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Dietary nucleotide rich yeast extract mitigated symptoms of colibacillosis in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli
Animal Feed Science and Technology 254 (2019) 114204
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Dietary nucleotide rich yeast extract mitigated symptoms of colibacillosis in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli R. Pattersona,1, J.M. Heob,c,1, S.S. Wickramasuriyac, Y.J. Yid, C.M. Nyachotib,
T
⁎
a
Canadian Bio-System Inc., Calgary, AB, T2C 0J7, Canada Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada c Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, 34134, Republic of Korea d Division of Biotechnology, Chonbuk National University, Iksan-si, Jeollabuk-do, 54596, Republic of Korea b
A R T IC LE I N F O
ABS TRA CT
Keywords: E. coli challenge Nucleotide rich extract Performance Pigs
A study was conducted to investigate the effect of a nucleotide rich yeast extract (NRYE) on the growth performance and the colibacillosis of weaned pigs. In Experiment. 1, a total of 168 mixedsex piglets weaned at 17 ± 2 days of age were fed diets formulated to meet or exceed nutrient requirements for 28 days. Diets consisted of i) Positive control (PC) containing antibiotics (110 mg/kg of chlortetracycline HCl and 31.2 mg/kg of tiamulin), ii) Negative control (NC) without antibiotics, iii) NC with the addition of 1 g/kg of NRYE (NRYE1), and iv) 2 g/kg of NRYE (NRYE2). No differences (P > 0.10) were found among treatments for average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR), whilst pigs fed NRYE1 weighed more (P < 0.05) than those fed NC on day 28. Pigs fed the NRYE2 diet had greater (P < 0.05) ADG and ADFI than those fed the NC diet from day 7 to 14. No differences (P > 0.10) were observed in ADFI among dietary treatments. In Experiment 2, a total of 144 barrows were randomly assigned to 4 treatments groups to give 6 replicates (6 pigs per pen) per group. Four treatment diets were similar to previous experiment. Pigs in PC fed a control diet without NYRE supplementation and also did not received an oral challenge. PC treatment was housed in a separate room within the same facility with a similar environment. Pigs in NC, NRYE1 and NRYE2 were fed the non-medicated control diet with 0g/kg, 1 g/kg or 2 g/kg NYRE, respectively and were orally challenged with enterotoxigenic Escherichia coli K88+ (ETEC) after the 3 days of weaning. From day 3 to 28 post-challenge, pigs fed diets NRYE1 or NRYE2 had greater (P < 0.05) ADG compared with those fed a NC diet. Average daily feed intake and FCR were not affected by dietary treatment throughout the study. Challenged pigs consuming NRYE1 or NRYE2 diets had less (P < 0.05) diarrhea compared with those fed NC diet. The mortality due to coliform infection tended to be higher (P < 0.10) in the challenged NC and NRYE2 pigs compared with PC and NRYE1 pigs. In conclusion, diets supplemented with NRYE enhanced BW, ADG and ADFI of weaned pigs and reduced the incidence of diarrhea in ETEC challenged pigs
Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; ETEC, Escherichia coli K88+; FCR, feed conversion ratio; HCl, hydrogen chloride; LT, heat labile toxins; NC, negative control; NRYE, nucleotide rich yeast extract; PC, positive control; PWD, post-weaning diarrhea; ST, heat stable toxins ⁎ Corresponding author. E-mail address: [email protected] (C.M. Nyachoti). 1 Authors have made equal contribution to this paper. https://doi.org/10.1016/j.anifeedsci.2019.114204 Received 29 October 2018; Received in revised form 11 June 2019; Accepted 19 June 2019 0377-8401/ © 2019 Published by Elsevier B.V.
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suggesting that NRYE could be effective in controlling post-weaning diarrhea in piglets, which could represent a major economic advantage to the swine industry.
1. Introduction Weaned piglets are susceptible to nutritional, psychological and environmental changes due to a naive immune system, which can lead to intestinal dysfunction and increased incidences of post-weaning diarrhea (PWD; Heo et al., 2015). In particular, enterotoxigenic Escherichia coli (ETEC) infection is a common disease in pre- and post-weaned pigs that leads to the incidences of PWD, poor growth performance, morbidity and mortality (Heo et al., 2013, 2015, Sun and Kim, 2017). Pigs infected with ETEC generally experience reduced integrity of the intestinal epithelium, premature enterocyte apoptosis, fluid exudation into the intestinal lumen and loss of digestive and absorptive capacity (Heo et al., 2013). Therefore, a large number of additives have attracted attention as approaches to maintain a healthy gut in an attempt to control post-weaning growth check and the symptoms of colibacillosis in weaned piglets (Heo et al., 2013; Pluske et al., 2018) Yeast extracts consist of bioactive compounds such as nucleotides and cell wall polysaccharides, which have the potential to be an alternative to growth promoting antibiotics (Waititu et al., 2016a, 2017). Recently, it has been proposed that yeast products might be beneficial for improving the ability of piglet to resist an ETEC challenge or improving post-challenge recovery, or the combination of both via a number of mechanisms including blocking mucosal attachment of the pathogen that could enhance the host immune response or promote intestinal mucosal integrity (Kiarie et al., 2012; Trckova et al., 2014; Waititu et al., 2016b; Rhouma et al., 2017). Importantly, dietary nucleotide helps to recover conditionally essential nucleotide requirement of newly weaner pigs to eliminate impedes nucleotide-dependent physiological processes (Waititu et al., 2016b). Necessities are existing to reveal the nucleotide rich yeast extract (NRYE) impact on mitigating symptoms of colibacillosis in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli. To this end, the present study was conducted to investigate the ability of a NRYE to improve growth performance of weaned piglets and to examine the effects of dietary NRYE supplementation in piglets orally challenged with a virulent strain of ETEC (E. coli K88+). In this study we hypothesized that nucleotide rich yeast extract supplementation in to diet may enhanced growth performance of newly-weaned pigs and showed positive effect on reducing colibacillosis along with the associated incidence of diarrhea.
Table 1 Composition of basal diet for Experiment 1 and Experiment 2 (as-fed basis). Item
Ingredient, g/kg Oat groats Skim milk powder Wheat Whey permeate Soybean meal Corn Fish meal Soybean oil Tallow Dicalcium phosphate Limestone Vitamin-mineral premixc Salt Lys-HCl L-Thr DL-Met Calculated analysisd CP, g/kg ME, MJ/kg SID Lys, g/kg SID Lys, g/Mcal ME
Exp. 1a
Exp. 2b
Phase 1
Phase 2
Phase 3
Phase 1
Phase 2
250.0 170.0 150.0 110.7 110.0 88.3 37.9 25.5 25.0 6.8 5.4 10.0 1.4 4.0 3.0 2.1
150.0 84.0 150.0 68.0 165.0 268.1 38.8 20.0 22.0 10.6 3.1 10.0 3.1 4.0 2.0 1.3
– 41.0 125.0 46.7 194.5 502.2 25.0 5.0 23.1 10.5 6.8 10.0 3.7 3.8 1.6 1.1
250.0 170.0 150.0 110.7 108.0 90.3 37.9 25.5 25.0 6.8 5.4 10.0 1.4 4.0 3.0 2.1
– 41.0 125.0 46.7 192.5 504.2 25.0 5.0 23.1 10.5 6.8 10.0 3.7 3.8 1.6 1.1
199.0 14.48 14.2 4.1
195.0 14.26 13.8 4.1
181.0 13.92 13.1 3.9
197.0 14.52 14.5 4.2
177.0 13.96 12.9 3.9
a
Experiment 1 Phase 1;1(17 ± 2 days of age) - 3 days after weaning, Phase 2; 4–7 days after weaning, Phase 3; 8–28 days after weaning. Experiment 2 Phase 1; −3 (17 ± 2 days of age) −18 days of post challenge, Phase 2; 19–28 days of post challenge. c Vitamin and mineral premix supplied the following per kilogram of complete diet: 8250 IU of vitamin A; 825 IU of vitamin D3; 40 IU of vitamin E; 4.0 mg of vitamin K as menadione; 500 mg of choline chloride; 22.5 mg of niacin; 15 mg of calcium pantothenate; 10 mg of riboflavin; 2 mg of thiamin; 25 μg of vitamin B12; 2000 μg biotin; 2 mg of folic acid; 50 mg Mn as manganese oxide; 100 mg Fe as iron sulfate; 150 mg of Zn as zinc oxide; 25 mg of Cu as copper sulfate; 0.4 mg of I as calcium iodate; 0.3 mg of Se as sodium selenite. d The values are calculated according to the values of feedstuffs in NRC (2012). b
2
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2. Materials and methods Experimental protocols used in the present study were reviewed and approved by Nutreco Canada Ag-research’s Animal Care Committee and pigs were cared for according to the guidelines of the Canadian Council on Animal Care (2009). 2.1. Experiment 1. – Growth performance 2.1.1. Animals, housing and experimental treatments A total of 168 mixed-sex piglets [(Yorkshire × Landrace) × Duroc; 1:1 gender ratio] were weaned at 17 ± 2 days of age from a high-health commercial sow herd. Upon arrival at the research facility (Nutreco AgResearch commercial research farm, Burford, Ontario, Canada), piglets were weighed and examined for general defects or signs of poor health. Eligible piglets were balanced for body weight (BW) and housed in pens (1.2 × 0.9 m) with solid plastic walls and plastic coated expanded steel flooring. Each pen housed 6 piglets (3 barrows and 3 gilts) and each treatment was randomly assigned to 7 pens for a total of 42 pigs per treatment. Each pen was equipped with a trough feeder and a height adjustable nipple drinker to which piglets had free access throughout the experiment. The ambient temperature was maintained at 29 ± 1 °C for the initial week and was then gradually decreased by 1 °C every week. All diets were corn and soybean meal-based and were manufactured at a commercial feed mill (Yantzi Feed and Seeds, Tavistock, Ontario, Canada). Diets were formulated to meet or exceed the NRC (2012) nutrient requirements for their respective BW (Table 1). Diet 1 was a positive control (PC) containing 110 mg/kg of Aueromycin (chlortetracycline hydrogen chloride; Bio Agri Mix LP, Mitchell, Ontario, Canada) and 31.2 mg/kg of Tiamulin (Bio Agri Mix LP, Mitchell, Ontario, Canada). Diet 2 was a negative control (NC) and contained no antibiotics. Diets 3 and 4 were equivalent to the NC but with the addition of 1 g/kg and 2 g/kg, respectively, of the nucleotide rich yeast extract (NRYE; Maxi-Gen Plus, Canadian Bio-Systems Inc., Calgary, Alberta, Canada). Nucleotide rich yeast extract (Maxi-Gen Plus) is a mixture of crude protein (327 g/kg), cell wall polysaccharides (216 g/kg), carbohydrates (143 g/kg), and a mixture of five nucleotides (11 g/kg; adenosine monophosphate, cytosine monophosphate, inosine monophosphate, uridine monophosphate, and guanosine monophosphate). Pigs were phase fed and received the phase one diet from 1 to 3-day after weaning, phase two diet from 4 to 7-day after weaning and phase three diet from 8 to 28-day after weaning. Feed intake was recoded as feed disappearance from the feeder, and pigs were weighed on a weekly basis for 4 weeks. Using body weight and feed intake, average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated. 2.2. Experiment 2. – Responses to enterotoxigenic Escherichia coli (ETEC) challenge 2.2.1. Animals, housing and experimental treatments A total of 144 barrows [(Yorkshire × Landrace) × Duroc] weaned at 17 ± 2 days of age were balanced for BW and randomly assigned to 24 pens (1.2 × 0.9 m; 6 pigs per pen). Each pen had raised, fully slatted, plastic coated expanded steel flooring and solid plastic walls, a stainless-steel feeder and a height adjustable nipple drinker. Pigs had free access feed and water throughout the experiment. The study consisted of 4 treatments similar to previous experiment with 6 replicate pens per group. Pigs in PC received a control diet and also did not received an oral challenge. This group was housed separately in a separate room within the same building with similar environmental condition as the challenged pigs. Pig in other three treatments received the control diet supplemented with 0 mg/kg (NC), 1 mg/kg (NRYE1) and 1 mg/kg (NRYE2) of a NRYE product (Maxi-Gen Plus, Canadian Bio-Systems Inc., Calgary, AB, Canada), respectively, and also received an oral challenge. Test diets were commercially manufactured (Yantzi Feed and Seeds, Tavistock, Ontario, Canada) and were formulated to meet or exceed NRC (2012) nutrient requirements for swine (Table 1). Pigs were phase fed and received the phase one diet from -3 (17 ± 2 days of age) to 18-day post challenge and the phase two diet from 19 to 28-day post challenge. 2.2.2. Oral challenge, animal observation and health status The ETEC β-hemolytic serotype O149; K91; K88 [toxins heat labile toxins (LT), heat stable toxins (ST; variants STa and STb)] was used for experimental infection of the pigs to clinically express PWD. After 3 days of weaning, designated pigs (NC, NRYE1 and NRYE2) received a 3 mL oral dose of inoculum containing 1.48 × 108 cfu/mL of ETEC following the procedure describe by Heo et al. (2009). The inoculum was prepared by trained personnel (Nutreco Canada Ag-Research laboratory, Mississauga, Ontario, Canada) according to standard in-house procedures with similar to the procedure followed by Heo et al. (2009). Designated non-challenge control pigs (PC) received a 3 mL oral dose of phosphate-buffered saline in a similar procedure (PBS) as a placebo. 2.2.3. Growth performance, fecal consistency scoring and mortality Feed intake was recoded as feed disappearance from the feeder, and pigs were weighed on a weekly basis for 4 weeks. Using body weight and feed intake, ADFI, ADG and FCR was calculated. Individual piglets were observed twice daily from arrival until completion of the study. Starting daily from post challenge to day 7 (3 to 10-day after weaning) severity of diarrhea was quantified by fecal consistency scoring (0, normal; 1, pasty feces; 2, liquid feces; 3, severe diarrhea) according to Heo et al. (2009) and Shin et al. (2017). All scoring was performed by trained individuals with no prior knowledge of dietary treatments. Data for the incidence of PWD were expressed as the mean percentage of pigs with diarrhea relative to the total number of pigs in each treatment (Heo et al., 2009). Mortality was recorded throughout the experiment when the 3
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dead occurred. Recorded mortality data were expressed as the mean percentage of days with mortality relative to the total 7 day after weaning. 2.3. Statistical analysis Data from both Exp. 1 and 2 were subjected to ANOVA as a completely randomized design (Statistix, 2008) and pen was considered the experimental unit for all growth performance response criteria. Individual pig was considered as the experimental unit for fecal consistency scoring and mortality data analysis. Statistical significance was accepted at P < 0.05 and trends were discussed at P < 0.10. When the treatment effect was significant (P < 0.05) Tukey’s multiple range test was used to differentiate significance between means. 3. Results 3.1. Experiment 1. – Growth performance There were no differences (P > 0.10) among dietary treatments for ADG, ADFI or FCR on day 1–7 after weaning (Table 2). Pigs fed the NYRE2 supplemented diet had greater ADG (P = 0.048) and ADFI (P = 0.034) than NC pigs but did not differ compared to PC or NYRE1 pigs (P > 0.10) from 8 to 14-day after weaning. Although ADG was not affected (P > 0.10) by dietary treatment from day 15 to 21, pigs fed the NYRE1 diet had a higher ADFI, which tended (P = 0.098) to be greater than for pigs in the NC group. Average daily feed intake was not affected (P > 0.10) by dietary treatment from 22 to 28-day after weaning, however, during this period, ADG of NYRE1 pigs was greater than that of the NC-fed pigs (P = 0.041) and was the same (P > 0.10) as NYRE2 and PC pigs, respectively. Overall, no differences were observed (P > 0.05) for ADFI among dietary treatments from weaning to 28-day after weaning. However, pigs fed the NYRE1 diet tended to be greater ADG than those fed the NC (P = 0.051) and the same (P > 0.10) ADG as the pigs fed the NYRE2 and the PC diets. No statistical differences were detected (P > 0.0.5) among dietary treatments for feed conversion during the entire experimental period. 3.2. Experiment 2. – Growth performance Unchallenged pigs fed the PC diet had greater ADG (P = 0.016) than challenged pigs fed the NC, NYRE1 or NYRE2 diets from 3 day pre-challenge to 7 day post-challenge (Table 3). However, ADG was greater (P < 0.05) in the challenged pigs fed NYRE1 and NYRE2 than in the pigs fed the NC diet from day 8 to 14 post-challenge (P = 0.012). No differences were found (P > 0.10) in ADG among dietary treatments from day 15 to 21, 22 to 28 and overall post challenge period. From 3 day pre- to 7 day post-challenge, ADFI was greater (P = 0.01) for unchallenged pigs fed the PC diet than challenged pigs fed the NC, NYRE1 and NYRE2 diets. There were no differences (P > 0.05) in ADFI among dietary treatments at any other stage during the study (Table 3). From challenge day 8 Table 2 Effect of the addition of the nucleotide rich yeast extract on growth performance of weaned pigs for Experiment 1.1,2,3,4 Item ADG, g/day day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28 ADFI, g/day day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28 FCR, g/g day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28
PC
NC
NRYE1
NRYE2
SEM
P-value
143 329ab 427 527ab 367ab
107 284b 412 537b 335b
124 294ab 432 623a 379a
140 332a 432 565ab 367ab
11.38 13.45 20.70 19.54 10.63
0.148 0.048 0.872 0.041 0.051
153 373ab 621ab 794 485
137 349b 569b 783 459
142 367ab 641a 858 497
153 406a 629ab 792 495
9.4 12.4 19.7 23.7 12.7
0.556 0.034 0.098 0.133 0.170
1.07 1.14 1.46 1.39 1.32
1.3 1.23 1.39 1.48 1.37
1.26 1.32 1.49 1.38 1.32
1.10 1.23 1.47 1.41 1.35
0.089 0.085 0.036 0.039 0.025
0.212 0.520 0.297 0.316 0.344
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (With antibiotics), NC; negative control (Without antibiotics), NRYE1; NC+1 g/kg nucleotide rich yeast extract, NRYE2; NC+2 g/kg nucleotide rich yeast extract, SEM; standard error of mean, ADG; average daily gain, ADFI; average daily feed intake, FCR; feed conversion ratio. 3 Values are the mean of 7 replicates per treatment. 4 Day 1= indicate the weaning day (17 ± 2 days of age). 2
4
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Table 3 Effect of the addition of the nucleotide rich yeast extract on growth performance of weaned pigs challenged with E. coli for Exp. 2.1,2,3 Item ADG, g/day day −34 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28 ADFI, g/day day −3 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28 FCR5, g/g day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28
PC
NC
NRYE1
NRYE2
SEM
P-value
120a 336a 407 536 289
27b 220b 288 533 249
55b 293a 419 534 271
49b 312a 407 550 262
18.3 21.9 33.4 28.4 17.7
0.016 0.012 0.930 0.970 0.460
168a 371 594 829 439
145b 409 602 825 410
146b 368 598 819 417
136b 401 602 850 413
5.90 24.5 33.2 32.3 19.7
0.010 0.560 0.990 0.910 0.710
1.79a 1.49 1.55 1.53
1.23b 1.58 1.55 1.67
1.25b 1.43 1.55 1.54
1.29b 1.49 1.55 1.60
0.082 0.060 0.051 0.053
0.001 0.420 0.990 0.250
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (Non-medicated diet and without ETEC challenged), NC; negative control (Non-medicated diet and Challenged with ETEC), NRYE1; NC+1 g/kg nucleotide rich yeast extract and challenged with ETEC, NRYE2; NC+2 g/kg nucleotide rich yeast extract and challenged with ETEC, SEM; standard error of mean, ADG; average daily gain, ADFI; average daily feed intake, FCR; feed conversion ratio. 3 Values are the mean of 6 replicates per treatment. 4 day -3= indicate the weaning day and 3 days pre-challenged (17 ± 2 days of age). 5 Negative FCR values occurred for day -3 to 7 post challange, and data were removed. 2
to 14, challenged pigs receiving the NC, NYRE1 and NYRE2 diets had higher FCR than unchallenged pigs receiving the PC diet (P = 0.001). However, FCR did not differ (P > 0.10) among dietary treatments during day 15 to 21, day 22 to 28 and overall post challenge period. 3.3. Mortality and incidence of diarrhea In experiment 2, mortality tended to be higher (P = 0.06) in challenged pigs fed the NC and NRYE2 diets than in unchallenged pigs fed the PC diet. On day 1 and day 3 post-challenge, the incidence of diarrhea was greater (P < 0.05) in challenged versus unchallenged pigs (Table 4), but no difference (P > 0.10) was observed between challenged pigs fed the NC diet and pigs fed either NRYE1 or NRYE2 diets. The proportion of pigs showing the incidence of diarrhea was greater in challenged pigs fed the NC and NYRE2 diet than unchallenged pigs fed the PC diet (P = 0.04) on day 1 post-challenge. However, no difference was observed (P > 0.10) between the Table 4 Effect of the addition of the nucleotide rich yeast extract on fecal consistency for 7-day after weaning for Exp. 2.1,2 Item Proportion > 0, % day 13 day 3 day 5 Proportion > 1, %4 day 1 day 3 day 5 Overall fecal score day 1–7 Mortality rate, %5
PC
NC
NRYE1
NRYE2
SEM1
P-value
11.1b 8.34b 2.78
50.0a 42.2a 22.22
38.9a 44.5a 20.83
44.4a 45.0a 12.50
7.17 7.55 7.920
0.008 0.009 0.320
2.78b 2.78 b 0.00
27.8a 26.7a 12.78
19.5ab 15.0ab 8.33
30.6a 13.9ab 2.78
6.601 4.310 5.210
0.040 0.010 0.340
0.096c 0.00b
0.47a 25.0a
0.34b 11.1ab
0.34b 19.5a
0.042 5.59
0.050 0.060
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (Non-medicated diet and without ETEC challenged), NC; negative control (Non-medicated diet and Challenged with ETEC), NRYE1; NC+1 g/kg nucleotide rich yeast extract and challenged with ETEC, NRYE2; NC+2 g/kg nucleotide rich yeast extract and challenged with ETEC, SEM; standard error of mean. 3 day 1= indicate the orally challenged day (20 ± 2 days of age). 4 Expressed as % cumulative score per day of pigs having fecal score greater than 1; higher values are associated with more liquid feces. 5 Expressed as the mean percentage of days with mortality relative to the total 7-day after weaning. 2
5
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unchallenged pigs fed thePC diet compared to those fed the diet supplemented with NRYE1. Three-day post-challenge, pigs fed the NC diet had a greater (P < 0.01) incidence of diarrhea compared with unchallenged pigs fed the PC diet, but the incidence of diarrhea was similar (P > 0.10) in pigs fed the PC and NRYE-supplemented NRYE1 or NRYE2 diets. Considering the overall fecal score, higher (P = 0.05) incidence of diarrhea was observed with challenged pigs fed the NC diets compared to pigs fed the PC, NRYE1 and NRYE2 diets. With regards to challenged pigs, lower incidence (P = 0.05) of diarrhea was observed for pigs fed NRYE1 and NRYE1 diets compared to pig fed the NC diet.
4. Discussion The purpose of the current study was to test the hypothesis that NRYE will improve growth performance commensurate with attenuated mortality rate and the incidence of PWD in piglets orally challenged with a virulent strain of ETEC. Nucleotides are constituents of DNA and RNA and participate in structural, metabolic, energetic, and regulatory functions in living cells (Lane and Fan, 2015; Waititu et al., 2016a), and these non-protein nitrogenous compounds regarded as ‘conditionally essential’ nutrients that modulate the immune system, promote intestinal growth and hepatic functions (Waititu et al., 2016b; Mosca and Gianni, 2017). Many articles have documented that weaning transition is a critical period for piglets because of the abrupt change in diet from milk to solid feed along with weaning-associated intestinal dysfunction (Heo et al., 2013). In addition, these are linked to a reduction in villous height and lowered brush border enzyme activity resulting in post-weaning growth stasis (Heo et al., 2013). Furthermore, the time around weaning is the most rapid growth period in the life of a pig, which creates a higher demand for nucleotides to support the rapidly dividing cells (i.e., blood and small and large intestinal cells). Domeneghini et al. (2004) demonstrated that piglets fed a diet containing 0.5 g/kg nucleotides showed numerical benefits during the first week post weaning, and intestinal tissues of piglets consuming nucleotide-containing diets indicated a greater content of mucosal protein and DNA, higher villous height and disaccharide activities, and improved recovery post injury (Domeneghini et al., 2006). Furthermore, Waititu et al. (2015) observed beneficial effect of NRYE on growth performance of weaned pigs and its comparative advantage over the antibiotic growth promoters. In the present study, greater ADG was observed in pigs fed diets supplemented with 1 g/kg NRYE during day 1–28 after weaning than those fed the NC and PC diets which suggests that NRYE supplementation had positive effects on the growth performance of weanling piglets (Table 2). However, the effect of dietary nucleotide supplementation on piglet growth and intestinal morphology is controversial. No improvements were observed with respect to growth performance when pigs (weaned at 26 day of age) were fed a diet containing 15 mg/kg nucleotide rich yeast extract (25% of free nucleotides) and carob pulp alone or in combination for 14 days (Andrés-Elias et al., 2007). A similar observation was reported by Mateo (2005) who fed 19- day old weaned pigs a control diet based on corn-soybean meal and dried whey or a control diet supplemented with 3 mg/kg nucleotides had no significant enhancement of growth performance. Furthermore, pigs fed corn–soybean meal–whey basal nursery diets supplemented with organic acids as well as 1 g/kg nucleotides had no effect on piglet performance over a 46- day period and no consistent effects on the immune response were observed (Lee et al., 2007). Meanwhile, Jiao et al. (2018) demonstrated that pigs fed a diet containing 5 g/kg and 1 g/kg nucleotide improved growth performance. Although piglets fed diets containing up to 2 times the amount of nucleotides as in sow milk had similar feed intake and growth as those fed control diets during the first week post-weaning, also nucleotide supplementation minimized the reduction of villous height following weaning (Martinez-Puig et al., 2007) which was consistent with the results of Domeneghini et al. (2004). Post-weaning diarrhea is associated with fecal shedding of β-hemolytic ETEC, which proliferate in the small intestine of both healthy and unhealthy weaned pigs (Heo et al., 2013; Liu et al., 2013; Rhouma et al., 2017; Girard et al., 2018) that can lead to ETEC infection, which is also known as ETEC colibacillosis (Luppi, 2017). Colibacillosis is characterized by increasing intestinal fluid loss and subsequent diarrhea (Luppi, 2017). In previous studies, dietary nucleotide supplementation effectively reduced incidences of diarrhea in piglets (Superchi et al., 2012; Waititu et al., 2016b). Additionally, nucleotide supplementation reduced the incidences of diarrhea in human infants (Lönnerdal, 2016) and inhibited endotoxin-induced intestinal translocation of bacteria in mice (Adjei et al., 1999). In the current study, pigs fed a diet supplemented with 1 g/kg or 2 g/kg NRYE showed numerically lower proportions of mild or moderate diarrhea after an oral ETEC challenge compared to control challenged pigs on day 1 and d 5 post-challenge. Moreover, significantly lower overall fecal scores were observed with NRYE-fed pigs. The observed lower incidences of diarrhea and mortality in NRYE treatment groups may have resulted from beneficial effects of modulating the intestinal microbiota (Andrés-Elias et al., 2007; Sauer et al., 2011) and the consequence of improving intestinal maturation (Martinez-Puig et al., 2007). For instance, dietary nucleotide supplementation maintained a stable microbial population in piglets post-weaning, which likely minimized enteric disorders (Andrés-Elias et al., 2007). Moreover, dietary nucleotides have been shown to promote the growth of lactobacilli and bifidobacteria, which could reduce the proliferation of pathogenic bacteria and incidences of diarrhea through competitive exclusion (Stein and Mateo, 2004). Thus, it may be possible that dietary nucleotide supplementation stabilized the level of microbiota, which may have supported the immune response within the intestine of pigs fed NRYE-supplemented diets. Additionally, it was reported by Lee et al. (2007) that NRYE supplementation improve the immunity of weaning pigs. They observed increased levels of plasma immunoglobulins in weaned piglets fed 1 g/kg nucleotides. In addition, it has recently been suggested that dietary nucleotides might eliminate the genotoxic effect of high polyunsaturated fatty acids intake on blood lymphocytes (Salobir et al., 2005), which indicates its potential immunomodulation properties (Sauer et al., 2011). In this study, orally ETEC challenged pigs fed 1 g/kg or 2 g/kg NRYE diets showed higher ADG up to day 14 after weaning and tended to lower mortality rate than challenged NC pigs (Tables 3 and 4), suggesting that nucleotides intake might help sustain growth and enhance immune response during an ETEC infection. 6
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5. Conclusion Dietary supplementation with nucleotide rich yeast extract enhanced growth performance of newly-weaned pigs and showed positive effect on reduced colibacillosis along with the associated incidence of diarrhea. This indicates that nucleotide rich yeast extract has the potential to modulate intestinal health and function in post-weanling pigs and may represents a potential alternative to antibiotic growth promotion technology for the swine industry. Acknowledgments Support for this research from Canadian Bio-System Inc., Canada is gratefully acknowledged. References Adjei, A.A., Jones, J.T., Enriquez, F.J., Yamamoto, S., 1999. Dietary nucleosides and nucleotides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice. Exp. Parasitol. 92, 199–208. Andrés-Elias, N., Pujols, J., Badiola, I., Torrallardona, D., 2007. Effect of nucleotides and carob pulp on gut health and performance of weanling piglets. Livest. Sci. 108, 280–283. Domeneghini, C., Di Giancamillo, A., Arrighi, S., Bosi, G., 2006. Gut-trophic feed additives and their effects upon the gut structure and intestinal metabolism. State of the art in the pig, and perspectives towards humans. Histol. Histopathol. 21, 273–283. Domeneghini, C., Di Giancamillo, A., Savoini, G., Paratte, R., Bontempo, V., Dell’Orto, V., 2004. Structural patterns of swine ileal mucosa following L-glutamine and nucleotide administration during the weaning period. An histochemical and histometrical study. Histol. Histopathol. 19, 49–58. Girard, M., Thanner, S., Pradervand, N., Hu, D., Ollagnier, C., Bee, G., 2018. Hydrolysable chestnut tannins for reduction of postweaning diarrhea: efficacy on an experimental ETEC F4 model. PLoS One 13, e0197878. Heo, J.M., Kim, J.C., Hansen, C.F., Mullan, B.P., Hampson, D.J., Pluske, J.R., 2009. Feeding a diet with decreased protein content reduces indices of protein fermentation and the incidence of postweaning diarrhoea in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli. J. Anim. Sci. 87, 2833–2843. Heo, J.M., Opapeju, F.O., Pluske, J.R., Kim, J.C., Hampson, D.J., Nyachoti, C.M., 2013. Gastrointestinal health and function in weaned pigs: a review of feeding strategies to control post‐weaning diarrhoea without usingin‐feed antimicrobial compounds. J. Anim. Physiol. Anim. Nutr. 97, 207–237. Heo, J.M., Kim, J.C., Yoo, J., Pluske, J.R., 2015. A between‐experiment analysis of relationships linking dietary protein intake and post‐weaning diarrhea in weanling pigs under conditions of experimental infection with an enterotoxigenic strain of Escherichia coli. Anim. Sci. J. 86, 286–293. Jiao, Y., Lee, D.J., Kim, J.K., Liang, X.X., Zhang, J.Y., Kim, I.H., 2018. Effect of nucleotides supplementation to corn soybean meal-based diet on growth performance, fecal microflora, and blood profiles in sows and their piglets. J. Anim. Sci. 96, 45–46. Kiarie, E., Scott, M., Krause, D.O., Khazanehei, H., Khafipour, E., Nyachoti, C.M., 2012. Interactions of fermentation product and in-feed antibiotic on gastrointestinal and immunological responses in piglets challenged with K88. J. Anim. Sci. 90, 1–3. Lane, A.N., Fan, T.W.M., 2015. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res. 43, 2466–2485. Lee, D.N., Liu, S.R., Chen, Y.T., Wang, R.C., Lin, S.Y., Weng, C.F., 2007. Effects of diets supplemented with organic acids and nucleotides on growth, immune responses and digestive tract development in weaned pigs. J. Anim. Physiol. Anim. Nutr. 91, 508–518. Liu, Y., Song, M., Che, T.M., Almeida, J.A.S., Lee, J.J., Bravo, D., Maddox, C.W., Pettigrew, J.E., 2013. Dietary plant extracts alleviate diarrhea and alter immune responses of weaned pigs experimentally infected with a pathogenic. J. Anim. Sci. 91, 5294–5306. Lönnerdal, B., 2016. Bioactive proteins in human milk: health, nutrition, and implications for infant formulas. J. Pediatr. 173, 4–9. Luppi, A., 2017. Swine enteric colibacillosis: diagnosis, therapy and antimicrobial resistance. Porcine Health Manag. 3, 16. Martinez-Puig, D., Manzanilla, E.G., Morales, J., Borda, E., Pérez, J.F., Piñeiro, C., Chetrit, C., 2007. Dietary nucleotide supplementation reduces occurrence of diarrhoea in early weaned pigs. Livest. Sci. 108, 276–279. Mateo, C.D., 2005. Aspects of Nucleotide Nutrition in Pigs. PhD. Dissertation. South Dakota State University, Brookings, South Dakota, USA. Mosca, F., Giannì, M.L., 2017. Human milk: composition and health benefits. Pediatr. Med. Chir. 39, 155. Pluske, J.R., Turpin, D.L., Kim, J.C., 2018. Gastrointestinal tract (gut) health in the young pig. Anim. Nutr. Feed Technol. 4, 187–196. Rhouma, M., Fairbrother, J.M., Beaudry, F., Letellier, A., 2017. Post weaning diarrhea in pigs: risk factors and non-colistin-based control strategies. Acta Vet. Scand. 59, 31. Salobir, J., Rezar, V., Pajk, T., Levart, A., 2005. Effect of nucleotide supplementation on lymphocyte DNA damage induced by dietary oxidative stress in pigs. Anim. Sci. 81, 135–140. Sauer, N., Mosenthin, R., Bauer, E., 2011. The role of dietary nucleotides in single-stomached animals. Nutr. Res. Rev. 24, 46–59. Shin, T.K., Yi, Y.J., Kim, J.C., Pluske, J.R., Cho, H.M., Wickramasuriya, S.S., Kim, E., Lee, S.M., Heo, J.M., 2017. Reducing the dietary omega-6 to omega-3 polyunsaturated fatty acid ratio attenuated inflammatory indices and sustained epithelial tight junction integrity in weaner pigs housed in a poor sanitation condition. Anim. Feed Sci. Technol. 234, 312–320. Statistix, 2008. Version 7.0, Analytical Software. Tallahassee, FL, USA. Stein, H.H., Mateo, C.D., 2004. Nucleotides may have a role in nutrition of young pigs. Feedstuffs 76, 11–14. Sun, Y., Kim, S.W., 2017. Intestinal challenge with enterotoxigenic Escherichia coli in pigs, and nutritional intervention to prevent postweaning diarrhea. Anim. Nutr. Feed Technol. 3, 322–330. Superchi, P., Saleri, R., Borghetti, P., De Angelis, E., Ferrari, L., Cavalli, V., Amicucci, P., Ossiprandi, M.C., Sabbioni, A., 2012. Effects of dietary nucleotide supplementation on growth performance and hormonal and immune responses of piglets. Animal 6, 902–908. Trckova, M., Faldyna, M., Alexa, P., Sramkova Zajacova, Z., Gopfert, E., Kumprechtova, D., Auclair, E., D’Inca, R., 2014. The effects of live yeast on postweaning diarrhea, immune response, and growth performance in weaned piglets. J. Anim. Sci. 92, 767–774. Waititu, S.M., Heo, J.M., Patterson, R., Nyachoti, C.M., 2015. Dose-response effects of in-feed antibiotics on growth performance and nutrient utilization in weaned pigs fed diets supplemented with yeast-based nucleotides. Anim. Nutr. Feed Technol. 3, 166–169. Waititu, S.M., Heo, J.M., Patterson, R., Nyachoti, C.M., 2016a. Dietary yeast-based nucleotides as an alternative to in-feed antibiotics in promoting growth performance and nutrient utilization in weaned pigs. Can. J. Anim. Sci. 96, 289–293. Waititu, S.M., Yin, F., Patterson, R., Rodriguez-Lecompte, J.C., Nyachoti, C.M., 2016b. Short-term effect of supplemental yeast extract without or with feed enzymes on growth performance, immune status and gut structure of weaned pigs challenged with Escherichia coli lipopolysaccharide. J. Anim. Sci. Biotechnol. 7, 64. Waititu, S.M., Yin, F., Patterson, R., Yitbarek, A., Rodriguez-Lecompte, J.C., Nyachoti, C.M., 2017. Dietary supplementation with a nucleotide-rich yeast extract modulates gut immune response and microflora in weaned pigs in response to a sanitary challenge. Animal 11, 2156–2164.
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Dietary nucleotide rich yeast extract mitigated symptoms of colibacillosis in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli R. Pattersona,1, J.M. Heob,c,1, S.S. Wickramasuriyac, Y.J. Yid, C.M. Nyachotib,
T
⁎
a
Canadian Bio-System Inc., Calgary, AB, T2C 0J7, Canada Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada c Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, 34134, Republic of Korea d Division of Biotechnology, Chonbuk National University, Iksan-si, Jeollabuk-do, 54596, Republic of Korea b
A R T IC LE I N F O
ABS TRA CT
Keywords: E. coli challenge Nucleotide rich extract Performance Pigs
A study was conducted to investigate the effect of a nucleotide rich yeast extract (NRYE) on the growth performance and the colibacillosis of weaned pigs. In Experiment. 1, a total of 168 mixedsex piglets weaned at 17 ± 2 days of age were fed diets formulated to meet or exceed nutrient requirements for 28 days. Diets consisted of i) Positive control (PC) containing antibiotics (110 mg/kg of chlortetracycline HCl and 31.2 mg/kg of tiamulin), ii) Negative control (NC) without antibiotics, iii) NC with the addition of 1 g/kg of NRYE (NRYE1), and iv) 2 g/kg of NRYE (NRYE2). No differences (P > 0.10) were found among treatments for average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR), whilst pigs fed NRYE1 weighed more (P < 0.05) than those fed NC on day 28. Pigs fed the NRYE2 diet had greater (P < 0.05) ADG and ADFI than those fed the NC diet from day 7 to 14. No differences (P > 0.10) were observed in ADFI among dietary treatments. In Experiment 2, a total of 144 barrows were randomly assigned to 4 treatments groups to give 6 replicates (6 pigs per pen) per group. Four treatment diets were similar to previous experiment. Pigs in PC fed a control diet without NYRE supplementation and also did not received an oral challenge. PC treatment was housed in a separate room within the same facility with a similar environment. Pigs in NC, NRYE1 and NRYE2 were fed the non-medicated control diet with 0g/kg, 1 g/kg or 2 g/kg NYRE, respectively and were orally challenged with enterotoxigenic Escherichia coli K88+ (ETEC) after the 3 days of weaning. From day 3 to 28 post-challenge, pigs fed diets NRYE1 or NRYE2 had greater (P < 0.05) ADG compared with those fed a NC diet. Average daily feed intake and FCR were not affected by dietary treatment throughout the study. Challenged pigs consuming NRYE1 or NRYE2 diets had less (P < 0.05) diarrhea compared with those fed NC diet. The mortality due to coliform infection tended to be higher (P < 0.10) in the challenged NC and NRYE2 pigs compared with PC and NRYE1 pigs. In conclusion, diets supplemented with NRYE enhanced BW, ADG and ADFI of weaned pigs and reduced the incidence of diarrhea in ETEC challenged pigs
Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; ETEC, Escherichia coli K88+; FCR, feed conversion ratio; HCl, hydrogen chloride; LT, heat labile toxins; NC, negative control; NRYE, nucleotide rich yeast extract; PC, positive control; PWD, post-weaning diarrhea; ST, heat stable toxins ⁎ Corresponding author. E-mail address: [email protected] (C.M. Nyachoti). 1 Authors have made equal contribution to this paper. https://doi.org/10.1016/j.anifeedsci.2019.114204 Received 29 October 2018; Received in revised form 11 June 2019; Accepted 19 June 2019 0377-8401/ © 2019 Published by Elsevier B.V.
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suggesting that NRYE could be effective in controlling post-weaning diarrhea in piglets, which could represent a major economic advantage to the swine industry.
1. Introduction Weaned piglets are susceptible to nutritional, psychological and environmental changes due to a naive immune system, which can lead to intestinal dysfunction and increased incidences of post-weaning diarrhea (PWD; Heo et al., 2015). In particular, enterotoxigenic Escherichia coli (ETEC) infection is a common disease in pre- and post-weaned pigs that leads to the incidences of PWD, poor growth performance, morbidity and mortality (Heo et al., 2013, 2015, Sun and Kim, 2017). Pigs infected with ETEC generally experience reduced integrity of the intestinal epithelium, premature enterocyte apoptosis, fluid exudation into the intestinal lumen and loss of digestive and absorptive capacity (Heo et al., 2013). Therefore, a large number of additives have attracted attention as approaches to maintain a healthy gut in an attempt to control post-weaning growth check and the symptoms of colibacillosis in weaned piglets (Heo et al., 2013; Pluske et al., 2018) Yeast extracts consist of bioactive compounds such as nucleotides and cell wall polysaccharides, which have the potential to be an alternative to growth promoting antibiotics (Waititu et al., 2016a, 2017). Recently, it has been proposed that yeast products might be beneficial for improving the ability of piglet to resist an ETEC challenge or improving post-challenge recovery, or the combination of both via a number of mechanisms including blocking mucosal attachment of the pathogen that could enhance the host immune response or promote intestinal mucosal integrity (Kiarie et al., 2012; Trckova et al., 2014; Waititu et al., 2016b; Rhouma et al., 2017). Importantly, dietary nucleotide helps to recover conditionally essential nucleotide requirement of newly weaner pigs to eliminate impedes nucleotide-dependent physiological processes (Waititu et al., 2016b). Necessities are existing to reveal the nucleotide rich yeast extract (NRYE) impact on mitigating symptoms of colibacillosis in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli. To this end, the present study was conducted to investigate the ability of a NRYE to improve growth performance of weaned piglets and to examine the effects of dietary NRYE supplementation in piglets orally challenged with a virulent strain of ETEC (E. coli K88+). In this study we hypothesized that nucleotide rich yeast extract supplementation in to diet may enhanced growth performance of newly-weaned pigs and showed positive effect on reducing colibacillosis along with the associated incidence of diarrhea.
Table 1 Composition of basal diet for Experiment 1 and Experiment 2 (as-fed basis). Item
Ingredient, g/kg Oat groats Skim milk powder Wheat Whey permeate Soybean meal Corn Fish meal Soybean oil Tallow Dicalcium phosphate Limestone Vitamin-mineral premixc Salt Lys-HCl L-Thr DL-Met Calculated analysisd CP, g/kg ME, MJ/kg SID Lys, g/kg SID Lys, g/Mcal ME
Exp. 1a
Exp. 2b
Phase 1
Phase 2
Phase 3
Phase 1
Phase 2
250.0 170.0 150.0 110.7 110.0 88.3 37.9 25.5 25.0 6.8 5.4 10.0 1.4 4.0 3.0 2.1
150.0 84.0 150.0 68.0 165.0 268.1 38.8 20.0 22.0 10.6 3.1 10.0 3.1 4.0 2.0 1.3
– 41.0 125.0 46.7 194.5 502.2 25.0 5.0 23.1 10.5 6.8 10.0 3.7 3.8 1.6 1.1
250.0 170.0 150.0 110.7 108.0 90.3 37.9 25.5 25.0 6.8 5.4 10.0 1.4 4.0 3.0 2.1
– 41.0 125.0 46.7 192.5 504.2 25.0 5.0 23.1 10.5 6.8 10.0 3.7 3.8 1.6 1.1
199.0 14.48 14.2 4.1
195.0 14.26 13.8 4.1
181.0 13.92 13.1 3.9
197.0 14.52 14.5 4.2
177.0 13.96 12.9 3.9
a
Experiment 1 Phase 1;1(17 ± 2 days of age) - 3 days after weaning, Phase 2; 4–7 days after weaning, Phase 3; 8–28 days after weaning. Experiment 2 Phase 1; −3 (17 ± 2 days of age) −18 days of post challenge, Phase 2; 19–28 days of post challenge. c Vitamin and mineral premix supplied the following per kilogram of complete diet: 8250 IU of vitamin A; 825 IU of vitamin D3; 40 IU of vitamin E; 4.0 mg of vitamin K as menadione; 500 mg of choline chloride; 22.5 mg of niacin; 15 mg of calcium pantothenate; 10 mg of riboflavin; 2 mg of thiamin; 25 μg of vitamin B12; 2000 μg biotin; 2 mg of folic acid; 50 mg Mn as manganese oxide; 100 mg Fe as iron sulfate; 150 mg of Zn as zinc oxide; 25 mg of Cu as copper sulfate; 0.4 mg of I as calcium iodate; 0.3 mg of Se as sodium selenite. d The values are calculated according to the values of feedstuffs in NRC (2012). b
2
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2. Materials and methods Experimental protocols used in the present study were reviewed and approved by Nutreco Canada Ag-research’s Animal Care Committee and pigs were cared for according to the guidelines of the Canadian Council on Animal Care (2009). 2.1. Experiment 1. – Growth performance 2.1.1. Animals, housing and experimental treatments A total of 168 mixed-sex piglets [(Yorkshire × Landrace) × Duroc; 1:1 gender ratio] were weaned at 17 ± 2 days of age from a high-health commercial sow herd. Upon arrival at the research facility (Nutreco AgResearch commercial research farm, Burford, Ontario, Canada), piglets were weighed and examined for general defects or signs of poor health. Eligible piglets were balanced for body weight (BW) and housed in pens (1.2 × 0.9 m) with solid plastic walls and plastic coated expanded steel flooring. Each pen housed 6 piglets (3 barrows and 3 gilts) and each treatment was randomly assigned to 7 pens for a total of 42 pigs per treatment. Each pen was equipped with a trough feeder and a height adjustable nipple drinker to which piglets had free access throughout the experiment. The ambient temperature was maintained at 29 ± 1 °C for the initial week and was then gradually decreased by 1 °C every week. All diets were corn and soybean meal-based and were manufactured at a commercial feed mill (Yantzi Feed and Seeds, Tavistock, Ontario, Canada). Diets were formulated to meet or exceed the NRC (2012) nutrient requirements for their respective BW (Table 1). Diet 1 was a positive control (PC) containing 110 mg/kg of Aueromycin (chlortetracycline hydrogen chloride; Bio Agri Mix LP, Mitchell, Ontario, Canada) and 31.2 mg/kg of Tiamulin (Bio Agri Mix LP, Mitchell, Ontario, Canada). Diet 2 was a negative control (NC) and contained no antibiotics. Diets 3 and 4 were equivalent to the NC but with the addition of 1 g/kg and 2 g/kg, respectively, of the nucleotide rich yeast extract (NRYE; Maxi-Gen Plus, Canadian Bio-Systems Inc., Calgary, Alberta, Canada). Nucleotide rich yeast extract (Maxi-Gen Plus) is a mixture of crude protein (327 g/kg), cell wall polysaccharides (216 g/kg), carbohydrates (143 g/kg), and a mixture of five nucleotides (11 g/kg; adenosine monophosphate, cytosine monophosphate, inosine monophosphate, uridine monophosphate, and guanosine monophosphate). Pigs were phase fed and received the phase one diet from 1 to 3-day after weaning, phase two diet from 4 to 7-day after weaning and phase three diet from 8 to 28-day after weaning. Feed intake was recoded as feed disappearance from the feeder, and pigs were weighed on a weekly basis for 4 weeks. Using body weight and feed intake, average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated. 2.2. Experiment 2. – Responses to enterotoxigenic Escherichia coli (ETEC) challenge 2.2.1. Animals, housing and experimental treatments A total of 144 barrows [(Yorkshire × Landrace) × Duroc] weaned at 17 ± 2 days of age were balanced for BW and randomly assigned to 24 pens (1.2 × 0.9 m; 6 pigs per pen). Each pen had raised, fully slatted, plastic coated expanded steel flooring and solid plastic walls, a stainless-steel feeder and a height adjustable nipple drinker. Pigs had free access feed and water throughout the experiment. The study consisted of 4 treatments similar to previous experiment with 6 replicate pens per group. Pigs in PC received a control diet and also did not received an oral challenge. This group was housed separately in a separate room within the same building with similar environmental condition as the challenged pigs. Pig in other three treatments received the control diet supplemented with 0 mg/kg (NC), 1 mg/kg (NRYE1) and 1 mg/kg (NRYE2) of a NRYE product (Maxi-Gen Plus, Canadian Bio-Systems Inc., Calgary, AB, Canada), respectively, and also received an oral challenge. Test diets were commercially manufactured (Yantzi Feed and Seeds, Tavistock, Ontario, Canada) and were formulated to meet or exceed NRC (2012) nutrient requirements for swine (Table 1). Pigs were phase fed and received the phase one diet from -3 (17 ± 2 days of age) to 18-day post challenge and the phase two diet from 19 to 28-day post challenge. 2.2.2. Oral challenge, animal observation and health status The ETEC β-hemolytic serotype O149; K91; K88 [toxins heat labile toxins (LT), heat stable toxins (ST; variants STa and STb)] was used for experimental infection of the pigs to clinically express PWD. After 3 days of weaning, designated pigs (NC, NRYE1 and NRYE2) received a 3 mL oral dose of inoculum containing 1.48 × 108 cfu/mL of ETEC following the procedure describe by Heo et al. (2009). The inoculum was prepared by trained personnel (Nutreco Canada Ag-Research laboratory, Mississauga, Ontario, Canada) according to standard in-house procedures with similar to the procedure followed by Heo et al. (2009). Designated non-challenge control pigs (PC) received a 3 mL oral dose of phosphate-buffered saline in a similar procedure (PBS) as a placebo. 2.2.3. Growth performance, fecal consistency scoring and mortality Feed intake was recoded as feed disappearance from the feeder, and pigs were weighed on a weekly basis for 4 weeks. Using body weight and feed intake, ADFI, ADG and FCR was calculated. Individual piglets were observed twice daily from arrival until completion of the study. Starting daily from post challenge to day 7 (3 to 10-day after weaning) severity of diarrhea was quantified by fecal consistency scoring (0, normal; 1, pasty feces; 2, liquid feces; 3, severe diarrhea) according to Heo et al. (2009) and Shin et al. (2017). All scoring was performed by trained individuals with no prior knowledge of dietary treatments. Data for the incidence of PWD were expressed as the mean percentage of pigs with diarrhea relative to the total number of pigs in each treatment (Heo et al., 2009). Mortality was recorded throughout the experiment when the 3
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dead occurred. Recorded mortality data were expressed as the mean percentage of days with mortality relative to the total 7 day after weaning. 2.3. Statistical analysis Data from both Exp. 1 and 2 were subjected to ANOVA as a completely randomized design (Statistix, 2008) and pen was considered the experimental unit for all growth performance response criteria. Individual pig was considered as the experimental unit for fecal consistency scoring and mortality data analysis. Statistical significance was accepted at P < 0.05 and trends were discussed at P < 0.10. When the treatment effect was significant (P < 0.05) Tukey’s multiple range test was used to differentiate significance between means. 3. Results 3.1. Experiment 1. – Growth performance There were no differences (P > 0.10) among dietary treatments for ADG, ADFI or FCR on day 1–7 after weaning (Table 2). Pigs fed the NYRE2 supplemented diet had greater ADG (P = 0.048) and ADFI (P = 0.034) than NC pigs but did not differ compared to PC or NYRE1 pigs (P > 0.10) from 8 to 14-day after weaning. Although ADG was not affected (P > 0.10) by dietary treatment from day 15 to 21, pigs fed the NYRE1 diet had a higher ADFI, which tended (P = 0.098) to be greater than for pigs in the NC group. Average daily feed intake was not affected (P > 0.10) by dietary treatment from 22 to 28-day after weaning, however, during this period, ADG of NYRE1 pigs was greater than that of the NC-fed pigs (P = 0.041) and was the same (P > 0.10) as NYRE2 and PC pigs, respectively. Overall, no differences were observed (P > 0.05) for ADFI among dietary treatments from weaning to 28-day after weaning. However, pigs fed the NYRE1 diet tended to be greater ADG than those fed the NC (P = 0.051) and the same (P > 0.10) ADG as the pigs fed the NYRE2 and the PC diets. No statistical differences were detected (P > 0.0.5) among dietary treatments for feed conversion during the entire experimental period. 3.2. Experiment 2. – Growth performance Unchallenged pigs fed the PC diet had greater ADG (P = 0.016) than challenged pigs fed the NC, NYRE1 or NYRE2 diets from 3 day pre-challenge to 7 day post-challenge (Table 3). However, ADG was greater (P < 0.05) in the challenged pigs fed NYRE1 and NYRE2 than in the pigs fed the NC diet from day 8 to 14 post-challenge (P = 0.012). No differences were found (P > 0.10) in ADG among dietary treatments from day 15 to 21, 22 to 28 and overall post challenge period. From 3 day pre- to 7 day post-challenge, ADFI was greater (P = 0.01) for unchallenged pigs fed the PC diet than challenged pigs fed the NC, NYRE1 and NYRE2 diets. There were no differences (P > 0.05) in ADFI among dietary treatments at any other stage during the study (Table 3). From challenge day 8 Table 2 Effect of the addition of the nucleotide rich yeast extract on growth performance of weaned pigs for Experiment 1.1,2,3,4 Item ADG, g/day day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28 ADFI, g/day day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28 FCR, g/g day 1 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day 1 to 28
PC
NC
NRYE1
NRYE2
SEM
P-value
143 329ab 427 527ab 367ab
107 284b 412 537b 335b
124 294ab 432 623a 379a
140 332a 432 565ab 367ab
11.38 13.45 20.70 19.54 10.63
0.148 0.048 0.872 0.041 0.051
153 373ab 621ab 794 485
137 349b 569b 783 459
142 367ab 641a 858 497
153 406a 629ab 792 495
9.4 12.4 19.7 23.7 12.7
0.556 0.034 0.098 0.133 0.170
1.07 1.14 1.46 1.39 1.32
1.3 1.23 1.39 1.48 1.37
1.26 1.32 1.49 1.38 1.32
1.10 1.23 1.47 1.41 1.35
0.089 0.085 0.036 0.039 0.025
0.212 0.520 0.297 0.316 0.344
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (With antibiotics), NC; negative control (Without antibiotics), NRYE1; NC+1 g/kg nucleotide rich yeast extract, NRYE2; NC+2 g/kg nucleotide rich yeast extract, SEM; standard error of mean, ADG; average daily gain, ADFI; average daily feed intake, FCR; feed conversion ratio. 3 Values are the mean of 7 replicates per treatment. 4 Day 1= indicate the weaning day (17 ± 2 days of age). 2
4
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Table 3 Effect of the addition of the nucleotide rich yeast extract on growth performance of weaned pigs challenged with E. coli for Exp. 2.1,2,3 Item ADG, g/day day −34 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28 ADFI, g/day day −3 to 7 day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28 FCR5, g/g day 8 to 14 day 15 to 21 day 22 to 28 day −3 to 28
PC
NC
NRYE1
NRYE2
SEM
P-value
120a 336a 407 536 289
27b 220b 288 533 249
55b 293a 419 534 271
49b 312a 407 550 262
18.3 21.9 33.4 28.4 17.7
0.016 0.012 0.930 0.970 0.460
168a 371 594 829 439
145b 409 602 825 410
146b 368 598 819 417
136b 401 602 850 413
5.90 24.5 33.2 32.3 19.7
0.010 0.560 0.990 0.910 0.710
1.79a 1.49 1.55 1.53
1.23b 1.58 1.55 1.67
1.25b 1.43 1.55 1.54
1.29b 1.49 1.55 1.60
0.082 0.060 0.051 0.053
0.001 0.420 0.990 0.250
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (Non-medicated diet and without ETEC challenged), NC; negative control (Non-medicated diet and Challenged with ETEC), NRYE1; NC+1 g/kg nucleotide rich yeast extract and challenged with ETEC, NRYE2; NC+2 g/kg nucleotide rich yeast extract and challenged with ETEC, SEM; standard error of mean, ADG; average daily gain, ADFI; average daily feed intake, FCR; feed conversion ratio. 3 Values are the mean of 6 replicates per treatment. 4 day -3= indicate the weaning day and 3 days pre-challenged (17 ± 2 days of age). 5 Negative FCR values occurred for day -3 to 7 post challange, and data were removed. 2
to 14, challenged pigs receiving the NC, NYRE1 and NYRE2 diets had higher FCR than unchallenged pigs receiving the PC diet (P = 0.001). However, FCR did not differ (P > 0.10) among dietary treatments during day 15 to 21, day 22 to 28 and overall post challenge period. 3.3. Mortality and incidence of diarrhea In experiment 2, mortality tended to be higher (P = 0.06) in challenged pigs fed the NC and NRYE2 diets than in unchallenged pigs fed the PC diet. On day 1 and day 3 post-challenge, the incidence of diarrhea was greater (P < 0.05) in challenged versus unchallenged pigs (Table 4), but no difference (P > 0.10) was observed between challenged pigs fed the NC diet and pigs fed either NRYE1 or NRYE2 diets. The proportion of pigs showing the incidence of diarrhea was greater in challenged pigs fed the NC and NYRE2 diet than unchallenged pigs fed the PC diet (P = 0.04) on day 1 post-challenge. However, no difference was observed (P > 0.10) between the Table 4 Effect of the addition of the nucleotide rich yeast extract on fecal consistency for 7-day after weaning for Exp. 2.1,2 Item Proportion > 0, % day 13 day 3 day 5 Proportion > 1, %4 day 1 day 3 day 5 Overall fecal score day 1–7 Mortality rate, %5
PC
NC
NRYE1
NRYE2
SEM1
P-value
11.1b 8.34b 2.78
50.0a 42.2a 22.22
38.9a 44.5a 20.83
44.4a 45.0a 12.50
7.17 7.55 7.920
0.008 0.009 0.320
2.78b 2.78 b 0.00
27.8a 26.7a 12.78
19.5ab 15.0ab 8.33
30.6a 13.9ab 2.78
6.601 4.310 5.210
0.040 0.010 0.340
0.096c 0.00b
0.47a 25.0a
0.34b 11.1ab
0.34b 19.5a
0.042 5.59
0.050 0.060
1
Means in the same row with different superscripts differ in (P < 0.05). PC; positive control (Non-medicated diet and without ETEC challenged), NC; negative control (Non-medicated diet and Challenged with ETEC), NRYE1; NC+1 g/kg nucleotide rich yeast extract and challenged with ETEC, NRYE2; NC+2 g/kg nucleotide rich yeast extract and challenged with ETEC, SEM; standard error of mean. 3 day 1= indicate the orally challenged day (20 ± 2 days of age). 4 Expressed as % cumulative score per day of pigs having fecal score greater than 1; higher values are associated with more liquid feces. 5 Expressed as the mean percentage of days with mortality relative to the total 7-day after weaning. 2
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unchallenged pigs fed thePC diet compared to those fed the diet supplemented with NRYE1. Three-day post-challenge, pigs fed the NC diet had a greater (P < 0.01) incidence of diarrhea compared with unchallenged pigs fed the PC diet, but the incidence of diarrhea was similar (P > 0.10) in pigs fed the PC and NRYE-supplemented NRYE1 or NRYE2 diets. Considering the overall fecal score, higher (P = 0.05) incidence of diarrhea was observed with challenged pigs fed the NC diets compared to pigs fed the PC, NRYE1 and NRYE2 diets. With regards to challenged pigs, lower incidence (P = 0.05) of diarrhea was observed for pigs fed NRYE1 and NRYE1 diets compared to pig fed the NC diet.
4. Discussion The purpose of the current study was to test the hypothesis that NRYE will improve growth performance commensurate with attenuated mortality rate and the incidence of PWD in piglets orally challenged with a virulent strain of ETEC. Nucleotides are constituents of DNA and RNA and participate in structural, metabolic, energetic, and regulatory functions in living cells (Lane and Fan, 2015; Waititu et al., 2016a), and these non-protein nitrogenous compounds regarded as ‘conditionally essential’ nutrients that modulate the immune system, promote intestinal growth and hepatic functions (Waititu et al., 2016b; Mosca and Gianni, 2017). Many articles have documented that weaning transition is a critical period for piglets because of the abrupt change in diet from milk to solid feed along with weaning-associated intestinal dysfunction (Heo et al., 2013). In addition, these are linked to a reduction in villous height and lowered brush border enzyme activity resulting in post-weaning growth stasis (Heo et al., 2013). Furthermore, the time around weaning is the most rapid growth period in the life of a pig, which creates a higher demand for nucleotides to support the rapidly dividing cells (i.e., blood and small and large intestinal cells). Domeneghini et al. (2004) demonstrated that piglets fed a diet containing 0.5 g/kg nucleotides showed numerical benefits during the first week post weaning, and intestinal tissues of piglets consuming nucleotide-containing diets indicated a greater content of mucosal protein and DNA, higher villous height and disaccharide activities, and improved recovery post injury (Domeneghini et al., 2006). Furthermore, Waititu et al. (2015) observed beneficial effect of NRYE on growth performance of weaned pigs and its comparative advantage over the antibiotic growth promoters. In the present study, greater ADG was observed in pigs fed diets supplemented with 1 g/kg NRYE during day 1–28 after weaning than those fed the NC and PC diets which suggests that NRYE supplementation had positive effects on the growth performance of weanling piglets (Table 2). However, the effect of dietary nucleotide supplementation on piglet growth and intestinal morphology is controversial. No improvements were observed with respect to growth performance when pigs (weaned at 26 day of age) were fed a diet containing 15 mg/kg nucleotide rich yeast extract (25% of free nucleotides) and carob pulp alone or in combination for 14 days (Andrés-Elias et al., 2007). A similar observation was reported by Mateo (2005) who fed 19- day old weaned pigs a control diet based on corn-soybean meal and dried whey or a control diet supplemented with 3 mg/kg nucleotides had no significant enhancement of growth performance. Furthermore, pigs fed corn–soybean meal–whey basal nursery diets supplemented with organic acids as well as 1 g/kg nucleotides had no effect on piglet performance over a 46- day period and no consistent effects on the immune response were observed (Lee et al., 2007). Meanwhile, Jiao et al. (2018) demonstrated that pigs fed a diet containing 5 g/kg and 1 g/kg nucleotide improved growth performance. Although piglets fed diets containing up to 2 times the amount of nucleotides as in sow milk had similar feed intake and growth as those fed control diets during the first week post-weaning, also nucleotide supplementation minimized the reduction of villous height following weaning (Martinez-Puig et al., 2007) which was consistent with the results of Domeneghini et al. (2004). Post-weaning diarrhea is associated with fecal shedding of β-hemolytic ETEC, which proliferate in the small intestine of both healthy and unhealthy weaned pigs (Heo et al., 2013; Liu et al., 2013; Rhouma et al., 2017; Girard et al., 2018) that can lead to ETEC infection, which is also known as ETEC colibacillosis (Luppi, 2017). Colibacillosis is characterized by increasing intestinal fluid loss and subsequent diarrhea (Luppi, 2017). In previous studies, dietary nucleotide supplementation effectively reduced incidences of diarrhea in piglets (Superchi et al., 2012; Waititu et al., 2016b). Additionally, nucleotide supplementation reduced the incidences of diarrhea in human infants (Lönnerdal, 2016) and inhibited endotoxin-induced intestinal translocation of bacteria in mice (Adjei et al., 1999). In the current study, pigs fed a diet supplemented with 1 g/kg or 2 g/kg NRYE showed numerically lower proportions of mild or moderate diarrhea after an oral ETEC challenge compared to control challenged pigs on day 1 and d 5 post-challenge. Moreover, significantly lower overall fecal scores were observed with NRYE-fed pigs. The observed lower incidences of diarrhea and mortality in NRYE treatment groups may have resulted from beneficial effects of modulating the intestinal microbiota (Andrés-Elias et al., 2007; Sauer et al., 2011) and the consequence of improving intestinal maturation (Martinez-Puig et al., 2007). For instance, dietary nucleotide supplementation maintained a stable microbial population in piglets post-weaning, which likely minimized enteric disorders (Andrés-Elias et al., 2007). Moreover, dietary nucleotides have been shown to promote the growth of lactobacilli and bifidobacteria, which could reduce the proliferation of pathogenic bacteria and incidences of diarrhea through competitive exclusion (Stein and Mateo, 2004). Thus, it may be possible that dietary nucleotide supplementation stabilized the level of microbiota, which may have supported the immune response within the intestine of pigs fed NRYE-supplemented diets. Additionally, it was reported by Lee et al. (2007) that NRYE supplementation improve the immunity of weaning pigs. They observed increased levels of plasma immunoglobulins in weaned piglets fed 1 g/kg nucleotides. In addition, it has recently been suggested that dietary nucleotides might eliminate the genotoxic effect of high polyunsaturated fatty acids intake on blood lymphocytes (Salobir et al., 2005), which indicates its potential immunomodulation properties (Sauer et al., 2011). In this study, orally ETEC challenged pigs fed 1 g/kg or 2 g/kg NRYE diets showed higher ADG up to day 14 after weaning and tended to lower mortality rate than challenged NC pigs (Tables 3 and 4), suggesting that nucleotides intake might help sustain growth and enhance immune response during an ETEC infection. 6
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5. Conclusion Dietary supplementation with nucleotide rich yeast extract enhanced growth performance of newly-weaned pigs and showed positive effect on reduced colibacillosis along with the associated incidence of diarrhea. This indicates that nucleotide rich yeast extract has the potential to modulate intestinal health and function in post-weanling pigs and may represents a potential alternative to antibiotic growth promotion technology for the swine industry. Acknowledgments Support for this research from Canadian Bio-System Inc., Canada is gratefully acknowledged. References Adjei, A.A., Jones, J.T., Enriquez, F.J., Yamamoto, S., 1999. Dietary nucleosides and nucleotides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice. Exp. Parasitol. 92, 199–208. Andrés-Elias, N., Pujols, J., Badiola, I., Torrallardona, D., 2007. Effect of nucleotides and carob pulp on gut health and performance of weanling piglets. Livest. Sci. 108, 280–283. 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