Han Y-K, Hwan Hwang IL, Thacker PA. Use of a micro-encapsulated eucalyptus-medium chain fatty acid product as an alternative to zinc oxide and antibiotics for weaned pigs. J Swine Health Prod. 2011;19(1):34–43.

Cite as: Han Y-K, Hwan Hwang IL, Thacker PA. Use of a micro-encapsulated eucalyptus-medium chain fatty acid product as an alternative to zinc oxide and antibiotics for weaned pigs. J Swine Health Prod. 2011;19(1):34–43.
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Keywords: swine, performance, zinc oxide, antibiotics, eucalyptus-medium chain fatty acids
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Received: February 16, 2010
Accepted: May 25, 2010

Weaning at 3 to 4 weeks of age exposes piglets to nutritional and environmental stresses, often resulting in suboptimal performance, increased risk of diarrhea, and sometimes death.1 In order to prevent diarrhea or poor performance, antimicrobial feed additives are often supplied in nursery pig diets.2 However, due to concerns about residues in meat and the potential for development of microbial resistance to antibiotics, government agencies in many pig-producing countries are considering implementation of a complete ban in the use of antibiotics in animal feeds.3 As a consequence, development of alternatives to antibiotics is receiving considerable attention.4

High levels of zinc in the form of zinc oxide (ZnO) are commonly added to nursery pig diets because they improve performance5-7 and reduce the incidence of diarrhea after weaning.8 However, a concern with feeding pharmacological levels of ZnO to pigs is that application of manure containing high levels of ZnO to agricultural lands has the potential to negatively impact the environment.9 Therefore, it would be desirable if the beneficial effects of feeding ZnO could be obtained at lower levels of supplementation,10 or if alternative growth promoters could be developed.

Eucalyptus oil is obtained from the leaves of the eucalyptus, a tree which belongs to the plant family Myrtaceae and is cultivated worldwide. In humans, eucalyptus oil has antibacterial effects on pathogenic bacteria in the respiratory tract.11 Eucalyptus oil also stimulates the immune system by affecting the phagocytic ability of monocyte-derived macrophages.12 Dietary inclusion of eucalyptus improves production performance and stimulates the immunity of commercial laying hens.13 However, the effect of eucalyptus on pig performance or immunity has not been determined.

Medium-chain fatty acids (MFCAs) have been suggested as an alternative feed additive to antibiotics for piglets.1,14 Unfortunately, MCFAs are a strong stimulus for the release of cholecystokinin, which has a pronounced satiating activity that could interfere with feed intake.14 In addition, MCFAs may reduce feed intake simply because of their unpleasant taste.15 To avoid these adverse side effects, Dierick et al14 attempted to use in situ generation of MCFAs by feeding a combination of triacylglycerol-containing MCFAs and exogenous lipolytic enzymes.

Micro-encapsulation of MCFAs is an alternative to in situ generation, which could be used to avoid the adverse effects of MCFAs on feed intake. In this process, the MCFAs are nano-micronized to extremely small particles to facilitate absorption and then encapsulated so that the negative effects on feed intake are avoided. A eucalyptus-MCFAs product (E-MCFAs) has recently been developed using this technology, but its effects on nursery pig performance have not been reported. Therefore, the objective of the present study was to compare the effects of feeding micro-encapsulated E-MCFAs, ZnO, and antibiotics on the performance, nutrient digestibility, and serum chemistry of weaned pigs.

Materials and methods

All procedures used in this experiment were approved by the Animal Ethics Committee of Sungkyunkwan University in Korea following the Guidelines for the Care and Use of Animals in Research16 published by the Korean Ministry for Food, Agriculture, Forestry and Fisheries.

Production of micro-encapsulated E-MCFAs

The process used to produce the micro-encapsulated E-MCFAs product has been patented by the Korean Intellectual Property Office (Daejeon, Korea) under patent number 10-2009-0025329. Briefly, the product was produced by mixing eucalyptus extract (Desert King International, San Diego, California), caprylic acid (C8:0), and carpric acid (C10:0) in a ratio of 2:1:1 and then subjecting the mixture to micronization (Ultra-Micro Pulverizer; GR Engineering Company, Seoul, Korea). Palm oil was then sprayed onto the extract and the two MCFAs, and the entire mixture was diluted 50:50 with a carrier consisting of calcium carbonate, rice-husk powder, and wheat powder (20:40:40). The product is marketed as a dry powder under the name Bye All CT and was obtained from Easybio Systems (Seoul, Korea).

Experiment One

In Experiment One, 120 pens were used to house 640 crossbred barrows (Landrace-Yorkshire female × Duroc sire), averaging 24 ± 3 days of age and 6.04 ± 0.88 kg. The pigs were blocked on the basis of initial weight and litter of origin and allotted to five dietary treatments in a randomized block design. The basal diet formula was based on corn (extruded and expanded), soybean meal (regular and fermented), milk-based products (lactose powder, whey powder, and milk powder complex), and spray-dried porcine plasma (Table 1). The five dietary treatments consisted of the basal diet fed without supplementation (negative control) or the basal diet supplemented with antibiotics (33 mg per kg tiamulin and 44 mg per kg lincomycin; positive control), ZnO (1500 or 2500 mg per kg), or 0.1% micro-encapsulated E-MCFAs (Easybio Systems). Prior to mixing in the diet, the antibiotics, ZnO, or micro-encapulated E-MCFAs were premixed with cellulose (Sigma, St Louis, Missouri) and were then added to the feed mixer.

The experiment was conducted in three phases, with the phase 1 diet (Days 0 to 7) formulated to provide 1.72% lysine, 1.06% methionine plus cystine, and 1.18% threonine; the phase 2 diet (Days 8 to 21) was formulated to provide 1.56% lysine, 0.89% methionine plus cystine, and 1.10% threonine; while the phase 3 diet (Days 22 to 28) was formulated to provide 1.49% lysine, 0.79% methionine plus cystine, and 0.99% threonine (Table 2). All amino acid levels reported are total, not digestible levels. All diets were fed in crumbled form and all nutrient levels met or exceeded NRC17 requirements for the nursery pig. The basal diet contained 100 mg per kg Zn as ZnSO4 (35.5% Zn).

The experiment was conducted on six separate commercial pig farms located in the provinces of Hallim, Icheon, Illzug, Namwon, Pocheon, and Seoguipo in South Korea. The piglets were housed in environmentally regulated barns in 1.2 × 1.4-m pens located over a plastic slatted floor. Air temperature was controlled at 30ºC during the first 7 days, and the temperature was decreased by 1ºC every 3 days until it reached 23ºC at the end of the experiment. There were four replicate pens per treatment in each of the six commercial pig farms (n = 24), and each pen housed four barrows (provinces of Hallim, Icheon, Illzug, and Seoguipo) or eight barrows (provinces of Namwon and Pocheon). Each pen had two feeders and two nipple waterers to provide free access to feed and water. Individual pig weights and pen feed disappearance were measured weekly and used to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion efficiency (FCE) on a pen basis. Feed wastage was negligible and was not considered.

Experiment Two

This experiment was conducted to further evaluate the effects of treatment with micro-encapsulated E-MCFAs, antibiotics, and ZnO addition on nursery pig performance. In Experiment Two, 90 pens were used to house 360 crossbred barrows (Landrace-Yorkshire × Duroc sire), averaging 28 ± 3 days of age and 7.34 ± 0.47 kg BW. The barrows were blocked on the basis of initial weight and litter of origin, and allotted to the five dietary treatments used in Experiment One in a randomized block design. The pigs were fed the phase 1 diets from Day 0 to 14 and the phase 2 diets from Day 15 to 28.

The 28-day experiment was conducted at two commercial farms located in the provinces of Namwon and Pocheon in South Korea. The pigs were housed in environmentally regulated barns in 1.0 × 1.2-m pens located over a slatted wire floor. There were nine pens per treatment in each of the two commercial farms (n = 18), and each pen housed four barrows. Each pen had two feeders and a nipple waterer to provide free access to feed and water. Air temperature in the barn was controlled at 32ºC during the first 7 days, and the temperature was decreased by 1ºC every 3 days until it reached 25ºC at the end of the experiment. Individual pig weights and pen feed disappearance were measured weekly and used to calculate ADG, ADFI, and FCE.

Experiment Three

This 14-day experiment was set up at a commercial farm in the province of Hallim and was conducted to evaluate the effects of treatment with microencapsulated E-MCFAs, antibiotics, and ZnO addition on performance, nutrient digestibility, and serum chemistry parameters of nursery pigs. In Experiment Three, 36 pens were used to house 504 crossbred barrows (Landrace-Yorkshire × Duroc sire), averaging 28 ± 3 days of age and 7.68 ± 0.64 kg. The barrows were blocked on the basis of initial weight and litter of origin and allotted to four dietary treatments in a randomized block design. The pigs were fed a mixture of the phase 1 and 2 diets (50:50) used in Experiment One. Because of severe diarrhea, feeding of the negative control diet was stopped after 10 days and all pigs on this treatment were removed from the experiment. All diets contained 1.0% diatomaceous earth (Celite 545, Fluka Chemika/Biochemika, Buchs, Switzerland) as a digestibility marker.

During the experiment, the pigs were housed in an environmentally regulated barn in 2.0 × 2.4-m pens located over a slatted wire floor. There were nine pens per treatment (n = 9), and each pen housed 14 barrows. Each pen had a three-hole feeder and two nipple waterers to provide free access to feed and water. Air temperature in the house was controlled at 32ºC during the first 7 days and the temperature was decreased by 1ºC every day until it reached 25ºC at the end of the experiment. Individual pig weights and pen feed disappearance were measured weekly and used to calculate ADG, ADFI, and FCE.

Fecal samples were collected from each pen on Days 11, 12, 13, and 14. The fecal samples from the four collections from each pen were pooled by placing the feces into a stainless steel pan and stirring with a teflon spatula so that there was a single sample from each pen representing the four collections. All fecal samples were stored in sealed plastic bags at -60ºC. Prior to analysis, the fecal samples were freeze-dried for 72 hours, allowed to equilibrate for 24 hours at room temperature, and then ground through a 1.0-mm screen with a Cyclotec Grinder (Model 1093; Foss Group, Hillerod, Denmark). Digestibility coefficients for nutrients were calculated using the equations for the indicator method described by Schneider and Flatt.18 Digestibility data was obtained from six randomly chosen pens per treatment.

On Day 14, one pig in each pen (n = 9), closest to the average body weight for that pen, was chosen for blood sampling. Blood samples (approximately 7 mL per sample) were collected by anterior vena cava puncture after a 6-hour fast, using plain vacuum-filled blood collection tubes (21-gauge × 1.5 inches; Serum Clot Activator; Greiner Bio-one, Kremsmunster, Austria). The samples were then centrifuged (Model VS-5000N; Vision Scientific, Bucheon, Korea) at 415g at 4ºC for 15 minutes. Serum was collected and stored at -25ºC until needed for analysis of serum parameters.

Chemical analysis

Samples of the diets and feces were analyzed in triplicate according to the methods of the Association of Official Analytical Chemists.19 Analyses were conducted for moisture (method 930.15), crude protein (method 984.13), ash (method 942.05), crude fiber (method 978.10), and ether extract (method 920.39). Calcium was determined by a Shimazu AA625 Atomic Absorption Spectrophotometer (Shimazu, Kyoto, Japan), and phosphorus was analyzed using a UV-Vis Spectrophotometer (Hitachi, Tokyo, Japan). An amino-acid analysis of the feeds and feces was performed using an L8500-Hitachi Amino Acid Analyzer (Hitachi) after hydrolysis for 24 hours in 6M HCl. Performic acid (85%) hydrolysis was performed prior to analysis of the sulfur-containing amino acids. Gross energy was measured using an Adiabatic Oxygen Bomb Calorimeter (Model 1241; Parr Instrument Co, Moline, Illinois). Diatomaceous earth (Acid Insoluble Ash) analysis was conducted according to the description provided by Prabucki et al.20 The analyzed chemical composition of the experimental diets is shown in Table 2.

The serum concentrations of total protein, albumin, glutamic-oxaloacetic transferase (GOT), glutamic-pyruvic transferase (GPT), urea nitrogen, total cholesterol, high density lipoprotein, low density lipoprotein, and triglyceride were determined using commercial kits (Bayer, Terrytown, New York) following the procedures recommended for an Automatic Biochemical Analyzer (Model ADVIA 1650, Bayer, Terrytown, New York). The concentration of immunoglobulin G was analyzed with a commercially available Nephelometry kit (Bade Behring, Schwalbach, Germany) and a Model BN II Nephelometer (Bade Behring). Zinc was analyzed using inductively coupled plasma mass spectrometry (ICP-MS Model Elan DRCe; Perkin Elmer, Wiesbaden, Germany). Serum cortisol, somatomedin-C, and tumor necrosis factor were analyzed using radioimmunoassay (Diagnostic Products Corporation, Los Angeles, California), chemiluminescent immunoassay (Diagnostic Products Corporation), and enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minnesota), respectively.

Statistical analysis

The performance and digestibility data were analyzed as a randomized block design using the analysis of variance procedures of the Statistix for Windows program (Analytical Software, Tallahassee, Florida). Pen was considered the experimental unit for analyses of performance and digestibility data. The animal was considered the experimental unit for analyses of data regarding serum parameters. Because of the severe diarrhea in Experiment Three, the data for the negative control group were deleted from the data set. The model included the effects of farm, treatment, farm × treatment, and error. There were no significant differences between the farms for any of the parameters measured. The significance of differences between means was determined by Student Newman Keul’s test for performance, digestibility, and serum parameters with the significance level set at α = .05.

Results

In Experiment One, ADG Days 8 to 21 and Days 0 to 28, and ADFI Days 8 to 21 and Days 0 to 28, were lower (P < .05) for pigs fed the negative control diet than for pigs fed diets supplemented with any of the four treatments (Table 3). However, during all experimental periods, ADG and ADFI did not differ for pigs fed diets supplemented with antibiotics, ZnO, or micro-encapsulated E-MCFAs (P > .05). There were no treatment effects on FCE during any experimental period (P > .05).

In Experiment Two, ADG (Days 0 to 14 and Days 0 to 28) was lower (P < .05) for pigs fed the negative control diet than for pigs fed diets supplemented with antibiotics, ZnO, or micro-encapsulated E-MCFAs (Table 4). The FCE of the negative control group measured Days 0 to 28 was higher (P < .05) than that of pigs fed diets supplemented with antibiotics or either of the ZnO supplemented diets, but did not differ from that of pigs fed the E-MCFA-supplemented diet. There were no treatment effects on ADFI during phases 1 and 2 or over the entire experimental period (P > .05).

In Experiment Three, there were no differences (P > .05) in ADG, ADFI, or FCE between pigs fed diets supplemented with antibiotics, ZnO, or E-MCFAs (Table 5).

The apparent fecal digestibility coefficients for various nutrients contained in the experimental diets are shown in Table 6. Digestibility coefficients of dry matter, calcium, phosphorus, energy, and methionine were lower (P < .05) in pigs fed the diets supplemented with ZnO than in pigs fed the diet supplemented with antibiotics. In contrast, digestibility coefficients for crude protein, calcium, phosphorus, energy, histidine, leucine, lysine, phenylalanine, and threonine were higher (P < .05) for pigs fed the diet supplemented with E-MCFAs than for pigs fed the diet supplemented with antibiotics. In addition, digestibility coefficients for dry matter, crude protein, calcium, phosphorus, energy, histidine, lysine, methionine, phenylalanine, threonine, and valine were higher in pigs fed the diet supplemented with E-MCFAs than in pigs fed the ZnO-supplemented diets (P < .05).

There were no differences (P > .05) between the treatments for serum concentrations of total protein, albumin, blood urea nitrogen, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, cortisol, somatomedin-C, immunoglobulin G, and tumor necrosis factor-α (Table 7). Serum zinc, glutamic-oxaloacetic transferase, and glutamic-pyruvic transferase concentrations were higher (P < .05) in pigs fed the diets supplemented with ZnO than in pigs fed the diets supplemented with antibiotics or E-MCFAs.

Discussion

The overall performance data (Days 0 to 28) demonstrated higher ADG and ADFI in Experiment One and higher ADG and FCE in Experiment Two for pigs fed ZnO-supplemented diets than for the negative control pigs. In both experiments, performance of the ZnO-supplemented pigs did not differ from that of the antibiotic-supplemented pigs, indicating that ZnO can substitute for antibiotics in diets fed to nursery pigs. Previous studies have also indicated that pharmacological doses of ZnO stimulate performance in weaned pigs,7,21,22 although there are some reports in which no growth-promoting benefits were observed.23,24

Our data indicate that the higher ADG due to ZnO supplementation can be at least partially attributed to an increase in voluntary ADFI. In a recent study by Yin et al,25 it was observed that ZnO supplementation stimulates ghrelin secretion from the stomach of young pigs, and ghrelin has been shown to act on the small intestine and the brain to stimulate feed intake via an unknown mechanism.25 Therefore, increased ghrelin secretion may explain the higher feed intake of the ZnO-supplemented pigs observed in Experiment One of the present study.

It has previously been suggested that ZnO promotes the growth of weaned pigs by controlling pathogenic bacterial scours.26-28 However, other work has indicated that ZnO promotes growth in early and conventionally weaned pigs regardless of diarrhea incidence or effects on intestinal microbial numbers.21,29 There was no evidence of diarrhea in Experiments One and Two, while severe diarrhea was observed in the control group in Experiment Three, to the extent that feeding of the control diet had to be terminated.

In comparison to the antibiotic treatment, lower digestibility of dry matter, calcium, phosphorus, energy, and methionine digestibility was observed as a result of feeding ZnO at either 1500 or 2500 mg per kg, while digestibility of histidine was lower for pigs fed ZnO at 1500 mg per kg but not at 2500 mg per kg. These findings are somewhat surprising, given the fact that Li et al29 and Li et al30 have shown that zinc supplementation improves gut morphology by increasing villous height and reducing crypt depth in the small intestine, thus potentially increasing the absorptive capacity of the small intestine. In addition, Hedemann et al31 reported that high dietary zinc increased the activity of several enzymes in the pancreatic tissue (amylase, carboxypeptidase, chymotrypsin, trypsin, and lipase) and it might reasonably be expected that such an increase would result in improvements in nutrient digestibility. However, in the present experiment, nutrient digestibility was either unaffected or reduced by zinc supplementation.

Serum zinc concentrations were elevated for pigs fed diets supplemented with ZnO at 1500 or 2500 mg per kg. This supports previous studies which have also reported elevated levels of serum zinc for zinc-supplemented pigs.21,32,33 Serum glutamic-oxaloacetic transferase and glutamic-pyruvic transferase concentrations were higher in ZnO-supplemented pigs, which supports our previous work.34 Elevated levels of these enzymes are typically associated with liver damage,35 and therefore future work should be conducted to determine whether or not pharmacological levels of dietary ZnO alter liver function.

The overall pig performance data from Experiments One and Two showed higher ADG and ADFI in Experiment One and higher ADG in Experiment Two for nursery pigs fed the diet supplemented with micro-encapsulated E-MCFAs than for pigs fed the negative control diet. In both experiments, the performance of pigs fed micro-encapsulated E-MCFAs did not differ from that of pigs fed antibiotics or pharmacological levels of ZnO, indicating that E-MCFAs can substitute for antibiotics or pharmacological levels of ZnO in diets fed to nursery pigs.

The performance-enhancing effects of micro-encapsulated E-MCFAs could be due to beneficial effects arising from inclusion of eucalyptus or MCFAs either alone or in combination. In poultry, dietary inclusion of eucalyptus has been shown to improve the production performance of commercial laying hens through mechanisms involving stimulation of the immune system.13 In humans, eucalyptus oil has been shown to stimulate the immune system by affecting the phagocytic ability of monocyte-derived macrophages.12 However, in the present study, there were no changes in serum parameters such as immunoglobulin G, tumor necrosis factor-α, or cortisol, suggesting that mechanisms other than immune-system stimulation are responsible for the performance enhancement observed in pigs fed eucalyptus. In humans, eucalyptus oil has been shown to have antibacterial effects on pathogenic bacteria in the respiratory tract,11 and it is possible that antibacterial effects of eucalyptus are responsible for at least some of the performance enhancement observed for pigs fed E-MCFAs.

Another possible explanation for the performance enhancement resulting from feeding E-MCFAs is direct effects of MCFAs. There is some evidence36 that their use in diets modulates antibacterial responses in pigs. Dierick et al1,14 showed strong in vitro and in vivo antibacterial effects of MCFAs in the pig proximal small intestine in the absence of growth-promoting antibiotics. It has been estimated that as much as 6% of the net energy in pig diets may be lost due to microbial fermentation in the intestine,37 and therefore a reduction in bacterial populations could leave more energy available for animal growth. In addition, caprylic acid has been used in the treatment of some bacterial infections. As a result of its relatively short chain length, it can penetrate fatty cell-wall membranes and is therefore effective in combating certain lipid-coated bacteria, such as Staphylococcus aureus and various species of Streptococcus.38

Our data suggest that the superior pig performance associated with supplementing the diet with micro-encapsulated E-MCFAs was primarily the result of higher nutrient digestibility. The results of Experiment Three demonstrated better digestibility of most major nutrients, including crude protein, calcium, phosphorus, and energy, as well as the amino acids histidine, lysine, phenyalanine, and threonine in the pigs supplemented with micro-encapsulated E-MCFAs. It has been reported that lipid may influence nutrient digestibility by altering intestinal morphology of young pigs.39-42

One concern with using MCFAs for growth promotion is that they are a strong stimulus for the release of cholecystokinin, which has a pronounced satiating activity that could interfere with feed intake.14 In addition, MCFAs are reported to be unpalatable.15 However, the results of the current experiment indicate that the process of micro-encapsulation overcomes any negative effects of E-MCFAs on feed intake, which was not adversely affected by dietary inclusion of E-MCFAs in any of the three experiments. The overall results of this study indicate that E-MCFAs may be an effective substitute for antibiotics and ZnO as a means of growth promotion in diets fed to weanling pigs.

An issue with feeding pharmacological levels of zinc to pigs is that application of their manure to soil can negatively impact the environment.9 Pigs fed zinc at 3000 mg per kg of feed excreted almost four times more zinc in their feces as pigs fed zinc at 500 mg per kg.33 Zinc accumulation in the soil has been implicated in reducing plant growth,43 while leaching of zinc from soils treated with swine manure may lead to pollution of lakes, streams, and coastal waters.44,45 The fact that 250 mg per kg of caprylic and carpric acids produced similar levels of growth promotion as 1500 or 2500 mg per kg zinc suggests that the use of E-MCFAs as a growth promoter could minimize the environmental impact of intensive swine operations and reduce the amount of minerals in swine manure that could potentially pollute the environment.

In conclusion, the performance of pigs fed diets supplemented with antibiotics or pharmacological levels of ZnO was better than that of pigs fed a negative control (unsupplemented) diet. The performance of pigs fed a diet supplemented with microencapsulated E-MCFAs did not differ from that of pigs fed diets supplemented with either antibiotics or pharmacological levels of ZnO, indicating that E-MCFAs can also be used as a growth promoter in diets fed to nursery pigs. The effects of E-MCFAs appeared to be mediated through greater nutrient digestibility.

Implications

• Under the conditions of this study, growth performance of pigs supplemented with microencapsulated E-MCFAs did not differ from that of pigs supplemented with antibiotics or pharmacological levels of ZnO.

• Eucalyptus-MCFAs can be used as a growth promoter in diets fed to nursery pigs.

Acknowledgments

This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

References

1. Dierick NA, Decuypere JA, Molly K, Van Beek E, Vanderbecke E. The combined use of triacyl-glycerols (TAGs) containing medium-chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition: II. In vivo release of MCFAs in gastric cannulated and slaughtered piglets by endogenous and exogenous lipases; effects on the luminal gut flora and growth performance. Livest Prod Sci. 2002;76:1–16.

2. Cromwell GL. Why and how antibiotics are used in swine production. Anim Biotechnol. 2002;13:7–27.

3. Adjiri-Awere A, van Lunen TA. Subtherapeutic use of antibiotics in pork production: Risks and alternatives. Can J Anim Sci. 2005;85:117–130.

4. Turner JL, Dritz SS, Minton JE. Review: Alternatives to conventional antimicrobials in swine diets. Prof Anim Sci. 2001;17:217–226.

5. Poulsen HD. Zinc oxide for weanling piglets. Acta Agric Scand Sect A Animal Sci. 1995;45:159–167.

6. Smith JW, Tokach MD, Goodband RD, Nelssen JL, Richert BT. Effects of the interrelationship between zinc oxide and copper sulphate on growth performance of early-weaned pigs. J Anim Sci. 1997;75:1861–1866.

7. Hill GM, Cromwell GL, Crenshaw TD, Dove CR, Ewan RC, Knabe DA, Lewis AJ, Libal GW, Mahan DC, Shurson GC, Southern LL, Veum TL. Growth promotion effects and plasma changes from feeding high dietary concentrations of zinc and copper to weanling pigs (regional study). J Anim Sci. 2000;78:1010–1016.

*8. Kavanagh NT. The effect of feed supplemented with zinc oxide on the performance of recently weaned pigs. Proc IPVS. The Hague, The Netherlands. 1992;616.

9. Berenguer P, Cela S, Santiveri F, Boixadera J, Lloveras J. Copper and zinc soil accumulation and plant concentration in irrigated maize fertilized with liquid swine manure. Agron J. 2008;100:1056–1061.

10. Han YK, Thacker PA. Performance, nutrient digestibility and nutrient balance in weaned pigs fed diets supplemented with antibiotics or zinc oxide. J Anim Vet Adv. 2009;8:868–875.

11. Salari MH, Amine G, Shirazi MH, Hafezi R, Mohammadypour M. Antibacterial effects of Eucalyptus globulus leaf extract on pathogenic bacteria isolated from specimens of patients with respiratory tract disorders. Clin Microbiol Infect. 2006;12:194–196.

12. Serafino A, Vallebona PS, Andreola F, Zonfrillo M, Mercuri L, Federici M, Rasi G, Garaci E, Pierimarchi P. Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response. BMC Immunol. 2008;9:17. doi:10.1086/1471–2172–9–7.

13. Abd El-Motaal AM, Ahmed AMH, Bahakaim ASA, Fathi MM. Productive performance and immunocompetence of commercial laying hens given diets supplemented with Eucalyptus. Intern J Poult Sci. 2008;7:445–449.

14. Dierick NA, Decuypere JA, Molly K, Van Beek E, Vanderbecke E. The combined use of triacylgly-cerols (TAGs) containing medium-chain fatty acids (MCFs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition: I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of piglets. Livest Prod Sci. 2002;75:129–142.

15. Cera KR, Mahan DC, Reinhart GA. Postweaning swine performance and serum profile responses to supplemental medium-chain free fatty acids and tallow. J Anim Sci. 1989;67:2048–2055.

16. Korean Ministry for Food, Agriculture, Forestry and Fisheries. 2008. Guidelines for the Care and Use of Animals in Research. Korean Ministry for Food, Agriculture, Forestry and Fisheries, Seoul, Korea.

17. National Research Council. Nutrient Requirements of Swine. 10th ed. Washington, DC: National Academy Press; 1998.

18. Schneider BH, Flatt WP. The Evaluation of Feeds Through Digestibility Experiments. Athens, Georgia: University of Georgia Press; 1975.

19. Association of Official Analytical Chemists. Official Methods of Analysis, 15th ed. Washington, DC: AOAC; 1990.

20. Prabucki AL, Rennerova L, Vogtmann H, Wenk C, Schuerch A. The use of 4-N HCL-insoluble ash as an indicator for determination of digestibility. Institute of Animal Nutrition Miscellaneous Papers. Wageningen, The Netherlands: Institute of Animal Nutrition. 1975;11:113–114.

21. Hahn JD, Baker DH. Growth and plasma zinc responses of young pigs fed pharmacologic levels of zinc. J Anim Sci. 1993;71:3020–3024.

22. Carlson MS, Hill GM, Link JE. Early- and traditionally weaned nursery pigs benefit from phase-feeding pharmacological concentrations of zinc oxide: Effect on metallothionein and mineral concentrations. J Anim Sci. 1999;77:1199–1207.

*23. Tokach LM, Tokach MD, Goodband RD, Nelssen JL, Henry SC, Marsteller TA. Influence of zinc oxide in starter diets on pig performance. Proc AASP. 1992;411.

24. Schell TC, Kornegay ET. Zinc concentration in tissues and performance of weanling pigs fed pharmacological levels of zinc from ZnO, Zn-methionine, Zn-lysine, or ZnSO4. J Anim Sci. 1996;74:1584–1593.

25. Yin J. Li X, Li D, Yue T, Fang Q, Ni J, Zhou X, Wu G. Dietary supplementation with zinc oxide stimulates ghrelin secretion from the stomach of young pigs. J Nutr Biochem. 2009;20:783–790.

26. Holm A, Poulsen HD. Zinc oxide in treating E. coli diarrhea in pigs after weaning. Compend Cont Educ Pract Vet Food Anim Med Manage. 1996;18:S26-S29, S48.

27. Katouli M, Melin L, Jensen-Waern M, Wallgren P, Mollby R. The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs. J Appl Microbiol. 1999;87:564–573.

28. Huang SX, McFall M, Cegielski AC, Kirkwood RN. Effect of dietary zinc supplementation on Escherichia coli septicemia in weaned pigs. Swine Health Prod. 1999;7:109–111.

29. Li BT, Van Kessel AG, Caine WR, Huang SX, Kirkwood RN. Small intestinal morphology and bacterial populations in ileal digesta and feces of newly weaned pigs receiving a high dietary level of zinc oxide. Can J Anim Sci. 2001;81:511–516.

30. Li X, Yin J, Li D, Chen X, Zang J, Zhou X. Dietary supplementation with zinc oxide increases IGF-I and IGF-I receptor gene expression in the small intestine of weanling piglets. J Nutr. 2006;136:1786–1791.

31. Hedemann MS, Jensen BB, Poulsen HD. Influence of dietary zinc and copper on digestive enzyme activity and intestinal morphology in weaned pigs. J Anim Sci. 2006;84:3310–3320.

32. Hill GM, Mahan DC, Carter SD, Cromwell GL, Ewan RC, Harrold RL, Lewis AJ, Miller PS, Shurson GC, Veum TL. Effect of pharmacological concentrations of zinc oxide with or without the inclusion of an antibacterial agent on nursery pig performance. J Anim Sci. 2001;79:934–941.

33. Case CL, Carlson MS. Effect of feeding organic and inorganic sources of additional zinc on growth performance and zinc balance in nursery pigs. J Anim Sci. 2002;80:1917–1924.

34. Han YK. Thacker PA. Effects of antibiotics, zinc oxide and lanthanum-yeast on performance, nutrient digestibility and blood parameters in weaned pigs. Asian-Aust J Anim Sci. 2010;23:1057–1065.

35. Giboney PT. Mildly elevated liver transaminase levels in the asymptomatic patient. Am Fam Physician. 2005;71:1105–1110.

36. Kabara J, Swieczkowski D, Conley A, Truant J. Fatty acids and derivates as antimicrobial agents. Antimicrob Agents Chemother. 1972;2:23–28.

37. Jensen BB. The impact of feed additives on the microbial ecology of the gut in young pigs. J Anim Feed Sci. 1998;7(Suppl 1):45–64.

38. Nair MK, Joy J, Vasudevan P, Hinckley L, Hoagland TA, Venkitanarayanan KS. Antibacterial effect of caprylic acid and monocaprylin on major bacterial mastitis pathogens. J Dairy Sci. 2005;88:3488–3495.

39. Li DF, Thaler RC, Nelssen JL, Harmon DL, Allee GL, Weeden TL. Effect of fat sources and combinations on starter pig performance, nutrient digestibility and intestinal morphology. J Anim Sci. 1990;68:3694–3704.

40. Cera KR, Mahan DC, Reinhart GA. Weekly digestibility of diets supplemented with corn oil, lard or tallow by weanling swine. J Anim Sci. 1988;66:1430–1437.

41. Cera KR, Mahan DC, Reinhart GA. Effect of dietary dried whey and corn oil on weanling pig performance, fat digestibility and nitrogen utilization. J Anim Sci. 1988;66:1438–1445.

42. Cera KR, Mahan DC, Reinhart GA. Apparent fat digestibility and performance responses of postweaning swine fed diets supplemented with coconut oil, corn oil or tallow. J Anim Sci. 1989;67:2040–2047.

43. Chaney RL, Zinc phytotoxicity. In: Robson AD, ed. Zinc in Soils and Plants. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1993:135–150.

44. Hsu JH, Lo SL. Effect of composting on characterization and leaching of copper, manganese and zinc from swine manure. Environ Pollution. 2001;114:119–127.

45. Martinez CE, Motto HL. Solubility of lead, zinc, and copper added to mineral soils. Environ Pollut. 2000;107:153–158.

*Non-refereed references.