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Effectiveness of dietary xylo-oligosaccharides for broilers fed a conventional corn-soybean meal diet
Journal of Integrative Agriculture 2015, 14(10): 2050–2057 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
Effectiveness of dietary xylo-oligosaccharides for broilers fed a conventional corn-soybean meal diet SUO Hai-qing1, LU Lin1, XU Guo-hui2, XIAO Lin2, CHEN Xiao-gang2, XIA Rui-rui3, ZHANG Li-yang1, LUO Xu-gang1 1
Mineral Nutrition Research Division, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 2 High-Technology Development Zone, Functional Sugar Engineering Research Center of Shandong Province,Yucheng 251200, P.R.China 3 High-Technology Development Zone, Shandong Key Laboratory of Straw and Stover Biorefinement Technologies, Yucheng 251200, P.R.China
Abstract An experiment was conducted to investigate the effect of dietary supplementation of xylo-oligosaccharides (XOS) on growth performance, meat quality, immune functions, duodenal morphology and intestinal microbial populations of broilers fed a conventional corn-soybean meal basal diet. A total of 450 1-day-old commercial Arbor Acres male broiler chicks were randomly allocated by bodyweight to 1 of 5 treatments with 6 replicate cages (15 broilers per cage) for each of 5 treatments in a completely randomized design. Chicks were fed the basal corn-soybean meal diets supplemented with 0, 25, 50, 75, or 100 mg of XOS kg–1 of diet, respectively, for an experimental duration of 42 days. The results showed that supplementation of XOS affected feed conversion rate (feed/gain, F/G) during days 22–42 and 1–42 (P<0.03), drip loss in thigh muscle (P=0.02), and duodenal crypt depth (P=0.005) on day 42, but had no effect (P>0.05) on all other measured indices. The chicks fed the diet supplemented with 100 mg of XOS kg–1 had the lowest (P<0.05) F/G and drip loss in thigh muscle. The drip loss in thigh muscle decreased linearly (P=0.003) as the supplemented XOS increased. Duodenal crypt depth decreased (P<0.05) at the supplemental level of 75 mg of XOS kg–1. The results indicate that dietary supplementations of 75 and 100 mg of XOS kg–1 are beneficial to broilers fed a conventional corn-soybean meal diet. Keywords: xylo-oligosaccharide, effectiveness, F/G, drip loss, broiler chick
1. Introduction
Received 24 March, 2015 Accepted 24 June, 2015 SUO Hai-qing, Mobile: +86-15652743313, E-mail: suohaiqing2008 @1 26.com; Correspondence LUO Xu-gang, Tel: +86-1062816012, Fax: +86-10-62810184, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61101-7
Xylo-oligosaccharides (XOS) are sugar oligomers mainly produced from the hydrolysis of xylan, the major component of plant hemicelluloses, which are heteropolysaccharides with homopolymeric backbone of xylopyranose moieties (Saha 2003). XOS have a ramified structure containing 2–7 xylose units linked by β-(1,4) bonds and with a variety of substituents such as acetyl groups, uronic
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acids, and arabinose units (Caparros et al. 2007). They are developed functional oligosaccharides because they could affect the host by selectively stimulating the growth or activity of beneficial bacteria in the colon. Thus XOS have been used as prebiotics for improving the intestinal health (Grbson and Roberfroid 1995). The main beneficial biomedical and biofunctional effects of XOS are: (1) reducing cholesterol level, (2) maintaining the gastrointestinal health, (3) having cytotoxic effects on human leukemia cells, and (4) having beneficial effect on type 2 diabetes mellitus (Swennen et al. 2006; Mussatto and Mancilha 2007; Sheu et al. 2008). In recent years, XOS have been studied extensively as prebiotics instead of antimicrobial agents in pigs and ruminants (Yoshioka et al. 1994; Howard et al. 1995; Younes et al. 1995; Hesta et al. 2001; Yang 2007; Wang et al. 2008). Results from the previous studies indicated that dietary supplementation of XOS could improve growth performance, immune functions, and modify the intestinal morphology and flora in animals. However, only few researches were carried out to evaluate the effectiveness of dietary supplementation of XOS as prebiotics for broilers, and inconsistent results were observed (Dang 2004; Chen 2009; Shi 2010). Dang (2004) reported that XOS at supplemental levels of 35–105 mg kg–1 increased cecal villus height and reduced its crypt depth, but had no effect on growth performance and the intestinal flora of broilers. Shi (2010) demonstrated that addition of 52.5 mg of XOS kg–1 in the diet decreased drip loss in breast and thigh muscles, but had no effect on growth performance and the intestinal morphology and flora of broilers. Chen (2009) observed no effect of XOS on growth performance, immune functions and intestinal flora of broilers at supplemental levels of 35–70 mg kg–1. The above disparities might be majorly due to the differences in the species of broilers and supplemental levels of XOS.
Therefore, the following study was conducted to further investigate the effect of dietary supplementation of XOS on growth performance, meat quality, immune functions, duodenal morphology and intestinal microbial populations of broilers.
2. Results 2.1. Growth performance of broilers Supplementation of XOS had no effect (P>0.24) on average daily gain (ADG), average daily feed intake (ADFI), mortality on days 1–21, 22–42 and 1–42, and feed conversion rate (feed/gain, F/G) on days 1–21, but affected (P<0.03) F/G on days 22–42 and 1–42 (Table 1). A linear response (P<0.05) was observed between supplementation of XOS and F/G. As supplemental XOS level increased, the F/G decreased linearly (P=0.02) on days 22–42 and 1–42. The chicks fed the diets supplemented with 100 mg of XOS kg–1 had the lowest (P<0.05) F/G on days 22–42 and 1–42 compared with those fed all other 4 treatment diets, and there were no differences (P>0.05) among all other 4 treatments (Table 1).
2.2. Meat quality of broilers Supplementation of XOS had no effect (P>0.34) on the meat quality of breast and thigh muscles except for drip loss (P=0.02) in leg muscle for broilers at 42 days of age (Table 2). A linear response (P=0.003) was observed between supplemental XOS level and drip loss in leg muscle. As supplemental XOS level increased, the drip loss in thigh muscle decreased linearly (P=0.003), with the lowest drip loss at the supplemental level of 100 mg of XOS kg–1 (Table 2).
Table 1 Effect of dietary xylo-oligosaccharides (XOS) level on the growth performance and mortality of broiler chicks1) Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value Linear2) Quadratic3) 1)
ADG (g d–1) 34 33.5 34.6 35.5 34.6 0.8 0.46
Days 1–21 ADFI F/G (g d–1) (g g–1) 48.4 1.41 48.7 1.45 50.3 1.46 51.3 1.44 49.4 1.43 0.9 0.02 0.25 0.64
Mortality (%) 1.11 5.56 0.00 1.11 3.33 0.36 0.66
ADG (g d–1) 78.9 80.3 78.4 79.3 79.6 1.5 0.92
Days 22–42 ADFI F/G (g d–1) (g g–1) 151 1.91 a 151 1.89 a 149 1.90 a 152 1.92 a 145 1.83 b 2 0.02 0.31 0.01 0.02 0.08
Mortality (%) 0 0 0 0 0 0 0
ADG (g d–1) 56.9 57.3 56.9 57.9 57.6 0.8 0.88
Days 1–42 ADFI F/G Mortality (g d–1) (g g–1) (%) 99.8 1.75 a 1.11 100.1 1.75 a 5.56 99.6 1.75 a 0.00 101.7 1.76 a 1.11 97.4 1.69 b 3.33 1.29 0.01 0.36 0.25 0.02 0.66 0.02 0.12
ADG, average daily gain; ADFI, average daily feed intake; F/G, feed/gain rate. 2) Linear effects of added XOS levels. 3) Quadratic effects of added XOS levels. Data represent the means of 6 replicate cages (n=6). Data with different letters within the same column differ significantly (P<0.05). The same as below.
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2.3. Immune functions of broilers Supplementation of XOS had no effect (P>0.08) on serum antibody titers against Newcastle disease and blood T-lymphocyte proliferation at both 21 and 42 days of age and serum antibody titers against H5N1 at 42 days of age (Table 3).
2.4. Duodenal morphology and intestinal microbial populations of broilers As shown in Table 4, supplemental XOS level had no effect
(P>0.05) on duodenal villus height and width, and villus height/crypt depth, but affected (P=0.005) duodenal crypt depth. Linear and quadratic responses (P<0.05) were observed between supplemental XOS level and duodenal crypt depth. The chicks fed the diets supplemented with 75 mg of XOS kg–1 had lower (P<0.05) duodenal crypt depth than those fed all other 4 treatment diets, and there were no differences (P>0.05) among all other 4 treatments. Since no flora was observed in the jejunum, the data only in the cecum were listed in Table 5. Supplemental XOS level had no effect (P>0.38) on the populations of
Table 2 Effect of dietary XOS level on meat quality of broiler chicks at 42 days of age Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value Linear Quadratic 1)
Breast muscle color1)
Thigh muscle color1)
L*
a*
b*
L*
a*
b*
36.0 35.7 35.3 35.4 35.6 0.7 0.96
2.09 3.14 3.46 2.69 2.58 0.49 0.35
5.19 4.56 4.81 4.70 4.76 0.28 0.59
41.4 40.7 42.0 41.2 41.0 0.8 0.81
3.62 4.15 4.01 4.68 4.08 0.56 0.77
6.28 6.00 6.75 6.13 6.26 0.36 0.64
Shear force (kg) Breast Thigh muscle muscle 2.66 1.14 3.11 1.03 3.22 1.08 2.78 1.11 2.80 0.98 0.50 0.91 0.92 0.78
Drip loss (%) Breast Thigh muscle muscle 3.92 6.32 a 3.56 5.57 ab 3.51 4.70 bc 3.64 5.33 abc 3.37 3.96 c 0.42 0.47 0.91 0.02 0.003 0.88
0-h pH value Breast Thigh muscle muscle 6.69 6.90 6.75 6.90 6.72 6.91 6.71 6.95 6.70 6.92 0.05 0.04 0.92 0.89
L*, lightness; a*, redness; b*, yellowness.
Table 3 Effect of dietary XOS level on immune functions of broiler chicks Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value 1)
Serum Newcastle disease (ND) antibody titers (Log2) Day 21 Day 42 5.3 2.5 5.0 2.3 5.2 2.3 4.7 2.7 4.3 2.5 0.65 0.27 0.82 0.90
Serum H5N1 antibody titers (Log2) Day 42 5.67 5.83 6.33 5.50 5.50 0.47 0.70
Blood T-lymphocyte proliferation (SI)1) Day 21 Day 42 2.22 1.31 2.73 1.65 1.68 2.11 1.38 1.57 1.36 1.38 0.37 0.30 0.09 0.48
SI was calculated by dividing the mean optional density (OD) of stimulated cells by OD of unstimulated cells.
Table 4 Effect of dietary XOS level on duodenal morphology and cecal flora amount of broiler chicks at 42 days of age Duodenal morphology Added XOS (mg kg–1)
Villus height (μm)
Villus width (μm)
Crypt depth (μm)
0 25 50 75 100 Pooled SE P-value Linear Quadratic
1 164 1 081 1 073 987 1 191 47 0.06
148 102 119 137 110 14 0.15
217 a 171 a 180 a 140 b 192 a 12 0.005 0.04 0.01
Cecal flora amount (wet basis)
Villus height/ Crypt depth 5.39 5.67 6.44 6.47 6.32 0.42 0.27
Escherichia Lactic acid (Lactic acid bacteria+ Salmonella Bifidobacterium coli bacteria Bifidobacterium)/Total –1 –1 (Log10CFU g ) (Log10CFU g ) (Log10CFU g–1) (Log10CFU g–1) analyzed bacteria 6.45 6.27 6.57 6.33 6.87 0.24 0.39
4.52 4.34 4.69 4.49 4.44 0.10 0.40
6.31 6.64 6.89 6.77 6.50 0.26 0.55
6.81 6.90 7.01 6.59 6.36 0.27 0.49
0.55 0.56 0.55 0.55 0.54 0.01 0.72
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Table 5 Composition and nutrient levels of the basal diets for broilers1) Item Ingredients (%) Corn Soybean meal Soybean oil CaHPO4 Limestone NaCl Methionine Micronutrients2, 3) Cornstarch+XOS4) Total Nutrient ME (MJ kg–1) Crude protein (%)5) Lysine (%) Methionine (%) Methionine+cysteine (%) Calcium (%)5) Total phosphorus (%)5) Nonphytate phosphorus (%)5) XOS
Days 1–21
Days 22–42
56.20 35.94 3.50 2.10 1.23 0.30 0.23 0.33 0.20 100
56.55 36.24 3.50 1.47 1.39 0.30 0.22 0.20 0.22 100
12.80 22.19 1.11 0.55 0.90 1.04 0.79 0.45 –
12.94 19.95 1.11 0.44 0.79 0.87 0.59 0.35 –
1)
As-fed basis. Provided per kilogram of diet for days 1–21: vitamin A (all-trans retinol acetate) 15 000 IU, cholecalciferol 4 500 IU, vitamin E (all-rac-α-tocopherol acetate) 24 IU, vitamin K3 3.0 mg, vitamin B1 3.0 mg, vitamin B2 9.6 mg, vitamin B6 3.0 mg, vitamin B12 0.018 mg, pantothenic acid calcium 15.0 mg, niacin 39.0 mg, folic acid 1.5 mg, biotin 0.15 mg, choline 700 mg, Cu (CuSO4·5H2O) 8 mg, Mn (MnSO4·H2O) 110 mg, Fe (FeSO4·7H2O) 60 mg, Zn (ZnSO4·7H2O) 60 mg, I (KI) 0.35 mg, Se (Na2SeO3) 0.15 mg. 3) Provided per kilogram of diet for days 22–42: vitamin A (all-trans retinol acetate) 10 000 IU, cholecalciferol 3 400 IU, vitamin E (all-rac-α-tocopherol acetate) 16 IU, vitamin K3 2.0 mg, vitamin B1 2.0 mg, vitamin B2 6.8 mg, vitamin B6 2.0 mg, vitamin B12 0.012 mg, pantothenic acid calcium 10 mg, niacin 26 mg, folic acid 1.0 mg, biotin 0.1 mg, choline 500 mg, Cu (CuSO4·5H2O) 8 mg, Zn (ZnSO4·7H2O) 40 mg, Mn (MnSO4·H2O) 80 mg, Fe (FeSO4·7H2O) 40 mg, I (KI) 0.35 mg, Se (Na2SeO3) 0.15 mg. 4) XOS product added in place of equivalent weights of corn starch. 5) Measured values. Each value based on triplicate determin ations. –, not detectable. 2)
E. coli, Salmonella, lactobacilli, Bifidobacterium or the ratio of microbiota (lactobacilli+bifidobacteria) to total analyzed bacteria in the cecum at 42 days of age.
3. Discussion In the present study, we found that dietary supplementation with 100 mg of XOS kg–1 improved feed conversion rate on days 22–42 and 1–42. This supported that XOS directly arrived at the mucosa of the small intestine to promote the absorption of nutrients (Orban et al. 1997), and also XOS could improve health status of animals (Grbson and Roberfroid 1995). However, no effect of XOS on growth performance of broilers was observed in previous studies
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(Dang 2004; Chen 2009; Shi 2010). Such disparities might be mainly due to the differences in supplemental levels of XOS, and XOS compositions. Higher XOS supplemental levels (0–105 mg kg–1) were used in our and Dang’s (2004) studies, while lower XOS supplemental levels (0–70 mg kg–1) were used in the studies of Chen (2009) and Shi (2010). The XOS used in the present study contain 2–7 xylose units, but the XOS used in the studies of Dang (2004), Chen (2009) and Shi (2010) contain 2–5 xylose units. Therefore, the results from our present study suggested that the XOS containing 2–7 xylose units and higher supplemental level of XOS might be beneficial for improving feed conversion rate of broilers. Sharon and Janssens (2001) showed that XOS could help to slow the absorption of antigen and increase the titer of antigen so as to improve cellular and humoral immunities. In the present study, we found that supplemental XOS level had no effect on immune function, perhaps because the chicks were in a relatively comfortable environment which did not exert a threat to their immune systems during the whole experiment. Water-holding capacity was an important index for evaluating meat quality (Otto et al. 2006). Savage et al. (1990) reported that moisture loss could cause the loss of soluble flavor substance in meat. Luciano et al. (2009) also found that moisture loss could take away the heme in meat. Therefore, the status of moisture is very important for the physical form, flavor and color of meat. In the present study, we found that the addition of 100 mg of XOS kg–1 improved the water-holding capacity of thigh muscle. Shi (2010) also reported that the addition of 52.5 mg of XOS kg–1 decreased drip loss in breast and thigh muscles of broilers. The improvement of the water-holding capacity of thigh muscle might be related to the increased antioxidant capability induced by XOS (Zhuang 2007). However, the mechanisms remain unclear and need to be further studied in the future. The intestine villus height, width and crypt depth were important indices for measuring the functions of intestinal digestion and absorption. It is reported that XOS could modify intestinal morphology that is associated with its functions in increasing the absorption of short-chain fatty acids (Yoshioka et al. 1994; Howard et al. 1995). Crypts are considered as villus factories as they contain stem cells. Deeper crypts indicate faster tissue turnover and higher nutrient demand in new tissue. Reduced crypt depth leads to a decreased endogenous secretion and an increased nutrient absorption, disease resistance and performance (Cook and Bird 1973). Yang (2007) found that XOS did not affect duodenal villus height and crypt depth of piglets. Dang (2004) reported that the addition of 52.5 mg of XOS kg–1 increased cecal villus height and reduced its crypt depth of broilers. In the
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present study, we found that the addition of 75 mg of XOS kg–1 decreased duodenal crypt depth of broilers, suggesting that XOS indeed have a beneficial effect on broilers. Recent studies have shown that XOS had a bifidogenic effect on the intestinal tract and could maintain the balance of bacteria in the intestinal tract (Chung et al. 2007; Grootaert et al. 2007). Moura et al. (2007) reported that XOS had a potential prebiotic effect on lactobacilli in the piglet intestinal tract but not on bifidobacteria, so as to produce an intestinal microbiota pattern more similar to the pattern of suckling piglets. In the present study, we found that XOS had no effect on the intestinal flora of broilers, which was consistent with the results reported in previous studies (Dang 2004; Chen 2009; Shi 2010). The highest dietary level of XOS supplemented in this study was 100 mg of XOS kg–1, and the effect of supplemental levels over 100 mg of XOS kg–1 was not studied. Based on our results, the effect of higher supplemental XOS levels on broilers may need to be further investigated in the future.
4. Conclusion In conclusion, the results from the present study indicate that the addition of 100 mg XOS kg–1 improves feed conversion rate and water-holding capacity of the thigh muscle of broilers; the addition of 75 mg of XOS kg–1 decreases duodenal crypt depth of broilers. Therefore, dietary supplementations with 75 and 100 mg of XOS kg–1 are beneficial to broilers fed a conventional corn-soybean meal diet.
5. Materials and methods 5.1. Birds All experimental procedures were approved by the Office of the Beijing Veterinarians, China (Li et al. 2011). A total of 450 1-day-old commercial Arbor Acres male broiler chicks were randomly allotted by bodyweight (BW) to 5 treatments. Each treatment contained 6 replicate cages and 15 birds per cage in a completely randomized design. Broilers were housed in electrically heated, thermostatically controlled rooms (100 cm×50 cm×45 cm) with fiberglass feeders and maintained on a 24-h constant light schedule. They were allowed ad libitum access to the experimental diets and tap water that contained no detectable levels of XOS. Broilers were vaccinated with the Newcastle vaccine VA/GA at 7 and 24 days of age, and with the H5N1 vaccine at 11 and 28 days of age, respectively. Feed consumption and BW gain of the chicks in each replicate cage were recorded at the end of 21 and 42 days of age. Whenever a bird was found dead, the feed for the cage of the dead bird was weighed immediately. According to the initial feed weight and the last weight for
the cage of dead bird, the feed intake of the dead bird could be calculated and deducted from the final feed intake of the cage of the dead bird.
5.2. Diets The basal corn–soybean meal diet (Table 5) was formulated to meet or exceed the nutrient requirements for starter broilers (NRC 1994). Dietary treatments included the basal corn-soybean meal diets supplemented with 0 (control), 25, 50, 75 or 100 mg of XOS kg–1. The XOS were supplied by Shangdong Longlive Bio-Technology Co. Ltd, China, and have 2–7 xylose units and a purity of about 35%. The remaining 65% are 15% monosaccharide plus 50% maltodextrin used as excipient, and they should have no effect on broilers. A single batch of basal mash feed was mixed at first and then divided into 5 aliquots according to the experimental treatment arrangement. Each supplemental XOS level was mixed with corn-starch to the same weight and then mixed with each aliquot of the basal diet. The diets were fed in mash form.
5.3. Sample collections and preparations The feed ingredients and diets samples from all the treatments were taken and submitted for CP, Ca, and P analyses before the initiation of the trial to confirm CP, Ca, and P contents in diets. At both 21 and 42 days of age, 2 chicks were chosen from each of 30 replicate cages according to average BW. Whole blood samples were taken into heparinized tubes from each of 2 birds via wing vein puncture after 12 h fasting for T-lymphocyte proliferation analysis. Another blood sample was collected also into tubes without anticoagulant, and then was centrifuged to harvest serum samples for analyses of antibody titers against Newcastle and H5N1 vaccines. At 42 days of age, the bled birds were weighed individually, and slaughtered according to the welfare of animal slaughter regulations of China. Carcass weight was measured after defeathering. Heads, necks, and feet were removed from the broilers, and then carcasses were eviscerated and weighted to calculate the percentage of eviscerated yield. Dressing percentage was calculated by dividing the carcass weight by live BW. Abdominal fat, the left breast and thigh muscles, thymus, bursa of fabricius and spleen were taken and weighed to determine the percentages of abdominal fat, breast and thigh muscles, thymus, bursa of fabricius and spleen. The breast and thigh muscles were used to determine the color of meat 45 min after slaughter, and they were then placed in a plastic bag and stored at 4°C for 24 h to measure the pH value, respectively. The percentages of abdominal fat, thymus, bursa of fabricius and spleen were calculated using the weights of abdominal fat,
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thymus, bursa of fabricius and spleen divided by the live BW; the percentages of breast and thigh muscles were calculated by dividing the breast or thigh weight by eviscerated weight. The right breast and thigh muscles were removed and weighed, placed separately in a plastic bag, and stored at 4°C for 24 h. They were subsequently taken out and wiped with filter paper and weighed to determine the drip loss of the muscle and then cooked to determine the shear force of the muscle. Samples were pooled together based on replicate cage. One chick was chosen also from each of 30 replicate cages according to the cage average BW. They were killed and their duodenums (0.5–1 cm) were taken and fixed in 10% formalin after washed off with physiological sodium chloride solution for the analyses of duodenal morphology. Their jejunum and cecum which contained contents were ligatured, removed, and then samples of the contents from jejunum or cecum were collected immediately into plastic bags under CO2, sealed, and stored in 4°C for immediate analyses of microbial populations.
5.4. Sample analyses Determinations of feed ingredients and diets The concentrations of crude protein (CP) and phosphorus (P) in feed ingredient and diet samples were determined as described by AOAC (1990). The concentrations of Ca in feed ingredient and diet samples were measured by inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions with HNO3 and HClO4 as described by Ma et al. (2014). The concentrations of XOS in the products and diet samples were measured by high performance liquid chromatograph on Aminex HPX 87H column (Greenherbs, Beijing, China) after hydrolysis with sulfuric acid as described by Pellerin et al. (1991). Muscle pH and color measurements The pH values of breast and thigh muscles at a depth of 2.5 cm below the surface were tested immediately after the birds were slaughtered using a Model pH-211 meter (Hanna Instruments Inc., Padova, Italy) equipped with a spear electrode. The color (L*, a*, and b*) of meat was determined using an automatic colorimeter (model SC-80C; Beijing Kangguang Instrument Co., Beijing, China). The color was measured at 3 points of every meat in triplicate after slaughter. Drip loss measurements Drip loss was measured as described by Liu et al. (2011). The breast muscle and thigh muscle were excised (1 cm×1 cm×5 cm) and weighed and then placed in a plastic bag and stored at 4°C. After 24 h the muscle was removed from the plastic bag wiped with filter paper, and weighed to evaluate drip loss and expressed as a percentage of initial muscle weight. Shear force measurements At 24 h after slaughter, the
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breast and thigh muscles were heated in plastic bags in a water bath at 80 to 85°C until the temperature of the meat center reached 75°C. They were then taken out and cooled to room temperature and excised (1 cm×1 cm×5 cm) to measure the shear force at 3 points of every meat in triplicate with a C-LM muscle tenderness instrument (Northeast Agricultural University, Heilongjiang, China) as described by Gwartney et al. (1992). Immune indices The antibody titers against Newcastle and H5N1 vaccines were measured using the haemagglut-ination inhibition test as described by Meulemans et al. (1987). Titers were expressed as the highest dilution of serum that completely inhibited viral hemagglutination. The analysis of whole blood T-lymphocyte proliferation was carried out by the methods described by Mosmann (1983). The mean optical density was determined for bipartite samples, and the proliferation responses were expressed as a mean stimulation index (SI) obtained by dividing the mean optical density (OD) of stimulated cells by OD of unstimulated cells. Duodenal morphology The duodenal villus height, width and crypt depth were determined by Olympus IX 71 automatic visualize (Olympus, Tokyo, Japan) after staining with hematoxylin and eosin using standard paraffin embedding procedures (Uni et al. 1998). Villus height was measured from the tip of the villi to the villus crypt junction, and crypt depth was defined as the depth of the invagination between adjacent villi. Intestinal microbial populations A 0.5-g of jejunal or cecal contents per sample was weighed out accurately after blended, dissolved into 4.5 mL of sterile saline solution, and homogenized for 5 min in a stomacher under CO2. Each jejunal or cecal homogenate was diluted exactly 10fold (10% w/v) with sterile ice-cold normal saline (pH 7.0). Diluted samples (0.1 mL) were inoculated into selective agar for further bacterial enumeration. Lactobacillus and Bifidobacterium were incubated using Luria-Bertani agar and Man-Rogosa-Sharpe agar, respectively, in an anaerobic incubator at 37°C for 24 h (Yang et al. 2012). E. coli and Salmonella were incubated using MacConkey agar and Salmonella Shigella agar (Land Bridge Technology, Beijing, China), respectively, in an aerobic incubator at 37°C for 24 h (Yang et al. 2012). Colonies were counted on the dilution with discernible numbers (30–300) (Kong et al. 2011). The microbial count data were expressed as colony forming units per gram wet sample (Cheng et al. 2004).
5.5. Statistical analyses Data of the experiment were analyzed by one-way analysis of variance (ANOVA) using the General Linear Models (GLM) procedure of the SAS Institute (1998). Differences among means were tested by the least significant difference
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(LSD) method. Orthogonal polynomials were used to assess linear and quadratic responses of dependent variables to dietary supplemental XOS levels. The replicate cage served as the experimental unit, and the P<0.05 was considered statistically significant.
Acknowledgements This work was supported by the Shandong Longlive Bio-Technology Co. Ltd., China, the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS08), the Special Fund for Agro-Scientific Research in the Public Interest, China (201403047), and the China Agriculture Research System (CARS-42).
References AOAC (Association of Official Analytical Chemists). 1990. Official Methods of Analysis. 16th ed. Association of Offiial Analytical Chemists, Arlington, Virginia, USA. Caparros S, Garrote G, Ariza J, Diaz M J, Lopez F. 2007. Xylooligosaccharides production from Arundo donax. Journal of Agricultural and Food Chemistry, 55, 5536–5543. Chen Y N. 2009. Effects of xylo-oligosaccharides on the immune function and intestinal health of AA broilers. MSc thesis, Nanjing Agricultural University, China. (in Chinese) Cheng K H, Liao J W, Chung Y C, Hsieh C P, Chan Y C. 2004. Effects of dietary supplementation with fructooligosaccharides on colonic microbiota populations and epithelial cell proliferation in neonatal pigs. Journal of Pediatric Gastroenterology and Nutrition, 21, 297–303. Chung, Y C, Hsu C K, Ko C Y, Chan Y C. 2007. Dietary intake of xylo-oligosaccharides improves the intestinal microbiota, fecal moisture, and pH value in the elderly. Nutrition Research, 27, 756–761. Cook R H, Bird F H. 1973. Duodenal villus area and epithelial cellular migration inconventional and germ-free chicks. Poultry Science, 52, 2276–2280. Dang G H. 2004. Effects of dietary xylo-oligosaccharides on performance of broilers and layers. MSc thesis, Nanjing Agricultural University, China. (in Chinese) Grbson G R, Roberfroid M B. 1995. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition, 125, 1401–1412. Grootaert C, Delcour J A, Courtin C M. 2007. Microbial metabolism and prebiotic potency of arabinoxylan oligosaccharides in the human intestine. Trends in Food Science and Technology, 18, 64–71. Gwartney B L, Jones S J, Calkins C R. 1992. Response time of broiler chickens to cimaterol: Meat tenderness, muscle composition, fiber size, and carcass characteristics. Journal of Animal Science, 70, 2144–2150. Howard M D, Gordon D T, Pace L W, Garleb K A, Kerley M S. 1995. Xylo-oligosaccharides and fructo-oligosaccharides affect the intestinal microbiota and precancerous colonic
lesion development in rats. The Journal of Nutrition, 134, 1523–1528. Kong Q, He G Q, Jia J L, Zhu Q L, Ruan H. 2011. Oral administration of clostridium butyricum for modulating gastrointestinal microflora in mice. Current Microbiology, 62, 512–517. Li S F, Lu L, Hao S F, Wang Y P, Zhang L Y, Liu S B, Liu B, Li K, Luo X G. 2011. Dietary manganese modulates expression of the manganese-containing superoxide dismutase gene in chickens. The Journal of Nutrition, 141, 189–194. Liu Z H, Lu L, Li S F, Zhang L Y, Xi L, Zhang K Y, Luo X G. 2011. Effects of supplemental zinc source and level on growth performance, carcass traits, and meat quality of broilers. Poultry Science, 90, 1782–1790. Luciano G, Monahan F J, Vasta V, Biondi L, Lanza M, Priolo A. 2009. Dietary tannins improve lamb meat colour stability. Meat Science, 81, 120–125. Ma X Y, Liu S B, Lu L, Li S F, Xie J J, Zhang L Y, Zhang J H, Luo X G. 2014. Relative bioavailability of iron proteinate for broilers fed a casein-dextrose diet. Poultry Science, 93, 556–563. Meulemans G, Carlier M C, Gonze M, Petit P. 1987. Comparison of hemagglutina-tioninhibition, agar gel precipitin, and enzyme linked immunosorbent assay for measuring antibodies against influenzaviruses in chickens. Avian Diseases, 31, 560–563. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. The Journal of Immunological Methods, 65, 55–63. Moura P, Marques S, Alves L, Freire J P B, Cunha L F, Esteves M P. 2007. Effect of xylo-oligosaccharides from corn cobs autohydrolysis on the intestinal microbiota of piglets after weaning. Livestock Science, 108, 244–248. Mussatto S I, Mancilha I M. 2007. Non-digestible oligosaccharides: A review. Carbohydrate Polymers, 68, 587–597. NRC (National Research Council). 1994. Nutrient Requirements of Poultry. 9th ed. National Academy of Sciences, Washington, D.C. Orban J I, Patterson J A, Adeola O. 1997. Growth performance and intestinal microbial populations of growing pigs fed diets containing sucrose chermal oligosaccharides caramel. Journal of Animal Science, 75, 170–175. Otto G, Roehe R, Looft H, Thoelking L, Henning M, Plastow G S, Kalm E. 2006. Drip loss of case-ready meat and of premium cuts and their association with earlier measured sample drip loss, meat quality and carcass traits in pigs. Meat Science, 72, 680–687. Pellerin P, Gosselin M, Lepoutre J, Samain E, Debeire P. 1991. Enzymic production of oligosaccharides from corncob xylan. Enzyme and Microbial Technology, 13, 617–621. Saha B C. 2003. Hemicellulose bioconversion. Journal of Industrial Microbiology and Biotechnology, 30, 279–291. SAS Institute. 1998. SAS User′s Guide: Statistics. SAS Institute, Cary, NC. Savage A W J, Warriss P D, Jolley P D. 1990. The amount and
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composition of the proteins in drip from stored pig meat. Meat Science, 27, 289–303. Hesta M, Debraekeleer J, Janssens G P, De Wilde R. 2001. The effect of a commercial high-fibre diet and an iso-maltooligosaccharide-supplemented diet on post-prandial glucose concentrations in dogs. Journal of Animal Physiology and Animal Nutrition (Berl), 85, 217–221. Sheu W H, Lee I T, Chan Y C. 2008. Effects of xylooligosaccharides in type 2 diabetes mellitus. Journal of Nutritional Science and Vitaminology, 54, 396–401. Shi N. 2010. Effects of bacillus licheniformins, xylooligosaccharides and lactic acid on intestinal villi and intestinal microflora in broilers. MSc thesis, Henan Polytechnic University, China. (in Chinese) Swennen K, Courtin C M, Delcour J A. 2006. Non-digestible oligosaccharides with prebiotic properties. Food Science and Nutrition, 46, 459–471. Uni Z, Noy Y, Sklan D. 1998. Posthatch development of mucosal function in the broiler small intestine. Poultry Science, 77, 75–82. Wang X M, Xu L, Yuan L, Wang J, Sun W. 2008. Effects of xylo-oligosaccharides on the growth performance and
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blood biochemical indices of calves. Journal of Northeast Agricultural University, 39, 61–65. (in Chinese) Yang C M, Cao G T, Ferket P R, Liu T T, Zhou L, Zhang L, Xiao Y P, Chen A G. 2012. Effects of probiotic, clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens. Poultry Science, 91, 2121–2129. Yang H Y. 2007. Study of probiotics and xylo-oligosaccharides on growth performance and mechanism of weanling pigs. MSc thesis, Shangdong Agricultural University, China. (in Chinese) Yoshioka M, Shimomura Y, Suzuki M. 1994. Dietary polydextrose affects the large intestine in rats. The Journal of Nutrition, 124, 539–544. Younes H, Garleb K, Behr S. 1995. Fermentable fibers or oligosaccharides reduce urinary nitrogen excretion by increasing urea disposal in the rat cecum. The Journal of Nutrition, 125, 1010–1016. Zhuang H Y. 2007. Effects of xylooligosaecharides on growth performance, meat quality and PRKAG3 gene expression in growing-finishing pig. MSc thesis, Chinese Academy of Agricultural Sciences, China. (in Chinese) (Managing editor ZHANG Juan)
ScienceDirect
RESEARCH ARTICLE
Effectiveness of dietary xylo-oligosaccharides for broilers fed a conventional corn-soybean meal diet SUO Hai-qing1, LU Lin1, XU Guo-hui2, XIAO Lin2, CHEN Xiao-gang2, XIA Rui-rui3, ZHANG Li-yang1, LUO Xu-gang1 1
Mineral Nutrition Research Division, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China 2 High-Technology Development Zone, Functional Sugar Engineering Research Center of Shandong Province,Yucheng 251200, P.R.China 3 High-Technology Development Zone, Shandong Key Laboratory of Straw and Stover Biorefinement Technologies, Yucheng 251200, P.R.China
Abstract An experiment was conducted to investigate the effect of dietary supplementation of xylo-oligosaccharides (XOS) on growth performance, meat quality, immune functions, duodenal morphology and intestinal microbial populations of broilers fed a conventional corn-soybean meal basal diet. A total of 450 1-day-old commercial Arbor Acres male broiler chicks were randomly allocated by bodyweight to 1 of 5 treatments with 6 replicate cages (15 broilers per cage) for each of 5 treatments in a completely randomized design. Chicks were fed the basal corn-soybean meal diets supplemented with 0, 25, 50, 75, or 100 mg of XOS kg–1 of diet, respectively, for an experimental duration of 42 days. The results showed that supplementation of XOS affected feed conversion rate (feed/gain, F/G) during days 22–42 and 1–42 (P<0.03), drip loss in thigh muscle (P=0.02), and duodenal crypt depth (P=0.005) on day 42, but had no effect (P>0.05) on all other measured indices. The chicks fed the diet supplemented with 100 mg of XOS kg–1 had the lowest (P<0.05) F/G and drip loss in thigh muscle. The drip loss in thigh muscle decreased linearly (P=0.003) as the supplemented XOS increased. Duodenal crypt depth decreased (P<0.05) at the supplemental level of 75 mg of XOS kg–1. The results indicate that dietary supplementations of 75 and 100 mg of XOS kg–1 are beneficial to broilers fed a conventional corn-soybean meal diet. Keywords: xylo-oligosaccharide, effectiveness, F/G, drip loss, broiler chick
1. Introduction
Received 24 March, 2015 Accepted 24 June, 2015 SUO Hai-qing, Mobile: +86-15652743313, E-mail: suohaiqing2008 @1 26.com; Correspondence LUO Xu-gang, Tel: +86-1062816012, Fax: +86-10-62810184, E-mail: [email protected] © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61101-7
Xylo-oligosaccharides (XOS) are sugar oligomers mainly produced from the hydrolysis of xylan, the major component of plant hemicelluloses, which are heteropolysaccharides with homopolymeric backbone of xylopyranose moieties (Saha 2003). XOS have a ramified structure containing 2–7 xylose units linked by β-(1,4) bonds and with a variety of substituents such as acetyl groups, uronic
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acids, and arabinose units (Caparros et al. 2007). They are developed functional oligosaccharides because they could affect the host by selectively stimulating the growth or activity of beneficial bacteria in the colon. Thus XOS have been used as prebiotics for improving the intestinal health (Grbson and Roberfroid 1995). The main beneficial biomedical and biofunctional effects of XOS are: (1) reducing cholesterol level, (2) maintaining the gastrointestinal health, (3) having cytotoxic effects on human leukemia cells, and (4) having beneficial effect on type 2 diabetes mellitus (Swennen et al. 2006; Mussatto and Mancilha 2007; Sheu et al. 2008). In recent years, XOS have been studied extensively as prebiotics instead of antimicrobial agents in pigs and ruminants (Yoshioka et al. 1994; Howard et al. 1995; Younes et al. 1995; Hesta et al. 2001; Yang 2007; Wang et al. 2008). Results from the previous studies indicated that dietary supplementation of XOS could improve growth performance, immune functions, and modify the intestinal morphology and flora in animals. However, only few researches were carried out to evaluate the effectiveness of dietary supplementation of XOS as prebiotics for broilers, and inconsistent results were observed (Dang 2004; Chen 2009; Shi 2010). Dang (2004) reported that XOS at supplemental levels of 35–105 mg kg–1 increased cecal villus height and reduced its crypt depth, but had no effect on growth performance and the intestinal flora of broilers. Shi (2010) demonstrated that addition of 52.5 mg of XOS kg–1 in the diet decreased drip loss in breast and thigh muscles, but had no effect on growth performance and the intestinal morphology and flora of broilers. Chen (2009) observed no effect of XOS on growth performance, immune functions and intestinal flora of broilers at supplemental levels of 35–70 mg kg–1. The above disparities might be majorly due to the differences in the species of broilers and supplemental levels of XOS.
Therefore, the following study was conducted to further investigate the effect of dietary supplementation of XOS on growth performance, meat quality, immune functions, duodenal morphology and intestinal microbial populations of broilers.
2. Results 2.1. Growth performance of broilers Supplementation of XOS had no effect (P>0.24) on average daily gain (ADG), average daily feed intake (ADFI), mortality on days 1–21, 22–42 and 1–42, and feed conversion rate (feed/gain, F/G) on days 1–21, but affected (P<0.03) F/G on days 22–42 and 1–42 (Table 1). A linear response (P<0.05) was observed between supplementation of XOS and F/G. As supplemental XOS level increased, the F/G decreased linearly (P=0.02) on days 22–42 and 1–42. The chicks fed the diets supplemented with 100 mg of XOS kg–1 had the lowest (P<0.05) F/G on days 22–42 and 1–42 compared with those fed all other 4 treatment diets, and there were no differences (P>0.05) among all other 4 treatments (Table 1).
2.2. Meat quality of broilers Supplementation of XOS had no effect (P>0.34) on the meat quality of breast and thigh muscles except for drip loss (P=0.02) in leg muscle for broilers at 42 days of age (Table 2). A linear response (P=0.003) was observed between supplemental XOS level and drip loss in leg muscle. As supplemental XOS level increased, the drip loss in thigh muscle decreased linearly (P=0.003), with the lowest drip loss at the supplemental level of 100 mg of XOS kg–1 (Table 2).
Table 1 Effect of dietary xylo-oligosaccharides (XOS) level on the growth performance and mortality of broiler chicks1) Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value Linear2) Quadratic3) 1)
ADG (g d–1) 34 33.5 34.6 35.5 34.6 0.8 0.46
Days 1–21 ADFI F/G (g d–1) (g g–1) 48.4 1.41 48.7 1.45 50.3 1.46 51.3 1.44 49.4 1.43 0.9 0.02 0.25 0.64
Mortality (%) 1.11 5.56 0.00 1.11 3.33 0.36 0.66
ADG (g d–1) 78.9 80.3 78.4 79.3 79.6 1.5 0.92
Days 22–42 ADFI F/G (g d–1) (g g–1) 151 1.91 a 151 1.89 a 149 1.90 a 152 1.92 a 145 1.83 b 2 0.02 0.31 0.01 0.02 0.08
Mortality (%) 0 0 0 0 0 0 0
ADG (g d–1) 56.9 57.3 56.9 57.9 57.6 0.8 0.88
Days 1–42 ADFI F/G Mortality (g d–1) (g g–1) (%) 99.8 1.75 a 1.11 100.1 1.75 a 5.56 99.6 1.75 a 0.00 101.7 1.76 a 1.11 97.4 1.69 b 3.33 1.29 0.01 0.36 0.25 0.02 0.66 0.02 0.12
ADG, average daily gain; ADFI, average daily feed intake; F/G, feed/gain rate. 2) Linear effects of added XOS levels. 3) Quadratic effects of added XOS levels. Data represent the means of 6 replicate cages (n=6). Data with different letters within the same column differ significantly (P<0.05). The same as below.
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2.3. Immune functions of broilers Supplementation of XOS had no effect (P>0.08) on serum antibody titers against Newcastle disease and blood T-lymphocyte proliferation at both 21 and 42 days of age and serum antibody titers against H5N1 at 42 days of age (Table 3).
2.4. Duodenal morphology and intestinal microbial populations of broilers As shown in Table 4, supplemental XOS level had no effect
(P>0.05) on duodenal villus height and width, and villus height/crypt depth, but affected (P=0.005) duodenal crypt depth. Linear and quadratic responses (P<0.05) were observed between supplemental XOS level and duodenal crypt depth. The chicks fed the diets supplemented with 75 mg of XOS kg–1 had lower (P<0.05) duodenal crypt depth than those fed all other 4 treatment diets, and there were no differences (P>0.05) among all other 4 treatments. Since no flora was observed in the jejunum, the data only in the cecum were listed in Table 5. Supplemental XOS level had no effect (P>0.38) on the populations of
Table 2 Effect of dietary XOS level on meat quality of broiler chicks at 42 days of age Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value Linear Quadratic 1)
Breast muscle color1)
Thigh muscle color1)
L*
a*
b*
L*
a*
b*
36.0 35.7 35.3 35.4 35.6 0.7 0.96
2.09 3.14 3.46 2.69 2.58 0.49 0.35
5.19 4.56 4.81 4.70 4.76 0.28 0.59
41.4 40.7 42.0 41.2 41.0 0.8 0.81
3.62 4.15 4.01 4.68 4.08 0.56 0.77
6.28 6.00 6.75 6.13 6.26 0.36 0.64
Shear force (kg) Breast Thigh muscle muscle 2.66 1.14 3.11 1.03 3.22 1.08 2.78 1.11 2.80 0.98 0.50 0.91 0.92 0.78
Drip loss (%) Breast Thigh muscle muscle 3.92 6.32 a 3.56 5.57 ab 3.51 4.70 bc 3.64 5.33 abc 3.37 3.96 c 0.42 0.47 0.91 0.02 0.003 0.88
0-h pH value Breast Thigh muscle muscle 6.69 6.90 6.75 6.90 6.72 6.91 6.71 6.95 6.70 6.92 0.05 0.04 0.92 0.89
L*, lightness; a*, redness; b*, yellowness.
Table 3 Effect of dietary XOS level on immune functions of broiler chicks Added XOS (mg kg–1) 0 25 50 75 100 Pooled SE P-value 1)
Serum Newcastle disease (ND) antibody titers (Log2) Day 21 Day 42 5.3 2.5 5.0 2.3 5.2 2.3 4.7 2.7 4.3 2.5 0.65 0.27 0.82 0.90
Serum H5N1 antibody titers (Log2) Day 42 5.67 5.83 6.33 5.50 5.50 0.47 0.70
Blood T-lymphocyte proliferation (SI)1) Day 21 Day 42 2.22 1.31 2.73 1.65 1.68 2.11 1.38 1.57 1.36 1.38 0.37 0.30 0.09 0.48
SI was calculated by dividing the mean optional density (OD) of stimulated cells by OD of unstimulated cells.
Table 4 Effect of dietary XOS level on duodenal morphology and cecal flora amount of broiler chicks at 42 days of age Duodenal morphology Added XOS (mg kg–1)
Villus height (μm)
Villus width (μm)
Crypt depth (μm)
0 25 50 75 100 Pooled SE P-value Linear Quadratic
1 164 1 081 1 073 987 1 191 47 0.06
148 102 119 137 110 14 0.15
217 a 171 a 180 a 140 b 192 a 12 0.005 0.04 0.01
Cecal flora amount (wet basis)
Villus height/ Crypt depth 5.39 5.67 6.44 6.47 6.32 0.42 0.27
Escherichia Lactic acid (Lactic acid bacteria+ Salmonella Bifidobacterium coli bacteria Bifidobacterium)/Total –1 –1 (Log10CFU g ) (Log10CFU g ) (Log10CFU g–1) (Log10CFU g–1) analyzed bacteria 6.45 6.27 6.57 6.33 6.87 0.24 0.39
4.52 4.34 4.69 4.49 4.44 0.10 0.40
6.31 6.64 6.89 6.77 6.50 0.26 0.55
6.81 6.90 7.01 6.59 6.36 0.27 0.49
0.55 0.56 0.55 0.55 0.54 0.01 0.72
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Table 5 Composition and nutrient levels of the basal diets for broilers1) Item Ingredients (%) Corn Soybean meal Soybean oil CaHPO4 Limestone NaCl Methionine Micronutrients2, 3) Cornstarch+XOS4) Total Nutrient ME (MJ kg–1) Crude protein (%)5) Lysine (%) Methionine (%) Methionine+cysteine (%) Calcium (%)5) Total phosphorus (%)5) Nonphytate phosphorus (%)5) XOS
Days 1–21
Days 22–42
56.20 35.94 3.50 2.10 1.23 0.30 0.23 0.33 0.20 100
56.55 36.24 3.50 1.47 1.39 0.30 0.22 0.20 0.22 100
12.80 22.19 1.11 0.55 0.90 1.04 0.79 0.45 –
12.94 19.95 1.11 0.44 0.79 0.87 0.59 0.35 –
1)
As-fed basis. Provided per kilogram of diet for days 1–21: vitamin A (all-trans retinol acetate) 15 000 IU, cholecalciferol 4 500 IU, vitamin E (all-rac-α-tocopherol acetate) 24 IU, vitamin K3 3.0 mg, vitamin B1 3.0 mg, vitamin B2 9.6 mg, vitamin B6 3.0 mg, vitamin B12 0.018 mg, pantothenic acid calcium 15.0 mg, niacin 39.0 mg, folic acid 1.5 mg, biotin 0.15 mg, choline 700 mg, Cu (CuSO4·5H2O) 8 mg, Mn (MnSO4·H2O) 110 mg, Fe (FeSO4·7H2O) 60 mg, Zn (ZnSO4·7H2O) 60 mg, I (KI) 0.35 mg, Se (Na2SeO3) 0.15 mg. 3) Provided per kilogram of diet for days 22–42: vitamin A (all-trans retinol acetate) 10 000 IU, cholecalciferol 3 400 IU, vitamin E (all-rac-α-tocopherol acetate) 16 IU, vitamin K3 2.0 mg, vitamin B1 2.0 mg, vitamin B2 6.8 mg, vitamin B6 2.0 mg, vitamin B12 0.012 mg, pantothenic acid calcium 10 mg, niacin 26 mg, folic acid 1.0 mg, biotin 0.1 mg, choline 500 mg, Cu (CuSO4·5H2O) 8 mg, Zn (ZnSO4·7H2O) 40 mg, Mn (MnSO4·H2O) 80 mg, Fe (FeSO4·7H2O) 40 mg, I (KI) 0.35 mg, Se (Na2SeO3) 0.15 mg. 4) XOS product added in place of equivalent weights of corn starch. 5) Measured values. Each value based on triplicate determin ations. –, not detectable. 2)
E. coli, Salmonella, lactobacilli, Bifidobacterium or the ratio of microbiota (lactobacilli+bifidobacteria) to total analyzed bacteria in the cecum at 42 days of age.
3. Discussion In the present study, we found that dietary supplementation with 100 mg of XOS kg–1 improved feed conversion rate on days 22–42 and 1–42. This supported that XOS directly arrived at the mucosa of the small intestine to promote the absorption of nutrients (Orban et al. 1997), and also XOS could improve health status of animals (Grbson and Roberfroid 1995). However, no effect of XOS on growth performance of broilers was observed in previous studies
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(Dang 2004; Chen 2009; Shi 2010). Such disparities might be mainly due to the differences in supplemental levels of XOS, and XOS compositions. Higher XOS supplemental levels (0–105 mg kg–1) were used in our and Dang’s (2004) studies, while lower XOS supplemental levels (0–70 mg kg–1) were used in the studies of Chen (2009) and Shi (2010). The XOS used in the present study contain 2–7 xylose units, but the XOS used in the studies of Dang (2004), Chen (2009) and Shi (2010) contain 2–5 xylose units. Therefore, the results from our present study suggested that the XOS containing 2–7 xylose units and higher supplemental level of XOS might be beneficial for improving feed conversion rate of broilers. Sharon and Janssens (2001) showed that XOS could help to slow the absorption of antigen and increase the titer of antigen so as to improve cellular and humoral immunities. In the present study, we found that supplemental XOS level had no effect on immune function, perhaps because the chicks were in a relatively comfortable environment which did not exert a threat to their immune systems during the whole experiment. Water-holding capacity was an important index for evaluating meat quality (Otto et al. 2006). Savage et al. (1990) reported that moisture loss could cause the loss of soluble flavor substance in meat. Luciano et al. (2009) also found that moisture loss could take away the heme in meat. Therefore, the status of moisture is very important for the physical form, flavor and color of meat. In the present study, we found that the addition of 100 mg of XOS kg–1 improved the water-holding capacity of thigh muscle. Shi (2010) also reported that the addition of 52.5 mg of XOS kg–1 decreased drip loss in breast and thigh muscles of broilers. The improvement of the water-holding capacity of thigh muscle might be related to the increased antioxidant capability induced by XOS (Zhuang 2007). However, the mechanisms remain unclear and need to be further studied in the future. The intestine villus height, width and crypt depth were important indices for measuring the functions of intestinal digestion and absorption. It is reported that XOS could modify intestinal morphology that is associated with its functions in increasing the absorption of short-chain fatty acids (Yoshioka et al. 1994; Howard et al. 1995). Crypts are considered as villus factories as they contain stem cells. Deeper crypts indicate faster tissue turnover and higher nutrient demand in new tissue. Reduced crypt depth leads to a decreased endogenous secretion and an increased nutrient absorption, disease resistance and performance (Cook and Bird 1973). Yang (2007) found that XOS did not affect duodenal villus height and crypt depth of piglets. Dang (2004) reported that the addition of 52.5 mg of XOS kg–1 increased cecal villus height and reduced its crypt depth of broilers. In the
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present study, we found that the addition of 75 mg of XOS kg–1 decreased duodenal crypt depth of broilers, suggesting that XOS indeed have a beneficial effect on broilers. Recent studies have shown that XOS had a bifidogenic effect on the intestinal tract and could maintain the balance of bacteria in the intestinal tract (Chung et al. 2007; Grootaert et al. 2007). Moura et al. (2007) reported that XOS had a potential prebiotic effect on lactobacilli in the piglet intestinal tract but not on bifidobacteria, so as to produce an intestinal microbiota pattern more similar to the pattern of suckling piglets. In the present study, we found that XOS had no effect on the intestinal flora of broilers, which was consistent with the results reported in previous studies (Dang 2004; Chen 2009; Shi 2010). The highest dietary level of XOS supplemented in this study was 100 mg of XOS kg–1, and the effect of supplemental levels over 100 mg of XOS kg–1 was not studied. Based on our results, the effect of higher supplemental XOS levels on broilers may need to be further investigated in the future.
4. Conclusion In conclusion, the results from the present study indicate that the addition of 100 mg XOS kg–1 improves feed conversion rate and water-holding capacity of the thigh muscle of broilers; the addition of 75 mg of XOS kg–1 decreases duodenal crypt depth of broilers. Therefore, dietary supplementations with 75 and 100 mg of XOS kg–1 are beneficial to broilers fed a conventional corn-soybean meal diet.
5. Materials and methods 5.1. Birds All experimental procedures were approved by the Office of the Beijing Veterinarians, China (Li et al. 2011). A total of 450 1-day-old commercial Arbor Acres male broiler chicks were randomly allotted by bodyweight (BW) to 5 treatments. Each treatment contained 6 replicate cages and 15 birds per cage in a completely randomized design. Broilers were housed in electrically heated, thermostatically controlled rooms (100 cm×50 cm×45 cm) with fiberglass feeders and maintained on a 24-h constant light schedule. They were allowed ad libitum access to the experimental diets and tap water that contained no detectable levels of XOS. Broilers were vaccinated with the Newcastle vaccine VA/GA at 7 and 24 days of age, and with the H5N1 vaccine at 11 and 28 days of age, respectively. Feed consumption and BW gain of the chicks in each replicate cage were recorded at the end of 21 and 42 days of age. Whenever a bird was found dead, the feed for the cage of the dead bird was weighed immediately. According to the initial feed weight and the last weight for
the cage of dead bird, the feed intake of the dead bird could be calculated and deducted from the final feed intake of the cage of the dead bird.
5.2. Diets The basal corn–soybean meal diet (Table 5) was formulated to meet or exceed the nutrient requirements for starter broilers (NRC 1994). Dietary treatments included the basal corn-soybean meal diets supplemented with 0 (control), 25, 50, 75 or 100 mg of XOS kg–1. The XOS were supplied by Shangdong Longlive Bio-Technology Co. Ltd, China, and have 2–7 xylose units and a purity of about 35%. The remaining 65% are 15% monosaccharide plus 50% maltodextrin used as excipient, and they should have no effect on broilers. A single batch of basal mash feed was mixed at first and then divided into 5 aliquots according to the experimental treatment arrangement. Each supplemental XOS level was mixed with corn-starch to the same weight and then mixed with each aliquot of the basal diet. The diets were fed in mash form.
5.3. Sample collections and preparations The feed ingredients and diets samples from all the treatments were taken and submitted for CP, Ca, and P analyses before the initiation of the trial to confirm CP, Ca, and P contents in diets. At both 21 and 42 days of age, 2 chicks were chosen from each of 30 replicate cages according to average BW. Whole blood samples were taken into heparinized tubes from each of 2 birds via wing vein puncture after 12 h fasting for T-lymphocyte proliferation analysis. Another blood sample was collected also into tubes without anticoagulant, and then was centrifuged to harvest serum samples for analyses of antibody titers against Newcastle and H5N1 vaccines. At 42 days of age, the bled birds were weighed individually, and slaughtered according to the welfare of animal slaughter regulations of China. Carcass weight was measured after defeathering. Heads, necks, and feet were removed from the broilers, and then carcasses were eviscerated and weighted to calculate the percentage of eviscerated yield. Dressing percentage was calculated by dividing the carcass weight by live BW. Abdominal fat, the left breast and thigh muscles, thymus, bursa of fabricius and spleen were taken and weighed to determine the percentages of abdominal fat, breast and thigh muscles, thymus, bursa of fabricius and spleen. The breast and thigh muscles were used to determine the color of meat 45 min after slaughter, and they were then placed in a plastic bag and stored at 4°C for 24 h to measure the pH value, respectively. The percentages of abdominal fat, thymus, bursa of fabricius and spleen were calculated using the weights of abdominal fat,
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thymus, bursa of fabricius and spleen divided by the live BW; the percentages of breast and thigh muscles were calculated by dividing the breast or thigh weight by eviscerated weight. The right breast and thigh muscles were removed and weighed, placed separately in a plastic bag, and stored at 4°C for 24 h. They were subsequently taken out and wiped with filter paper and weighed to determine the drip loss of the muscle and then cooked to determine the shear force of the muscle. Samples were pooled together based on replicate cage. One chick was chosen also from each of 30 replicate cages according to the cage average BW. They were killed and their duodenums (0.5–1 cm) were taken and fixed in 10% formalin after washed off with physiological sodium chloride solution for the analyses of duodenal morphology. Their jejunum and cecum which contained contents were ligatured, removed, and then samples of the contents from jejunum or cecum were collected immediately into plastic bags under CO2, sealed, and stored in 4°C for immediate analyses of microbial populations.
5.4. Sample analyses Determinations of feed ingredients and diets The concentrations of crude protein (CP) and phosphorus (P) in feed ingredient and diet samples were determined as described by AOAC (1990). The concentrations of Ca in feed ingredient and diet samples were measured by inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions with HNO3 and HClO4 as described by Ma et al. (2014). The concentrations of XOS in the products and diet samples were measured by high performance liquid chromatograph on Aminex HPX 87H column (Greenherbs, Beijing, China) after hydrolysis with sulfuric acid as described by Pellerin et al. (1991). Muscle pH and color measurements The pH values of breast and thigh muscles at a depth of 2.5 cm below the surface were tested immediately after the birds were slaughtered using a Model pH-211 meter (Hanna Instruments Inc., Padova, Italy) equipped with a spear electrode. The color (L*, a*, and b*) of meat was determined using an automatic colorimeter (model SC-80C; Beijing Kangguang Instrument Co., Beijing, China). The color was measured at 3 points of every meat in triplicate after slaughter. Drip loss measurements Drip loss was measured as described by Liu et al. (2011). The breast muscle and thigh muscle were excised (1 cm×1 cm×5 cm) and weighed and then placed in a plastic bag and stored at 4°C. After 24 h the muscle was removed from the plastic bag wiped with filter paper, and weighed to evaluate drip loss and expressed as a percentage of initial muscle weight. Shear force measurements At 24 h after slaughter, the
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breast and thigh muscles were heated in plastic bags in a water bath at 80 to 85°C until the temperature of the meat center reached 75°C. They were then taken out and cooled to room temperature and excised (1 cm×1 cm×5 cm) to measure the shear force at 3 points of every meat in triplicate with a C-LM muscle tenderness instrument (Northeast Agricultural University, Heilongjiang, China) as described by Gwartney et al. (1992). Immune indices The antibody titers against Newcastle and H5N1 vaccines were measured using the haemagglut-ination inhibition test as described by Meulemans et al. (1987). Titers were expressed as the highest dilution of serum that completely inhibited viral hemagglutination. The analysis of whole blood T-lymphocyte proliferation was carried out by the methods described by Mosmann (1983). The mean optical density was determined for bipartite samples, and the proliferation responses were expressed as a mean stimulation index (SI) obtained by dividing the mean optical density (OD) of stimulated cells by OD of unstimulated cells. Duodenal morphology The duodenal villus height, width and crypt depth were determined by Olympus IX 71 automatic visualize (Olympus, Tokyo, Japan) after staining with hematoxylin and eosin using standard paraffin embedding procedures (Uni et al. 1998). Villus height was measured from the tip of the villi to the villus crypt junction, and crypt depth was defined as the depth of the invagination between adjacent villi. Intestinal microbial populations A 0.5-g of jejunal or cecal contents per sample was weighed out accurately after blended, dissolved into 4.5 mL of sterile saline solution, and homogenized for 5 min in a stomacher under CO2. Each jejunal or cecal homogenate was diluted exactly 10fold (10% w/v) with sterile ice-cold normal saline (pH 7.0). Diluted samples (0.1 mL) were inoculated into selective agar for further bacterial enumeration. Lactobacillus and Bifidobacterium were incubated using Luria-Bertani agar and Man-Rogosa-Sharpe agar, respectively, in an anaerobic incubator at 37°C for 24 h (Yang et al. 2012). E. coli and Salmonella were incubated using MacConkey agar and Salmonella Shigella agar (Land Bridge Technology, Beijing, China), respectively, in an aerobic incubator at 37°C for 24 h (Yang et al. 2012). Colonies were counted on the dilution with discernible numbers (30–300) (Kong et al. 2011). The microbial count data were expressed as colony forming units per gram wet sample (Cheng et al. 2004).
5.5. Statistical analyses Data of the experiment were analyzed by one-way analysis of variance (ANOVA) using the General Linear Models (GLM) procedure of the SAS Institute (1998). Differences among means were tested by the least significant difference
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(LSD) method. Orthogonal polynomials were used to assess linear and quadratic responses of dependent variables to dietary supplemental XOS levels. The replicate cage served as the experimental unit, and the P<0.05 was considered statistically significant.
Acknowledgements This work was supported by the Shandong Longlive Bio-Technology Co. Ltd., China, the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS08), the Special Fund for Agro-Scientific Research in the Public Interest, China (201403047), and the China Agriculture Research System (CARS-42).
References AOAC (Association of Official Analytical Chemists). 1990. Official Methods of Analysis. 16th ed. Association of Offiial Analytical Chemists, Arlington, Virginia, USA. Caparros S, Garrote G, Ariza J, Diaz M J, Lopez F. 2007. Xylooligosaccharides production from Arundo donax. Journal of Agricultural and Food Chemistry, 55, 5536–5543. Chen Y N. 2009. Effects of xylo-oligosaccharides on the immune function and intestinal health of AA broilers. MSc thesis, Nanjing Agricultural University, China. (in Chinese) Cheng K H, Liao J W, Chung Y C, Hsieh C P, Chan Y C. 2004. Effects of dietary supplementation with fructooligosaccharides on colonic microbiota populations and epithelial cell proliferation in neonatal pigs. Journal of Pediatric Gastroenterology and Nutrition, 21, 297–303. Chung, Y C, Hsu C K, Ko C Y, Chan Y C. 2007. Dietary intake of xylo-oligosaccharides improves the intestinal microbiota, fecal moisture, and pH value in the elderly. Nutrition Research, 27, 756–761. Cook R H, Bird F H. 1973. Duodenal villus area and epithelial cellular migration inconventional and germ-free chicks. Poultry Science, 52, 2276–2280. Dang G H. 2004. Effects of dietary xylo-oligosaccharides on performance of broilers and layers. MSc thesis, Nanjing Agricultural University, China. (in Chinese) Grbson G R, Roberfroid M B. 1995. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. The Journal of Nutrition, 125, 1401–1412. Grootaert C, Delcour J A, Courtin C M. 2007. Microbial metabolism and prebiotic potency of arabinoxylan oligosaccharides in the human intestine. Trends in Food Science and Technology, 18, 64–71. Gwartney B L, Jones S J, Calkins C R. 1992. Response time of broiler chickens to cimaterol: Meat tenderness, muscle composition, fiber size, and carcass characteristics. Journal of Animal Science, 70, 2144–2150. Howard M D, Gordon D T, Pace L W, Garleb K A, Kerley M S. 1995. Xylo-oligosaccharides and fructo-oligosaccharides affect the intestinal microbiota and precancerous colonic
lesion development in rats. The Journal of Nutrition, 134, 1523–1528. Kong Q, He G Q, Jia J L, Zhu Q L, Ruan H. 2011. Oral administration of clostridium butyricum for modulating gastrointestinal microflora in mice. Current Microbiology, 62, 512–517. Li S F, Lu L, Hao S F, Wang Y P, Zhang L Y, Liu S B, Liu B, Li K, Luo X G. 2011. Dietary manganese modulates expression of the manganese-containing superoxide dismutase gene in chickens. The Journal of Nutrition, 141, 189–194. Liu Z H, Lu L, Li S F, Zhang L Y, Xi L, Zhang K Y, Luo X G. 2011. Effects of supplemental zinc source and level on growth performance, carcass traits, and meat quality of broilers. Poultry Science, 90, 1782–1790. Luciano G, Monahan F J, Vasta V, Biondi L, Lanza M, Priolo A. 2009. Dietary tannins improve lamb meat colour stability. Meat Science, 81, 120–125. Ma X Y, Liu S B, Lu L, Li S F, Xie J J, Zhang L Y, Zhang J H, Luo X G. 2014. Relative bioavailability of iron proteinate for broilers fed a casein-dextrose diet. Poultry Science, 93, 556–563. Meulemans G, Carlier M C, Gonze M, Petit P. 1987. Comparison of hemagglutina-tioninhibition, agar gel precipitin, and enzyme linked immunosorbent assay for measuring antibodies against influenzaviruses in chickens. Avian Diseases, 31, 560–563. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. The Journal of Immunological Methods, 65, 55–63. Moura P, Marques S, Alves L, Freire J P B, Cunha L F, Esteves M P. 2007. Effect of xylo-oligosaccharides from corn cobs autohydrolysis on the intestinal microbiota of piglets after weaning. Livestock Science, 108, 244–248. Mussatto S I, Mancilha I M. 2007. Non-digestible oligosaccharides: A review. Carbohydrate Polymers, 68, 587–597. NRC (National Research Council). 1994. Nutrient Requirements of Poultry. 9th ed. National Academy of Sciences, Washington, D.C. Orban J I, Patterson J A, Adeola O. 1997. Growth performance and intestinal microbial populations of growing pigs fed diets containing sucrose chermal oligosaccharides caramel. Journal of Animal Science, 75, 170–175. Otto G, Roehe R, Looft H, Thoelking L, Henning M, Plastow G S, Kalm E. 2006. Drip loss of case-ready meat and of premium cuts and their association with earlier measured sample drip loss, meat quality and carcass traits in pigs. Meat Science, 72, 680–687. Pellerin P, Gosselin M, Lepoutre J, Samain E, Debeire P. 1991. Enzymic production of oligosaccharides from corncob xylan. Enzyme and Microbial Technology, 13, 617–621. Saha B C. 2003. Hemicellulose bioconversion. Journal of Industrial Microbiology and Biotechnology, 30, 279–291. SAS Institute. 1998. SAS User′s Guide: Statistics. SAS Institute, Cary, NC. Savage A W J, Warriss P D, Jolley P D. 1990. The amount and
SUO Hai-qing et al. Journal of Integrative Agriculture 2015, 14(10): 2050–2057
composition of the proteins in drip from stored pig meat. Meat Science, 27, 289–303. Hesta M, Debraekeleer J, Janssens G P, De Wilde R. 2001. The effect of a commercial high-fibre diet and an iso-maltooligosaccharide-supplemented diet on post-prandial glucose concentrations in dogs. Journal of Animal Physiology and Animal Nutrition (Berl), 85, 217–221. Sheu W H, Lee I T, Chan Y C. 2008. Effects of xylooligosaccharides in type 2 diabetes mellitus. Journal of Nutritional Science and Vitaminology, 54, 396–401. Shi N. 2010. Effects of bacillus licheniformins, xylooligosaccharides and lactic acid on intestinal villi and intestinal microflora in broilers. MSc thesis, Henan Polytechnic University, China. (in Chinese) Swennen K, Courtin C M, Delcour J A. 2006. Non-digestible oligosaccharides with prebiotic properties. Food Science and Nutrition, 46, 459–471. Uni Z, Noy Y, Sklan D. 1998. Posthatch development of mucosal function in the broiler small intestine. Poultry Science, 77, 75–82. Wang X M, Xu L, Yuan L, Wang J, Sun W. 2008. Effects of xylo-oligosaccharides on the growth performance and
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blood biochemical indices of calves. Journal of Northeast Agricultural University, 39, 61–65. (in Chinese) Yang C M, Cao G T, Ferket P R, Liu T T, Zhou L, Zhang L, Xiao Y P, Chen A G. 2012. Effects of probiotic, clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens. Poultry Science, 91, 2121–2129. Yang H Y. 2007. Study of probiotics and xylo-oligosaccharides on growth performance and mechanism of weanling pigs. MSc thesis, Shangdong Agricultural University, China. (in Chinese) Yoshioka M, Shimomura Y, Suzuki M. 1994. Dietary polydextrose affects the large intestine in rats. The Journal of Nutrition, 124, 539–544. Younes H, Garleb K, Behr S. 1995. Fermentable fibers or oligosaccharides reduce urinary nitrogen excretion by increasing urea disposal in the rat cecum. The Journal of Nutrition, 125, 1010–1016. Zhuang H Y. 2007. Effects of xylooligosaecharides on growth performance, meat quality and PRKAG3 gene expression in growing-finishing pig. MSc thesis, Chinese Academy of Agricultural Sciences, China. (in Chinese) (Managing editor ZHANG Juan)