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Animal Science Journal (2014) ••, ••–••
doi: 10.1111/asj.12277
ORIGINAL ARTICLE Effects of essential oil supplementation of a low-energy diet on performance, intestinal morphology and microflora, immune properties and antioxidant activities in weaned pigs Zhikai ZENG, Xiao XU, Qiang ZHANG, Ping LI, Panfeng ZHAO, Qingyun LI, Jundi LIU and XiangShu PIAO State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing, China
ABSTRACT A total of 144 weaned piglets were used to evaluate the effects of essential oil (EO) supplementation of a low-energy diet on performance, apparent nutrient digestibility, small intestinal morphology, intestinal microflora, immune properties and antioxidant activities in weaned pigs. Pigs received a low-energy diet (negative control, NC, digestible energy = 3250 kcal/ kg), NC plus 0.025% EO or a positive control diet (PC, digestible energy = 3400 kcal/kg) for 28 days. Growth performance was similar between the EO group and PC group. However, EO supplementation increased (P < 0.05) average daily gain and the apparent digestibility of dry matter, crude protein and energy compared with pigs fed the NC diet. Greater (P < 0.05) villus height and lower (P < 0.05) counts of Escherichia coli and total anaerobes in the rectum in the EO group were observed compared with NC or PC groups. Pigs fed EO diet had higher (P < 0.05) concentrations of albumin, immunoglobulin A (IgA), IgG and total antioxidant capacity and lower fecal score than pigs fed the PC and NC diets. Above all, this study indicates that supplementation of EO to a low-energy pig diet has beneficial results and obtains similar performance compared with normal energy (PC) diet.
Key words: antioxidant activity, essential oils, microflora, performance, pig.
INTRODUCTION Essential oils (EO), also referred to as volatile or ethereal oils, are mixtures of volatile, lipophilic, largely terpenoid compounds extracted from plant materials such as flowers, buds, seed, leaves, twigs, bark, herbs, woods, fruits and roots (Brenes & Roura 2010). EO are secondary metabolites of plants and have several advantages over commercial antibiotics since they are residue free, broad-spectrum and are generally recognized as safe and beneficial by the Food and Drug Administration (Yan et al. 2010). The trend of ban on the use of antibiotics has led to many investigations conducted to identify alternatives to commercial antibiotics as growth promoters for use in the animal industry (Jang et al. 2007; Yan et al. 2010). EO have received increased attention as one of the potential alternatives to improve performance in intensive management systems (Brenes & Roura 2010). A growing number of studies have indicated that EO have beneficial effects, such as antiviral (Bishop 1995), antimicrobial (Rota et al. 2004), antioxidant © 2014 Japanese Society of Animal Science
(Botsoglou et al. 2004) properties, and can enhance the production of digestive secretions (Jang et al. 2004) and stimulate immune function (Wenk 2003). Although in vitro, the antimicrobial effect of some plant extracts is as consistent as the effect of antibiotics (Cowan 1999). However, the effect of plant extracts in vivo is inconsistent, even for the same product used in similar but not equal conditions (Windisch et al. 2008). Giannenas et al. (2003) reported that the efficacy of dietary EO was affected by intrinsic and extrinsic factors, such as nutritional status, infection, diet composition and environment. Diet is one of the main factors that can affect the efficacy of EO (Si et al. 2006a). For instance, Manzanilla et al. (2009)
Correspondence: XiangShu Piao, State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing 100193, China. (Email: [email protected]) Received 6 March 2014; accepted for publication 6 June 2014.
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demonstrated that dietary protein can modify the effect of plant extracts in the intestinal ecosystem of weaning pigs. In addition, EO supplementation significantly improved the digestibility of energy in pigs during the grower period (Yan et al. 2010) and can increase ileal digestibility of energy in broilers (Cao et al. 2010). Therefore, theis experiment was conducted to evaluate the effects of EO supplementation to a low-energy diet on performance, apparent digestibility of nutrients, antioxidant activities, immune properties, small intestinal morphology and intestinal microflora in weaned pigs.
MATERIALS AND METHODS Essential oil product The commercial EO product used in this study was provided by Danisco Co., Ltd., Kunshan, China. The product contained at least 4.5% cinnamaldehyde and 13.5% thymol, which have been demonstrated to possess anti-bacterial and antioxidant activities (Didry et al. 1993; Aeschbach et al. 1994; Gill & Holley 2004).
Animals and facilities All the procedures in this study were approved by the China Agricultural University Animal Care and Use Committee (Beijing, China). A total of 144 crossbred piglets (Duroc × Landrace × Large White), weighing on average 7.26 ± 1.57 kg and weaned at 28 ± 2 days of age were used in this experiment. Piglets were allotted to one of the three dietary treatments on the basis of weight, genetics and gender in a randomized complete block design. Each dietary treatment was fed to 12 pens with four pigs per pen (two barrows and two gilts). The experimental diets were a low-energy diet (negative control, digestible energy (DE) = 3250 kcal/kg) supplemented with 0% or 0.025% EO product as well as a positive control diet (PC, DE = 3400 kcal/kg) (Table 1). All diets were fed in mash form and contained 0.25% chromic oxide as an indigestible marker. All pigs had free access to feed and water throughout the 4-week feeding trial. The experimental piglets were housed in an environmentally controlled nursery room in 2 × 3 m raised pens equipped with a mesh floor. The temperature of the pig barn was controlled between 25 and 32°C.
Growth performance and fecal digestibility Pigs and feeders were weighed at the beginning and the end of the experiment in order to determine weight gain, feed intake and feed efficiency. Fecal consistency was visually assessed every morning by observers unaware of the dietary treatments, using a modification of the method described by Sherman et al. (1983). Fresh excreta were ranked using the following scale: 1, normal (feces firm and well formed); 2, soft consistency (feces soft and formed); 3, mild diarrhea (fluid feces, usually yellowish); and 4, severe diarrhea (feces watery and projectile). Fresh fecal grab samples were collected from the anus of pigs on days 26 to 28 and pooled by pen. Approximately 50 g of fresh feces were collected from each pen into sterile plastic bags and immediately stored at −20°C until analysis. © 2014 Japanese Society of Animal Science
Table 1
Ingredients of the experimental diets† (as-fed basis)
Ingredients, %
PC
Wheat Corn Extruded full-fat soybean Fish meal Whey power Wheat middling Soybean oil Dicalcium phosphate Limestone Salt L-Lys-HCl, 78% DL-Methionine hydroxyl analogue, 84% L-Threonine L-Tryptophan Vitamin-mineral premix‡ Essential oil Chromic oxide Total Nutrient levels, %§ Digestible energy, Mcal/kg Crude protein Calcium Phosphorus
NC
EO
54.00 5.10 16.20 4.40 4.00 10.00 2.70 0.45 0.68 0.25 0.56 0.10
54.00 8.50 15.50 4.40 4.00 10.00 – 0.45 0.68 0.25 0.56 0.10
54.00 8.48 15.50 4.40 4.00 10.00 – 0.45 0.68 0.25 0.56 0.10
0.25 0.06 1.00 – 0.25 100.00
0.25 0.06 1.00 – 0.25 100.00
0.25 0.06 1.00 0.025 0.25 100.00
3.40 19.01 0.69 0.55
3.25 18.94 0.72 0.58
3.25 19.06 0.68 0.56
†The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Vitamin and mineral premix provides the following per kg of feed: vitamin A, 9000 IU; vitamin D3, 2400 IU; vitamin E, 20 IU; vitamin K3, 3 mg; thiamine, 1.4 mg; riboflavin, 4 mg; vitamin B6, 3 mg; vitamin B12, 12 μg; nicotinic acid, 30 mg; pantothenic acid, 14 mg; folic acid, 0.8 mg; biotin, 44 μg; choline chloride, 0.5 g; Fe, 76 mg; Cu, 200 mg; Zn, 76 mg; Mn, 20 mg; I, 0.48 mg; Se, 0.4 mg. §The digestible energy is calculated while the other nutrients are analyzed.
On day 28, blood samples (about 5 mL) were obtained from the jugular vein by vena cava puncture from one barrow (weighing closest to the average body weight for each pen) per pen, using a 10 mL anticoagulant-free vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) after an overnight fast. Blood samples were centrifuged at 3000 × g (Heraeus Biofuge 22R Centrifuge, Hanau, Germany) for 10 min at 4°C and the serum samples were obtained and stored at −80°C for later analysis. At 08.30 hours on day 29, the 18 pigs (six pens from each treatment and one pig per pen) which supplied blood samples were humanely killed by exsanguination after electrical stunning to obtain intestinal tissues and digesta samples as described by Shen et al. (2009). Intestinal tissue from middle sections of duodenum, jejunum and ileum were aseptically isolated, flushed with 0.9% salt solution, fixed in 10% formaldehyde-phosphate buffer, and kept at 4°C for microscopic assessment of mucosal morphology. The digesta in the cecum, colon and rectum were collected and immediately immersed in liquid nitrogen and then stored at −80°C for subsequent microbial counting.
Chemical analysis of feed and feces Feed samples were collected at the start of the experiment. Fecal samples were thawed, dried in an oven (65°C) and ground to pass through a 1-mm sieve. Animal Science Journal (2014) ••, ••–••
ESSENTIAL OIL FOR WEANED PIGS
Feed and fecal samples were analyzed for dry matter, crude protein (N × 6.25) and calcium, according to the methods of AOAC (1990). Gross energy was determined by an automatic adiabatic oxygen bomb calorimeter (Parr 1281 Automatic Energy Analyzer, Parr Instruments Co., Moline, IL, USA). Chromium content was analyzed using an atomic absorption spectrophotometer (Z-5000 Automatic Absorption Spectrophotometer; Hitachi, Tokyo, Japan) according to Williams et al. (1962). Phosphorus content was analyzed using a UV-visible spectrophotometer (U-1000; Hitachi, Tokyo, Japan).
Small intestinal morphology Fixed intestinal segments were embedded in paraffin wax. Cross-sections at 6 μm thickness of each intestinal segment were stained with hematoxylin and eosin. Intestinal morphological measurements including villus height and crypt depth were determined at 40 × magnification using an Olympus CK 40 microscope (Olympus Optical Company, Shenzhen, China). Lengths of 10 well-oriented intact villi and their associated crypt were measured in triplicate for each segment of each pig according to the procedure of Touchette et al. (2002).
Intestinal microbiology analysis The microbiological counts in the large intestinal digesta were determined by the plate-count technique (Wang et al. 2011). One gram of mixed digesta was homogenized in salt solution (0.9% NaCl) and serially diluted from 10−1 to 10−9 for analysis of Escherichia coli (MacConkey agar, Beijing Haidian Microbiological Culture Factory, Beijing, China), lactobacilli (MRS agar, De Man, Rogosa, Sharpe, Oxoid Ltd., Basingstoke, UK; CM0361. anaerobic chamber), total anaerobes (plate-count agar, anaerobic chamber), and total aerobes (plate-count agar). All plates were incubated for 48 h in 37°C. The microbial populations were log transformed before statistical analysis.
Measurement of immune and antioxidant indices in serum Serum concentrations of immunoglobulins (IgA, IgG and IgM) were quantified using an enzyme-linked immunosorbent assay specific for swine (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. Assays were counted in duplicate using a TEACAN Plate Reader (TEACAN Asia, Shanghai, China). Plasma albumin level was measured with an Automatic Biochemical Analyzer (RA-1000; Bayer Corp., Tarrytown, NY, USA) using colorimetric methods and the instructions from the manufacturer’s corresponding reagent kit (Zhongsheng Biochemical Company, Beijing, China). Determination of serum total antioxidant capacity, superoxide dismutase, glutathione peroxidase and malondialdehyde levels were conducted by spectrophotometric methods using a spectrophotometer (Leng Guang SFZ1606017568, Shanghai, China) following the instructions of the kit’s manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis All data were analyzed as a completely randomized block design using the GLM procedure of SAS (1999). Each pen or individual pig was considered as the experimental unit. Statistical differences among treatments are separated by Animal Science Journal (2014) ••, ••–••
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Table 2 Effect of dietary essential oil on the performance and fecal consistency of weaned pigs†
Item
PC
NC
EO
SEM§
Average daily 382¶ 348†† 383¶ 4.50 gain, g*** Average daily feed 633 636 631 11.98 intake, g Feed conversion 1.65¶ 1.82†† 1.64¶ 0.04 ratio*** Fecal 1.42†† 1.44†† 1.29¶ 0.07 consistency‡** ¶††Means in the same row with different superscripts are significantly different (P < 0.05). **Treatments effect (P < 0.05); ***Treatments effect (P < 0.01). †Value represents mean of 12 pens with four pigs per pen. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Fecal score was assessed visually every morning, and fresh excreta were ranked using the following scale: 1, normal (feces firm and well formed); 2, soft consistency (feces soft and formed); 3, mild diarrhea (fluid feces, usually yellowish); and 4, severe diarrhea (feces watery and projectile). §Pooled standard error mean.
Student-Neuman-Keul’s multiple range test. Significance was taken at P ≤ 0.05, with a trend of P ≤ 0.10.
RESULTS Performance and fecal consistency Pigs fed EO or the PC diets had greater (P < 0.05) average daily gain (ADG) and lower (P < 0.05) feed conversion ratio (FCR) compared with pigs fed the NC diet (Table 2). However, no difference was observed in ADG and FCR between pigs fed EO or the PC diets. Average daily feed intake was not affected by dietary treatments. Fecal consistency score was significantly reduced (P < 0.05) in pigs fed EO diet compared with pigs fed the PC or NC diets.
Apparent nutrient digestibility Pigs fed EO or the PC diets had greater (P < 0.01) total tract apparent digestibility of dry matter, crude protein and energy than pigs fed the NC diet (Table 3). However, no difference was observed in these measurements between pigs fed EO or the PC diets. The digestibility of calcium and phosphorus did not differ among treatments.
Intestinal morphology Histological evaluation of duodenum and ileum indicated no effect of dietary treatments on villus height, crypt depth, and ratio of villus height and crypt depth (Table 4). However, jejunal villus height and villus height to crypt depth ratio of pigs fed EO diet were greater (P < 0.05) than those pigs fed the NC diet. Jejunal villus height was greater in pigs fed EO diet (P < 0.05), but smaller in pigs fed NC diet compared with pigs fed the PC diet. © 2014 Japanese Society of Animal Science
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Table 3 Effect of dietary essential oil on apparent total tract nutrient digestibility (%) in weaned pigs†
Item
PC
NC
EO
SEM‡
Dry matter*** Crude protein*** Energy*** Calcium Phosphorus
81.2§ 79.3§ 79.9§ 56.3 56.3
79.2¶ 73.3¶ 76.3¶ 57.0 56.0
81.2§ 79.2§ 81.1§ 59.5 60.0
0.48 0.85 0.57 1.65 1.61
§¶Means in the same row with different superscripts are significantly different (P < 0.05). ***Treatments effect (P < 0.01). †Value represents mean of 12 pens with four pigs per pen. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
Table 4 Effect of dietary essential oil on small intestinal morphology of weaned pigs†
Item Duodenum Villus height (μm) Crypt depth (μm) Villus height/crypt depth Jejunum Villus height (μm)*** Crypt depth (μm) Villus height/crypt depth*** Ileum Villus height (μm) Crypt depth (μm) Villus height/crypt depth
PC
NC
593 188 3.16
586 192 3.06
EO 597 190 3.15
SEM‡ 6.57 3.87 0.07
512¶ 495†† 529§ 5.20 163 165 165 2.25 3.14§¶ 3.01¶ 3.21§ 0.05
471 132 3.57
473 138 3.45
480 134 3.60
4.98 2.14 0.07
§–††Means in the same row with different superscripts are significantly different (P < 0.05). ***Treatment effect (P < 0.01). †Value represents mean of six pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
Microbial counts in large intestine No significant difference was observed in the microbial counts among treatments in the cecum (Table 5). The counts of E. coli tended to decrease (P < 0.10), while the number of lactobacilli was slightly increased (P < 0.10) in the colon in EO treatment compared with pigs fed the PC and NC diets. In addition, total anaerobe numbers in the colon were significantly reduced (P < 0.05) by the addition of EO compared with pigs fed the PC and NC diets. In the rectum, the counts of E. coli and total anaerobes were significantly reduced (P < 0.05) in pigs fed the EO-supplemented diet compared with pigs fed the PC and NC diets. Furthermore, there was also a tendency (P < 0.10) for increased number of lactobacilli in the rectum in pigs fed the diet supplemented with EO. Finally, total aerobes number © 2014 Japanese Society of Animal Science
Table 5 Effect of dietary essential oil on microbial counts (log10 colony-forming units/g of digesta) in the large intestine of weaned pigs†
Item Cecum E. coli Lactobacilli Total anaerobes Total aerobes Colon E. coli* Lactobacilli* Total anaerobes** Total aerobes Rectum E. coli** Lactobacilli* Total anaerobes** Total aerobes
PC
NC
EO
SEM‡
5.13 6.79 6.75 6.11
5.00 6.84 6.79 6.05
4.73 6.73 6.66 6.02
0.13 0.23 0.05 0.12
5.06 7.02 7.06§¶ 5.94
5.16 6.92 7.19§ 5.92
4.70 7.27 6.88¶ 5.77
0.13 0.09 0.06 0.08
4.88§ 7.19 7.68§ 6.39
5.00§ 7.30 7.64§ 6.29
4.67¶ 7.46 7.37¶ 6.32
0.08 0.07 0.07 0.08
§¶Means in the same row with different superscripts are significantly different (P < 0.05). *Treatments effect (P < 0.10); **Treatments effect (P < 0.05). †Value represents mean of six pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
in different segments of the hindgut was not affected by dietary treatment.
Immunity and antioxidant indices in serum The concentrations of albumin, IgA and IgG were significantly increased (P < 0.05) in response to EO supplementation compared with pigs fed the PC and NC diets (Table 6). Dietary EO significantly increased (P < 0.05) total antioxidant capacity, while leading to a slight improvement (P < 0.10) in the level of superoxide dismutase and glutathione peroxidase. There was no significant difference in concentrations of IgM and malondialdehyde among dietary treatments.
DISCUSSION In the present study, our results demonstrate that EO supplementation improved ADG and FCR, which agrees with Li et al. (2012b), who reported that the addition of EO at 100 and 150 ppm significantly increased ADG and FCR in weaned pigs. Hong et al. (2004) found similar results that 0.1% or 0.2% plant extract supplementation significantly increased ADG in weaned pigs compared with pigs fed diets without botanical supplementation. Evidently, the improved performance in pigs fed EO diet compared with those pigs fed the NC diet in the present study were associated with greater apparent total tract digestibility of dry matter, crude protein and energy, as well as Animal Science Journal (2014) ••, ••–••
ESSENTIAL OIL FOR WEANED PIGS
Table 6 Effect of dietary essential oil on immune indices and antioxidant capacity in plasma of weaned pigs†
Item
PC
NC
EO
SEM‡
Immune indices Albumin, g/L** 26.3¶ 25.9¶ 27.6§ 0.42 Immunoglobulin A, mg/L*** 46.4¶ 46.8¶ 51.0§ 0.68 Immunoglobulin G, mg/L*** 245¶ 236¶ 273§ 3.64 Immunoglobulin M, mg/L 49.7 50.1 52.5 0.97 Antioxidant indices Total antioxidant capacity, 11.2¶ 11.2¶ 12.1§ 0.22 U/mL** Superoxide dismutase, U/mL* 73.5 76.7 77.0 1.13 Glutathione peroxidase, U/mL* 933 941 949 4.76 Malondialdehyde, nmol/mL 4.5 4.5 4.4 0.07 §¶Means in the same row with different superscripts are significantly different (P < 0.05). *Treatments effect (P < 0.10); **treatments effect (P < 0.05); ***treatments effect (P < 0.01). †Value represents mean of 12 pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
improved intestinal morphology in jejunum, microbial composition in large intestine and total antioxidant capacity in serum. The significant improvement in apparent total tract digestibility of dry matter, crude protein and energy in this study is in agreement with Yan et al. (2010), who reported that the digestibility was increased by EO supplementation. Similar results were reported by Cho et al. (2006). The beneficial effect of EO (blend of thymol and cinnamaldehyde) on energy and nutrient utilization was also observed in broilers (Amerah et al. 2011). Improved nutrient utilization might be due to enhanced small morphology or increased secretion of digestive enzymes (Jang et al. 2007). It is well documented that weaning stress in piglets has caused significant reduction in villus height (Tang et al. 1999; Shen et al. 2009; Wang et al. 2011). Villus atrophy is mainly caused by an increased rate of cell apoptosis or programmed death and decreased rate of renewal, which can be affected by cellular factors or endogenous stressors (van der Peet-Schwering et al. 2007). An increase in villus height and villus : crypt ratio in the jejunum was observed in this study in pigs fed EO after weaning when compared with pigs fed the NC diet. These results agree with those from a study conducted by Li et al. (2012a), in which dietary supplementation of essential oil improved the villus : crypt ratio in the jejunum in weaned pigs. Decreased number of pathogenic bacteria in the gut may improve proliferation of epithelial cells to build villus in the gut and thus enhance intestinal morphology (Moura¯o et al. 2005). Antioxidants may fix the intestine impairment caused by oxidative stress and improve intestinal morphology in weaned pigs (Han et al. 2012). In this study, the change of microflora Animal Science Journal (2014) ••, ••–••
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composition in the colon and rectum and increased serum total antioxidant capacity may partly explain the enhanced intestinal morphology. It is generally known that post-weaning diarrhea is one of the critical factors causing high mortality in piglets (Osek 1999). In the present study, the fecal consistency score was significantly reduced in pigs fed EO diets compared with pigs fed the PC and NC diets, which is in agreement with Hong et al. (2004) and Manzanilla et al. (2004). Si et al. (2006b) indicated that EO compounds have a strong antimicrobial action against gut pathogens while not harming beneficial bacteria such as bifidobacteria and lactobacilli. Meanwhile, increased number of lactobacilli and reduction of E. coli in the gut microbiota might result in a decreased incidence of diarrhea in piglets (Manzanilla et al. 2004; Li et al. 2012a). In the present study, the counts of E. coli and total anaerobes in the rectum were significantly reduced (P < 0.05) in pigs fed EO, while the number of lactobacilli was slightly increased in the colon and rectum in pigs fed EO. The antimicrobial mode of action of EO supplementation is considered to arise mainly from the potential of the hydrophobic EO to intrude into the bacterial cell membrane, disintegrate membrane structures and cause ion leakage (Windisch et al. 2008). In addition, phytogenic feed additives were also reported to stimulate intestinal secretion of mucus in broilers, an effect that was assumed to impair adhesion of pathogens such as E. coli and thus to contribute to stabilizing the microbial eubiosis in the gut of the animals (Jamroz et al. 2006). The results in this study suggest that EO have potential for suppressing harmful intestinal microflora and improving the intestinal ecosystem in weaned pigs. The maturation and optimal development of the immune system is highly associated with the development and composition of the indigenous microflora and vice versa (Insoft et al. 1996; de Vrese & Marteau 2007). In this study, EO supplementation increased the concentrations of serum albumin, IgA and IgG, which may be highly related to the improved microflora composition in the large intestine. Similar results were reported by Yan et al. (2012), who demonstrated that plant extracts played a positive role in enhancing fecal microflora composition and immune status. Cho et al. (2006) also reported an increased serum IgG concentration in pigs fed diets supplemented with EO. The newly weaned piglet is very susceptible to diseases, stressors and digestive disorders during the first week post-weaning (Yoon et al. 2012). At this stage, an increased concentration of the serum immunoglobulin is required to regulate and enhance immune function, which provides health benefits, diminishes weaning stress and improves health status and performance of weanling pigs (Turner et al. 2002). © 2014 Japanese Society of Animal Science
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Oxidative stress is caused by excessive oxidative radicals, including reactive oxygen species, which damage DNA, bio-membrane lipids, and proteins, as well as causing a variety of impairments to tissue (Zhao & Shen 2005; Zhang et al. 2011). However, excessive oxidative radicals can be eliminated by the use of an antioxidant system, including nonenzymatic components and a series of antioxidant enzymes. In the enzyme antioxidant system, superoxide dismutase and glutathione peroxidase are the most important antioxidants (Lu et al. 2010). They work together to detoxify superoxide anions and hydrogen peroxide in cells (Zhang et al. 2011). Superoxide dismutase is able to catalyze superoxide anions to produce hydrogen peroxide and molecular oxygen. Glutathione peroxidase normally converts H2O2 to water (Lu et al. 2010). In the present study, the level of superoxide dismutase and glutathione peroxidase in serum tended to increase by the addition of EO. Total antioxidant capacity, which reflects the non-enzymatic antioxidant defense system (Wang et al. 2008), was significantly increased by EO in the present study, indicating that EO may play an important role in preventing endogenous lipids from peroxidation and oxidation. Furthermore, the level of malondialdehyde, which is a major product of lipid peroxidation, was an effective marker in oxidative stress in sepsis (Lu et al. 2010). In conclusion, EO supplementation to a low-energy diet (DE = 3250 kJ/kg) improved performance compared with those pigs fed NC diet and obtained similar performance compared with those pigs fed PC diet (DE = 3400 kJ/kg). The beneficial results may be explained by improved intestinal morphology, microflora composition in the large intestine and improved health status measured by fecal scores, immune indices and antioxidant indices.
ACKNOWLEDGMENTS The financial support was received from the National Natural Science Foundation of China (No.31372316), the State Key Laboratory of Animal Nutrition of China (No. 2004DA125184F1211), and the Danisco Co. Ltd. (China). We thank Novus International for supplying the methionine hydroxyl analogue.
REFERENCES Aeschbach R, Löliger J, Scott BC, Murcia A, Butler J, Halliwell B, Aruoma OI. 1994. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food and Chemical Toxicology 32, 31–36. Amerah AM, Peron A, Zaefarian F, Ravindran V. 2011. Influence of whole wheat inclusion and a blend of essential oils on the performance, nutrient utilisation, digestive tract development and ileal microbiota profile of broiler chickens. British Poultry Science 52, 124–132.
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Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Washington, DC. Bishop CD. 1995. Antiviral activity of the essential oil of Melaleuca alternifolia (Maiden and Betche) Cheel (tea tree) against tobacco mosaic virus. Journal of Essential Oil Research 7, 641–644. Botsoglou NA, Christaki E, Florou-Paneri P, Giannenas I, Papageorgiou G, Spais AB. 2004. The effect of a mixture of herbal essential oils or α-tocopheryl acetate on performance parameters and oxidation of body lipid in broilers. South African Journal of Animal Science 34, 52–61. Brenes A, Roura E. 2010. Essential oils in poultry nutrition: main effects and modes of action. Animal Feed Science and Technology 158, 1–14. Cao PH, Li FD, Li YF, Ru YJ, Peron A, Schulze H, Bento H. 2010. Effect of essential oils and feed enzymes on performance and nutrient utilization in broilers fed a corn/ soy-based diet. International Journal of Poultry Science 9, 749–755. Cho JH, Chen YJ, Min BJ, Kim HJ, Kwon OS, Shon KS, et al. 2006. Effects of essential oils supplementation on growth performance, IgG concentration and fecal noxious gas concentration of weaned pigs. Asian-Australasian Journal of Animal Sciences 19, 80–85. Cowan MM. 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12, 564–582. de Vrese M, Marteau PR. 2007. Probiotics and prebiotics: effects on diarrhea. The Journal of Nutrition 137, 803–811. Didry N, Dubreuil L, Pinkas M. 1993. Antibacterial activity of thymol, carvacrol and cinnamaldehyde alone or in combination. Die Pharmazie 48, 301–304. Giannenas I, Florou-Paneri P, Papazahariadou M, Christaki E, Botsoglou NA, Spais AB. 2003. Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition 57, 99–106. Gill AO, Holley RA. 2004. Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei. Applied and Environmental Microbiology 70, 5750–5755. Han X, Piao XS, Zhang HY, Li PF, Yi JQ, Zhang Q, Li P. 2012. Forsythia suspensa extract has the potential to substitute antibiotic in broiler chicken. Asian-Australasian Journal of Animal Sciences 25, 569–576. Hong JW, Kim IH, Kwon OS, Min BJ, Lee WB, Shon KS. 2004. Influences of plant extract supplementation on performance and blood characteristics in weaned pigs. AsianAustralasian Journal of Animal Sciences 17, 374–378. Insoft RM, Sanderson IR, Walker WA. 1996. Development of immune function in the intestine and its role in neonatal diseases. Pediatric Clinics of North America 43, 551–571. Jamroz D, Wertelecki T, Houszka M, Kamel C. 2006. Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90, 255– 268. Jang IS, Ko YH, Kang SY, Lee CY. 2007. Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Animal Feed Science and Technology 134, 304–315. Jang IS, Ko YH, Yang HY, Ha JS, Kim JY, Kim JY, et al. 2004. Influence of essential oil components on growth perfor-
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mance and the functional activity of the pancreas and small intestine in broiler chickens. Asian-Australasian Journal of Animal Sciences 17, 394–400. Li PF, Piao XS, Ru YJ, Han X, Xue LF, Zhang HY. 2012a. Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. Asian-Australasian Journal of Animal Sciences 25, 1617–1626. Li SY, Ru YJ, Liu M, Xu B, Péron A, Shi XG. 2012b. The effect of essential oils on performance, immunity and gut microbial population in weaner pigs. Livestock Science 145, 119–123. Lu T, Piao XL, Zhang Q, Wang D, Piao XS, Kim SW. 2010. Protective effects of Forsythia suspensa extract against oxidative stress induced by diquat in rats. Food and Chemical Toxicology 48, 764–770. Manzanilla EG, Perez JF, Martin M, Kamel C, Baucells F, Gasa J. 2004. Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 82, 3210–3218. Manzanilla EG, Pérez JF, Martín M, Blandón JC, Baucells F, Kamel C, Gasa J. 2009. Dietary protein modifies effect of plant extracts in the intestinal ecosystem of the pig at weaning. Journal of Animal Science 87, 2029–2037. Moura¯o JL, Pinheiro V, Alves A, Guedes CM, Pinto L, Saavedra MJ, et al. 2005. Effect of mannan oligosaccharides on the performance, intestinal morphology and cecal fermentation of fattening rabbits. Animal Feed Science and Technology 126, 107–120. Osek J. 1999. Prevalence of virulence factors of Escherichia coli strains isolated from diarrheic and healthy piglets after weaning. Veterinary Microbiology 68, 209–217. Rota C, Carraminana JJ, Burillo J, Herrera A. 2004. In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens. Journal of Food Protection 67, 1252–1256. SAS Inst. 1999. SAS User’s Guide: Statistics, Version 8.01 edn. SAS Inst., Cary, NC. Shen YB, Piao XS, Kim SW, Wang L, Liu P, Yoon I, Zhen YG. 2009. Effects of yeast culture supplementation on growth performance, intestinal health, and immune response of nursery pigs. Journal of Animal Science 87, 2614–2624. Sherman DM, Acres SD, Sadowski PL, Springer JA, Bray B, Raybould TJ, Muscoplat CC. 1983. Protection of calves against fatal enteric colibacillosis by orally administered Escherichia coli K99-specific monoclonal antibody. Infection and Immunity 42, 653–658. Si W, Gong J, Chanas C, Cui S, Yu H, Caballero C, Friendship RM. 2006a. In vitro assessment of antimicrobial activity of carvacrol, thymol and cinnamaldehyde towards Salmonella serotype Typhimurium DT104: effects of pig diets and emulsification in hydrocolloids. Journal of Applied Microbiology 101, 1282–1291. Si W, Gong J, Tsao R, Zhou T, Yu H, Poppe C, et al. 2006b. Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology 100, 296–305. Tang M, Laarveld B, Van Kessel AG, Hamilton DL, Estrada A, Patience JF. 1999. Effect of segregated early weaning on
Animal Science Journal (2014) ••, ••–••
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postweaning small intestinal development in pigs. Journal of Animal Science 77, 3191–3200. Touchette KJ, Carroll JA, Allee GL, Matteri RL, Dyer CJ, Beausang LA, Zannelli ME. 2002. Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs. Journal of Animal Science 80, 494–501. Turner JL, Dritz SS, Higgins JJ, Minton JE. 2002. Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium. Journal of Animal Science 80, 1947– 1953. van der Peet-Schwering CMC, Jansman AJM, Smidt H, Yoon I. 2007. Effects of yeast culture on performance, gut integrity, and blood cell composition of weaning pigs. Journal of Animal Science 85, 3099–3109. Wang D, Piao XS, Zeng ZK, Lu T, Zhang Q, Li PF, et al. 2011. Effects of keratinase on performance, nutrient utilization, intestinal morphology, intestinal ecology and inflammatory response of weaned piglets fed diets with different levels of crude protein. Asian-Australasian Journal of Animal Sciences 24, 1718–1728. Wang YZ, Xu CL, An ZH, Liu JX, Feng J. 2008. Effect of dietary bovine lactoferrin on performance and antioxidant status of piglets. Animal Feed Science and Technology 140, 326–336. Wenk C. 2003. Herbs and botanicals as feed additives in monogastric animals. Asian-Australasian Journal of Animal Sciences 16, 282–289. Williams CH, David DJ, Iismaa O. 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. The Journal of Agricultural Science 59, 381–385. Windisch W, Schedle K, Plitzner C, Kroismayr A. 2008. Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86, E140– E148. Yan L, Meng QW, Kim IH. 2012. The effect of an herb extract mixture on growth performance, nutrient digestibility, blood characteristics and fecal microbial shedding weanling pigs. Livestock Science 145, 189–195. Yan L, Wang JP, Kim HJ, Meng QW, Ao X, Hong SM, Kim IH. 2010. Influence of essential oil supplementation and diets with different nutrient densities on growth performance, nutrient digestibility, blood characteristics, meat quality and fecal noxious gas content in grower – finisher pigs. Livestock Science 128, 115–122. Yoon JH, Ingale SL, Kim JS, Kim KH, Lee SH, Park YK, et al. 2012. Effects of dietary supplementation of antimicrobial peptide-A3 on growth performance, nutrient digestibility, intestinal and fecal microflora and intestinal morphology in weanling pigs. Animal Feed Science and Technology 177, 98–107. Zhang Q, Piao XL, Piao XS, Lu T, Wang D, Kim SW. 2011. Preventive effect of Coptis chinensis and berberine on intestinal injury in rats challenged with lipopolysaccharides. Food and Chemical Toxicology 49, 61–69. Zhao R, Shen GX. 2005. Functional modulation of antioxidant enzymes in vascular endothelial cells by glycated LDL. Atherosclerosis 179, 277–284.
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doi: 10.1111/asj.12277
ORIGINAL ARTICLE Effects of essential oil supplementation of a low-energy diet on performance, intestinal morphology and microflora, immune properties and antioxidant activities in weaned pigs Zhikai ZENG, Xiao XU, Qiang ZHANG, Ping LI, Panfeng ZHAO, Qingyun LI, Jundi LIU and XiangShu PIAO State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing, China
ABSTRACT A total of 144 weaned piglets were used to evaluate the effects of essential oil (EO) supplementation of a low-energy diet on performance, apparent nutrient digestibility, small intestinal morphology, intestinal microflora, immune properties and antioxidant activities in weaned pigs. Pigs received a low-energy diet (negative control, NC, digestible energy = 3250 kcal/ kg), NC plus 0.025% EO or a positive control diet (PC, digestible energy = 3400 kcal/kg) for 28 days. Growth performance was similar between the EO group and PC group. However, EO supplementation increased (P < 0.05) average daily gain and the apparent digestibility of dry matter, crude protein and energy compared with pigs fed the NC diet. Greater (P < 0.05) villus height and lower (P < 0.05) counts of Escherichia coli and total anaerobes in the rectum in the EO group were observed compared with NC or PC groups. Pigs fed EO diet had higher (P < 0.05) concentrations of albumin, immunoglobulin A (IgA), IgG and total antioxidant capacity and lower fecal score than pigs fed the PC and NC diets. Above all, this study indicates that supplementation of EO to a low-energy pig diet has beneficial results and obtains similar performance compared with normal energy (PC) diet.
Key words: antioxidant activity, essential oils, microflora, performance, pig.
INTRODUCTION Essential oils (EO), also referred to as volatile or ethereal oils, are mixtures of volatile, lipophilic, largely terpenoid compounds extracted from plant materials such as flowers, buds, seed, leaves, twigs, bark, herbs, woods, fruits and roots (Brenes & Roura 2010). EO are secondary metabolites of plants and have several advantages over commercial antibiotics since they are residue free, broad-spectrum and are generally recognized as safe and beneficial by the Food and Drug Administration (Yan et al. 2010). The trend of ban on the use of antibiotics has led to many investigations conducted to identify alternatives to commercial antibiotics as growth promoters for use in the animal industry (Jang et al. 2007; Yan et al. 2010). EO have received increased attention as one of the potential alternatives to improve performance in intensive management systems (Brenes & Roura 2010). A growing number of studies have indicated that EO have beneficial effects, such as antiviral (Bishop 1995), antimicrobial (Rota et al. 2004), antioxidant © 2014 Japanese Society of Animal Science
(Botsoglou et al. 2004) properties, and can enhance the production of digestive secretions (Jang et al. 2004) and stimulate immune function (Wenk 2003). Although in vitro, the antimicrobial effect of some plant extracts is as consistent as the effect of antibiotics (Cowan 1999). However, the effect of plant extracts in vivo is inconsistent, even for the same product used in similar but not equal conditions (Windisch et al. 2008). Giannenas et al. (2003) reported that the efficacy of dietary EO was affected by intrinsic and extrinsic factors, such as nutritional status, infection, diet composition and environment. Diet is one of the main factors that can affect the efficacy of EO (Si et al. 2006a). For instance, Manzanilla et al. (2009)
Correspondence: XiangShu Piao, State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing 100193, China. (Email: [email protected]) Received 6 March 2014; accepted for publication 6 June 2014.
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demonstrated that dietary protein can modify the effect of plant extracts in the intestinal ecosystem of weaning pigs. In addition, EO supplementation significantly improved the digestibility of energy in pigs during the grower period (Yan et al. 2010) and can increase ileal digestibility of energy in broilers (Cao et al. 2010). Therefore, theis experiment was conducted to evaluate the effects of EO supplementation to a low-energy diet on performance, apparent digestibility of nutrients, antioxidant activities, immune properties, small intestinal morphology and intestinal microflora in weaned pigs.
MATERIALS AND METHODS Essential oil product The commercial EO product used in this study was provided by Danisco Co., Ltd., Kunshan, China. The product contained at least 4.5% cinnamaldehyde and 13.5% thymol, which have been demonstrated to possess anti-bacterial and antioxidant activities (Didry et al. 1993; Aeschbach et al. 1994; Gill & Holley 2004).
Animals and facilities All the procedures in this study were approved by the China Agricultural University Animal Care and Use Committee (Beijing, China). A total of 144 crossbred piglets (Duroc × Landrace × Large White), weighing on average 7.26 ± 1.57 kg and weaned at 28 ± 2 days of age were used in this experiment. Piglets were allotted to one of the three dietary treatments on the basis of weight, genetics and gender in a randomized complete block design. Each dietary treatment was fed to 12 pens with four pigs per pen (two barrows and two gilts). The experimental diets were a low-energy diet (negative control, digestible energy (DE) = 3250 kcal/kg) supplemented with 0% or 0.025% EO product as well as a positive control diet (PC, DE = 3400 kcal/kg) (Table 1). All diets were fed in mash form and contained 0.25% chromic oxide as an indigestible marker. All pigs had free access to feed and water throughout the 4-week feeding trial. The experimental piglets were housed in an environmentally controlled nursery room in 2 × 3 m raised pens equipped with a mesh floor. The temperature of the pig barn was controlled between 25 and 32°C.
Growth performance and fecal digestibility Pigs and feeders were weighed at the beginning and the end of the experiment in order to determine weight gain, feed intake and feed efficiency. Fecal consistency was visually assessed every morning by observers unaware of the dietary treatments, using a modification of the method described by Sherman et al. (1983). Fresh excreta were ranked using the following scale: 1, normal (feces firm and well formed); 2, soft consistency (feces soft and formed); 3, mild diarrhea (fluid feces, usually yellowish); and 4, severe diarrhea (feces watery and projectile). Fresh fecal grab samples were collected from the anus of pigs on days 26 to 28 and pooled by pen. Approximately 50 g of fresh feces were collected from each pen into sterile plastic bags and immediately stored at −20°C until analysis. © 2014 Japanese Society of Animal Science
Table 1
Ingredients of the experimental diets† (as-fed basis)
Ingredients, %
PC
Wheat Corn Extruded full-fat soybean Fish meal Whey power Wheat middling Soybean oil Dicalcium phosphate Limestone Salt L-Lys-HCl, 78% DL-Methionine hydroxyl analogue, 84% L-Threonine L-Tryptophan Vitamin-mineral premix‡ Essential oil Chromic oxide Total Nutrient levels, %§ Digestible energy, Mcal/kg Crude protein Calcium Phosphorus
NC
EO
54.00 5.10 16.20 4.40 4.00 10.00 2.70 0.45 0.68 0.25 0.56 0.10
54.00 8.50 15.50 4.40 4.00 10.00 – 0.45 0.68 0.25 0.56 0.10
54.00 8.48 15.50 4.40 4.00 10.00 – 0.45 0.68 0.25 0.56 0.10
0.25 0.06 1.00 – 0.25 100.00
0.25 0.06 1.00 – 0.25 100.00
0.25 0.06 1.00 0.025 0.25 100.00
3.40 19.01 0.69 0.55
3.25 18.94 0.72 0.58
3.25 19.06 0.68 0.56
†The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Vitamin and mineral premix provides the following per kg of feed: vitamin A, 9000 IU; vitamin D3, 2400 IU; vitamin E, 20 IU; vitamin K3, 3 mg; thiamine, 1.4 mg; riboflavin, 4 mg; vitamin B6, 3 mg; vitamin B12, 12 μg; nicotinic acid, 30 mg; pantothenic acid, 14 mg; folic acid, 0.8 mg; biotin, 44 μg; choline chloride, 0.5 g; Fe, 76 mg; Cu, 200 mg; Zn, 76 mg; Mn, 20 mg; I, 0.48 mg; Se, 0.4 mg. §The digestible energy is calculated while the other nutrients are analyzed.
On day 28, blood samples (about 5 mL) were obtained from the jugular vein by vena cava puncture from one barrow (weighing closest to the average body weight for each pen) per pen, using a 10 mL anticoagulant-free vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) after an overnight fast. Blood samples were centrifuged at 3000 × g (Heraeus Biofuge 22R Centrifuge, Hanau, Germany) for 10 min at 4°C and the serum samples were obtained and stored at −80°C for later analysis. At 08.30 hours on day 29, the 18 pigs (six pens from each treatment and one pig per pen) which supplied blood samples were humanely killed by exsanguination after electrical stunning to obtain intestinal tissues and digesta samples as described by Shen et al. (2009). Intestinal tissue from middle sections of duodenum, jejunum and ileum were aseptically isolated, flushed with 0.9% salt solution, fixed in 10% formaldehyde-phosphate buffer, and kept at 4°C for microscopic assessment of mucosal morphology. The digesta in the cecum, colon and rectum were collected and immediately immersed in liquid nitrogen and then stored at −80°C for subsequent microbial counting.
Chemical analysis of feed and feces Feed samples were collected at the start of the experiment. Fecal samples were thawed, dried in an oven (65°C) and ground to pass through a 1-mm sieve. Animal Science Journal (2014) ••, ••–••
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Feed and fecal samples were analyzed for dry matter, crude protein (N × 6.25) and calcium, according to the methods of AOAC (1990). Gross energy was determined by an automatic adiabatic oxygen bomb calorimeter (Parr 1281 Automatic Energy Analyzer, Parr Instruments Co., Moline, IL, USA). Chromium content was analyzed using an atomic absorption spectrophotometer (Z-5000 Automatic Absorption Spectrophotometer; Hitachi, Tokyo, Japan) according to Williams et al. (1962). Phosphorus content was analyzed using a UV-visible spectrophotometer (U-1000; Hitachi, Tokyo, Japan).
Small intestinal morphology Fixed intestinal segments were embedded in paraffin wax. Cross-sections at 6 μm thickness of each intestinal segment were stained with hematoxylin and eosin. Intestinal morphological measurements including villus height and crypt depth were determined at 40 × magnification using an Olympus CK 40 microscope (Olympus Optical Company, Shenzhen, China). Lengths of 10 well-oriented intact villi and their associated crypt were measured in triplicate for each segment of each pig according to the procedure of Touchette et al. (2002).
Intestinal microbiology analysis The microbiological counts in the large intestinal digesta were determined by the plate-count technique (Wang et al. 2011). One gram of mixed digesta was homogenized in salt solution (0.9% NaCl) and serially diluted from 10−1 to 10−9 for analysis of Escherichia coli (MacConkey agar, Beijing Haidian Microbiological Culture Factory, Beijing, China), lactobacilli (MRS agar, De Man, Rogosa, Sharpe, Oxoid Ltd., Basingstoke, UK; CM0361. anaerobic chamber), total anaerobes (plate-count agar, anaerobic chamber), and total aerobes (plate-count agar). All plates were incubated for 48 h in 37°C. The microbial populations were log transformed before statistical analysis.
Measurement of immune and antioxidant indices in serum Serum concentrations of immunoglobulins (IgA, IgG and IgM) were quantified using an enzyme-linked immunosorbent assay specific for swine (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. Assays were counted in duplicate using a TEACAN Plate Reader (TEACAN Asia, Shanghai, China). Plasma albumin level was measured with an Automatic Biochemical Analyzer (RA-1000; Bayer Corp., Tarrytown, NY, USA) using colorimetric methods and the instructions from the manufacturer’s corresponding reagent kit (Zhongsheng Biochemical Company, Beijing, China). Determination of serum total antioxidant capacity, superoxide dismutase, glutathione peroxidase and malondialdehyde levels were conducted by spectrophotometric methods using a spectrophotometer (Leng Guang SFZ1606017568, Shanghai, China) following the instructions of the kit’s manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis All data were analyzed as a completely randomized block design using the GLM procedure of SAS (1999). Each pen or individual pig was considered as the experimental unit. Statistical differences among treatments are separated by Animal Science Journal (2014) ••, ••–••
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Table 2 Effect of dietary essential oil on the performance and fecal consistency of weaned pigs†
Item
PC
NC
EO
SEM§
Average daily 382¶ 348†† 383¶ 4.50 gain, g*** Average daily feed 633 636 631 11.98 intake, g Feed conversion 1.65¶ 1.82†† 1.64¶ 0.04 ratio*** Fecal 1.42†† 1.44†† 1.29¶ 0.07 consistency‡** ¶††Means in the same row with different superscripts are significantly different (P < 0.05). **Treatments effect (P < 0.05); ***Treatments effect (P < 0.01). †Value represents mean of 12 pens with four pigs per pen. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Fecal score was assessed visually every morning, and fresh excreta were ranked using the following scale: 1, normal (feces firm and well formed); 2, soft consistency (feces soft and formed); 3, mild diarrhea (fluid feces, usually yellowish); and 4, severe diarrhea (feces watery and projectile). §Pooled standard error mean.
Student-Neuman-Keul’s multiple range test. Significance was taken at P ≤ 0.05, with a trend of P ≤ 0.10.
RESULTS Performance and fecal consistency Pigs fed EO or the PC diets had greater (P < 0.05) average daily gain (ADG) and lower (P < 0.05) feed conversion ratio (FCR) compared with pigs fed the NC diet (Table 2). However, no difference was observed in ADG and FCR between pigs fed EO or the PC diets. Average daily feed intake was not affected by dietary treatments. Fecal consistency score was significantly reduced (P < 0.05) in pigs fed EO diet compared with pigs fed the PC or NC diets.
Apparent nutrient digestibility Pigs fed EO or the PC diets had greater (P < 0.01) total tract apparent digestibility of dry matter, crude protein and energy than pigs fed the NC diet (Table 3). However, no difference was observed in these measurements between pigs fed EO or the PC diets. The digestibility of calcium and phosphorus did not differ among treatments.
Intestinal morphology Histological evaluation of duodenum and ileum indicated no effect of dietary treatments on villus height, crypt depth, and ratio of villus height and crypt depth (Table 4). However, jejunal villus height and villus height to crypt depth ratio of pigs fed EO diet were greater (P < 0.05) than those pigs fed the NC diet. Jejunal villus height was greater in pigs fed EO diet (P < 0.05), but smaller in pigs fed NC diet compared with pigs fed the PC diet. © 2014 Japanese Society of Animal Science
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Table 3 Effect of dietary essential oil on apparent total tract nutrient digestibility (%) in weaned pigs†
Item
PC
NC
EO
SEM‡
Dry matter*** Crude protein*** Energy*** Calcium Phosphorus
81.2§ 79.3§ 79.9§ 56.3 56.3
79.2¶ 73.3¶ 76.3¶ 57.0 56.0
81.2§ 79.2§ 81.1§ 59.5 60.0
0.48 0.85 0.57 1.65 1.61
§¶Means in the same row with different superscripts are significantly different (P < 0.05). ***Treatments effect (P < 0.01). †Value represents mean of 12 pens with four pigs per pen. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
Table 4 Effect of dietary essential oil on small intestinal morphology of weaned pigs†
Item Duodenum Villus height (μm) Crypt depth (μm) Villus height/crypt depth Jejunum Villus height (μm)*** Crypt depth (μm) Villus height/crypt depth*** Ileum Villus height (μm) Crypt depth (μm) Villus height/crypt depth
PC
NC
593 188 3.16
586 192 3.06
EO 597 190 3.15
SEM‡ 6.57 3.87 0.07
512¶ 495†† 529§ 5.20 163 165 165 2.25 3.14§¶ 3.01¶ 3.21§ 0.05
471 132 3.57
473 138 3.45
480 134 3.60
4.98 2.14 0.07
§–††Means in the same row with different superscripts are significantly different (P < 0.05). ***Treatment effect (P < 0.01). †Value represents mean of six pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
Microbial counts in large intestine No significant difference was observed in the microbial counts among treatments in the cecum (Table 5). The counts of E. coli tended to decrease (P < 0.10), while the number of lactobacilli was slightly increased (P < 0.10) in the colon in EO treatment compared with pigs fed the PC and NC diets. In addition, total anaerobe numbers in the colon were significantly reduced (P < 0.05) by the addition of EO compared with pigs fed the PC and NC diets. In the rectum, the counts of E. coli and total anaerobes were significantly reduced (P < 0.05) in pigs fed the EO-supplemented diet compared with pigs fed the PC and NC diets. Furthermore, there was also a tendency (P < 0.10) for increased number of lactobacilli in the rectum in pigs fed the diet supplemented with EO. Finally, total aerobes number © 2014 Japanese Society of Animal Science
Table 5 Effect of dietary essential oil on microbial counts (log10 colony-forming units/g of digesta) in the large intestine of weaned pigs†
Item Cecum E. coli Lactobacilli Total anaerobes Total aerobes Colon E. coli* Lactobacilli* Total anaerobes** Total aerobes Rectum E. coli** Lactobacilli* Total anaerobes** Total aerobes
PC
NC
EO
SEM‡
5.13 6.79 6.75 6.11
5.00 6.84 6.79 6.05
4.73 6.73 6.66 6.02
0.13 0.23 0.05 0.12
5.06 7.02 7.06§¶ 5.94
5.16 6.92 7.19§ 5.92
4.70 7.27 6.88¶ 5.77
0.13 0.09 0.06 0.08
4.88§ 7.19 7.68§ 6.39
5.00§ 7.30 7.64§ 6.29
4.67¶ 7.46 7.37¶ 6.32
0.08 0.07 0.07 0.08
§¶Means in the same row with different superscripts are significantly different (P < 0.05). *Treatments effect (P < 0.10); **Treatments effect (P < 0.05). †Value represents mean of six pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
in different segments of the hindgut was not affected by dietary treatment.
Immunity and antioxidant indices in serum The concentrations of albumin, IgA and IgG were significantly increased (P < 0.05) in response to EO supplementation compared with pigs fed the PC and NC diets (Table 6). Dietary EO significantly increased (P < 0.05) total antioxidant capacity, while leading to a slight improvement (P < 0.10) in the level of superoxide dismutase and glutathione peroxidase. There was no significant difference in concentrations of IgM and malondialdehyde among dietary treatments.
DISCUSSION In the present study, our results demonstrate that EO supplementation improved ADG and FCR, which agrees with Li et al. (2012b), who reported that the addition of EO at 100 and 150 ppm significantly increased ADG and FCR in weaned pigs. Hong et al. (2004) found similar results that 0.1% or 0.2% plant extract supplementation significantly increased ADG in weaned pigs compared with pigs fed diets without botanical supplementation. Evidently, the improved performance in pigs fed EO diet compared with those pigs fed the NC diet in the present study were associated with greater apparent total tract digestibility of dry matter, crude protein and energy, as well as Animal Science Journal (2014) ••, ••–••
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Table 6 Effect of dietary essential oil on immune indices and antioxidant capacity in plasma of weaned pigs†
Item
PC
NC
EO
SEM‡
Immune indices Albumin, g/L** 26.3¶ 25.9¶ 27.6§ 0.42 Immunoglobulin A, mg/L*** 46.4¶ 46.8¶ 51.0§ 0.68 Immunoglobulin G, mg/L*** 245¶ 236¶ 273§ 3.64 Immunoglobulin M, mg/L 49.7 50.1 52.5 0.97 Antioxidant indices Total antioxidant capacity, 11.2¶ 11.2¶ 12.1§ 0.22 U/mL** Superoxide dismutase, U/mL* 73.5 76.7 77.0 1.13 Glutathione peroxidase, U/mL* 933 941 949 4.76 Malondialdehyde, nmol/mL 4.5 4.5 4.4 0.07 §¶Means in the same row with different superscripts are significantly different (P < 0.05). *Treatments effect (P < 0.10); **treatments effect (P < 0.05); ***treatments effect (P < 0.01). †Value represents mean of 12 pigs per treatment. The dietary treatments were: PC (positive control); NC (negative control, 150 kJ/kg digestible energy lower than PC diet); EO (NC diet supplemented with 0.025% EO product which contained at least 4.5% cinnamaldehyde and 13.5% thymol). ‡Pooled standard error mean.
improved intestinal morphology in jejunum, microbial composition in large intestine and total antioxidant capacity in serum. The significant improvement in apparent total tract digestibility of dry matter, crude protein and energy in this study is in agreement with Yan et al. (2010), who reported that the digestibility was increased by EO supplementation. Similar results were reported by Cho et al. (2006). The beneficial effect of EO (blend of thymol and cinnamaldehyde) on energy and nutrient utilization was also observed in broilers (Amerah et al. 2011). Improved nutrient utilization might be due to enhanced small morphology or increased secretion of digestive enzymes (Jang et al. 2007). It is well documented that weaning stress in piglets has caused significant reduction in villus height (Tang et al. 1999; Shen et al. 2009; Wang et al. 2011). Villus atrophy is mainly caused by an increased rate of cell apoptosis or programmed death and decreased rate of renewal, which can be affected by cellular factors or endogenous stressors (van der Peet-Schwering et al. 2007). An increase in villus height and villus : crypt ratio in the jejunum was observed in this study in pigs fed EO after weaning when compared with pigs fed the NC diet. These results agree with those from a study conducted by Li et al. (2012a), in which dietary supplementation of essential oil improved the villus : crypt ratio in the jejunum in weaned pigs. Decreased number of pathogenic bacteria in the gut may improve proliferation of epithelial cells to build villus in the gut and thus enhance intestinal morphology (Moura¯o et al. 2005). Antioxidants may fix the intestine impairment caused by oxidative stress and improve intestinal morphology in weaned pigs (Han et al. 2012). In this study, the change of microflora Animal Science Journal (2014) ••, ••–••
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composition in the colon and rectum and increased serum total antioxidant capacity may partly explain the enhanced intestinal morphology. It is generally known that post-weaning diarrhea is one of the critical factors causing high mortality in piglets (Osek 1999). In the present study, the fecal consistency score was significantly reduced in pigs fed EO diets compared with pigs fed the PC and NC diets, which is in agreement with Hong et al. (2004) and Manzanilla et al. (2004). Si et al. (2006b) indicated that EO compounds have a strong antimicrobial action against gut pathogens while not harming beneficial bacteria such as bifidobacteria and lactobacilli. Meanwhile, increased number of lactobacilli and reduction of E. coli in the gut microbiota might result in a decreased incidence of diarrhea in piglets (Manzanilla et al. 2004; Li et al. 2012a). In the present study, the counts of E. coli and total anaerobes in the rectum were significantly reduced (P < 0.05) in pigs fed EO, while the number of lactobacilli was slightly increased in the colon and rectum in pigs fed EO. The antimicrobial mode of action of EO supplementation is considered to arise mainly from the potential of the hydrophobic EO to intrude into the bacterial cell membrane, disintegrate membrane structures and cause ion leakage (Windisch et al. 2008). In addition, phytogenic feed additives were also reported to stimulate intestinal secretion of mucus in broilers, an effect that was assumed to impair adhesion of pathogens such as E. coli and thus to contribute to stabilizing the microbial eubiosis in the gut of the animals (Jamroz et al. 2006). The results in this study suggest that EO have potential for suppressing harmful intestinal microflora and improving the intestinal ecosystem in weaned pigs. The maturation and optimal development of the immune system is highly associated with the development and composition of the indigenous microflora and vice versa (Insoft et al. 1996; de Vrese & Marteau 2007). In this study, EO supplementation increased the concentrations of serum albumin, IgA and IgG, which may be highly related to the improved microflora composition in the large intestine. Similar results were reported by Yan et al. (2012), who demonstrated that plant extracts played a positive role in enhancing fecal microflora composition and immune status. Cho et al. (2006) also reported an increased serum IgG concentration in pigs fed diets supplemented with EO. The newly weaned piglet is very susceptible to diseases, stressors and digestive disorders during the first week post-weaning (Yoon et al. 2012). At this stage, an increased concentration of the serum immunoglobulin is required to regulate and enhance immune function, which provides health benefits, diminishes weaning stress and improves health status and performance of weanling pigs (Turner et al. 2002). © 2014 Japanese Society of Animal Science
6 Z. ZENG et al.
Oxidative stress is caused by excessive oxidative radicals, including reactive oxygen species, which damage DNA, bio-membrane lipids, and proteins, as well as causing a variety of impairments to tissue (Zhao & Shen 2005; Zhang et al. 2011). However, excessive oxidative radicals can be eliminated by the use of an antioxidant system, including nonenzymatic components and a series of antioxidant enzymes. In the enzyme antioxidant system, superoxide dismutase and glutathione peroxidase are the most important antioxidants (Lu et al. 2010). They work together to detoxify superoxide anions and hydrogen peroxide in cells (Zhang et al. 2011). Superoxide dismutase is able to catalyze superoxide anions to produce hydrogen peroxide and molecular oxygen. Glutathione peroxidase normally converts H2O2 to water (Lu et al. 2010). In the present study, the level of superoxide dismutase and glutathione peroxidase in serum tended to increase by the addition of EO. Total antioxidant capacity, which reflects the non-enzymatic antioxidant defense system (Wang et al. 2008), was significantly increased by EO in the present study, indicating that EO may play an important role in preventing endogenous lipids from peroxidation and oxidation. Furthermore, the level of malondialdehyde, which is a major product of lipid peroxidation, was an effective marker in oxidative stress in sepsis (Lu et al. 2010). In conclusion, EO supplementation to a low-energy diet (DE = 3250 kJ/kg) improved performance compared with those pigs fed NC diet and obtained similar performance compared with those pigs fed PC diet (DE = 3400 kJ/kg). The beneficial results may be explained by improved intestinal morphology, microflora composition in the large intestine and improved health status measured by fecal scores, immune indices and antioxidant indices.
ACKNOWLEDGMENTS The financial support was received from the National Natural Science Foundation of China (No.31372316), the State Key Laboratory of Animal Nutrition of China (No. 2004DA125184F1211), and the Danisco Co. Ltd. (China). We thank Novus International for supplying the methionine hydroxyl analogue.
REFERENCES Aeschbach R, Löliger J, Scott BC, Murcia A, Butler J, Halliwell B, Aruoma OI. 1994. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food and Chemical Toxicology 32, 31–36. Amerah AM, Peron A, Zaefarian F, Ravindran V. 2011. Influence of whole wheat inclusion and a blend of essential oils on the performance, nutrient utilisation, digestive tract development and ileal microbiota profile of broiler chickens. British Poultry Science 52, 124–132.
© 2014 Japanese Society of Animal Science
Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Washington, DC. Bishop CD. 1995. Antiviral activity of the essential oil of Melaleuca alternifolia (Maiden and Betche) Cheel (tea tree) against tobacco mosaic virus. Journal of Essential Oil Research 7, 641–644. Botsoglou NA, Christaki E, Florou-Paneri P, Giannenas I, Papageorgiou G, Spais AB. 2004. The effect of a mixture of herbal essential oils or α-tocopheryl acetate on performance parameters and oxidation of body lipid in broilers. South African Journal of Animal Science 34, 52–61. Brenes A, Roura E. 2010. Essential oils in poultry nutrition: main effects and modes of action. Animal Feed Science and Technology 158, 1–14. Cao PH, Li FD, Li YF, Ru YJ, Peron A, Schulze H, Bento H. 2010. Effect of essential oils and feed enzymes on performance and nutrient utilization in broilers fed a corn/ soy-based diet. International Journal of Poultry Science 9, 749–755. Cho JH, Chen YJ, Min BJ, Kim HJ, Kwon OS, Shon KS, et al. 2006. Effects of essential oils supplementation on growth performance, IgG concentration and fecal noxious gas concentration of weaned pigs. Asian-Australasian Journal of Animal Sciences 19, 80–85. Cowan MM. 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12, 564–582. de Vrese M, Marteau PR. 2007. Probiotics and prebiotics: effects on diarrhea. The Journal of Nutrition 137, 803–811. Didry N, Dubreuil L, Pinkas M. 1993. Antibacterial activity of thymol, carvacrol and cinnamaldehyde alone or in combination. Die Pharmazie 48, 301–304. Giannenas I, Florou-Paneri P, Papazahariadou M, Christaki E, Botsoglou NA, Spais AB. 2003. Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition 57, 99–106. Gill AO, Holley RA. 2004. Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei. Applied and Environmental Microbiology 70, 5750–5755. Han X, Piao XS, Zhang HY, Li PF, Yi JQ, Zhang Q, Li P. 2012. Forsythia suspensa extract has the potential to substitute antibiotic in broiler chicken. Asian-Australasian Journal of Animal Sciences 25, 569–576. Hong JW, Kim IH, Kwon OS, Min BJ, Lee WB, Shon KS. 2004. Influences of plant extract supplementation on performance and blood characteristics in weaned pigs. AsianAustralasian Journal of Animal Sciences 17, 374–378. Insoft RM, Sanderson IR, Walker WA. 1996. Development of immune function in the intestine and its role in neonatal diseases. Pediatric Clinics of North America 43, 551–571. Jamroz D, Wertelecki T, Houszka M, Kamel C. 2006. Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrition 90, 255– 268. Jang IS, Ko YH, Kang SY, Lee CY. 2007. Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Animal Feed Science and Technology 134, 304–315. Jang IS, Ko YH, Yang HY, Ha JS, Kim JY, Kim JY, et al. 2004. Influence of essential oil components on growth perfor-
Animal Science Journal (2014) ••, ••–••
ESSENTIAL OIL FOR WEANED PIGS
mance and the functional activity of the pancreas and small intestine in broiler chickens. Asian-Australasian Journal of Animal Sciences 17, 394–400. Li PF, Piao XS, Ru YJ, Han X, Xue LF, Zhang HY. 2012a. Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. Asian-Australasian Journal of Animal Sciences 25, 1617–1626. Li SY, Ru YJ, Liu M, Xu B, Péron A, Shi XG. 2012b. The effect of essential oils on performance, immunity and gut microbial population in weaner pigs. Livestock Science 145, 119–123. Lu T, Piao XL, Zhang Q, Wang D, Piao XS, Kim SW. 2010. Protective effects of Forsythia suspensa extract against oxidative stress induced by diquat in rats. Food and Chemical Toxicology 48, 764–770. Manzanilla EG, Perez JF, Martin M, Kamel C, Baucells F, Gasa J. 2004. Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science 82, 3210–3218. Manzanilla EG, Pérez JF, Martín M, Blandón JC, Baucells F, Kamel C, Gasa J. 2009. Dietary protein modifies effect of plant extracts in the intestinal ecosystem of the pig at weaning. Journal of Animal Science 87, 2029–2037. Moura¯o JL, Pinheiro V, Alves A, Guedes CM, Pinto L, Saavedra MJ, et al. 2005. Effect of mannan oligosaccharides on the performance, intestinal morphology and cecal fermentation of fattening rabbits. Animal Feed Science and Technology 126, 107–120. Osek J. 1999. Prevalence of virulence factors of Escherichia coli strains isolated from diarrheic and healthy piglets after weaning. Veterinary Microbiology 68, 209–217. Rota C, Carraminana JJ, Burillo J, Herrera A. 2004. In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens. Journal of Food Protection 67, 1252–1256. SAS Inst. 1999. SAS User’s Guide: Statistics, Version 8.01 edn. SAS Inst., Cary, NC. Shen YB, Piao XS, Kim SW, Wang L, Liu P, Yoon I, Zhen YG. 2009. Effects of yeast culture supplementation on growth performance, intestinal health, and immune response of nursery pigs. Journal of Animal Science 87, 2614–2624. Sherman DM, Acres SD, Sadowski PL, Springer JA, Bray B, Raybould TJ, Muscoplat CC. 1983. Protection of calves against fatal enteric colibacillosis by orally administered Escherichia coli K99-specific monoclonal antibody. Infection and Immunity 42, 653–658. Si W, Gong J, Chanas C, Cui S, Yu H, Caballero C, Friendship RM. 2006a. In vitro assessment of antimicrobial activity of carvacrol, thymol and cinnamaldehyde towards Salmonella serotype Typhimurium DT104: effects of pig diets and emulsification in hydrocolloids. Journal of Applied Microbiology 101, 1282–1291. Si W, Gong J, Tsao R, Zhou T, Yu H, Poppe C, et al. 2006b. Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology 100, 296–305. Tang M, Laarveld B, Van Kessel AG, Hamilton DL, Estrada A, Patience JF. 1999. Effect of segregated early weaning on
Animal Science Journal (2014) ••, ••–••
7
postweaning small intestinal development in pigs. Journal of Animal Science 77, 3191–3200. Touchette KJ, Carroll JA, Allee GL, Matteri RL, Dyer CJ, Beausang LA, Zannelli ME. 2002. Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs. Journal of Animal Science 80, 494–501. Turner JL, Dritz SS, Higgins JJ, Minton JE. 2002. Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium. Journal of Animal Science 80, 1947– 1953. van der Peet-Schwering CMC, Jansman AJM, Smidt H, Yoon I. 2007. Effects of yeast culture on performance, gut integrity, and blood cell composition of weaning pigs. Journal of Animal Science 85, 3099–3109. Wang D, Piao XS, Zeng ZK, Lu T, Zhang Q, Li PF, et al. 2011. Effects of keratinase on performance, nutrient utilization, intestinal morphology, intestinal ecology and inflammatory response of weaned piglets fed diets with different levels of crude protein. Asian-Australasian Journal of Animal Sciences 24, 1718–1728. Wang YZ, Xu CL, An ZH, Liu JX, Feng J. 2008. Effect of dietary bovine lactoferrin on performance and antioxidant status of piglets. Animal Feed Science and Technology 140, 326–336. Wenk C. 2003. Herbs and botanicals as feed additives in monogastric animals. Asian-Australasian Journal of Animal Sciences 16, 282–289. Williams CH, David DJ, Iismaa O. 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. The Journal of Agricultural Science 59, 381–385. Windisch W, Schedle K, Plitzner C, Kroismayr A. 2008. Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86, E140– E148. Yan L, Meng QW, Kim IH. 2012. The effect of an herb extract mixture on growth performance, nutrient digestibility, blood characteristics and fecal microbial shedding weanling pigs. Livestock Science 145, 189–195. Yan L, Wang JP, Kim HJ, Meng QW, Ao X, Hong SM, Kim IH. 2010. Influence of essential oil supplementation and diets with different nutrient densities on growth performance, nutrient digestibility, blood characteristics, meat quality and fecal noxious gas content in grower – finisher pigs. Livestock Science 128, 115–122. Yoon JH, Ingale SL, Kim JS, Kim KH, Lee SH, Park YK, et al. 2012. Effects of dietary supplementation of antimicrobial peptide-A3 on growth performance, nutrient digestibility, intestinal and fecal microflora and intestinal morphology in weanling pigs. Animal Feed Science and Technology 177, 98–107. Zhang Q, Piao XL, Piao XS, Lu T, Wang D, Kim SW. 2011. Preventive effect of Coptis chinensis and berberine on intestinal injury in rats challenged with lipopolysaccharides. Food and Chemical Toxicology 49, 61–69. Zhao R, Shen GX. 2005. Functional modulation of antioxidant enzymes in vascular endothelial cells by glycated LDL. Atherosclerosis 179, 277–284.
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