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Animal Science Journal (2015) 86, 617–623
doi: 10.1111/asj.12329
ORIGINAL ARTICLE Effects of dietary supplementation of modified zinc oxide on growth performance, nutrient digestibility, blood profiles, fecal microbial shedding and fecal score in weanling pigs Jin Ho CHO,1 Santi Devi UPADHAYA2 and In Ho KIM2 1
Department of Animal Science, Chungbuk National University, Cheongju and 2Department of Animal Resource & Science, Dankook University, Cheonan, Korea
ABSTRACT One hundred and forty piglets ((Landrace × Yorkshire) × Duroc, 21 day of age) with an initial weight of 6.50 ± 0.71 kg, were randomly allotted into four treatments to determine the effects of a modified form of zinc oxide (ZnO) on growth performance, nutrient digestibility, blood profiles, fecal microbial shedding and fecal score in weanling pigs. Dietary treatments were: (i) NC, negative control, basal diet containing zinc (Zn) from the premix; (ii) PC, positive control, basal diet containing Zn-free premix + 3000 ppm ZnO; (iii) H1, basal diet containing Zn-free premix + 3000 ppm ZnO (phase 1, days 1 to 14)/200 ppm modified ZnO (phase 2, days 15 to 42); (iv) H2, basal diet containing Zn-free premix + 300 ppm modified ZnO (phase 1)/200 ppm modified ZnO (phase 2). During days 1 to 14, average daily gains (ADG) were higher (P = 0.04) in PC, H1 and H2 groups than that in NC group. Overall, H1 treatment increased the ADG compared with NC (P = 0.05). On day 14, the alkaline phosphatase and plasma Zn concentration were increased (P = 0.01 and 0.04, respectively) in PC, H1 and H2 treatments compared with NC treatment. On days 14 and 42, the fecal Lactobacillus counts in NC group were lowest (P = 0.01, P = 0.04 respectively) among treatments. All supplemented groups showed lower (P = 0.03) fecal score than NC treatment on days 21 and 28. In conclusion, dietary supplementation with modified ZnO increased growth rates and reduced fecal scores in weanling pig. Modified ZnO could be used as a substitute to ZnO as a growth promoter and reduce Zn excretion to the environment because of the lower dosage. [Correction added on 3 February 2015, after first online publication: the initial weight of ‘6.50 ± 1.11 kg’ has been replaced with ‘6.50 ± 0.71 kg’ in the abstract.]
Key words: blood profiles, fecal microbial shedding, growth performance, modified zinc oxide, weanling pig.
INTRODUCTION Zinc (Zn) is the second most abundant transition metal in biological systems, and serves as a cofactor for more than 300 enzymes in the body, particularly those involved in protein, carbohydrate and fat metabolism (Coleman 1992; Huang 1997; Suhy et al. 1999). According to the nutrient requirement of swine (NRC 2012), the recommended zinc level is 80 ppm in piglets fed with conventional diet. The use of 3000 ppm Zn from zinc oxide (ZnO) is widely used by researchers in weanling diets to promote growth performance and reduce the incidence of diarrhea (Hahn & Baker 1993; Jensen-Waern et al. 1998; Hill et al. 2001; Melin & Wallgren 2002). Li et al. (2001) and Li et al. (2006) also demonstrated that ZnO supplementation improved gut health thus potentially increasing the absorptive capacity of the small intestine. However, the growth promoting effect was inconsist© 2015 Japanese Society of Animal Science
ent (Fryer et al. 1992; Tokach et al. 1992) and the high zinc excretion in animal waste poses environmental challenges (Jondreville et al. 2003). A modified form of ZnO has stronger efficacy than conventional ZnO, which is small particle ZnO as it further increases the possible sites of interaction among the molecules of ZnO, the animal, and microbes in the gastrointestinal tract (Mavromichalis 2011). It showed a higher ability to inhibit bacterial growth under ex vivo conditions, and its concentration in feed could be reduced and therefore decrease zinc
Correspondence: In Ho Kim, Department of Animal Resource & Science, Dankook University, #29 Anseodong, Cheonan, Choongnam 330-714, Korea. (Email: inhokim@ dankook.ac.kr) Received 29 April 2014; accepted for publication 15 August 2014.
618 J. H. CHO et al.
excretion (Vahjen et al. 2012). Morales et al. (2012) reported that dietary supplementation with 110 ppm of the same potentiated ZnO improved the growth performance of piglets (42 to 63 days of age). However, the mechanism of this form of ZnO was not well studied. Therefore, this study was conducted to investigate the effects of conventional ZnO and modified ZnO with enhanced surface area in weanling pigs.
overnight at 0°C. Performic acid is an oxidizing reagent that converts cysteine quantitatively to cysteic acid and methionine to methionine sulfone (Moore 1963). Nitrogen was determined by a Kjectec 2300 Nitrogen Analyzer (Foss Tecator AB, Hoeganaes, Sweden). The gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 Oxygen Bomb Calorimeter (Parr instrument Co., Moline, IL, USA).
Sampling and measurements MATERIALS AND METHODS The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University.
Animals and facilities A total of 140 weanling pigs ((Yorkshire × Landrace) × Duroc, 21 days of age) with an average body weight (BW) of 6.50 ± 0.71 kg were used in a 6-week experiment. Pigs were randomly allotted to one of four treatments with seven replicate pens and five pigs per pen (two barrows and three gilts) according to their initial BW and sex. All pigs were housed in an environmentally controlled room. The ambient temperature within the room was approximately 30°C at the start of the experiment and decreased by 1°C each week. Each pen was equipped with a one-sided, stainless steel self-feeder and a nipple drinker; pigs were allowed unlimited access to feed and water. On days 1, 14 and 42, individual pig BW and feed disappearance were recorded to determine average daily gain (ADG), average daily feed intake (ADFI) and gain/feed ratio (G/F).
Dietary treatments Dietary treatments were as follows. Phase 1 (for 14 days): (i) NC (negative control, basal diet containing Zn from premix); (ii) PC (positive control, basal diet containing Zn-free premix + 3000 ppm ZnO); (iii) H1 (basal diet containing Zn-free premix + 3000 ppm ZnO); (iv) H2 (basal diet containing Zn-free premix + 300 ppm modified ZnO). Phase 2 (for 28 days): (i) NC (negative control, basal diet containing Zn from premix); (ii) PC (positive control, basal diet containing Zn-free premix + 3000 ppm ZnO); (iii) H1 (basal diet containing Zn-free premix + 200 ppm modified ZnO); (iv) H2 (basal diet containing Zn-free premix + 200 ppm potential ZnO). Conventional ZnO (96% ZnO; screen size: 50 mesh) used in the present study was obtained from DongWoo (Anyang, Korea) and the modified form of ZnO (76% ZnO; screen size: 200 mesh) was produced by Animine (HiZox, Sillingy, France). All diets were formulated to meet or exceed the NRC (2012) requirements for weanling pigs, and were fed in a mash form (Table 1). Zinc oxide concentration in the experimental diets is shown in Table 2.
Chemical analysis Samples of diets were analyzed using standard methods (Association of Official Analytical Chemists 2000) for dry matter (DM; method 934.01) and nitrogen (N; method 968.06). Individual amino acid composition was measured using an amino acid analyzer (Beckman 6300; Beckman Coulter, Inc., Fullerton, CA, USA) after 24-h 6 N-HCl hydrolysis at 110°C (Association of Official Analytical Chemists 2000). For the determination of cysteine and methionine, the samples were oxidized with performic acid © 2015 Japanese Society of Animal Science
From days 8 to 14 and 36 to 42, chromium oxide was added to the diet at 0.2% as an indigestible marker for determination of the apparent total tract digestibility (ATTD) of DM, N and gross energy (GE). At 17.00 hours on days 12, 13, 14 as well as days 40, 41, and 42, fresh fecal grab samples were collected from two pigs per pen, then mixed and pooled, and a representative sample was stored in a freezer at −20°C until analyzed. Before chemical analysis, the fecal samples 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 screen. All fecal samples were analyzed following the procedures outlined by the Association of Official Analytical Chemists (2000). Chromium was analyzed by UV absorption spectrophotometry (UV-1201; Shimadzu, Tokyo, Japan) following the method described by Williams et al. (1962). Nutrient digestibility was then calculated using the chromium technique (Sauer et al. 2000). For the blood profiles, 14 pigs (two pigs/pen) from each treatment were randomly selected and blood samples were collected via anterior vena cava puncture on days 1, 14 and 42. At the time of collection, blood samples were collected into both nonheparinized tubes and vacuum tubes containing tripotassium ethylenediaminetetraacetic acid (K3EDTA; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) to obtain serum and whole blood, respectively. After collection, blood samples were centrifuged (3000 × g) for 15 min at 4°C. The white blood cells (WBC), red blood cells (RBC) and lymphocyte levels in the whole blood were determined using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA). The alkaline phosphatase (ALP) activity was measured according to the method described by Low and Finean (1977). The plasma zinc was determined according to the method described by Hill et al. (2000). The immunoglobulin G (IgG) concentration was determined using an automatic biochemistry blood analyzer (HITACHI 747; Hitachi, Tokyo, Japan).
Procedures of microbial shedding Fresh fecal samples were collected directly on days 14 and 42 via massaging the rectum of two pigs in each pen, and then pooled and placed on ice for transportation to the lab. One gram of the composite fecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and then homogenized. Viable counts of bacteria in the fecal samples were then conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and Lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the Escherichia coli and Lactobacillus, respectively. The lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. The E. coli and Lactobacillus colonies were counted immediately after Animal Science Journal (2015) 86, 617–623
EFFECTS OF MODIFIED ZINC OXIDE IN PIGLET
Table 1
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Feed compositions (%, as-fed basis)
Item
Ingredient Extruded corn Soybean meal (48% CP) Fermented soybean meal (45% CP) Fish meal (66% CP, Brazil) Soy oil Lactose Whey Dicalcium phosphate Sucrose Plasma powder (AP 920) L-Lys HCl (78%) DL-Met (50%) L-Thr (89%) Choline chloride (25%) Vitamin premix† Mineral premix (normal)‡ Mineral premix (Zn-free)§ Limestone Salt Analyzed nutritional content: ME, MJ/kg CP, % Lys, % Met, % Met + Cys % Ca, % Total P, % Crude fat,% Crude fiber, %
Phase 1 (0 to 14 days)
Phase 2 (14 to 42 days)
Basal diet
Zn-free diet
Basal diet
Zn-free diet
44.49 16.20 5.00 3.50 2.55 8.30 10.00 1.50 3.00 3.00 0.39 0.30 0.19 0.10 0.10 0.20 – 0.98 0.20
44.49 16.20 5.00 3.50 2.55 8.30 10.00 1.50 3.00 3.00 0.39 0.30 0.19 0.10 0.10 – 0.20 0.98 0.20
61.97 25.30 2.50 – 1.05 – 5.00 1.50 – – 0.46 0.24 0.20 0.10 0.10 0.20 – 1.13 0.25
61.97 25.30 2.50 – 1.05 – 5.00 1.50 – – 0.46 0.24 0.20 0.10 0.10 – 0.20 1.13 0.25
14.80 20.00 1.50 0.62 0.97 0.95 0.75 5.02 1.87
– – – – – – – – –
14.26 19.00 1.35 0.53 0.84 0.90 0.70 3.98 2.45
– – – – – – – – –
†Provided per kg of complete diet: vitamin A, 11 025 IU; vitamin D3, 1103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; d-pantothenic, 29 mg; choline, 166 mg; and vitamin B12, 33 μg. ‡Provided per kg of complete diet: Cu (as CuSO4·5H2O), 12 mg; Zn (as ZnSO4), 85 mg; Mn (as MnO2), 8 mg; I (as KI), 0.28 mg; and Se (as Na2SeO3 · 5H2O), 0.15 mg. §Provided per kg of complete diet: Cu (as CuSO4 · 5H2O), 12 mg; Mn (as MnO2), 8 mg; I (as KI), 0.28 mg; and Se (as Na2SeO3 · 5H2O), 0.15 mg. CP, crude protein; Lys, lysine; Met, methionine; Thr, threonine, Zn, zinc; ME, metabolizable energy.
removal from the incubator. A single colony was removed from selective media plates and cultivated in peptone yeast glucose broth. Subsequently, the bacteria was characterized to genus level on the basis of colonial appearance, Gram reaction, spore production, cell morphology and fermentation end-product formation.
Table 2
Fecal score
NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Znfree premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn-free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days).
On 08.00 and 20.00 hours on days 1, 7, 14, 21, 28, 35 and 42, the fecal score was evaluated and recorded. The fecal score was determined by the average value of five pigs of each pen by using a 5-grade score system (Hu et al. 2012). The standard of this system is as following: 1 = hard, dry pellets in a small, hard mass; 2 = hard, formed stool that remains firm and soft; 3 = soft, formed and moist stool that retains its shape; 4 = soft, unformed stool that assumes the shape of the container; 5 = watery, liquid stool that can be poured. Scores were recorded on a pen basis following observations of individual pig and signs of stool consistency in the pen.
Statistical analysis Data were subjected to analysis of variance (ANOVA) of SAS (SAS Inst. Inc., Cary, NC, USA), with the diet as the main Animal Science Journal (2015) 86, 617–623
Zinc oxide concentration in experimental diets
Item
NC
PC
H1
H2
Zinc oxide, ppm Phase 1 Phase 2
148 131
2,483 2,429
2,498 237
351 225
factor. The fecal score was compared with a chi-squared test, using the FREQ procedure of SAS. All other data were analyzed with the GLM procedure of SAS, according to a complete block design, with the pen being considered as the experimental unit. Before carrying out statistical analysis of the microbial counts, logarithmic conversion of the data was performed. Differences among treatment means were determined using Tukey’s multiple range tests with a P < 0.05 indicating significance. © 2015 Japanese Society of Animal Science
620 J. H. CHO et al.
RESULTS Growth performance and apparent total tract digestibility of nutrient The ADG was lower (P = 0.04) in NC treatment than other treatments during days 1 to 14 as presented in Table 3. Overall, the ADG in H1 treatment was higher (P = 0.05) compared with the NC treatment. The ADFI and G/F were not affected by dietary treatments. At
days 14 and 42, the ATTD digestibility of DM, N and GE was unaffected by dietary treatments (data not shown).
Blood profiles The ALP activity and plasma Zn concentrations were lower (ALP, P = 0.01; plasma Zn, P = 0.04; Table 4) in the NC treatment compared with H1 and H2
Table 3 Effect of modified zinc oxide supplementation on growth performance in weanling pigs
Item No.of replicates 1 to 14 days ADG, g ADFI, g G/F 15 to 42 days ADG, g ADFI, g G/F 1 to 42 days ADG, g ADFI, g G/F
NC
PC
7
H1
7
H2
7
SE
P-value
7
283b 371 0.763
310a 373 0.828
316a 377 0.838
318a 380 0.837
6.0 22.0 0.046
0.04 0.99 0.49
474 676 0.701
504 681 0.740
513 682 0.752
506 696 0.727
15.0 25.0 0.041
0.33 0.95 0.82
411b 495 0.830
437ab 500 0.874
447a 499 0.896
443ab 503 0.881
9.0 20.0 0.045
0.05 0.92 0.59
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Znfree premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; ADG, average daily gain; ADFI, average daily feed intake; G/F, gain/feed ratio.
Table 4 Effect of modified zinc oxide supplementation on blood profiles in weanling pigs
Item No. of replicates 1 day IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL 14 days IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL 42 days IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL
NC
PC
H1
H2
SE
P-value
7
7
7
7
250 610 5.32 8.03 58.0 134
227 619 5.32 8.66 60.4 138
232 663 5.21 7.99 58.2 139
242 643 5.49 7.71 61.2 145
22.0 48.0 0.25 0.51 5.2 4.0
0.89 0.86 0.89 0.62 0.96 0.28
440 357b 6.49 19.06 49.7 140b
390 441a 6.24 19.00 47.5 166ab
408 459a 6.36 18.01 51.7 170a
402 426a 6.37 18.70 55.2 176a
74.0 18.0 0.22 1.84 4.2 8.0
0.97 0.01 0.88 0.97 0.63 0.04
571 208 6.75 22.18 49.7 138
453 234 6.35 21.61 50.3 133
506 219 6.71 21.48 51.0 136
525 223 6.62 22.91 51.8 137
74.0 24.0 0.41 1.77 1.5 11.0
0.73 0.88 0.90 0.94 0.77 0.97
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn- free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn-free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; IgG, immunoglobulin G; ALP, alkaline phosphatase; RBC, red blood cells; WBC, white blood cells; Zn, zinc.
© 2015 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 617–623
EFFECTS OF MODIFIED ZINC OXIDE IN PIGLET
Table 5
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Effect of modified zinc oxide supplementation on fecal microflora in weanling pigs
Items
NC
PC
H1
H2
SE
P-value
No. of replicates 14 days Lactobacillus, log10cfu/g E. coli, log10cfu/g 42 days Lactobacillus, log10cfu/g E. coli, log10cfu/g
7
7
7
7
6.37b 5.49
7.41a 5.31
7.46a 5.30
7.66a 5.24
0.19 0.18
0.01 0.79
5.61b 4.92
6.53a 5.00
6.46a 4.70
6.78a 4.96
0.52 0.27
0.47 0.99
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn-free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Znfree premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; cfu, colony-forming units.
treatments at day 14. There was no difference in IgG, RBC, WBC, lymphocyte and plasma Zn concentrations and activity of ALP among treatments on days 1 and 42.
Fecal microbial shedding The fecal Lactobacillus concentration was higher (P = 0.01, 0.04) in PC, H1 and H2 treatments than that in the NC treatment on days 14 and 42 (Table 5).There was no difference in fecal Lactobacillus and E. coli concentrations among treatments.
Fecal score The fecal score was lower (P = 0.04) in H2 treatment than that in the NC treatment on day 7 (Table 6). The fecal score was higher (day 21, P = 0.03; day 28, P = 0.03) in the NC treatment than in the other treatments on days 21 and 28. The fecal score was not affected by dietary Zn supplementation at on days 1, 14, 35 and 42.
DISCUSSION It has been demonstrated that high concentrations (2000 to 3000 ppm) of ZnO are added to nursery diets as a growth promoter. In the current study, dietary supplementation with 3000 ppm conventional ZnO and 200 ppm of modified ZnO improved growth performance during days 1 to 14. These results are in agreement with Poulsen (1995) and Smith et al. (1997) who reported that growth rate was increased by 10-26% when nursery pigs were fed with 25004000 ppm ZnO. Other studies also demonstrated that pharmacological dose of ZnO improved growth performance in weanling pigs during the first 2 weeks postweaning (Katouli et al. 1999; Mavromichalis et al. 2000; Case & Carlson 2002; Pérez et al. 2010). There is only one study conducted by Morales et al. (2012) who reported that dietary supplementation with 110 ppm of the modified form of ZnO improved the growth performance of 42- to 63-day-old pigs. According to our present study, no significant difference was observed in blood profiles except for ALP and Animal Science Journal (2015) 86, 617–623
Table 6 Effect of modified zinc oxide supplementation on fecal score in weanling pigs
Item†
NC
PC
H1
H2
SE
P-value
No.of replicates 1 day 7 days 14 days 21 days 28 days 35 days 42 days
7 3.7 4.0a 3.7 3.7a 3.7a 3.0 2.7
7 4.0 3.3ab 3.3 3.0b 3.0b 3.0 2.3
7 4.0 3.3ab 3.0 3.0b 3.0b 2.7 2.0
7 3.7 3.0b 3.0 3.0b 3.0b 2.3 2.0
0.2 0.3 0.2 0.2 0.2 0.2 0.2
0.45 0.04 0.19 0.03 0.03 0.19 0.19
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn-free premix + 3000 ppm ZnO; H1, basal diet containing Zn-free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn- free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). †Fecal score: 1 = hard, dry pellets in a small, hard mass; 2 = hard, formed stool that remains firm and soft; 3 = soft, formed, and moist stool that retains its shape; 4 = soft, unformed stool that assumes the shape of the container; 5 = watery, liquid stool that can be poured. Scores were recorded on a pen basis following observations of individual pig and signs of stool consistency in the pen. SE, standard error of the means.
plasma zinc concentration during day 14 of the experiment. The increase in plasma zinc concentration in the current study with the dietary supplementation of conventional or modified ZnO is in line with the result of Wang et al. (2012) who indicated that 3000 ppm of ZnO or 150 ppm of encapsulated ZnO supplementation increased serum Zn concentration in weaned piglets. Increase in the plasma Zn concentration in H1 and H2 treatments compared with NC group in the current study indicates that dietary supplementation of conventional or modified ZnO promoted the absorption of zinc which might have activated many enzymes. The increase in ALP could be due to increase in Zn absorption since it has direct stimulatory effect on ALP and osteoclastin (Imamoglu et al. 2005). It has been shown that Zn acts as a cofactor for ALP, thereby promoting bone mineralization (Yamaguchi & Yamaguchi 1986). © 2015 Japanese Society of Animal Science
622 J. H. CHO et al.
The biological mechanism of growth-promoting effects of ZnO is still unclear. Katouli et al. (1999) reported that ZnO supplementation did not affect the stability of the intestinal microflora and the diversity of coliforms after 2 weeks post-weaning, which may indicate that the promoting effect of ZnO will be diminished with the growth of pigs. In our study, the gain/feed ratio was not affected by supplementing with a pharmacological dose of conventional ZnO or modified ZnO, which is consistent with the results of the previous studies. Vahjen et al. (2012) demonstrated that the new form of modified ZnO showed higher solubility, and reduced bacterial growth in the stomach and jejunum more rapidly than analytical grade ZnO. King et al. (1995) found that feeding 3000 ppm ZnO to weaned pigs has efficacy for controlling E. coli-induced diarrhea and increasing daily voluntary feed intake and body weight. In contrast, Hojberg et al. (2005) reported that lactic acid bacteria were decreased, but no effect on E. coli (Broom et al. 2006) with high doses of ZnO. In the present study, increased Lactobacillus counts were observed on day 14. Therefore, the beneficial effect of modified ZnO on piglet growth performance was probably due to the improved gut health. A previous study has demonstrated that high concentration of ZnO reduced the diarrhea rate of weaning pig (Carlson et al. 2008). In our study, the fecal score, which could represent the diarrhea status, was decreased in pigs fed with the modified ZnO compared with pigs fed the NC diet at days 21 and 28. Huang et al. (1999) found that supplementing with 3000 ppm ZnO reduced the number of bacteria reaching the ileal mesenteric lymph nodes of weaned pigs. Hahn and Baker (1993) and Carlson et al. (1999) demonstrated that plasma Zn concentrations increased as dietary Zn concentration increased, particularly when diets contained more than 1000 mg Zn/kg. Case and Carlson (2002) indicated that the major factor affecting nutrient excretion appears to be dietary concentration, independent of source. However, the form of Zn in the diet can affect its bioavailability and subsequent plasma Zn concentration. Wedekind and Baker (1990) and Wedekind et al. (1994) reported that ZnO had lower bioavailability, which resulted in lower plasma Zn concentrations compared with zinc sulfate, zinc-lysine and zinc-methionine. Our results showed that ADG was increased in pigs fed with 3000 ppm of conventional ZnO as well as with 200 ppm of modified ZnO. However, dietary supplementation of 250 or 500 ppm of conventional ZnO failed to improve growth rate of weaning pigs according to Hollis et al. (2005) indicating that conventional ZnO below pharmacological dose is not effective. The plasma Zn concentration in the present study was increased when pigs were fed the diets supplemented with 3000 ppm ZnO, as well as 200 ppm modified ZnO © 2015 Japanese Society of Animal Science
compared with NC treatment. However, no difference was observed between conventional ZnO and modified ZnO treatments despite the difference in the inclusion level. This result suggests that lower level of modified ZnO could be replaced with higher level of conventional ZnO. The lower level of modified ZnO in weaning pig diet would be more cost effective and it would help reduce the environmental pollution problem by reducing the fecal excretion of Zn. In conclusion, dietary supplementation with modified ZnO increased growth rate, bioavailability and reduced fecal scores in weanling pig. Thus, modified ZnO could be used as a substitute to conventional ZnO as a growth promoter.
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Hollis GR, Carter SD, Cline TR, Crenshaw TD, Cromwell GL, Hill GM, et al. 2005. Effects of replacing pharmacological levels of dietary zinc oxide with lower dietary levels of various organic zinc sources for weanling pigs. Journal of Animal Science 83, 2123–2129. Hu CH, Gu LY, Luan ZS, Song J, Zhu K. 2012. Effects of montmorillonite–zinc oxide hybrid on performance, diarrhea, intestinal permeability and morphology of weanling pigs. Animal Feed Science and Technology 177, 103–115. Huang EP. 1997. Metal ions and synaptic transmission: think zinc. Proceedings of the National Academy of Sciences of the United States of America 94, 13386–13387. Huang SX, McFall M, Cegielski AC, Kirkwood RN. 1999. Effect of dietary zinc supplementation on Escherichia coli septicemia in weaned pigs. Journal of Swine Health and Production 7, 109–111. Imamoglu S, Bereket A, Turan S, Taga Y, Haklar G. 2005. Effect of zinc supplementation on growth hormone secretion, IGF-1, IGFBP-3, somatomed in generation, alkaline phosphatase, osteocalcin and growth in prepubertal children with idiopathic short stature. Journal of Pediatric Endocrinology and Metabolism 18, 69–74. Jensen-Waern M, Melin L, Lindberg R, Johannisson A. 1998. Dietary zinc oxide in weaned pigs-effects on performance, tissue concentrations, morphology, neutrophil functions and faecal microflora. Research in Veterinary Science 64, 225–231. Jondreville C, Revy PS, Dourmad JY. 2003. Dietary means to better control the environmental impact of copper and zinc by pigs from weaning to slaughter. Livestock Production Science 84, 147–156. Katouli M, Melin L, Jensen-Waern M, Wallgren P, Möllby R. 1999. The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs. Journal of Applied Microbiology 87, 564–573. King LE, Osati-Ashtiani F, Fraker PJ. 1995. Depletion of cells of the B lineage in the bone marrow of zinc-deficient mice. Immunology 85, 69–73. Li BT, Van Kessel AG, Caine WR, Huang SX, Kirkwood RN. 2001. Small intestinal morphology and bacterial populations in ileal digesta and feces of newly weaned pigs receiving a high dietary level of zinc oxide. Canadian Journal of Animal Science 81, 511–516. Li X, Yin J, Li D, Chen X, Zang J, Zhou X. 2006. Dietary supplementation with zinc oxide increases IGF-I and IGF-I receptor gene expression in the small intestine of weanling piglets. Journal of Nutrition 136, 1786–1791. Low MG, Finean JB. 1977. Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. Biochemical Journal 167, 281–284. Mavromichalis I. 2011. The search for alternatives to zinc oxide. Pig Progress 27, 2. Mavromichalis I, Peter CM, Parr TM, Ganessunker D, Baker DH. 2000. Growth promoting efficacy in young pigs of two sources of zinc oxide having either a high or a low bioavailability of zinc. Journal of Animal Science 78, 2896– 2902. Melin L, Wallgren P. 2002. Aspects on feed related prophylactic measures aiming to prevent post weaning diarrhea in pigs. Acta Veterinaria Scandinavica 43, 231–245.
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Moore S. 1963. On the determination of cystine as cysteic acid. Journal of Biological Chemisry 238, 235–237. Morales J, Cordero G, Piñeiro C, Durosoy S. 2012. Zinc oxide at low supplementation level improves productive performance and health status of piglets. Journal of Animal Science 90, 436–438. National Requirements Council. 2012. Nutrient Requirement of Swine, 11th rev edn. National Academy Press, Washington, DC. Pérez VG, Waguespack AM, Bidner TD, Southern LL, Fakler TM, Ward TL, et al. 2010. Additivity of effects from dietary copper and zinc on growth performance and fecal microbiota of pigs after weaning. Journal of Animal Science 89, 414–425. Poulsen HD. 1995. Zinc oxide for weanling piglets. Acta Agriculturae Scandinavica, Section A – Animal Science 45, 159–167. Sauer WC, Fan MZ, Mosenthin R, Drochner W. 2000. Methods for measuring ileal amino acid digestibility in pigs. In: D’Mello JPF (ed.), Farm Animal Metabolism and Nutrition, pp. 279–306. CABI Publishing, Oxfordshire. Smith JW II, Tokach MD, Goodband RD, Nelssen JL, Richert BT. 1997. Effects of the interrelationship between zinc oxide and copper sulfate on growth performance of early-weaned pigs. Journal of Animal Science 75, 1861– 1866. Suhy DA, Simon KD, Linzer DIH, O’Halloran TV. 1999. Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation. The Journal of Biological Chemistry 247, 9183– 9192. Tokach LM, Tokach MD, Goodband RD, Nelssen JL, Henry SC, Marsteller TA. 1992. Influence of zinc oxide in starter diets on pig performance. Proceedings of the American Association of Swine Practitioners p. 411. Vahjen W, Zentek J, Durosoy S. 2012. Inhibitory action of two zinc oxide sources on the ex vivo growth of porcine small intestine bacteria. Journal of Animal Science 90, 334– 336. Wang C, Xie P, Liu LL, Dong XY, Lu JJ, Zou TX. 2012. Use of lower level of capsulated zinc oxide as an alternative to pharmacological dose of zinc oxide for weaned piglets. Asian Journal of Animal and Veterinary Advance 7, 1290– 1300. Wedekind KJ, Baker DH. 1990. Zinc bioavailability in feedgrade sources of zinc. Journal of Animal Science 68, 684– 689. Wedekind KJ, Lewis AJ, Gieseman MA, Miller PS. 1994. Bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean meal diets. Journal of Animal Science 72, 2681–2689. Williams CH, David DJ, Iismaa O. 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. Journal of Agricultural Science 59, 381– 385. Yamaguchi M, Yamaguchi R. 1986. Action of zinc on bone metabolism in rats. Increases in alkaline phosphatase activity and DNA content. Biochemical Pharmacology 5, 773–777.
© 2015 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 617–623
doi: 10.1111/asj.12329
ORIGINAL ARTICLE Effects of dietary supplementation of modified zinc oxide on growth performance, nutrient digestibility, blood profiles, fecal microbial shedding and fecal score in weanling pigs Jin Ho CHO,1 Santi Devi UPADHAYA2 and In Ho KIM2 1
Department of Animal Science, Chungbuk National University, Cheongju and 2Department of Animal Resource & Science, Dankook University, Cheonan, Korea
ABSTRACT One hundred and forty piglets ((Landrace × Yorkshire) × Duroc, 21 day of age) with an initial weight of 6.50 ± 0.71 kg, were randomly allotted into four treatments to determine the effects of a modified form of zinc oxide (ZnO) on growth performance, nutrient digestibility, blood profiles, fecal microbial shedding and fecal score in weanling pigs. Dietary treatments were: (i) NC, negative control, basal diet containing zinc (Zn) from the premix; (ii) PC, positive control, basal diet containing Zn-free premix + 3000 ppm ZnO; (iii) H1, basal diet containing Zn-free premix + 3000 ppm ZnO (phase 1, days 1 to 14)/200 ppm modified ZnO (phase 2, days 15 to 42); (iv) H2, basal diet containing Zn-free premix + 300 ppm modified ZnO (phase 1)/200 ppm modified ZnO (phase 2). During days 1 to 14, average daily gains (ADG) were higher (P = 0.04) in PC, H1 and H2 groups than that in NC group. Overall, H1 treatment increased the ADG compared with NC (P = 0.05). On day 14, the alkaline phosphatase and plasma Zn concentration were increased (P = 0.01 and 0.04, respectively) in PC, H1 and H2 treatments compared with NC treatment. On days 14 and 42, the fecal Lactobacillus counts in NC group were lowest (P = 0.01, P = 0.04 respectively) among treatments. All supplemented groups showed lower (P = 0.03) fecal score than NC treatment on days 21 and 28. In conclusion, dietary supplementation with modified ZnO increased growth rates and reduced fecal scores in weanling pig. Modified ZnO could be used as a substitute to ZnO as a growth promoter and reduce Zn excretion to the environment because of the lower dosage. [Correction added on 3 February 2015, after first online publication: the initial weight of ‘6.50 ± 1.11 kg’ has been replaced with ‘6.50 ± 0.71 kg’ in the abstract.]
Key words: blood profiles, fecal microbial shedding, growth performance, modified zinc oxide, weanling pig.
INTRODUCTION Zinc (Zn) is the second most abundant transition metal in biological systems, and serves as a cofactor for more than 300 enzymes in the body, particularly those involved in protein, carbohydrate and fat metabolism (Coleman 1992; Huang 1997; Suhy et al. 1999). According to the nutrient requirement of swine (NRC 2012), the recommended zinc level is 80 ppm in piglets fed with conventional diet. The use of 3000 ppm Zn from zinc oxide (ZnO) is widely used by researchers in weanling diets to promote growth performance and reduce the incidence of diarrhea (Hahn & Baker 1993; Jensen-Waern et al. 1998; Hill et al. 2001; Melin & Wallgren 2002). Li et al. (2001) and Li et al. (2006) also demonstrated that ZnO supplementation improved gut health thus potentially increasing the absorptive capacity of the small intestine. However, the growth promoting effect was inconsist© 2015 Japanese Society of Animal Science
ent (Fryer et al. 1992; Tokach et al. 1992) and the high zinc excretion in animal waste poses environmental challenges (Jondreville et al. 2003). A modified form of ZnO has stronger efficacy than conventional ZnO, which is small particle ZnO as it further increases the possible sites of interaction among the molecules of ZnO, the animal, and microbes in the gastrointestinal tract (Mavromichalis 2011). It showed a higher ability to inhibit bacterial growth under ex vivo conditions, and its concentration in feed could be reduced and therefore decrease zinc
Correspondence: In Ho Kim, Department of Animal Resource & Science, Dankook University, #29 Anseodong, Cheonan, Choongnam 330-714, Korea. (Email: inhokim@ dankook.ac.kr) Received 29 April 2014; accepted for publication 15 August 2014.
618 J. H. CHO et al.
excretion (Vahjen et al. 2012). Morales et al. (2012) reported that dietary supplementation with 110 ppm of the same potentiated ZnO improved the growth performance of piglets (42 to 63 days of age). However, the mechanism of this form of ZnO was not well studied. Therefore, this study was conducted to investigate the effects of conventional ZnO and modified ZnO with enhanced surface area in weanling pigs.
overnight at 0°C. Performic acid is an oxidizing reagent that converts cysteine quantitatively to cysteic acid and methionine to methionine sulfone (Moore 1963). Nitrogen was determined by a Kjectec 2300 Nitrogen Analyzer (Foss Tecator AB, Hoeganaes, Sweden). The gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 Oxygen Bomb Calorimeter (Parr instrument Co., Moline, IL, USA).
Sampling and measurements MATERIALS AND METHODS The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University.
Animals and facilities A total of 140 weanling pigs ((Yorkshire × Landrace) × Duroc, 21 days of age) with an average body weight (BW) of 6.50 ± 0.71 kg were used in a 6-week experiment. Pigs were randomly allotted to one of four treatments with seven replicate pens and five pigs per pen (two barrows and three gilts) according to their initial BW and sex. All pigs were housed in an environmentally controlled room. The ambient temperature within the room was approximately 30°C at the start of the experiment and decreased by 1°C each week. Each pen was equipped with a one-sided, stainless steel self-feeder and a nipple drinker; pigs were allowed unlimited access to feed and water. On days 1, 14 and 42, individual pig BW and feed disappearance were recorded to determine average daily gain (ADG), average daily feed intake (ADFI) and gain/feed ratio (G/F).
Dietary treatments Dietary treatments were as follows. Phase 1 (for 14 days): (i) NC (negative control, basal diet containing Zn from premix); (ii) PC (positive control, basal diet containing Zn-free premix + 3000 ppm ZnO); (iii) H1 (basal diet containing Zn-free premix + 3000 ppm ZnO); (iv) H2 (basal diet containing Zn-free premix + 300 ppm modified ZnO). Phase 2 (for 28 days): (i) NC (negative control, basal diet containing Zn from premix); (ii) PC (positive control, basal diet containing Zn-free premix + 3000 ppm ZnO); (iii) H1 (basal diet containing Zn-free premix + 200 ppm modified ZnO); (iv) H2 (basal diet containing Zn-free premix + 200 ppm potential ZnO). Conventional ZnO (96% ZnO; screen size: 50 mesh) used in the present study was obtained from DongWoo (Anyang, Korea) and the modified form of ZnO (76% ZnO; screen size: 200 mesh) was produced by Animine (HiZox, Sillingy, France). All diets were formulated to meet or exceed the NRC (2012) requirements for weanling pigs, and were fed in a mash form (Table 1). Zinc oxide concentration in the experimental diets is shown in Table 2.
Chemical analysis Samples of diets were analyzed using standard methods (Association of Official Analytical Chemists 2000) for dry matter (DM; method 934.01) and nitrogen (N; method 968.06). Individual amino acid composition was measured using an amino acid analyzer (Beckman 6300; Beckman Coulter, Inc., Fullerton, CA, USA) after 24-h 6 N-HCl hydrolysis at 110°C (Association of Official Analytical Chemists 2000). For the determination of cysteine and methionine, the samples were oxidized with performic acid © 2015 Japanese Society of Animal Science
From days 8 to 14 and 36 to 42, chromium oxide was added to the diet at 0.2% as an indigestible marker for determination of the apparent total tract digestibility (ATTD) of DM, N and gross energy (GE). At 17.00 hours on days 12, 13, 14 as well as days 40, 41, and 42, fresh fecal grab samples were collected from two pigs per pen, then mixed and pooled, and a representative sample was stored in a freezer at −20°C until analyzed. Before chemical analysis, the fecal samples 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 screen. All fecal samples were analyzed following the procedures outlined by the Association of Official Analytical Chemists (2000). Chromium was analyzed by UV absorption spectrophotometry (UV-1201; Shimadzu, Tokyo, Japan) following the method described by Williams et al. (1962). Nutrient digestibility was then calculated using the chromium technique (Sauer et al. 2000). For the blood profiles, 14 pigs (two pigs/pen) from each treatment were randomly selected and blood samples were collected via anterior vena cava puncture on days 1, 14 and 42. At the time of collection, blood samples were collected into both nonheparinized tubes and vacuum tubes containing tripotassium ethylenediaminetetraacetic acid (K3EDTA; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) to obtain serum and whole blood, respectively. After collection, blood samples were centrifuged (3000 × g) for 15 min at 4°C. The white blood cells (WBC), red blood cells (RBC) and lymphocyte levels in the whole blood were determined using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA). The alkaline phosphatase (ALP) activity was measured according to the method described by Low and Finean (1977). The plasma zinc was determined according to the method described by Hill et al. (2000). The immunoglobulin G (IgG) concentration was determined using an automatic biochemistry blood analyzer (HITACHI 747; Hitachi, Tokyo, Japan).
Procedures of microbial shedding Fresh fecal samples were collected directly on days 14 and 42 via massaging the rectum of two pigs in each pen, and then pooled and placed on ice for transportation to the lab. One gram of the composite fecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and then homogenized. Viable counts of bacteria in the fecal samples were then conducted by plating serial 10-fold dilutions (in 1% peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and Lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the Escherichia coli and Lactobacillus, respectively. The lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. The E. coli and Lactobacillus colonies were counted immediately after Animal Science Journal (2015) 86, 617–623
EFFECTS OF MODIFIED ZINC OXIDE IN PIGLET
Table 1
619
Feed compositions (%, as-fed basis)
Item
Ingredient Extruded corn Soybean meal (48% CP) Fermented soybean meal (45% CP) Fish meal (66% CP, Brazil) Soy oil Lactose Whey Dicalcium phosphate Sucrose Plasma powder (AP 920) L-Lys HCl (78%) DL-Met (50%) L-Thr (89%) Choline chloride (25%) Vitamin premix† Mineral premix (normal)‡ Mineral premix (Zn-free)§ Limestone Salt Analyzed nutritional content: ME, MJ/kg CP, % Lys, % Met, % Met + Cys % Ca, % Total P, % Crude fat,% Crude fiber, %
Phase 1 (0 to 14 days)
Phase 2 (14 to 42 days)
Basal diet
Zn-free diet
Basal diet
Zn-free diet
44.49 16.20 5.00 3.50 2.55 8.30 10.00 1.50 3.00 3.00 0.39 0.30 0.19 0.10 0.10 0.20 – 0.98 0.20
44.49 16.20 5.00 3.50 2.55 8.30 10.00 1.50 3.00 3.00 0.39 0.30 0.19 0.10 0.10 – 0.20 0.98 0.20
61.97 25.30 2.50 – 1.05 – 5.00 1.50 – – 0.46 0.24 0.20 0.10 0.10 0.20 – 1.13 0.25
61.97 25.30 2.50 – 1.05 – 5.00 1.50 – – 0.46 0.24 0.20 0.10 0.10 – 0.20 1.13 0.25
14.80 20.00 1.50 0.62 0.97 0.95 0.75 5.02 1.87
– – – – – – – – –
14.26 19.00 1.35 0.53 0.84 0.90 0.70 3.98 2.45
– – – – – – – – –
†Provided per kg of complete diet: vitamin A, 11 025 IU; vitamin D3, 1103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; d-pantothenic, 29 mg; choline, 166 mg; and vitamin B12, 33 μg. ‡Provided per kg of complete diet: Cu (as CuSO4·5H2O), 12 mg; Zn (as ZnSO4), 85 mg; Mn (as MnO2), 8 mg; I (as KI), 0.28 mg; and Se (as Na2SeO3 · 5H2O), 0.15 mg. §Provided per kg of complete diet: Cu (as CuSO4 · 5H2O), 12 mg; Mn (as MnO2), 8 mg; I (as KI), 0.28 mg; and Se (as Na2SeO3 · 5H2O), 0.15 mg. CP, crude protein; Lys, lysine; Met, methionine; Thr, threonine, Zn, zinc; ME, metabolizable energy.
removal from the incubator. A single colony was removed from selective media plates and cultivated in peptone yeast glucose broth. Subsequently, the bacteria was characterized to genus level on the basis of colonial appearance, Gram reaction, spore production, cell morphology and fermentation end-product formation.
Table 2
Fecal score
NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Znfree premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn-free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days).
On 08.00 and 20.00 hours on days 1, 7, 14, 21, 28, 35 and 42, the fecal score was evaluated and recorded. The fecal score was determined by the average value of five pigs of each pen by using a 5-grade score system (Hu et al. 2012). The standard of this system is as following: 1 = hard, dry pellets in a small, hard mass; 2 = hard, formed stool that remains firm and soft; 3 = soft, formed and moist stool that retains its shape; 4 = soft, unformed stool that assumes the shape of the container; 5 = watery, liquid stool that can be poured. Scores were recorded on a pen basis following observations of individual pig and signs of stool consistency in the pen.
Statistical analysis Data were subjected to analysis of variance (ANOVA) of SAS (SAS Inst. Inc., Cary, NC, USA), with the diet as the main Animal Science Journal (2015) 86, 617–623
Zinc oxide concentration in experimental diets
Item
NC
PC
H1
H2
Zinc oxide, ppm Phase 1 Phase 2
148 131
2,483 2,429
2,498 237
351 225
factor. The fecal score was compared with a chi-squared test, using the FREQ procedure of SAS. All other data were analyzed with the GLM procedure of SAS, according to a complete block design, with the pen being considered as the experimental unit. Before carrying out statistical analysis of the microbial counts, logarithmic conversion of the data was performed. Differences among treatment means were determined using Tukey’s multiple range tests with a P < 0.05 indicating significance. © 2015 Japanese Society of Animal Science
620 J. H. CHO et al.
RESULTS Growth performance and apparent total tract digestibility of nutrient The ADG was lower (P = 0.04) in NC treatment than other treatments during days 1 to 14 as presented in Table 3. Overall, the ADG in H1 treatment was higher (P = 0.05) compared with the NC treatment. The ADFI and G/F were not affected by dietary treatments. At
days 14 and 42, the ATTD digestibility of DM, N and GE was unaffected by dietary treatments (data not shown).
Blood profiles The ALP activity and plasma Zn concentrations were lower (ALP, P = 0.01; plasma Zn, P = 0.04; Table 4) in the NC treatment compared with H1 and H2
Table 3 Effect of modified zinc oxide supplementation on growth performance in weanling pigs
Item No.of replicates 1 to 14 days ADG, g ADFI, g G/F 15 to 42 days ADG, g ADFI, g G/F 1 to 42 days ADG, g ADFI, g G/F
NC
PC
7
H1
7
H2
7
SE
P-value
7
283b 371 0.763
310a 373 0.828
316a 377 0.838
318a 380 0.837
6.0 22.0 0.046
0.04 0.99 0.49
474 676 0.701
504 681 0.740
513 682 0.752
506 696 0.727
15.0 25.0 0.041
0.33 0.95 0.82
411b 495 0.830
437ab 500 0.874
447a 499 0.896
443ab 503 0.881
9.0 20.0 0.045
0.05 0.92 0.59
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Znfree premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; ADG, average daily gain; ADFI, average daily feed intake; G/F, gain/feed ratio.
Table 4 Effect of modified zinc oxide supplementation on blood profiles in weanling pigs
Item No. of replicates 1 day IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL 14 days IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL 42 days IgG, mg/dL ALP, U/L RBC, 106/μL WBC, 103/μL Lymphocyte, % Plasma Zn, μg/dL
NC
PC
H1
H2
SE
P-value
7
7
7
7
250 610 5.32 8.03 58.0 134
227 619 5.32 8.66 60.4 138
232 663 5.21 7.99 58.2 139
242 643 5.49 7.71 61.2 145
22.0 48.0 0.25 0.51 5.2 4.0
0.89 0.86 0.89 0.62 0.96 0.28
440 357b 6.49 19.06 49.7 140b
390 441a 6.24 19.00 47.5 166ab
408 459a 6.36 18.01 51.7 170a
402 426a 6.37 18.70 55.2 176a
74.0 18.0 0.22 1.84 4.2 8.0
0.97 0.01 0.88 0.97 0.63 0.04
571 208 6.75 22.18 49.7 138
453 234 6.35 21.61 50.3 133
506 219 6.71 21.48 51.0 136
525 223 6.62 22.91 51.8 137
74.0 24.0 0.41 1.77 1.5 11.0
0.73 0.88 0.90 0.94 0.77 0.97
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn- free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn- free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn-free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; IgG, immunoglobulin G; ALP, alkaline phosphatase; RBC, red blood cells; WBC, white blood cells; Zn, zinc.
© 2015 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 617–623
EFFECTS OF MODIFIED ZINC OXIDE IN PIGLET
Table 5
621
Effect of modified zinc oxide supplementation on fecal microflora in weanling pigs
Items
NC
PC
H1
H2
SE
P-value
No. of replicates 14 days Lactobacillus, log10cfu/g E. coli, log10cfu/g 42 days Lactobacillus, log10cfu/g E. coli, log10cfu/g
7
7
7
7
6.37b 5.49
7.41a 5.31
7.46a 5.30
7.66a 5.24
0.19 0.18
0.01 0.79
5.61b 4.92
6.53a 5.00
6.46a 4.70
6.78a 4.96
0.52 0.27
0.47 0.99
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn-free premix+ 3000 ppm ZnO; H1, basal diet containing Zn- free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Znfree premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). SE, standard error of the means; cfu, colony-forming units.
treatments at day 14. There was no difference in IgG, RBC, WBC, lymphocyte and plasma Zn concentrations and activity of ALP among treatments on days 1 and 42.
Fecal microbial shedding The fecal Lactobacillus concentration was higher (P = 0.01, 0.04) in PC, H1 and H2 treatments than that in the NC treatment on days 14 and 42 (Table 5).There was no difference in fecal Lactobacillus and E. coli concentrations among treatments.
Fecal score The fecal score was lower (P = 0.04) in H2 treatment than that in the NC treatment on day 7 (Table 6). The fecal score was higher (day 21, P = 0.03; day 28, P = 0.03) in the NC treatment than in the other treatments on days 21 and 28. The fecal score was not affected by dietary Zn supplementation at on days 1, 14, 35 and 42.
DISCUSSION It has been demonstrated that high concentrations (2000 to 3000 ppm) of ZnO are added to nursery diets as a growth promoter. In the current study, dietary supplementation with 3000 ppm conventional ZnO and 200 ppm of modified ZnO improved growth performance during days 1 to 14. These results are in agreement with Poulsen (1995) and Smith et al. (1997) who reported that growth rate was increased by 10-26% when nursery pigs were fed with 25004000 ppm ZnO. Other studies also demonstrated that pharmacological dose of ZnO improved growth performance in weanling pigs during the first 2 weeks postweaning (Katouli et al. 1999; Mavromichalis et al. 2000; Case & Carlson 2002; Pérez et al. 2010). There is only one study conducted by Morales et al. (2012) who reported that dietary supplementation with 110 ppm of the modified form of ZnO improved the growth performance of 42- to 63-day-old pigs. According to our present study, no significant difference was observed in blood profiles except for ALP and Animal Science Journal (2015) 86, 617–623
Table 6 Effect of modified zinc oxide supplementation on fecal score in weanling pigs
Item†
NC
PC
H1
H2
SE
P-value
No.of replicates 1 day 7 days 14 days 21 days 28 days 35 days 42 days
7 3.7 4.0a 3.7 3.7a 3.7a 3.0 2.7
7 4.0 3.3ab 3.3 3.0b 3.0b 3.0 2.3
7 4.0 3.3ab 3.0 3.0b 3.0b 2.7 2.0
7 3.7 3.0b 3.0 3.0b 3.0b 2.3 2.0
0.2 0.3 0.2 0.2 0.2 0.2 0.2
0.45 0.04 0.19 0.03 0.03 0.19 0.19
a,b Means in the same row with different superscripts differ (P < 0.05). NC, negative control, basal diet containing Zn from the premix; PC, positive control, basal diet containing Zn-free premix + 3000 ppm ZnO; H1, basal diet containing Zn-free premix + 3000 ppm ZnO (phase 1, 1 to 14 days), basal diet containing Zn-free premix + 200 ppm modified zinc oxide (phase 2, 15 to 42 days); H2, basal diet containing Zn- free premix + 300 ppm modified zinc oxide (phase 1, 1 to 14 days), NC + 200 ppm modified zinc oxide (phase 2, 15 to 42 days). †Fecal score: 1 = hard, dry pellets in a small, hard mass; 2 = hard, formed stool that remains firm and soft; 3 = soft, formed, and moist stool that retains its shape; 4 = soft, unformed stool that assumes the shape of the container; 5 = watery, liquid stool that can be poured. Scores were recorded on a pen basis following observations of individual pig and signs of stool consistency in the pen. SE, standard error of the means.
plasma zinc concentration during day 14 of the experiment. The increase in plasma zinc concentration in the current study with the dietary supplementation of conventional or modified ZnO is in line with the result of Wang et al. (2012) who indicated that 3000 ppm of ZnO or 150 ppm of encapsulated ZnO supplementation increased serum Zn concentration in weaned piglets. Increase in the plasma Zn concentration in H1 and H2 treatments compared with NC group in the current study indicates that dietary supplementation of conventional or modified ZnO promoted the absorption of zinc which might have activated many enzymes. The increase in ALP could be due to increase in Zn absorption since it has direct stimulatory effect on ALP and osteoclastin (Imamoglu et al. 2005). It has been shown that Zn acts as a cofactor for ALP, thereby promoting bone mineralization (Yamaguchi & Yamaguchi 1986). © 2015 Japanese Society of Animal Science
622 J. H. CHO et al.
The biological mechanism of growth-promoting effects of ZnO is still unclear. Katouli et al. (1999) reported that ZnO supplementation did not affect the stability of the intestinal microflora and the diversity of coliforms after 2 weeks post-weaning, which may indicate that the promoting effect of ZnO will be diminished with the growth of pigs. In our study, the gain/feed ratio was not affected by supplementing with a pharmacological dose of conventional ZnO or modified ZnO, which is consistent with the results of the previous studies. Vahjen et al. (2012) demonstrated that the new form of modified ZnO showed higher solubility, and reduced bacterial growth in the stomach and jejunum more rapidly than analytical grade ZnO. King et al. (1995) found that feeding 3000 ppm ZnO to weaned pigs has efficacy for controlling E. coli-induced diarrhea and increasing daily voluntary feed intake and body weight. In contrast, Hojberg et al. (2005) reported that lactic acid bacteria were decreased, but no effect on E. coli (Broom et al. 2006) with high doses of ZnO. In the present study, increased Lactobacillus counts were observed on day 14. Therefore, the beneficial effect of modified ZnO on piglet growth performance was probably due to the improved gut health. A previous study has demonstrated that high concentration of ZnO reduced the diarrhea rate of weaning pig (Carlson et al. 2008). In our study, the fecal score, which could represent the diarrhea status, was decreased in pigs fed with the modified ZnO compared with pigs fed the NC diet at days 21 and 28. Huang et al. (1999) found that supplementing with 3000 ppm ZnO reduced the number of bacteria reaching the ileal mesenteric lymph nodes of weaned pigs. Hahn and Baker (1993) and Carlson et al. (1999) demonstrated that plasma Zn concentrations increased as dietary Zn concentration increased, particularly when diets contained more than 1000 mg Zn/kg. Case and Carlson (2002) indicated that the major factor affecting nutrient excretion appears to be dietary concentration, independent of source. However, the form of Zn in the diet can affect its bioavailability and subsequent plasma Zn concentration. Wedekind and Baker (1990) and Wedekind et al. (1994) reported that ZnO had lower bioavailability, which resulted in lower plasma Zn concentrations compared with zinc sulfate, zinc-lysine and zinc-methionine. Our results showed that ADG was increased in pigs fed with 3000 ppm of conventional ZnO as well as with 200 ppm of modified ZnO. However, dietary supplementation of 250 or 500 ppm of conventional ZnO failed to improve growth rate of weaning pigs according to Hollis et al. (2005) indicating that conventional ZnO below pharmacological dose is not effective. The plasma Zn concentration in the present study was increased when pigs were fed the diets supplemented with 3000 ppm ZnO, as well as 200 ppm modified ZnO © 2015 Japanese Society of Animal Science
compared with NC treatment. However, no difference was observed between conventional ZnO and modified ZnO treatments despite the difference in the inclusion level. This result suggests that lower level of modified ZnO could be replaced with higher level of conventional ZnO. The lower level of modified ZnO in weaning pig diet would be more cost effective and it would help reduce the environmental pollution problem by reducing the fecal excretion of Zn. In conclusion, dietary supplementation with modified ZnO increased growth rate, bioavailability and reduced fecal scores in weanling pig. Thus, modified ZnO could be used as a substitute to conventional ZnO as a growth promoter.
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