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Animal Science Journal (2014) ••, ••–••
doi: 10.1111/asj.12331
ORIGINAL ARTICLE Effect of probiotic bacteria-fermented medicinal plants (Gynura procumbens, Rehmannia glutinosa, Scutellaria baicalensis) as performance enhancers in growing pigs Jin Suk JEONG and In Ho KIM Department of Animal Resource and Science, Dankook University, Cheonan, South Korea
ABSTRACT This study was conducted to investigate the effect of dietary supplementation of mixed fermented medicinal plants (FMP) obtained from exudates of Gynura procumbens, Rehmannia glutinosa and Scutellaria baicalensis fermented with Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus licheniformis, respectively, on growth performance in growing pigs in order to assess the feasibility of using FMP as an alternative to antibiotic growth promoters (AGP), such as tiamulin. A total of 150 growing pigs (body wieght 25.50 ± 2.50 kg) were used in a 6 weeks experiment and randomly divided into five groups with six replicates of five growing pigs each. The treatments were NC (basal diet), basal diet with 33 ppm tiamulin (PC), basal diet with FMP 0.05% (FMP 0.05), basal diet with FMP 0.1% (FMP 0.1) and basal diet with FMP 0.2% (FMP 0.2). Overall, body weight gain, feed conversion rate, the digestibility of dry matter and gross energy, noxious gas emission all improved with FMP supplementation as compared to NC. Taken together, these results suggest the feasibility of using FMP as an alternative to AGP for enhancing the growth performance, nutrient digestibility and excreta noxious gas emission of growing pigs.
Key words: excreta noxious gas, fermented medicinal plant, growing pig, growth performance, nutrient digestibility.
INTRODUCTION Tiamulin is a member of the pleuromutilin family of antimicrobial agents which are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. Tiamulin is commonly used to treat swine dysentery, spirochete-associated diarrhea, porcine proliferative enteropathy, enzootic pneumonia and other infections where mycoplasma is involved (van Duijkeren et al. 2014). Only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to tiamulin is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions presents a considerable threat to pig health, welfare and productivity. Due to the negative effects of antibiotic growth promoters (AGP), such as inducing microbial antibiotic resistance, public demand for natural, organic products has pressured and expedited the removal of all chemical additives from animal foodstuff in many countries (Sean 2002; Diarra et al. 2011). As a result, natural alternatives, such as medicinal plants and © 2014 Japanese Society of Animal Science
herbs, have attracted and gained widespread attention, due to their wide range of potential beneficial effects and increasing industry demands for maximizing net returns and carcass quality (Jenkins & Atwal 1995; Manesh et al. 2012; Costa et al. 2013). Gynura procumbens (also known as African mistletoe) is a wellknown tropical Asian medicinal herbal plant, containing flavonoid sterol and steroid, as well as some proteins (Hew & Gam 2010). Rehmannia glutinosa has been extensively used in traditional Chinese medicine to treat rheumatoid arthritis, asthma, urticaria and chronic nephritis (Zhang et al. 2008). Lastly, the genus Lamiaceae comprises over 360 species, many of which are medically important. Some Scutellaria baicalensis is used to treat neurological disorders, cancer, inflammatory diseases and viral and bacterial infections (Shang et al. 2010).
Correspondence: In Ho Kim, Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam 330-714, South Korea. (Email: inhokim@ dankook.ac.kr) Received 29 April 2014; accepted for publication 7 August 2014.
2 J. S. JEONG and I. H. KIM
Another alternative approach to sub-therapeutic antibiotics in livestock is the use of probiotic microorganisms. Probiotic is a generic term and products using this label tend to contain bacterial cultures capable of stimulating micro-organisms capable of modifying the gastrointestinal environment to favor health status and improve feed efficiency (Dierck 1989). Interestingly, fermentation of plant materials with a microbial inoculum has been widely used to promote novel functional ingredients because its various activities such as antioxidant and antiinflammatory activity may be promoted by the fermentation process (Lee et al. 2008; Wang et al. 2011a,b; Cao et al. 2012). For example, using exudates of medicinal plants and herbs fermented by probiotics could be better than medicinal plants and herbs or probiotics alone in theory, since pigs would benefit from the active ingredients found in medicinal plant materials in addition to the presence of probiotic bacteria in their intestines. However, to the best of our knowledge, there is limited data pertaining to the effect of dietary supplementation of mixed-fermented medicinal plants (FMP) obtained from exudates of Lactobacillus plantarum-fermented G. procumbens, Saccharomyses cereviseae-fermented R. glutinosa and Bacillus licheniformis-fermented S. baicalensis as an alternative to AGP on growing pigs. Therefore, the objective of this study was to investigate whether FMP can match or even surpass an AGP as a viable alternative in feed, regarding relevant parameters, such as growth performance, nutrient digestibility, blood profiles and excreta noxious gas emission of growing pigs as compared to basal diet supplemented with tiamulin.
MATERIALS AND METHODS The experimental protocols used in this study were approved by the Animal Care and Use Committee of Dankook University (Anseodong, Cheonan, Choongnam, Korea).
Preparation of fermented medicinal plants The solid culture of the FMP product used in this study contained a mixture of lactic acid bacteria at 3.0 × 108 colony forming units (CFU)/g (Lactobacillus planetarium species), yeasts at 7.5 × 107 CFU/g (Saccharomyces cerevisiae species) and fermenting bacteria at 8.0 × 108 CFU/g (Bacillus licheniformis species). The FMPs were prepared as follows: the upper leaves of the G. procumbens plant were taken and thoroughly washed, and then cut to approximately 1 × 1 cm pieces. Processed leaves were mixed with L. plantarum inoculants which were previously fermented with soybean meal (1:1, w/w). The fermentation condition of L. plantarum had an estimated 40% moisture for 2 days at 30°C under solid-state fermentation (SSF). R. glutinosa roots were thoroughly washed and inoculated with 5% (v/w) S. cerevisiae which were fermented with soybean meal (1:1, w/w) for 2 months at low temperature and then kept for 5 days at 25°C under SSF. S. baicalensis © 2014 Japanese Society of Animal Science
roots were thoroughly washed and inoculated with 5% (v/w) B. licheniformis which were fermented with soybean meal (1:1, w/w) for 2 months at low temperature and then kept for 5 days at 30°C under SSF.
Experimental design and diets A total of 150 growing pigs ((Landrace × Yorkshire) × Duroc)) with an average body weight (BW) of 25.50 ± 2.50 kg were randomly allocated to one of five treatments with six replicates of growing pigs with five pigs per pen, according to initial BW for a 42-day experiment. The experimental design was a randomized complete block design. Dietary treatments included: (i) negative control (NC, basal diet); (ii) positive control (PC, basal diet with 33 ppm tiamulin); (iii) basal diet with fermented medicinal plant (FMP) 0.05% (FMP 0.05); (iv) basal diet with FMP 0.1% (FMP 0.1); and (v) basal diet with FMP 0.2% (FMP 0.2). The additive was added at the expense of soybean meal and dietary nutrients were provided to exceed or meet National Requirements Council (1998) recommendations (Table 1). All pigs were housed in an environmentally controlled nursery facility, with slatted plastic flooring in 20 adjacent pens (1.8 × 1.8 m), and provided with ad libitum access to feed and water.
Table 1 Ingredient and composition of the basal diet (as-fed basis)
Item
FMP (%) 0
Ingredient, % Corn Soybean meal (47.5% CP) Animal fat Molasses Dicalcium phosphate Salt Limestone Vitamin premix† FMP‡ Trace mineral premix§ L-Lys·HCl Ethoxyquin, 25.0% Analyzed composition, % DE, MJ/kg CP Lys Met Ca Total P
0.05
0.10
0.20
66.00 66.00 66.00 66.00 23.96 23.91 23.86 23.76 4.24 4.24 4.24 4.24 3.00 3.00 3.00 3.00 1.26 1.26 1.26 1.26 0.25 0.25 0.25 0.25 1.01 1.01 1.01 1.01 0.12 0.12 0.12 0.12 0.05 0.10 0.20 0.10 0.10 0.10 0.10 0.01 0.01 0.01 0.01 0.05 0.05 0.05 0.05 14.50 14.50 14.50 14.50 17.60 17.60 17.60 17.60 1.11 1.11 1.11 1.11 0.31 0.31 0.31 0.31 0.76 0.76 0.76 0.76 0.64 0.64 0.64 0.64
†Provided per kg of complete diet: 10 000 IU vitamin A, 2000 IU vitamin D3, 40 IU vitamin E, 100 mg vitamin C, 10 mg vitamin K3, 10 mg riboflavin, 2 mg vitamin B6, 25 mg pantothenic acid, 50 mg niacin, and 0.04 mg biotin. ‡The experimental diets were formulated by replacing soybean meal with 0 (control), 0.05%, 0.1%, and 0.2% fermented medicinal plants (FMP) extract. §Provided per kg of complete diet: 179 mg Zn (as zinc oxide); 12.5 mg Mn (as manganese oxide); 5 mg Cu (as copper sulfate); 0.5 mg I (as potassium iodide); and 0.4 mg Se (as sodium selenite). Lys, lysine; DE, digestible energy; CP, crude protein, Met, methionine.
Animal Science Journal (2014) ••, ••–••
FERMENTED MEDICINAL PLANTS IN PIGS
Growth performance and nutrient digestibility analysis Body weight gain (BWG) and feed intake (FI) were recorded on day 0, 2 weeks, 4 weeks and end of the 6 weeks feeding trial, which was then used to calculate gain/feed (G/F) ratio after correcting for mortality. From days 38 to 42, chromic oxide (0.2%) was added to diets as an indigestible marker for determination of apparent total tract digestibility (ATTD) of dry matter (DM), nitrogen (N) and gross energy (GE). Fresh fecal samples were obtained by massaging the rectum from at least two pigs per pen at days 41 and 42. Fecal samples from the same pen were pooled and mixed immediately, after which samples were stored in a freezer at −20°C until analysis. Before chemical analysis, feces were thawed and dried at 70°C for 72 h, after which they were finely ground to a size that could pass through a 1 mm sieve. Feed samples were ground to pass through a 1 mm screen, after which they were analyzed for N (Method 968.06; Association of Official Analytical Chemists 2000), Ca (Method 984.01; Association of Official Analytical Chemists 1995) and P (Method 965.17; Association of Official Analytical Chemists 1995). Individual amino acid (AA) composition was measured using an AA analyzer (Beckman 6300; Beckman Coulter, Inc., Fullerton, CA, USA) after 24 h hydrolysis with HCl (Spackman et al. 1958). Nitrogen content was determined (Kjectec 2300 Nitrogen; Foss Tecator AB, Hoeganaes, Sweden) and crude protein (CP) basis was calculated as N × 6.25. All feed and feces samples were analyzed for DM (Method 934.01; Association of Official Analytical Chemists 2000). Chromium was analyzed using UV absorption spectrophotometry (Shimadzu UV-1201; Shimadzu, Kyoto, Japan), according to the method described by Williams et al. (1962). GE was determined by measuring heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA).
Blood profile analysis Blood samples (10 mL) were collected, via anterior vena cava puncture, randomly from one pig in each pen at the end of this experiment. Blood samples were collected into both nonheparinized tubes (5 mL) and vacuum tubes (5 mL), containing tripotassium ethylenediaminetetraacetic acid (K3EDTA; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and stored at 4°C. Samples for serum analysis were then centrifuged at 3000 × g for 15 min, and then separated and stored at 4°C. White blood cells (WBC), red blood cells (RBC) and lymphocyte percentages were analyzed using an automatic blood analyzer (ADVIA 120; Bayer, Tarrytown, NY, USA).
Excreta gas emission analysis Fresh excreta samples were collected from each pen on the last 2 days of the experiment and then mixed well for each respective pen. Analysis was then conducted using excreta samples (150 g feces and 150 g of urine were well mixed; 1:1 on the wet weight basis) obtained from each pen then stored in 2.6 L sealed plastic boxes. Each box had a small hole in the middle of one side wall, which was sealed with adhesive plaster. The samples were permitted to ferment for 7 days at room temperature (25°C). The concentrations of gas were determined on days 1, 3, 5 and 7 during the fermentation period. A gas sampling pump (Model GV-100; Gastec Corp., Ayase, Japan) was utilized for gas detection (Gastec detector Animal Science Journal (2014) ••, ••–••
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tube no. 3La for ammonia; no. 4 LK for hydrogen sulfide; no. 70 for mercaptan; Gastec Corp.). Before the measurements, slurry samples were shaken manually for approximately 30 s to disrupt any crust formation on the surface of the slurry sample and to homogenize them. The adhesive plasters were punctured and 100 mL of headspace air was sampled approximately 2.0 cm above the slurry surface. Two samples from each pen were measured and then the mean was calculated.
Statistical analysis All experimental data were analyzed as a completely randomized block design using the GLM procedure of SAS (2003). The pen was used as the experimental unit. Orthogonal contrasts were used to test the overall effect of growth promoting materials supplementation (NC vs. FMPs) and the effect of the difference between effect of PC and FMPs (PC vs. FMPs); additionally, orthogonal comparison were conducted using polynomial regression to measure the linear and quadratic effects of increasing concentrations of FMPs. Variability in the data was expressed as the SEM and P < 0.05 was considered statistically significant, whereas P < 0.10 was considered a tendency.
RESULTS Growth performance In the phase 2 period, the supplementation of FMP improved BWG (P = 0.0243 and linear, P = 0.0265) and G/F (P = 0.0200 and linear, P = 0.0150) as observed with the comparison of NC versus FMPs and dose response of FMP, respectively (Table 2). In the phase 3 period, the supplementation of FMP improved BWG (P = 0.0041 and linear, P = 0.0021) and G/F (P = 0.0181 and linear, P = 0.0136) as observed with treatment comparison of NC versus FMP and dose response of FMPs, respectively. FMP treatment had no effect on FI as compared with NC and PC treatments; however, dose response of FMPs tended to increase according to the FMP concentration (linear, P = 0.0220) in the phase 3 period. Consequently, during the overall experiment, FMP supplementation improved BWG (P = 0.0228 and linear, P = 0.0182) and G/F (P = 0.0299 and linear, P = 0.0241), but not FI, with the treatment comparison of NC versus FMP and dose response of FMP, respectively.
Nutrient digestibility and blood profiles FMP supplementation improved ATTD of DM (P = 0.0299 and linear, P = 0.0179) and GE (P = 0.0790 and linear, P = 0.0628) with the treatment comparison of NC versus FMP and dose response of FMP, respectively. There was no significant effect on the ATTD of N (Table 3). FMP supplementation also increased WBC counts (P = 0.0201) with the treatment comparison of NC versus FMP and dose response of FMP tended to increase according to the FMP concentration (linear, P = 0.0878) (Table 4). Alternately, RBC counts were © 2014 Japanese Society of Animal Science
4 J. S. JEONG and I. H. KIM
Table 2 Effect of fermented medicinal plants (FMP) supplementation on growth performance in growing pigs
Treatment items
NC
PC
FMP 0.05 FMP 0.1
FMP 0.2
SEM
P-value NC vs. FMPs
Phase 1 (0–2 weeks) BWG (g) 583 FI (g) 1343 G/F 0.434 Phase 2 (3–4 weeks) BWG (g) 677 FI (g) 1598 G/F 0.424 Phase 3 (4–6 weeks) BWG (g) 707 FI (g) 2204 G/F 0.321 Overall BWG (g) 656 FI (g) 1715 G/F 0.383
PC vs. FMPs
Linear
Quadratic
608 595 1348 1326 0.451 0.449
611 622 7.94 0.2314 0.9638 0.1463 0.9302 1336 1332 4.37 0.2134 0.0987 0.5686 0.3802 0.457 0.467 0.006 0.1678 0.7077 0.1298 0.9525
709 702 1592 1606 0.445 0.437
712 717 5.92 0.0243 0.8993 0.0265 0.8301 1590 1583 4.89 0.6580 0.9327 0.2540 0.5511 0.448 0.453 0.004 0.0200 0.8870 0.0150 0.9929
749 743 2201 2210 0.240 0.336
759 770 6.55 0.0041 0.5903 0.0021 0.8127 2233 2235 5.86 0.1361 0.0863 0.0220 0.4046 0.340 0.345 0.003 0.0181 0.9942 0.0136 0.6378
689 680 1713 1714 0.402 0.397
694 703 6.10 0.0228 0.8046 0.0182 0.9234 1720 1717 1.81 0.7575 0.4785 0.6174 0.4432 0.403 0.409 0.004 0.0299 0.8690 0.0241 0.8639
BWG, body weight gain; FI, feed intake; G/F, gain/feed; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
Table 3 Effect of fermented medicinal plants (FMP) supplementation on nutrient digestibility in growing pigs
Treatment items
DM, % N, % GE, %
NC
78.14 78.47 79.29
PC
80.05 79.58 82.36
FMP 0.05
79.93 78.94 80.62
FMP 0.1
80.78 80.36 82.77
FMP 0.2
81.71 81.16 83.42
SEM
0.510 0.799 0.737
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
0.0299 0.3876 0.0790
0.5134 0.7663 0.9540
0.0179 0.2544 0.0628
0.8176 0.8478 0.7560
DM, dry matter; N, nitrogen; GE, gross energy; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
Table 4 Effect of fermented medicinal plants (FMP) supplementation on blood profiles in growing pigs
Treatment items
WBC, 103/ul RBC, 106/ul Lymphocyte, %
NC
19.01 7.27 47.03
PC
19.86 7.68 48.48
FMP 0.05
19.43 7.85 48.55
FMP 0.1
20.02 7.71 47.38
FMP 0.2
19.43 7.82 53.22
SEM
0.085 0.209 0.980
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
0.0201 0.2808 0.3308
0.5778 0.6792 0.6518
0.0923 0.4053 0.0878
0.2717 0.5442 0.3293
WBC, white blood cell; RBC, red blood cell; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
not affected by FMP supplementation as compared with NC and PC treatments (P > 0.05).
with treatment comparison of NC versus FMP and dose response of FMP, respectively.
Excreta noxious gas emission
DISCUSSION
The effect of FMP supplementation on the emission of ammonia, hydrogen sulfide and total mercaptans is shown in Table 5. Overall, FMP supplementation reduced ammonia (P = 0.0039 and linear, P = 0.0025 for day 7), hydrogen sulfide (P = 0.0076 and linear, P = 0.0038 for day 5) and total mercaptans (P = 0.0402 and linear, P = 0.0716 for day 5) emissions as observed
In the current study, BWG, FI and G/F of pigs eating a diet supplemented with FMP appeared to be comparable to that of pigs eating a diet supplemented with tiamulin, an AGP, thus illustrating the potential of FMP as an alternative to AGPs. In particular, dietary supplementation with FMP improved BWG and G/F in phase 2, phase 3 and overall periods in growing pigs.
© 2014 Japanese Society of Animal Science
Animal Science Journal (2014) ••, ••–••
FERMENTED MEDICINAL PLANTS IN PIGS
Table 5
5
Effect of fermented medicinal plants (FMP) supplementation on fecal noxious gas emission in growing pigs
Treatment Items
Ammonia, ppm 1 day 3 days 5 days 7 days Hydrogen sulfide, ppm 1 day 3 days 5 days 7 days Total mercaptans, ppm 1 day 3 days 5 days 7 days
NC
PC
FMP 0.05
FMP 0.1
FMP 0.2
SEM
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
4.9 15.2 19.3 22.9
4.6 15.7 18.9 20.7
4.3 15.0 18.3 20.6
4.2 14.6 18.1 19.3
4.0 14.4 17.6 18.8
0.264 0.417 0.357 0.444
0.3060 0.5907 0.1610 0.0039
0.5654 0.3405 0.3204 0.2552
0.3025 0.5497 0.1750 0.0025
0.8453 0.9589 0.7891 0.9900
5.5 10.8 14.8 16.4
4.7 10.3 14.0 16.0
4.8 10.4 13.4 15.0
5.6 10.4 13.4 15.2
5.2 10.0 13.1 14.3
0.058 0.060 0.124 0.076
0.8079 0.1152 0.0076 0.8145
0.4478 0.9137 0.2835 0.4373
0.2114 0.0922 0.0038 0.6916
0.5704 0.8359 0.8236 0.7859
0.5 1.3 2.2 1.9
0.4 1.1 1.6 1.7
0.3 1.2 1.7 1.8
0.5 1.0 1.3 1.9
0.7 1.1 1.0 1.8
0.205 0.329 0.281 0.516
0.6539 0.5146 0.0402 0.2897
0.3438 0.9297 0.3335 0.4292
0.9362 0.4728 0.0716 0.2531
0.2095 0.7728 0.5725 0.6239
NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
G. procumbens, R. glutinosa and S. baicalensis FMPderived improvements to BWG and G/F might be attributed to bioactive chemicals (peroxidase, osmotin-like protein, thaumatin-like protein (Hew & Gam 2010); catalpol, mannit, mannitol, stachyose (Zhang et al. 2008); baicalein, baicalin, wogonin, wogonoside (Shang et al. 2010), respectively) found in medicinal plants. Further supporting this premise is corroborating results originating from comparable studies using plant extracts (PE). PE supplementation to the diet has resulted in a large variation in growth performance of grower-finisher period pigs, with numerous examples of positive experimental results indicating that PE additives, in general, may actually exert growth promoting activity in pigs. Cullen et al. (2005) reported that pigs fed a garlic-treated diet improved BWG, FI and G/F as compared with control diets. Grela et al. (1998) and Li et al. (2012) also observed improvement in BWG and G/F, followed by growing pigs fed with PE (great nettle, garlic and wheat grass) and by weaned pigs fed with essential oil (thymol and cinnamaldehyde) in the diet, respectively. Meanwhile, some other researchers have reported that no beneficial effects were observed in BWG and G/F throughout the overall period (Manzanilla et al. 2004; Neill et al. 2006). These contradictory results or trends among studies may be due to: (i) the variation in the quality as well as the quantity of active chemicals derived from plant extracts, and (ii) inadequacies or failures in selecting the proper plants, active components and efficacious dietary doses. Nonetheless, many PE studies reflect the suitability of PE alternatives to an AGP. The improved growth performance by FMP supplementation appears to be mediated by improvements in Animal Science Journal (2014) ••, ••–••
DM and GE digestibility, arising from the modulation of the gastrointestinal environment and subsequent improved nutrient utilization in growing pigs. Interest in the use of PE as a potential replacement for AGP in pigs has been generated as a result of studies showing that PE has antimicrobial activity against microflora commonly present in the pig gut (Michiels et al. 2009). Li et al. (2012) demonstrated that PE supplementation, consisting of thymol and cinnamaldehyde, improved apparent digestibility of DM and CP of the weaned pig diet. Huang et al. (2010) also observed that weaning pig diets supplemented with a mixture of blended essential oils, containing cinnamaldehyde, carvacrol, eugenol and thymol, presented similar DM and GE digestibilities as our results, which were significantly higher than the control diet. The exact mode of action of PE has yet to be established, but their mode of action may be considered to be mediated through four possible modes: (i) PE may modulate the permeability of the cytoplasmic membrane, resulting in loss of chemiosmotic control of the affected cell, leading to cell death (Michiels et al. 2009); (ii) monoterpenes derived from PE can cross the lipid bilayer, penetrating the cell and interacting in specific sites, exerting their antimicrobial activity intracellularly (Brenes & Roura 2010); (iii) PE may stimulate the production of saliva, and favoring the secretion of enzymes of pancreatic and intestinal origin, improving nutrient digestibility (Platel & Srinivasan 2004); and (iv) the plant matrix may contain different molecules that have intrinsic bioactivities on animal physiology and metabolism, similar to prebiotics (Dorman & Deans 2000). Based on the results of the present study, it may be considered that particular bioactive chemicals derived from medicinal plants and microflora grown in fermented © 2014 Japanese Society of Animal Science
6 J. S. JEONG and I. H. KIM
medicinal plants matrix modulate synergistically by suppressing intestinal harmful microflora and favoring beneficial microflora, ultimately enhancing the gut. Modulating the intestinal microflora, as a result of FMP supplementation, reduced the overall amount of excreta noxious gas emission, with reductions in excreta ammonia, hydrogen sulfide and total mercaptans gas emissions as compared with NC treatment in growing pigs. This is consistent with the findings of Lee et al. (2009) and Vondruskova et al. (2010) who reported that PE and probiotics can beneficially influence intestinal microflora, improving dietary nutrient digestibility resulting from increased enzymatic activity induced from a shift in intestinal microflora, and enhancing nitrogen absorption, thereby indirectly reducing excreta noxious gas emission. Excreta noxious gas emission has been suggested to be related to nutrient utilization and the intestinal microbial ecosystem (Ferket et al. 2002). Lastly, concerning blood profiles, the present study demonstrated that FMP supplementation in growing pig diets induced increases in WBC counts as compared with NC treatment, indicating the potency of FMP immune stimulation. These results are consistent with Lien et al. (2007) who reported that the Chinese medicinal plant Bazhen improves immune ability by increasing WBC counts, interleukin-6, and tumor necrosis factor-α levels in weanling pigs. In agreement with these findings, Wang et al. (2011b) reported that dietary fermented garlic powder improves WBC counts of pigs. One reason may be that bioactive chemical compounds and secondary plant metabolites, a product of FMP biodegradation, may be responsible for the immune stimulation since it possesses strong antimicrobial activity (Michiels et al. 2009) and antioxidant activity (Frankic et al. 2009), in addition to anti-inflammatory activity (Wang et al. 2011b) on pigs. However, the exact immune stimulation mechanism of FMP in growing pigs is still poorly understood and warrants further investigation.
Conclusion The current study demonstrates that FMP supplementation derived from G. procumbens, R. glutinosa and S. baicalensis in growing pig diets has the potential to promote growth performance, digestibility of DM and GE, and reduce excreta noxious gas emission as compared with NC treatment. We speculate that the fermentation process used in this study improved: (i) the extraction efficiency and amount of materials derived from medicinal plants, raising it from 40–50% to 90%, resulting from smaller particles created by microbial digestion (Kim et al. 2012), and (ii) nutrient utilization, due to manipulation of the gastrointestinal flora, thus potentially affecting digestive and absorptive processes in growing pigs (Canibe & Jensen 2003). As a consequence, growth enhancement through FMP sup© 2014 Japanese Society of Animal Science
plementation is most likely a result of the synergistic beneficial effects of PE bioactive molecules and intrinsic probiotic activity, which may be related to the increase in digestive secretions, improvement of digestibility and absorption of nutrients, modification of intestinal microflora and stimulation of the immune system. As such, it would appear that a combination of two different AGP alternatives may be more beneficial than each single alternative alone for maintaining health and improving performance of growing pigs as compared to an AGP. Overall, based on the results observed in our study, FMP appears to be a viable possible alternative to AGP in growing pigs, while obtaining most of the same benefits without any of the negatives associated with an AGP. However, further investigation is warranted. Because the source of FMP included three different microorganism (L. plantarum, S. cereviseae and B. licheniformis) and three different types of medicinal plants (G. procumbens, R. glutinosa and S. baicalensis), great amounts of variation may be observed when producing batches of FMP. In future studies, we plan to test and compare individual FMPs and combinations thereof, used in this study to determine the effect of each type of plant alone.
ACKNOWLEDGMENTS The authors wish to thank Peter Lee for critical reading and discussion, in addition for his help with manuscript preparation.
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doi: 10.1111/asj.12331
ORIGINAL ARTICLE Effect of probiotic bacteria-fermented medicinal plants (Gynura procumbens, Rehmannia glutinosa, Scutellaria baicalensis) as performance enhancers in growing pigs Jin Suk JEONG and In Ho KIM Department of Animal Resource and Science, Dankook University, Cheonan, South Korea
ABSTRACT This study was conducted to investigate the effect of dietary supplementation of mixed fermented medicinal plants (FMP) obtained from exudates of Gynura procumbens, Rehmannia glutinosa and Scutellaria baicalensis fermented with Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus licheniformis, respectively, on growth performance in growing pigs in order to assess the feasibility of using FMP as an alternative to antibiotic growth promoters (AGP), such as tiamulin. A total of 150 growing pigs (body wieght 25.50 ± 2.50 kg) were used in a 6 weeks experiment and randomly divided into five groups with six replicates of five growing pigs each. The treatments were NC (basal diet), basal diet with 33 ppm tiamulin (PC), basal diet with FMP 0.05% (FMP 0.05), basal diet with FMP 0.1% (FMP 0.1) and basal diet with FMP 0.2% (FMP 0.2). Overall, body weight gain, feed conversion rate, the digestibility of dry matter and gross energy, noxious gas emission all improved with FMP supplementation as compared to NC. Taken together, these results suggest the feasibility of using FMP as an alternative to AGP for enhancing the growth performance, nutrient digestibility and excreta noxious gas emission of growing pigs.
Key words: excreta noxious gas, fermented medicinal plant, growing pig, growth performance, nutrient digestibility.
INTRODUCTION Tiamulin is a member of the pleuromutilin family of antimicrobial agents which are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. Tiamulin is commonly used to treat swine dysentery, spirochete-associated diarrhea, porcine proliferative enteropathy, enzootic pneumonia and other infections where mycoplasma is involved (van Duijkeren et al. 2014). Only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to tiamulin is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions presents a considerable threat to pig health, welfare and productivity. Due to the negative effects of antibiotic growth promoters (AGP), such as inducing microbial antibiotic resistance, public demand for natural, organic products has pressured and expedited the removal of all chemical additives from animal foodstuff in many countries (Sean 2002; Diarra et al. 2011). As a result, natural alternatives, such as medicinal plants and © 2014 Japanese Society of Animal Science
herbs, have attracted and gained widespread attention, due to their wide range of potential beneficial effects and increasing industry demands for maximizing net returns and carcass quality (Jenkins & Atwal 1995; Manesh et al. 2012; Costa et al. 2013). Gynura procumbens (also known as African mistletoe) is a wellknown tropical Asian medicinal herbal plant, containing flavonoid sterol and steroid, as well as some proteins (Hew & Gam 2010). Rehmannia glutinosa has been extensively used in traditional Chinese medicine to treat rheumatoid arthritis, asthma, urticaria and chronic nephritis (Zhang et al. 2008). Lastly, the genus Lamiaceae comprises over 360 species, many of which are medically important. Some Scutellaria baicalensis is used to treat neurological disorders, cancer, inflammatory diseases and viral and bacterial infections (Shang et al. 2010).
Correspondence: In Ho Kim, Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam 330-714, South Korea. (Email: inhokim@ dankook.ac.kr) Received 29 April 2014; accepted for publication 7 August 2014.
2 J. S. JEONG and I. H. KIM
Another alternative approach to sub-therapeutic antibiotics in livestock is the use of probiotic microorganisms. Probiotic is a generic term and products using this label tend to contain bacterial cultures capable of stimulating micro-organisms capable of modifying the gastrointestinal environment to favor health status and improve feed efficiency (Dierck 1989). Interestingly, fermentation of plant materials with a microbial inoculum has been widely used to promote novel functional ingredients because its various activities such as antioxidant and antiinflammatory activity may be promoted by the fermentation process (Lee et al. 2008; Wang et al. 2011a,b; Cao et al. 2012). For example, using exudates of medicinal plants and herbs fermented by probiotics could be better than medicinal plants and herbs or probiotics alone in theory, since pigs would benefit from the active ingredients found in medicinal plant materials in addition to the presence of probiotic bacteria in their intestines. However, to the best of our knowledge, there is limited data pertaining to the effect of dietary supplementation of mixed-fermented medicinal plants (FMP) obtained from exudates of Lactobacillus plantarum-fermented G. procumbens, Saccharomyses cereviseae-fermented R. glutinosa and Bacillus licheniformis-fermented S. baicalensis as an alternative to AGP on growing pigs. Therefore, the objective of this study was to investigate whether FMP can match or even surpass an AGP as a viable alternative in feed, regarding relevant parameters, such as growth performance, nutrient digestibility, blood profiles and excreta noxious gas emission of growing pigs as compared to basal diet supplemented with tiamulin.
MATERIALS AND METHODS The experimental protocols used in this study were approved by the Animal Care and Use Committee of Dankook University (Anseodong, Cheonan, Choongnam, Korea).
Preparation of fermented medicinal plants The solid culture of the FMP product used in this study contained a mixture of lactic acid bacteria at 3.0 × 108 colony forming units (CFU)/g (Lactobacillus planetarium species), yeasts at 7.5 × 107 CFU/g (Saccharomyces cerevisiae species) and fermenting bacteria at 8.0 × 108 CFU/g (Bacillus licheniformis species). The FMPs were prepared as follows: the upper leaves of the G. procumbens plant were taken and thoroughly washed, and then cut to approximately 1 × 1 cm pieces. Processed leaves were mixed with L. plantarum inoculants which were previously fermented with soybean meal (1:1, w/w). The fermentation condition of L. plantarum had an estimated 40% moisture for 2 days at 30°C under solid-state fermentation (SSF). R. glutinosa roots were thoroughly washed and inoculated with 5% (v/w) S. cerevisiae which were fermented with soybean meal (1:1, w/w) for 2 months at low temperature and then kept for 5 days at 25°C under SSF. S. baicalensis © 2014 Japanese Society of Animal Science
roots were thoroughly washed and inoculated with 5% (v/w) B. licheniformis which were fermented with soybean meal (1:1, w/w) for 2 months at low temperature and then kept for 5 days at 30°C under SSF.
Experimental design and diets A total of 150 growing pigs ((Landrace × Yorkshire) × Duroc)) with an average body weight (BW) of 25.50 ± 2.50 kg were randomly allocated to one of five treatments with six replicates of growing pigs with five pigs per pen, according to initial BW for a 42-day experiment. The experimental design was a randomized complete block design. Dietary treatments included: (i) negative control (NC, basal diet); (ii) positive control (PC, basal diet with 33 ppm tiamulin); (iii) basal diet with fermented medicinal plant (FMP) 0.05% (FMP 0.05); (iv) basal diet with FMP 0.1% (FMP 0.1); and (v) basal diet with FMP 0.2% (FMP 0.2). The additive was added at the expense of soybean meal and dietary nutrients were provided to exceed or meet National Requirements Council (1998) recommendations (Table 1). All pigs were housed in an environmentally controlled nursery facility, with slatted plastic flooring in 20 adjacent pens (1.8 × 1.8 m), and provided with ad libitum access to feed and water.
Table 1 Ingredient and composition of the basal diet (as-fed basis)
Item
FMP (%) 0
Ingredient, % Corn Soybean meal (47.5% CP) Animal fat Molasses Dicalcium phosphate Salt Limestone Vitamin premix† FMP‡ Trace mineral premix§ L-Lys·HCl Ethoxyquin, 25.0% Analyzed composition, % DE, MJ/kg CP Lys Met Ca Total P
0.05
0.10
0.20
66.00 66.00 66.00 66.00 23.96 23.91 23.86 23.76 4.24 4.24 4.24 4.24 3.00 3.00 3.00 3.00 1.26 1.26 1.26 1.26 0.25 0.25 0.25 0.25 1.01 1.01 1.01 1.01 0.12 0.12 0.12 0.12 0.05 0.10 0.20 0.10 0.10 0.10 0.10 0.01 0.01 0.01 0.01 0.05 0.05 0.05 0.05 14.50 14.50 14.50 14.50 17.60 17.60 17.60 17.60 1.11 1.11 1.11 1.11 0.31 0.31 0.31 0.31 0.76 0.76 0.76 0.76 0.64 0.64 0.64 0.64
†Provided per kg of complete diet: 10 000 IU vitamin A, 2000 IU vitamin D3, 40 IU vitamin E, 100 mg vitamin C, 10 mg vitamin K3, 10 mg riboflavin, 2 mg vitamin B6, 25 mg pantothenic acid, 50 mg niacin, and 0.04 mg biotin. ‡The experimental diets were formulated by replacing soybean meal with 0 (control), 0.05%, 0.1%, and 0.2% fermented medicinal plants (FMP) extract. §Provided per kg of complete diet: 179 mg Zn (as zinc oxide); 12.5 mg Mn (as manganese oxide); 5 mg Cu (as copper sulfate); 0.5 mg I (as potassium iodide); and 0.4 mg Se (as sodium selenite). Lys, lysine; DE, digestible energy; CP, crude protein, Met, methionine.
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Growth performance and nutrient digestibility analysis Body weight gain (BWG) and feed intake (FI) were recorded on day 0, 2 weeks, 4 weeks and end of the 6 weeks feeding trial, which was then used to calculate gain/feed (G/F) ratio after correcting for mortality. From days 38 to 42, chromic oxide (0.2%) was added to diets as an indigestible marker for determination of apparent total tract digestibility (ATTD) of dry matter (DM), nitrogen (N) and gross energy (GE). Fresh fecal samples were obtained by massaging the rectum from at least two pigs per pen at days 41 and 42. Fecal samples from the same pen were pooled and mixed immediately, after which samples were stored in a freezer at −20°C until analysis. Before chemical analysis, feces were thawed and dried at 70°C for 72 h, after which they were finely ground to a size that could pass through a 1 mm sieve. Feed samples were ground to pass through a 1 mm screen, after which they were analyzed for N (Method 968.06; Association of Official Analytical Chemists 2000), Ca (Method 984.01; Association of Official Analytical Chemists 1995) and P (Method 965.17; Association of Official Analytical Chemists 1995). Individual amino acid (AA) composition was measured using an AA analyzer (Beckman 6300; Beckman Coulter, Inc., Fullerton, CA, USA) after 24 h hydrolysis with HCl (Spackman et al. 1958). Nitrogen content was determined (Kjectec 2300 Nitrogen; Foss Tecator AB, Hoeganaes, Sweden) and crude protein (CP) basis was calculated as N × 6.25. All feed and feces samples were analyzed for DM (Method 934.01; Association of Official Analytical Chemists 2000). Chromium was analyzed using UV absorption spectrophotometry (Shimadzu UV-1201; Shimadzu, Kyoto, Japan), according to the method described by Williams et al. (1962). GE was determined by measuring heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA).
Blood profile analysis Blood samples (10 mL) were collected, via anterior vena cava puncture, randomly from one pig in each pen at the end of this experiment. Blood samples were collected into both nonheparinized tubes (5 mL) and vacuum tubes (5 mL), containing tripotassium ethylenediaminetetraacetic acid (K3EDTA; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and stored at 4°C. Samples for serum analysis were then centrifuged at 3000 × g for 15 min, and then separated and stored at 4°C. White blood cells (WBC), red blood cells (RBC) and lymphocyte percentages were analyzed using an automatic blood analyzer (ADVIA 120; Bayer, Tarrytown, NY, USA).
Excreta gas emission analysis Fresh excreta samples were collected from each pen on the last 2 days of the experiment and then mixed well for each respective pen. Analysis was then conducted using excreta samples (150 g feces and 150 g of urine were well mixed; 1:1 on the wet weight basis) obtained from each pen then stored in 2.6 L sealed plastic boxes. Each box had a small hole in the middle of one side wall, which was sealed with adhesive plaster. The samples were permitted to ferment for 7 days at room temperature (25°C). The concentrations of gas were determined on days 1, 3, 5 and 7 during the fermentation period. A gas sampling pump (Model GV-100; Gastec Corp., Ayase, Japan) was utilized for gas detection (Gastec detector Animal Science Journal (2014) ••, ••–••
3
tube no. 3La for ammonia; no. 4 LK for hydrogen sulfide; no. 70 for mercaptan; Gastec Corp.). Before the measurements, slurry samples were shaken manually for approximately 30 s to disrupt any crust formation on the surface of the slurry sample and to homogenize them. The adhesive plasters were punctured and 100 mL of headspace air was sampled approximately 2.0 cm above the slurry surface. Two samples from each pen were measured and then the mean was calculated.
Statistical analysis All experimental data were analyzed as a completely randomized block design using the GLM procedure of SAS (2003). The pen was used as the experimental unit. Orthogonal contrasts were used to test the overall effect of growth promoting materials supplementation (NC vs. FMPs) and the effect of the difference between effect of PC and FMPs (PC vs. FMPs); additionally, orthogonal comparison were conducted using polynomial regression to measure the linear and quadratic effects of increasing concentrations of FMPs. Variability in the data was expressed as the SEM and P < 0.05 was considered statistically significant, whereas P < 0.10 was considered a tendency.
RESULTS Growth performance In the phase 2 period, the supplementation of FMP improved BWG (P = 0.0243 and linear, P = 0.0265) and G/F (P = 0.0200 and linear, P = 0.0150) as observed with the comparison of NC versus FMPs and dose response of FMP, respectively (Table 2). In the phase 3 period, the supplementation of FMP improved BWG (P = 0.0041 and linear, P = 0.0021) and G/F (P = 0.0181 and linear, P = 0.0136) as observed with treatment comparison of NC versus FMP and dose response of FMPs, respectively. FMP treatment had no effect on FI as compared with NC and PC treatments; however, dose response of FMPs tended to increase according to the FMP concentration (linear, P = 0.0220) in the phase 3 period. Consequently, during the overall experiment, FMP supplementation improved BWG (P = 0.0228 and linear, P = 0.0182) and G/F (P = 0.0299 and linear, P = 0.0241), but not FI, with the treatment comparison of NC versus FMP and dose response of FMP, respectively.
Nutrient digestibility and blood profiles FMP supplementation improved ATTD of DM (P = 0.0299 and linear, P = 0.0179) and GE (P = 0.0790 and linear, P = 0.0628) with the treatment comparison of NC versus FMP and dose response of FMP, respectively. There was no significant effect on the ATTD of N (Table 3). FMP supplementation also increased WBC counts (P = 0.0201) with the treatment comparison of NC versus FMP and dose response of FMP tended to increase according to the FMP concentration (linear, P = 0.0878) (Table 4). Alternately, RBC counts were © 2014 Japanese Society of Animal Science
4 J. S. JEONG and I. H. KIM
Table 2 Effect of fermented medicinal plants (FMP) supplementation on growth performance in growing pigs
Treatment items
NC
PC
FMP 0.05 FMP 0.1
FMP 0.2
SEM
P-value NC vs. FMPs
Phase 1 (0–2 weeks) BWG (g) 583 FI (g) 1343 G/F 0.434 Phase 2 (3–4 weeks) BWG (g) 677 FI (g) 1598 G/F 0.424 Phase 3 (4–6 weeks) BWG (g) 707 FI (g) 2204 G/F 0.321 Overall BWG (g) 656 FI (g) 1715 G/F 0.383
PC vs. FMPs
Linear
Quadratic
608 595 1348 1326 0.451 0.449
611 622 7.94 0.2314 0.9638 0.1463 0.9302 1336 1332 4.37 0.2134 0.0987 0.5686 0.3802 0.457 0.467 0.006 0.1678 0.7077 0.1298 0.9525
709 702 1592 1606 0.445 0.437
712 717 5.92 0.0243 0.8993 0.0265 0.8301 1590 1583 4.89 0.6580 0.9327 0.2540 0.5511 0.448 0.453 0.004 0.0200 0.8870 0.0150 0.9929
749 743 2201 2210 0.240 0.336
759 770 6.55 0.0041 0.5903 0.0021 0.8127 2233 2235 5.86 0.1361 0.0863 0.0220 0.4046 0.340 0.345 0.003 0.0181 0.9942 0.0136 0.6378
689 680 1713 1714 0.402 0.397
694 703 6.10 0.0228 0.8046 0.0182 0.9234 1720 1717 1.81 0.7575 0.4785 0.6174 0.4432 0.403 0.409 0.004 0.0299 0.8690 0.0241 0.8639
BWG, body weight gain; FI, feed intake; G/F, gain/feed; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
Table 3 Effect of fermented medicinal plants (FMP) supplementation on nutrient digestibility in growing pigs
Treatment items
DM, % N, % GE, %
NC
78.14 78.47 79.29
PC
80.05 79.58 82.36
FMP 0.05
79.93 78.94 80.62
FMP 0.1
80.78 80.36 82.77
FMP 0.2
81.71 81.16 83.42
SEM
0.510 0.799 0.737
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
0.0299 0.3876 0.0790
0.5134 0.7663 0.9540
0.0179 0.2544 0.0628
0.8176 0.8478 0.7560
DM, dry matter; N, nitrogen; GE, gross energy; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
Table 4 Effect of fermented medicinal plants (FMP) supplementation on blood profiles in growing pigs
Treatment items
WBC, 103/ul RBC, 106/ul Lymphocyte, %
NC
19.01 7.27 47.03
PC
19.86 7.68 48.48
FMP 0.05
19.43 7.85 48.55
FMP 0.1
20.02 7.71 47.38
FMP 0.2
19.43 7.82 53.22
SEM
0.085 0.209 0.980
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
0.0201 0.2808 0.3308
0.5778 0.6792 0.6518
0.0923 0.4053 0.0878
0.2717 0.5442 0.3293
WBC, white blood cell; RBC, red blood cell; NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
not affected by FMP supplementation as compared with NC and PC treatments (P > 0.05).
with treatment comparison of NC versus FMP and dose response of FMP, respectively.
Excreta noxious gas emission
DISCUSSION
The effect of FMP supplementation on the emission of ammonia, hydrogen sulfide and total mercaptans is shown in Table 5. Overall, FMP supplementation reduced ammonia (P = 0.0039 and linear, P = 0.0025 for day 7), hydrogen sulfide (P = 0.0076 and linear, P = 0.0038 for day 5) and total mercaptans (P = 0.0402 and linear, P = 0.0716 for day 5) emissions as observed
In the current study, BWG, FI and G/F of pigs eating a diet supplemented with FMP appeared to be comparable to that of pigs eating a diet supplemented with tiamulin, an AGP, thus illustrating the potential of FMP as an alternative to AGPs. In particular, dietary supplementation with FMP improved BWG and G/F in phase 2, phase 3 and overall periods in growing pigs.
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Table 5
5
Effect of fermented medicinal plants (FMP) supplementation on fecal noxious gas emission in growing pigs
Treatment Items
Ammonia, ppm 1 day 3 days 5 days 7 days Hydrogen sulfide, ppm 1 day 3 days 5 days 7 days Total mercaptans, ppm 1 day 3 days 5 days 7 days
NC
PC
FMP 0.05
FMP 0.1
FMP 0.2
SEM
P-value NC vs. FMPs
PC vs. FMPs
Linear
Quadratic
4.9 15.2 19.3 22.9
4.6 15.7 18.9 20.7
4.3 15.0 18.3 20.6
4.2 14.6 18.1 19.3
4.0 14.4 17.6 18.8
0.264 0.417 0.357 0.444
0.3060 0.5907 0.1610 0.0039
0.5654 0.3405 0.3204 0.2552
0.3025 0.5497 0.1750 0.0025
0.8453 0.9589 0.7891 0.9900
5.5 10.8 14.8 16.4
4.7 10.3 14.0 16.0
4.8 10.4 13.4 15.0
5.6 10.4 13.4 15.2
5.2 10.0 13.1 14.3
0.058 0.060 0.124 0.076
0.8079 0.1152 0.0076 0.8145
0.4478 0.9137 0.2835 0.4373
0.2114 0.0922 0.0038 0.6916
0.5704 0.8359 0.8236 0.7859
0.5 1.3 2.2 1.9
0.4 1.1 1.6 1.7
0.3 1.2 1.7 1.8
0.5 1.0 1.3 1.9
0.7 1.1 1.0 1.8
0.205 0.329 0.281 0.516
0.6539 0.5146 0.0402 0.2897
0.3438 0.9297 0.3335 0.4292
0.9362 0.4728 0.0716 0.2531
0.2095 0.7728 0.5725 0.6239
NC, negative control (basal diet); PC, positive control (basal diet + 33 ppm tiamulin); FMP 0.05, basal diet with 0.05% FMP extract; FMP 0.1, basal diet with 0.1% FMP extract; FMP 0.2, basal diet with 0.2% FMP extract; SEM, standard error of the mean.
G. procumbens, R. glutinosa and S. baicalensis FMPderived improvements to BWG and G/F might be attributed to bioactive chemicals (peroxidase, osmotin-like protein, thaumatin-like protein (Hew & Gam 2010); catalpol, mannit, mannitol, stachyose (Zhang et al. 2008); baicalein, baicalin, wogonin, wogonoside (Shang et al. 2010), respectively) found in medicinal plants. Further supporting this premise is corroborating results originating from comparable studies using plant extracts (PE). PE supplementation to the diet has resulted in a large variation in growth performance of grower-finisher period pigs, with numerous examples of positive experimental results indicating that PE additives, in general, may actually exert growth promoting activity in pigs. Cullen et al. (2005) reported that pigs fed a garlic-treated diet improved BWG, FI and G/F as compared with control diets. Grela et al. (1998) and Li et al. (2012) also observed improvement in BWG and G/F, followed by growing pigs fed with PE (great nettle, garlic and wheat grass) and by weaned pigs fed with essential oil (thymol and cinnamaldehyde) in the diet, respectively. Meanwhile, some other researchers have reported that no beneficial effects were observed in BWG and G/F throughout the overall period (Manzanilla et al. 2004; Neill et al. 2006). These contradictory results or trends among studies may be due to: (i) the variation in the quality as well as the quantity of active chemicals derived from plant extracts, and (ii) inadequacies or failures in selecting the proper plants, active components and efficacious dietary doses. Nonetheless, many PE studies reflect the suitability of PE alternatives to an AGP. The improved growth performance by FMP supplementation appears to be mediated by improvements in Animal Science Journal (2014) ••, ••–••
DM and GE digestibility, arising from the modulation of the gastrointestinal environment and subsequent improved nutrient utilization in growing pigs. Interest in the use of PE as a potential replacement for AGP in pigs has been generated as a result of studies showing that PE has antimicrobial activity against microflora commonly present in the pig gut (Michiels et al. 2009). Li et al. (2012) demonstrated that PE supplementation, consisting of thymol and cinnamaldehyde, improved apparent digestibility of DM and CP of the weaned pig diet. Huang et al. (2010) also observed that weaning pig diets supplemented with a mixture of blended essential oils, containing cinnamaldehyde, carvacrol, eugenol and thymol, presented similar DM and GE digestibilities as our results, which were significantly higher than the control diet. The exact mode of action of PE has yet to be established, but their mode of action may be considered to be mediated through four possible modes: (i) PE may modulate the permeability of the cytoplasmic membrane, resulting in loss of chemiosmotic control of the affected cell, leading to cell death (Michiels et al. 2009); (ii) monoterpenes derived from PE can cross the lipid bilayer, penetrating the cell and interacting in specific sites, exerting their antimicrobial activity intracellularly (Brenes & Roura 2010); (iii) PE may stimulate the production of saliva, and favoring the secretion of enzymes of pancreatic and intestinal origin, improving nutrient digestibility (Platel & Srinivasan 2004); and (iv) the plant matrix may contain different molecules that have intrinsic bioactivities on animal physiology and metabolism, similar to prebiotics (Dorman & Deans 2000). Based on the results of the present study, it may be considered that particular bioactive chemicals derived from medicinal plants and microflora grown in fermented © 2014 Japanese Society of Animal Science
6 J. S. JEONG and I. H. KIM
medicinal plants matrix modulate synergistically by suppressing intestinal harmful microflora and favoring beneficial microflora, ultimately enhancing the gut. Modulating the intestinal microflora, as a result of FMP supplementation, reduced the overall amount of excreta noxious gas emission, with reductions in excreta ammonia, hydrogen sulfide and total mercaptans gas emissions as compared with NC treatment in growing pigs. This is consistent with the findings of Lee et al. (2009) and Vondruskova et al. (2010) who reported that PE and probiotics can beneficially influence intestinal microflora, improving dietary nutrient digestibility resulting from increased enzymatic activity induced from a shift in intestinal microflora, and enhancing nitrogen absorption, thereby indirectly reducing excreta noxious gas emission. Excreta noxious gas emission has been suggested to be related to nutrient utilization and the intestinal microbial ecosystem (Ferket et al. 2002). Lastly, concerning blood profiles, the present study demonstrated that FMP supplementation in growing pig diets induced increases in WBC counts as compared with NC treatment, indicating the potency of FMP immune stimulation. These results are consistent with Lien et al. (2007) who reported that the Chinese medicinal plant Bazhen improves immune ability by increasing WBC counts, interleukin-6, and tumor necrosis factor-α levels in weanling pigs. In agreement with these findings, Wang et al. (2011b) reported that dietary fermented garlic powder improves WBC counts of pigs. One reason may be that bioactive chemical compounds and secondary plant metabolites, a product of FMP biodegradation, may be responsible for the immune stimulation since it possesses strong antimicrobial activity (Michiels et al. 2009) and antioxidant activity (Frankic et al. 2009), in addition to anti-inflammatory activity (Wang et al. 2011b) on pigs. However, the exact immune stimulation mechanism of FMP in growing pigs is still poorly understood and warrants further investigation.
Conclusion The current study demonstrates that FMP supplementation derived from G. procumbens, R. glutinosa and S. baicalensis in growing pig diets has the potential to promote growth performance, digestibility of DM and GE, and reduce excreta noxious gas emission as compared with NC treatment. We speculate that the fermentation process used in this study improved: (i) the extraction efficiency and amount of materials derived from medicinal plants, raising it from 40–50% to 90%, resulting from smaller particles created by microbial digestion (Kim et al. 2012), and (ii) nutrient utilization, due to manipulation of the gastrointestinal flora, thus potentially affecting digestive and absorptive processes in growing pigs (Canibe & Jensen 2003). As a consequence, growth enhancement through FMP sup© 2014 Japanese Society of Animal Science
plementation is most likely a result of the synergistic beneficial effects of PE bioactive molecules and intrinsic probiotic activity, which may be related to the increase in digestive secretions, improvement of digestibility and absorption of nutrients, modification of intestinal microflora and stimulation of the immune system. As such, it would appear that a combination of two different AGP alternatives may be more beneficial than each single alternative alone for maintaining health and improving performance of growing pigs as compared to an AGP. Overall, based on the results observed in our study, FMP appears to be a viable possible alternative to AGP in growing pigs, while obtaining most of the same benefits without any of the negatives associated with an AGP. However, further investigation is warranted. Because the source of FMP included three different microorganism (L. plantarum, S. cereviseae and B. licheniformis) and three different types of medicinal plants (G. procumbens, R. glutinosa and S. baicalensis), great amounts of variation may be observed when producing batches of FMP. In future studies, we plan to test and compare individual FMPs and combinations thereof, used in this study to determine the effect of each type of plant alone.
ACKNOWLEDGMENTS The authors wish to thank Peter Lee for critical reading and discussion, in addition for his help with manuscript preparation.
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