Effects of a lipid-encapsulated zinc oxide dietary supplement, on growth parameters and intestinal morphology in weanling pigs artificially infected with enterotoxigenic Escherichia coli
© Kim et al.; licensee BioMed Central. 2015
Received: 17 October 2014
Accepted: 29 December 2014
Published: 24 January 2015
The study was performed to investigate the effect of dietary supplementation of a lipid-encapsulated Zinc oxide on growth parameters and intestinal mucosal morphology piglets born to Duroc-sired Landrace × Yorkshire dams. Twenty-four 30-day-old piglets weaned at 25 days of age were orally challenged with 5 × 108 colony forming units of enterotoxigenic Escherichia coli (ETEC) K88 and fed one of the four diets for 7 days: (i) a nursery basal diet containing 100-ppm ZnO (referred to as BASAL), (ii) BASAL supplemented with 120-ppm apramycin (referred to as ANTIBIO), (iii) BASAL with 2,400-ppm ZnO (referred to as HIGH), and BASAL containing 100-ppm lipid-encapsulated ZnO (referred to as LE). All piglets were killed at the end of the experiment for histological examination on the intestine. The results showed that the average daily gain (ADG), the villus height: crypt depth (CD) ratio in the ileum, and the goblet cell density of the villus and crypt in the duodenum, jejunum, and colon were greater in the LE-fed group that those of the BASAL (p < 0.05). Fecal consistency score (FCS) and the CD ratio in the ileum were less in the LE-fed group, compared to the BASAL-fed one (p < 0.05). The effects observed in the LE-fed group were almost equal to those of the HIGH-fed group as well as even superior to those of the ANTIBIO-fed group. Taken together, our results imply that dietary supplementation of 100-ppm lipid-encapsulated ZnO is as effective as that of 2,400-ppm ZnO for promoting growth diarrhea and intestinal morphology caused by ETEC infection.
Dietary supplementation of 2,000- to 3,000-ppm of zinc oxide (ZnO) alleviates diarrhea and improves growth performance and the integrity of intestinal morphology of post-weaning piglets [1-4]. Moreover, dietary ZnO at those pharmacological doss exhibits the similar effects in the enterotoxigenic Escherichia coli (ETEC)-challenged piglets, which have been used as a model host for post-weaning diarrhea caused by ETEC infection [5-7].
A lipid-encapsulated proprietary Zn supplement whose active component ZnO is supposedly released after digestion of the lipid coating by lipase in the intestinal lumen. The ZnO particle of the mineral supplement is more efficiently delivered to the intestine than native ZnO because ZnO contained in the former is not ionized in the stomach owing to the lipid coating .
We have previously reported that dietary supplementation of 100-ppm of the lipid-encapsulated ZnO (LE ZnO) was as effective as 2,500-ppm of native ZnO in alleviating diarrhea and growth retardation of weanling pigs artificially challenged with 3 × 1010 colony forming units (CFU) of ETEC K88 . It is questionable, however, whether or not the supplemental LE ZnO would exhibit such effects comparable to those of pharmacological ZnO as well as antibiotics in weanling pigs with infection of ETEC, because the challenge dose of ETEC in our previous study was greater than that necessary to induce a mild clinical infection . The present study was therefore undertaken to determine the effect of dietary supplementation of LE ZnO on growth promotion and intestinal morphology in weanling pigs after infection with a low infectious dose (5 × 108 CFU) of ETEC, compared to that of antibiotics and/or pharmacological concentration of ZnO.
Animals and dietary treatments
Composition of the basal diet (as-fed basis)
Calculated chemical composition
Digestible energy, MJ/kg
Crude protein, %
Ether extract, %
Collection of blood samples and intestinal tissues
All the piglets were killed at the end of the feeding trial. Upon opening the abdominal cavity, the intestinal tract was dissected as described previously [9,13]. For morphological examination of the intestinal mucosa, cross-sectional segments (approximately 3 cm in length) were cut approximately at a 10-cm location from the pylorus (duodenum), at 50% (jejunum) and 90% (ileum) of the length of the small intestine, and at the transverse colon. Serum was collected after centrifugation and stored in aliquots at −20°C until used.
Quantification of fecal shedding of ETEC K88
The bacterial number of ETEC K88 shed in the feces was determined by quantitative real-time PCR targeting a 70-bp fragment of the K88 fimbrial gene as described by West et al. . Briefly, the genomic DNAs in the feces were extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The PCR mixture contained 2 μL of DNA template, 100 pmol of the forward primer (5′-GGTTCAGTGAAAGTCAATGCATCT-3′), 100 pmol of the reverse primer (5′-CCCCGTCCGCAGAAGTAAC-3′), 0.5 μL of the probe (Cy5-5′-CCACCTCTCCCTAACACACCGGCAT-3′-BHQ2; GenoTech, Daejeon, Korea), and 12.5 μL of Premix EX Taq DNA polymerase (Takara Bio, Shiga, Japan) in a total volume of 25 μL. Thermal cycling was performed with an initial denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 20 sec, 62°C for 30 sec, and 72°C for 30 sec. The cfu of ETEC K88 in each fecal sample was assessed from the standard curve for the ETEC K88 standard solution plotted against the threshold (Ct) value using the Smartcycler software (Cepheid, Sunnyvale, CA).
The cross-sectional segments of the small and large intestines were fixed for 48 hrs in a 10% neutral formalin solution. The fixed tissues were embedded in paraffin and sliced to a thickness of 4 μm, after which the thin sections were mounted on glass slides and stained with hematoxylin/eosin for microscopic examination of the mucosal structure as previously described . For determination of the goblet cell density, the specimens were stained with periodic acid-Schiff and Alcian Blue to count the neutral mucin- and acid mucin-secreting goblet cells, respectively, as described by Uni et al. . The villus height (VH), crypt depth (CD), and the goblet cell number were determined on four well-oriented structures under a 400-fold magnified microscopic field using the Diagnostic Insight visual analysis program (Olympus, Tokyo, Japan) as described previously . In each variable, the average of four measurements was taken as an observation.
Data were analyzed as a completely randomized design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The model included the dietary treatment as the main effect as well as the day and its interaction with treatment in the analysis of repeated measurements. Effects of the treatment and day including its interaction with treatment were tested using the animal and day × animal nested within treatment as error terms, respectively. Means were separated by the probability difference (PDIFF) option.
Effects of dietary supplementations of 100 ppm of ZnO without (BASAL) or with 120 ppm of apramycin (ANTIBIO), 2,500 ppm of ZnO (HIGH), and 100 ppm of the lipid-encapsulated ZnO (LE) on clinical signs of post-weaning pigs challenged with 5 × 10 8 cfu of ETEC K88
Rectal temperature, °C
Fecal consistency score2
Fecal shedding of E. coli, Log10 cfu/g feces
The FCS was also elevated on Day 1 and 2 above that on Day 0, but returned to the pre-challenge level by d 7 (Table 2). The mean FCS was less in the LE-fed group than in the BASAL- and ANTIBIO-fed groups, but it did not differ between the former and HIGH-fed groups. The mean level of excretion of ETEC K88 to feces expressed as log cfu also was less in the LE-fed group, compared to the BASAL- and ANTIBIO-fed groups, not being different between the former and the HIGH-fed group.
Effects of dietary supplementations of 100 ppm of ZnO without (BASAL) or with 120 ppm of apramycin (ANTIBIO), 2,500 ppm of ZnO (HIGH), and 100 ppm of the lipid-encapsulated ZnO (LE) on growth performance of weanling pigs challenged with 5 × 10 8 cfu of ETEC K88
Before the ETEC K88 challenge (d −5 to d 0)1
Initial wt, kg
Final wt, kg
After the ETEC K88 challenge (d 0 to d 7)1
Final wt, kg
Effects of dietary supplementations of 100 ppm of ZnO without (BASAL) or with 120 ppm of apramycin (ANTIBIO), 2,500 ppm of ZnO (HIGH), and 100 ppm of the lipid-encapsulated ZnO (LE) on histological measurements of the intestine in weanling pigs challenged with 5 × 10 8 cfu of ETEC K88
Goblet cell density, cells/mm2
The VH, CD and VH:CD ratio in the duodenum were not influenced significantly by the dietary treatment (Table 4). The CD in the jejunum was less in the LE-fed group than in the BASAL- and ANTIBIO-fed groups whereas it did differ between the LE- and HIGH-fed groups. In the ileum, the VH:CD ratio was greater in the LE-fed group than in the BASAL- and ANTIBIO-fed groups, but it did not differ between the former and the HIGH-fed group.
The piglets challenged with 5 × 108 cfu of ETEC K88 exhibited expected responses to both ANTIBIO and HIGH, with decreases in rectal temperature, FCS, and fecal ETEC shedding and increases in ADG and goblet cell density. Moreover, the effects of LE were virtually equal to those of HIGH, which were equal to or greater than those of ANTIBIO. The ADG of the experimental groups were thus reflective of their ETEC infection status, suggesting that the greater weight gain in the LE- and HIGH-fed groups, compared to the BASAL, resulted from the antibacterial activities of the former. Overall, the present results for the ANTIBIO- and HIGH-fed groups were consistent with published reports regarding the effects of apramycin with or without other antibiotics [12,16,17] as well as pharmacological ZnO [1,7,18]. Furthermore, the effects of HIGH and LE-BASAL, compared to BASAL, observed in the present study were very similar to our previous results in post-weaning pigs challenged with 3 × 1010 cfu of ETEC K88 , suggesting that LE and HIGH are equally effective for alleviating the ETEC infection.
The VH, CD, and VH:CD of the intestinal mucosa of the ETEC-infected piglets did not change in response to HIGH or LE, except for a decrease in CD in the jejunum and an increase in the VH:CD ratio in the ileum. These results were substantially different from the almost consistent increase in VH and VH:CD and decrease in CD in response to both HIGH and LE in the jejunum and ileum in post-weaning pigs challenged with a high dose of ETEC K88 in our previous study . Regarding the effects of supplemental ZnO on intestinal mucosal morphology of the piglets in the literature, the VH and VH:CD increased and CD decreased inconsistently in response to pharmacological ZnO in Li et al. [19,20] and Owusu-Adiedu et al. , but in Hedemann et al. , the VH decreased due to supplemental ZnO. Collectively, these results suggest that the beneficial effects of supplemental ZnO on the structural integrity of intestinal mucosa are more pronounced when the piglets are severely infected with ETEC than when moderately infected.
The present results indicated that dietary supplementation of 72 ppm Zn as LE ZnO was as effective as that of 2,000 to 2,500 ppm Zn provided as native ZnO or antibiotics for increasing growth performance, goblet cell density in the intestine as well as reducing diarrhea in weanling pigs challenged with a low dose of ETEC K88. More studies are necessary, however, to determine the effects of LE ZnO relative to those of native ZnO on those responses examined in the present study in naïve weanling pigs under production conditions.
This study was financially supported by CTCBIO, Inc. and the Regional Animal Industry Center at Gyeongnam National University of Science and Technology.
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