary mannan oligosaccharide, bacitracin methylene disalicylate, or both on the live performance and intestinal microbiology of turkeys. Poult. Sci. 83:1148–1154. Singer, R. S., and C. L. Hofacre. 2006. Potential impacts of antibiotic use in poultry production. Avian Dis. 50:161–172. Steinberg, D., M. Feldman, I. Ofek, and E. I. Weiss. 2004. Effect of a high-molecular-weight component of cranberry on constituents of dental biofilm. J. Antimicrob. Chemother. 54:86–89. Strompfová, V., and A. Lauková. 2007. In vitro study on bacteriocin production of enterococci associated with chickens. Anaerobe 13:228–237. Sun, J., Y.-F. Chu, X. Wu, and R. H. Liu. 2002. Antioxidant and antiproliferative activities of common fruits. J. Agric. Food Chem. 50:7449–7454. Vinson, J. A., X. Su, L. Zubik, and P. Bose. 2001. Phenol antioxidant quantity and quality in foods: Fruits. J. Agric. Food Chem. 49:5315–5321.
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Wood, J. D., and M. Enser. 1997. Factors influencing fatty acids in meat and the role of antioxidants in improving meat quality. Br. J. Nutr. 78:S49–S60. Yamanaka, A., R. Kimizuka, T. Kato, and K. Okuda. 2004. Inhibitory effects of cranberry juice on attachment of oral streptococci and biofilm formation. Oral Microbiol. Immunol. 19:150–154. Young, J. F., J. Stagsted, I. S. Jensen, A. H. Karlsson, and P. Henckel. 2003. Ascorbic acid, α-tocopherol and oregano supplements reduce stress-induced deterioration of chicken meat quality. Poult. Sci. 82:1343–1351. Zhang, L., J. Ma, K. Pan, V. Liang, W. Go, J. Chen, and W.C. You. 2005. Efficacy of cranberry juice on Helicobacter pylori infection: A double-blind, randomized placebo-controlled trial. Helicobacter 10:139–145. Zhou, T. X., Y. J. Chen, J. S. Yoo, Y. Huang, J. H. Lee, H. D. Jang, S. O. Shin, H. J. Kim, J. H. Cho, and I. H. Kim. 2009. Effects of chitooligosaccharide supplementation on performance, blood characteristics, relative organ weight, and meat quality in broiler chickens. Poult. Sci. 88:593–600.
respectively. Feed intake ranged from 2.7 to 2.8 kg and feed efficiency from 1.8 to 1.9 g of feed/g of BW. However, the treatment effects on bird performance were not statistically significant (P > 0.05). The mortality rate tended to be lower (P = 0.09) in birds fed 40 mg of CFE/kg of feed. Feed supplementation with CFE did not significantly alter any broiler meat properties evaluated when compared with the control diet (P > 0.05). At d 28, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower (P < 0.05) in birds receiving CFE at 160 mg/kg of feed than the other groups. No significant differences were noted between the control and the treatment groups for general health and intestinal integrity (P > 0.05). These findings suggest that more studies are needed to investigate potential beneficial effects of CFE or its derivatives in broiler production.
Key words: cranberry fruit extract, broiler performance, bacteria, breast meat quality 2010 Poultry Science 89:1514–1523 doi:10.3382/ps.2009-00364
INTRODUCTION Antimicrobial agents are used in broiler chicken feed for growth promotion and to prevent infectious diseases (Butaye et al., 2003; Singer and Hofacre, 2006). These agents improve feed conversion and BW gain presumably by altering the composition and activities of gut microflora (Knarreborg et al., 2002; Collier et al., 2003). Antimicrobial feed supplementation may create a selective pressure in favor of antibiotic-resistant bacteria (Singer and Hofacre, 2006). Recently, we reported the presence of multi-antibiotic-resistant bacteria on commercial broiler chicken farms (Diarrassouba et al., 2007) and that the phenotype and distribution of antibiotic resistance genes in chicken gut bacteria can be
©2010 Poultry Science Association Inc. Received July 17, 2009. Accepted February 10, 2010. 1 Pacific Agri-Food Research Centre contribution number 790. 2 Corresponding author: [email protected]
modulated by feed supplementation with some antimicrobial agents (Diarra et al., 2007). In response to the emergence of antibiotic resistance, several European countries have restricted or banned the use of antibiotics as growth promoters (Anonymous, 2007). Several alternatives to antibiotics in poultry are under investigation (Doyle, 2001; Dahiya et al., 2006). None of these alternatives have yielded an efficient control equivalent to in-feed antibiotic supplementation. Consequently, there is an urgent need to develop effective approaches that would preclude changes to existing poultry production practices. Consumers are becoming increasingly health conscious, and poultry meat quality is receiving considerable attention recently due to the emergence of problems associated with poor water-holding capacity, poor texture, and pale color called pale, soft, and exudative (Fletcher, 1999; Baéza, 2004). The ultimate cause of pale, soft, and exudative meat needs to be identified. However, in commercial broiler operations, it creates value-added processing problems and affects consumer
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EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
satisfaction. Nutritional methods including the use of antioxidants have been investigated (Wood and Enser, 1997; Gladine et al., 2007; Jang et al., 2008) to maximize growth and yield and to maintain or improve breast meat functional properties. Cranberry (Vaccinium macrocarpon Ait.) is an important commercial crop in the United States and Canada. About 500 million tons are produced every year and cranberries are used as ingredients in more than 700 products for human consumption (Puupponen-Pimiä et al., 2005). Cranberry fruit has received considerable attention for its putative human health benefits. Most of the focus is on the flavonoid constituents due to their relatively high antioxidant activity in various assays. In vitro chemical assays have rated cranberries as having the highest antioxidant values of over 21 fruits, and the overall phenolic content appears to correlate with the level of antioxidant activity (Vinson et al., 2001; Sun et al., 2002). The phenolic classes identified in cranberries include phenolic acids, anthocyanins, flavonols, and flavan-3-ols, which consist of both monomers and the polymer classes of procyanidins and proanthocyanidins (Vinson et al., 2001; Sun et al., 2002). The proanthocyanidins in plants are well known to improve nitrogen nutrition in ruminant animals and as powerful antioxidants with beneficial effects on human health and immunity (Lin et al., 2002). Particular interest is being shown for the proanthocyanidins from cranberry (Foo et al., 2000). Cranberry constituents are known to exert antiadhesion and antimicrobial activities against several pathogenic bacteria and have been suggested to prevent urinary tract infections (Foo et al., 2000; Nogueira et al., 2003; Howell, 2007). The cranberry A-type proanthocyanidins have been recognized for their antiadherence activities against uropathogenic P-type Escherichia coli (Foo et al., 2000) and may play a role in urinary tract health. Cranberry juice extracts were found to inhibit the specific adhesion of Helicobacter pylori to immobilized human mucus, erythrocytes, and cultured gastric epithelial cells (Burger et al., 2000; Zhang et al., 2005; Shmuely et al., 2007). In addition, cranberry may affect biofilm formation via inhibition of extracellular polysaccharide synthesis, which promotes Streptococcus sobrinus (Steinberg et al., 2004; Yamanaka et al., 2004; Labrecque et al., 2006). Limited laboratory research has examined the biological effect of cranberry extract using animal models. The purpose of the present work is to evaluate the growth performance, breast meat quality, gut bacterial numbers, and intestinal health in broiler chickens receiving a commercial cranberry extract through feed.
MATERIALS AND METHODS Broiler Chickens and Housing Twelve hundred Ross 308 one-day-old male broiler chicks were obtained from a local commercial hatchery
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and placed in 24 pens (50 birds per pen). Before placement, all chicks were visually examined for health and inferior chicks were not included in the trial. The clean and disinfected concrete floor was covered with approximately 3 in. (7.6 cm) of clean softwood shavings and the bird density was approximately 0.75 ft2 (0.07 m2) per bird. Ventilation was provided by negative pressure with fans. Heat was provided by gas-fired brooders; water and feed were offered ad libitum through nipple drinkers and tube feeders, respectively. The temperature was initially set at 32°C at d 0 and then was progressively reduced by 1.7°C each week to reach 23°C at 35 d of age. Chicks were exposed to light for 24 h for the first day, 23 h for the second and third days, then 18 h thereafter. The composition of the feed used in this study is presented in Table 1. The starter, grower, and finisher diets were formulated in accordance with the broiler diet used in Western Canada with wheat, barley, and corn as the principal cereals and soybean and canola meals as protein concentrates to meet the NRC nutrient requirements for broiler chickens (NRC, 1994). Analysis for DM, total proteins, soluble carbohydrates, fatty acids, and some of the most common minerals was performed at the Centre de Recherche en Sciences Animales de Deschambault (CRESAD, Deschambault, Quebec, Canada). All experimental procedures performed in this study were approved by the Animal Care Committee of the Pacific Agri-Food Research Center (Agassiz, British Columbia, Canada) according to guidelines described by the Canadian Council on Animal Care (CCAC, 1993).
Study Design The chicks were assigned at random to 4 treatments (6 pens per treatment): a control untreated group without antibiotics and 3 groups fed rations containing the following per kilogram of feed: 160, 80, and 40 mg of a commercial cranberry fruit extract (CFE) supplied by Decas Botanical Synergies (Wareham, MA). No additional anticoccidials or antibiotics were administrated to the birds throughout the trial. The concentrations of CFE were chosen to have a comparative range of the concentrations of some approved antimicrobial agents used in broiler feed (i.e., bacitracin, 55 to 110 mg/kg; salinomycin, 60 mg/kg; and virginiamycin, 11 to 22 mg/ kg). As described by the manufacturer, the CFE used in this study contained at least 30% organic acids, 2 to 3.8% total phenolic compounds, 0.3 to 1% anthocyanins, 0.8 to 1.5% proanthocyanidins, 300 to 435 µg/g of quercetin, and were 100% soluble in water.
Data Collection Chicks were weighed at the start of the trial (d 0) and every week thereafter; feed intake was measured at d 14, 28, and 35 from each pen and was used to determine feed efficiency (g of gain per g of feed). During the trial, all mortalities were removed from the pens
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Leusink et al. Table 1. Composition of the feed used in this study Item, % of inclusion in diet Ingredient Wheat Soy Barley Canola Canola oil Corn Corn gluten Limestone Dicalcium phosphate1 Vitamin-mineral mix2 Lysine Methionine Salt Avizyme3 Calculated energy (kJ/kg) Analyzed nutrient4 DM Ash Protein Fat Glucose Fructose Acid detergent fiber Neutral detergent fiber Ca Mg K P Na Fe Zn Mn
Starter (d 0 to 14)
Grower (d 14 to 28)
Finisher (d 28 to 35)
34.96 23.00 10.00 9.00 8.60 7.00 2.30 1.60 1.60 1.00 0.40 0.18 0.31 0.05 13.16
35.03 0.00 0.00 22.00 7.00 25.00 6.00 1.30 1.50 1.00 0.71 0.09 0.32 0.05 13.35
40.79 0.51 0.00 18.00 7.00 25.00 4.00 1.20 1.40 1.00 0.63 0.10 0.32 0.05 13.44
89.01 6.42 24.84 10.58 19.25 21.95 7.42 13.35 0.99 0.22 0.89 0.80 0.22 0.04 0.02 0.02
88.70 5.75 21.34 10.35 17.28 22.68 7.18 13.72 0.99 0.22 0.55 0.81 0.26 0.04 0.02 0.02
88.02 5.81 19.81 10.35 18.58 24.43 6.62 14.31 1.01 0.22 0.53 0.79 0.26 0.04 0.02 0.02
1A
mixture of mono- and dicalcium phosphate containing 18% calcium and 21% phosphate. per kilogram of diet: vitamin A, 9,000 IU; cholecalciferol, 1,500 IU; vitamin E, 10 IU; vitamin K, 0.5 mg; vitamin B12, 0.007 mg; thiamine, 0.4 mg; riboflavin, 6 mg; folic acid, 1 mg; biotin, 0.15 mg; niacin, 135 mg; pyridoxine, 4 mg; choline chloride, 1,000 mg; dl-methionine, 1,184 mg; ethoxyquin, 125 mg; NaCl, 2 g; manganese sulfate, 60 mg; copper sulfate, 5 mg; selenium (sodium selenium), 0.1 mg; iodine, 0.35 mg; and zinc sulfate, 50 mg. 3Multi-Enzyme System for Wheat-Based Poultry Feed (Halchemix Canada Inc., Toronto, Ontario, Canada) containing 5,000 U/g of xylanase and 1,600 U/g of protease. 4Analyzed nutrient contents on a DM basis determined at the Centre de Recherche en Sciences Animales de Deschambault (CRESAD, Deschambault, Quebec, Canada). 2Supplied
and recorded. Necropsies were performed on all mortalities, which were categorized as early mortality (due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration), physiological (due to sudden death syndrome, ascites, heart failure, and unknown reasons), and culls (due to spondylolisthesis, valgus, and varus deviations of tibiotarsi, rotated tibia, and runts). At d 36 (in the Fraser Valley of British Columbia, most of the broilers are marketed between 35 and 40 d), the broilers were fasted for 12 h and 18 birds (3 per pen) from each treatment were randomly selected (a total of 72 birds) and transported to a commercial slaughterhouse situated at a distance of 80 km. Birds were scalded, plucked, and manually eviscerated. The circumference of the breast and the carcass weight before and after evisceration were obtained. The individual carcasses were then vacuum-packaged and stored at −20°C until they were used for quality tests.
Meat Quality Chickens were partially thawed in a 2°C refrigerator overnight and the left and right breast muscles (pectoralis major and pectoralis minor) were removed and weighed. The left breast muscle samples were used for pH, percentage of lipid, and water content analyses according to the official methods of analysis (Association of Official Analytical Chemists, 1995). Raw finely chopped middle sections of the breasts were used for pH measurement. Two-gram samples were homogenized with 10 mL of distilled deionized water in a blender then transferred to a 50-mL beaker. The pH was measured and recorded in triplicate for every sample at room temperature. Fat was analyzed using the BlighDyer lipid extraction method (Bligh and Dyer, 1959). Samples (4 ± 0.001 g) of chicken meat were cut from the lower tip of the breast. Muscle moisture was de-
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
termined using a vacuum oven (model number 1430, VWR Scientific Products, Batavia, IL) at 70°C under pressure (<100 mmHg) for 18 h (Park and Bell, 2004). The fleshiest part near the top of the right breast muscle was excised and analyzed for color and texture. Color (lightness = L*; redness = a*; and yellowness = b*) of each sample was measured in triplicate using a Hunterlab LabScan XE colorimeter (Hunter Associates Laboratory Inc., Reston, VA) using a D65 illuminant. The instrument was calibrated with a white-and-black tile before analysis in agreement with the International Commission on Illumination (Fletcher et al., 2000). After the color measurements, breast muscle samples were then wrapped in aluminum foil and cooked at 95°C for 20 min and cooled at room temperature. Then, 2 × 2 × 1 cm samples were cut for texture (shear force and hardness) analyses using a TA/XT2 texture analyzer (Stable Microsystems, Surrey, UK) as described previously (Fletcher, 1999). A 3-mm blade was used for texture measurement.
Bacteriological Analyses Ten chicks at d 0 and 2 birds per pen at each sampling time (7, 14, 21, 28, and 35 d of age) were killed by cervical dislocation. Cloacae samples and cecal contents from the 2 killed birds were aseptically collected and transferred to peptone buffer in test tubes and sterile Whirl-Pak plastic bags (Nasco, Fort Atkinson, WI), respectively, for bacteriological culture. The samples were placed on ice and transported to the microbiology laboratory for bacteriological analysis that was carried out the same day. Sample weights were estimated by subtracting the weight of the container without sample from the weight with the samples. From d 7 to 35, bacteriological analyses were performed on a total of 120 cloacae and 120 cecal samples (pooled samples from 2 birds of the same pen constituting 1 sample). The generic E. coli population was estimated using E. coli and coliform Petrifilms (3M, St. Paul, MN) as described previously (Diarra et al., 2007). Enterococcus spp. populations were determined by spreading 10-fold dilutions of samples on KF Streptococcal Agar CM0701 (Oxoid, Nepean, Ontario, Canada) and incubating at 37°C for 48 h (Hayes et al., 2003). Clostridium perfringens was enumerated according to Knarreborg et al. (2002). Briefly, samples were spread on tryptose sulfite agar (Oxoid) supplemented with cycloserine (SR088E, Oxoid) and incubated anaerobically for 24 h at 37°C. Lactobacillus spp. populations were quantified using Lactobacilli MRS Agar (Oxoid) according to the manufacturer’s methods.
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coccidiosis and necrotic enteritis because this disease occurs most commonly in broiler chickens of 2 to 5 wk of age. The intestines were longitudinally opened and intestinal mucosa were scored on a scale of 0 to 3 for necrotic enteritis lesions for each of the upper gut, mid gut, lower gut, and ceca according to the method of Collier et al. (2003). Body weights of killed birds were determined.
Statistical Analysis Statistical analyses were conducted according to a randomized complete block design using the GLM procedure of SAS (2000) with treatment groups as sources of variation and the individual pens as experimental units (6 pens per treatment group). Data from bacterial counts were log-transformed before analysis and the sample origins (ceca and cloacae) and bird ages (d) were included as sources of variation. Least significance difference was used to separate treatments means whenever the F-value was significant. The 0.05 P-value was used to declare significance.
RESULTS Broiler Performance The effects of diet supplementation with CFE on BW, feed intake, feed efficiency, and mortality estimated are presented in Table 2. No significant differences were noted between the treatment groups for all parameters evaluated from d 0 to 28 (P > 0.05). At d 35, the highest feed intake and BW values for birds fed 160 mg of CFE were not statistically significant (P > 0.05). All mortalities and culls were necropsied and on d 35, fatal necrotic enteritis was observed in 1 bird from one pen receiving 80 mg/kg. Most of the mortalities were culls (runts or leg disorders especially in CFE, 80 and 160 mg/kg) or routine physiological conditions including acute death syndrome or ascites (Figure 1). The percentage of mortality in birds in the 40 mg of CFE treatment tended to be lower than for chickens from the other treatments or the control (P = 0.09), possibly due to the decrease of early mortality in this treatment. Early mortality was decreased by 50% with diet containing CFE at 40 mg/kg of feed when compared with the control untreated bird (Figure 1). The highest mortality (11.1%) observed in CFE 160 mg/kg treatment may be due to the increased physiological mortality (sudden death syndrome, ascites, heart failure, and unknown reasons) or an increased number of culled birds of this treatment, or both.
Intestinal Health
Carcass and Meat Quality
At each of 21, 28, and 35 d of age, the intestines of killed birds (2 per pen) were examined for evidence of
Chickens fed CFE at 80 and 160 mg/kg of feed had higher breast circumference and carcass weight (Figure
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Figure 1. Categories of mortalities: early mortality due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration; physiological due to sudden death syndrome, ascites, heart failure, and unknown; culls due to spondylolisthesis, valgus and varus deviations of tibiotarsi, rotated tibia, and runts.
2 A and B), but these effects were not statistically significant (P > 0.05). The characteristics of the breast meat from the broilers fed different concentrations of CFE are presented in Table 3. The means of pH values, fat content, and moisture were 6.39, 2.11, and 76.25%, respectively.
The lightness (L*) values ranged from 48.7 (control) to 50.38 (40 mg/kg), the yellowness (b*) values ranged from 13.50 (40 mg/kg) to 14.14 (160 mg/kg), and the redness (a*) values ranged from 2.71 (40 mg/kg) to 3.56 (80 mg/kg). Meat from the control birds and birds fed 160 mg of CFE/kg of feed had the highest values
Table 2. Performance of broiler chickens fed diets containing cranberry fruit extract (CFE) at different concentrations (0 to 160 mg/kg of feed) Concentration of CFE (mg/kg of feed) Parameter BW (g) Initial 0 to 14 d 14 to 28 d 28 to 35 d Feed intake (g) 0 to 14 d 14 to 28 d 28 to 35 d 0 to 35 d Feed efficiency3 (feed:gain) 0 to 14 d 14 to 28 d 28 to 35 d 0 to 35 d Total mortality (%) 0 to 35 d
0
40
80
160
SEM1
P-value2
41.5 399.8 1,208.3 1,619.8
41.3 395.9 1,183.1 1,573.8
41.2 402.9 1,199.1 1,611.7
41.3 402.9 1,200.4 1,637.9
0.185 5.466 19.771 32.764
0.65 0.77 0.83 0.56
430.7 1,386.0 951.2 2,767.8 1.2 2.0 2.4 1.8 6.9
425.7 1,369.4 950.5 2,745.8 1.2 2.0 2.8 1.9 5.2
420.5 1,387.4 961.3 2,769.2 1.2 2.0 2.6 1.9 9.5
425.4 1,389.0 1,003.4 2,817.8 1.2 2.0 2.5 1.9 11.1
6.748 23.805 20.463 42.181 0.023 0.026 0.019 0.028 1.803
0.77 0.93 0.23 0.67 0.59 0.55 0.52 0.67 0.09
1Cranberry fruit extract was administrated via feed from 0 to 35 d. Data represent means ± SE of 6 replicates/ treatment (n = 6 pens of at least 30 chickens/pen) arranged in a completely randomized block design. 2P-value obtained by ANOVA. 3Grams of feed per gram of BW gain.
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
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Bacteriological Analyses In general, the populations of generic E. coli, C. perfringens, Enterococcus spp., and Lactobacillus spp. were higher (P < 0.05) in the cecal samples than in the cloacal samples (Table 4). The log C. perfringens numbers per gram of sample in ceca were greater (P < 0.05) at d 7 (4.5), 28 (4.2), and 35 (4.5) than at d 14 (2.1) to 21 (2.4), but no significant treatment effects were noted (P > 0.05). The population of this bacterium remained lower than those of other bacteria (P < 0.05). The populations of E. coli or Enterococcus spp. were highest on d 7 and slowly declined thereafter. There was no significant effect of any treatment on E. coli population in any sample (cloacae or ceca, P > 0.05). At d 28, counts of Enterococcus spp. in cecal and cloacae samples were significantly lower (P < 0.05) in birds receiving 80 mg of CFE/kg of feed than the other groups. The counts of Lactobacillus were not influenced by the treatments and remained relatively stable in ceca and cloacae from d 7 to 35.
Intestinal and General Health
Figure 2. Breast circumference and carcass weight of broiler chicken fed 0 to 160 mg/kg of cranberry fruit extract in feed.
for texture. However, no statistically significant differences in fat content (%), moisture content (%), pH, texture (hardness and shear force), and color (L*, b*, and a*) were observed between the treatment groups (P > 0.05).
Gross observations of the health status and intestinal mucosa were performed weekly, beginning at 21 d of age. In general, health and body condition were good, and bursal size and anatomical features were normal in all birds examined. In most birds, intestinal mucosa was normal on gross observation. However, on d 28 and 35, some birds were seen with rough, pitted mucosa involving the duodenum and the upper part of the jejunum, proximal to Meckel’s diverticulum. In birds examined on d 21, 28, and 35, small foci of necrosis from approximately 1 to 5 mm in diameter (lesions score of 1) in the upper intestine were observed in 7 (4.9%) of the 144 sample birds, none on d 21, 2 (1 in each of 80 and 160 mg/kg treatments) on d 28, and 5 (2, 1, and 2 in the control, 80, and 160 mg/kg treatments, respectively). None of the birds receiving the lower CFE concentration (40 mg/kg) were found to have intestinal lesions.
Table 3. Characteristics of breast meat from broiler chickens fed diets containing cranberry fruit extract (CFE) at different concentrations (0 to 160 mg/kg of feed) Concentration of CFE (mg/kg of feed) Meat characteristic Fat (%) pH Moisture (%) Lightness (L*) Yellowness (b*) Redness (a*) Hardness (N) Shear force (Nm)
0
40
80
160
SEM1
P-value2
2.01 6.39 76.19 48.70 14.03 2.88 35.74 0.29
2.03 6.37 76.26 50.38 13.50 2.71 33.49 0.26
2.2 6.37 76.17 48.65 13.83 3.56 33.40 0.26
2.22 6.41 76.38 49.90 14.14 3.47 35.82 0.30
0.189 0.025 1.110 0.728 0.315 0.314 2.100 0.023
0.35 0.63 0.76 0.14 0.79 0.18 0.41 0.81
1Cranberry fruit extract was administrated via feed from 0 to 35 d. Data represent means ± SE of 6 replicates/ treatment (n = 6 pens of 3 carcasses/pen) arranged in a completely randomized block design. 2P-value obtained by ANOVA.
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Table 4. Log10 bacterial number per gram of sample obtained from ceca and cloacae samples from broiler chickens fed diets containing cranberry fruit extract (CFE) at 0, 40, 80, and 160 mg/kg of feed1 Concentration of CFE (mg/kg) Bacteria2 Escherichia coli d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Enterococcus spp. d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Lactobacillus spp. d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL Clostridium perfringens d 7 CE CL d 14 CE CL d 21 CE CL d 28 CE CL d 35 CE CL a,bValues
0
40
80
160
SEM3
P-value4
8.9 7.1
8.9 7.2
8.9 7.1
8.8 7.3
0.26
0.989
8.2 6.3
8.2 6.4
8.1 6.8
8.3 6.4
0.244
0.879
7.4 6.4
7.1 6.4
6.6 6.7
7.5 6.3
0.34
0.849
6.3 6.1
6.7 6.5
6.6 6.2
7.2 6.2
0.269
0.288
7.0 6.4
6.8 6.6
7.2 6.2
7.0 6.5
0.302
0.997
8.5 7.6
8.4 7.8
8.4 7.7
8.4 7.6
0.256
0.986
6.8 5.9
6.5 5.6
7.2 5.8
6.7 5.7
0.301
0.44
6.2 5.8
5.6 5.4
5.6 5.4
6 5
0.263
0.175
5.3a 5.4
5.5ab 5.7
5.0a 5.2
5.9b 5.7
0.182
0.001
5.7 5.6
5.0 5.5
5.9 5.3
5.9 5.9
0.256
0.129
8.6 7.9
8.5 7.6
8.6 7.5
8.6 7.5
0.082
0.064
8.9 7.9
8.9 8.1
8.4 7.8
9.1 7.9
0.193
0.159
8.0 7.3
7.8 7.1
8.2 7.1
8.2 7.4
0.217
0.589
8.2 7.0
8.3 8.2
7.9 8.3
8.5 8.4
0.59
0.56
8.4 8.3
8.6 8.5
8.5 7.7
8.6 8.2
0.178
0.17
4.5 1.8
4.7 2.6
4.8 2.1
4.4 1.8
0.512
0.626
2.1 1.1
2.6 0.5
2.7 1.5
2.7 0.6
0.589
0.828
2.4 1.8
1.7 2.2
1.1 1.4
2.9 2.1
0.506
0.1
4.2 3.7
4.4 3.7
4.4 3.1
4.5 3.4
0.626
0.961
4.5 3.1
4.4 3.2
4.9 4.1
4.4 3.1
0.848
0.769
within a row with different superscripts are statistically different (P < 0.05). of colony-forming units per gram of sample. 2CE = ceca sample; CL = cloacae sample. According to ANOVA, statistically significant differences (P < 0.05) were observed between bacterial numbers from different locations (ceca vs. cloacae). 3Standard error of means of 6 replicates/treatment (n = 6 pens of 2 chickens/pen) arranged in a completely randomized block design. 4P-value obtained by ANOVA. 1Mean
EFFECT OF DIETARY CRANBERRY FRUIT EXTRACT IN BROILER CHICKEN
No mortality due to necrotic enteritis was seen during necropsies. There was no clinical evidence of infectious disease during the feeding trial. No significant effect of treatments on intestinal and general health was noted.
DISCUSSION Because cranberries have received considerable attention for their putative human health benefits (Vinson et al., 2001; Sun et al., 2002), we evaluated the use of cranberry extracts as alternatives to traditional antibiotics in broiler chicken production. We did not find that growth performance of broiler chickens was statistically improved when they were fed diets containing commercial CFE at concentrations of 40, 80, and 160 mg/kg of feed. The lack of significance of our observed effects on growth performance was not surprising because we previously reported no significant effects of traditional approved individual antimicrobial agents including salinomycin and bacitracin on performance using our broiler model (Diarra et al., 2007). This may be due to the high hygienic and biosecurity practices we used before and throughout the experimental protocol. Mortality in research trials is often high, possibly related to handling. The CFE used at 40 mg/kg seemed to decrease early mortality due to yolk sac infection, omphalitis, crossbeak, starve-out, and dehydration. For animal care concerns, higher mortalities were due to culled birds and acute deaths mainly in treatment with 80 and 160 mg/kg of CFE. The severity of necrotic enteritis within the population was not extensive. Only about 5% of birds examined from d 21 to 35 presented a lesion score of 1. We can speculate that most cases observed during sampling likely would have healed and the affected birds would have survived with some loss of performance. Only 1 case of fatal necrotic enteritis was seen. Further work is needed to confirm and explain the effect of CFE used at different concentrations in diets on the mortality and the overall bird health. We did not estimate the cost (which was beyond the scope of the study) of the use of CFE in broiler diets. However, it would be interesting to investigate the effects on production parameters of similar or greater doses of CFE in commercial farming conditions because the beneficial effects of growth-promoting agents have been reported under stress and poor management conditions often found in commercial poultry production (Sims et al., 2004). In recent years, consumers have become concerned about poultry meat quality. Several factors including bird sex, age at slaughter, strain, method of processing, exposure to chemicals, irradiation, and freezing have been shown to affect poultry meat quality (Froning, 1995; Le Bihan-Duval et al., 1999; Zhou et al., 2009). It has been reported that proximate composition of nonfrozen breast meat from broilers fed diets supplemented with medicinal herb extracts mix was not affected, but the total phenol content of the breast meat from these birds receiving supplemented diets was significantly dif-
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ferent and displayed higher antioxidant activities when compared with those from the control diet (Jang et al., 2008). In our study, we used frozen samples and the CFE in feed was not found to affect the broiler meat quality parameters that were evaluated. Whether freezing might have any adverse effects on tested meat samples is unknown; however, it would be interesting to evaluate fresh meat characteristics. Feed supplementation with oregano extracts resulted in greater a* and b* values in chicken meat than in the meat of control chickens (Young et al., 2003). Larrain et al. (2008) showed in familial hypercholesterolemic pigs that continuous feeding with cranberry juice powder altered the time-course changes of bacon color. These authors reported that lipid oxidation, but not TBA reactive substances, were significantly high in frozen meat samples from cranberry-fed pigs. In our study, lipid peroxidation was not investigated. However, our findings suggest the need for more rigorous studies on the effects of cranberry feeding on overall broiler meat quality including sensory tests after processing and storage. In our study, the cranberry extracts used did not affect intestinal health when compared with the nonsupplemented diet. Escherichia coli, Enterococcus spp., Clostridium spp., and Lactobacillus spp. are normal inhabitants of the gastrointestinal tract of the chicken. Feed supplementation with cranberry extract had no effect on the population of these commensal bacteria species in the ceca and cloacae of the broiler chickens at d 7 to 21 and 35. However, at d 28, in birds receiving CFE at 80 mg/kg of feed, the populations of Enterococcus spp. in cecal and cloacal samples were significantly lower, whereas greater numbers of this bacterium were observed in chickens receiving feed containing CFE at 160 mg/kg. The mechanism underlining this observation on the enterococci number is not known. However, because enterococci are lactic acid-producing bacteria, we can speculate that the increase in their population might translate into elevated lactic acid levels that might affect the growth of some other bacteria. Some Enterococcus spp. are used in meat and dairy processing as starter cultures (Giraffa, 2003). Bacteriocin producing Enterococcus was isolated from chickens, suggesting the possible development of such isolates as a probiotic that could reduce the burden of pathogens in poultry (Strompfová and Lauková, 2007). On the other hand, Enterococcus spp. are ubiquitous commensal bacteria, but some strains of enterococci, particularly Enterococcus faecium and Enterococcus faecalis, are important in food safety and public health due to their potential to cause disease and their ability to acquire multiple antibiotic-resistant genes. Much more work is needed to investigate the potential in vivo effects of cranberry extract on the gut microflora of poultry. The consequences of poultry production for the environment, food safety, and animal welfare issues are now part of consumers’ opinions and demands. Replacement strategies based on novel or alternative feeding practices without antibiotics are favored by the industry.
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This study did not find significant effects of 3 levels of cranberry extract on bird performance. We evaluated 1 raw commercial sample. It would be interesting to evaluate samples from different sources as well as subfractions (derivatives) of such samples. Although our studied cranberry extract did not show significant beneficial effects in broilers, more investigation of cranberry fruit compounds could lead to the development of feeding strategies for chicken to improve bird health and on-farm food safety that will reduce the use of antibiotics as growth promoters.
ACKNOWLEDGMENTS This work was financially supported by Agriculture and Agri-Food Canada and the Canadian Cranberry Growers Coalition (Abbotsford, British Columbia, Canada). We thank L. Struthers and M. Fraser (Pacific Agri-Food Research Centre) and William R. Cox (Canadian Animal Health Management Services Ltd., Chilliwack, British Columbia, Canada) for assistance. We also thank Decas Botanicals for providing cranberry extract and acknowledge the help of D. Mah and S. Li.
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