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Dietary omega-3 polyunsaturated fatty acid does not influence the intestinal microbial communities of broiler chickens
Dietary omega-3 polyunsaturated fatty acid does not influence the intestinal microbial communities of broiler chickens M. S. Geier,*†1 V. A. Torok,†‡ G. E. Allison,§ K. Ophel-Keller,†‡ R. A. Gibson,# C. Munday,§ and R. J. Hughes*† *Pig and Poultry Production Institute, South Australian Research and Development Institute, University of Adelaide, Roseworthy Campus, 5371, South Australia; †Australian Poultry Cooperative Research Centre, University of New England, Armidale 2351, New South Wales, Australia; ‡Plant and Soil Health, South Australian Research and Development Institute, Waite Campus, Urrbrae 5001, South Australia; §School of Biochemistry and Molecular Biology, and ANU Medical School, The Australian National University, Canberra 0200, Australian Capital Territory, Australia; and #School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae 5064, South Australia ABSTRACT The capacity for n-3 polyunsaturated fatty acids (PUFA) to improve broiler chicken growth, influence the intestinal microbial communities, and modify the PUFA content of meat was studied. Male Cobb 500 chickens were fed 1 of 4 diets from hatch: control (standard diet with no additives), ZnB (standard diet with added antibiotics), 2% SALmate (standard diet with 2% SALmate, which is composed of 42% fish oil and 58% starch), and 5% SALmate (standard diet with 5% SALmate). A 7-d energy metabolism study was conducted between d 15 and 22 posthatch. Birds were killed at d 25 and intestinal samples were collected to assess microbial communities by terminal restriction fragment length polymorphism and Lactobacillus PCRdenaturing gradient gel electrophoresis. Diet did not affect BW, feed intake, feed conversion, or ileal digestible energy (P > 0.05). Apparent ME was greater in ZnB-fed birds compared with all other diets (P < 0.05).
Breast tissue levels of eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, and total n-3 PUFA were elevated significantly in 2% SALmate- and 5% SALmate-fed chickens compared with control and ZnB diets (P < 0.05). No significant differences in overall microbial communities were observed in the ileum or cecum as assessed by terminal RFLP (P > 0.05). Birds fed 2% SALmate had a significantly different cecal Lactobacillus species profile compared with birds fed the control diet (P < 0.05); however, no differences were observed in birds fed 5% SALmate compared with birds fed all other diets. In addition to the expected increase in breast tissue n-3 fatty acid levels, a low level of dietary n-3 PUFA also altered the intestinal Lactobacillus species profiles. However, n-3 PUFA supplementation did not alter the overall microbial communities or broiler performance.
Key words: broiler, denaturing gradient gel electrophoresis, polyunsaturated fatty acid, terminal restriction fragment length polymorphism 2009 Poultry Science 88:2399–2405 doi:10.3382/ps.2009-00126
INTRODUCTION Supplemental dietary antibiotics are used in poultry production to improve the growth and performance of broiler chickens and to reduce the incidence of infectious diseases. However, the risk of the development of antibiotic-resistant bacteria as a result of long-term antibiotic exposure led to their ban in the European Union in 2006. Other regions have not yet implement©2009 Poultry Science Association Inc. Received March 10, 2009. Accepted July 15, 2009. 1 Corresponding author: [email protected]
ed such a ban; however, consumer and environmental pressure may lead to future changes in legislation. Debate continues as to the exact mechanisms of action of antibiotics, with antimicrobial and antiinflammatory mechanisms postulated (Niewold, 2007). The need for natural antibiotic alternatives has led to the investigation of a wide range of compounds including probiotics, prebiotics, and plant extracts (Yang et al., 2009). The immunomodulatory and antiinflammatory properties of n-3 polyunsaturated fatty acids (PUFA) suggest a possible role as a replacement for antibiotics. The health-promoting effects of n-3 PUFA are well documented in humans (Calder, 2006). In poultry, the consumption of diets high in fish-derived n-3 PUFA,
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particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been demonstrated to improve BW gain (Lopez-Ferrer et al., 2001; Schreiner et al., 2005), reduce the inflammatory response (Korver and Klasing, 1997), and alter immune parameters including antibody production and lymphocyte proportion and responsiveness (Wang et al., 2000). It has also been reported that n-3 PUFA influence intestinal bacteria in vitro and in vivo (Kankaanpaa et al., 2001; Hekmatdoost et al., 2008). The aim of the current study was to compare the effects of dietary fish oil on the composition of the intestinal microbial communities, with particular focus on Lactobacillus species due to their known healthpromoting properties. Additionally, broiler growth and production traits and the fatty acid composition of breast tissue were studied.
MATERIALS AND METHODS Birds, Management, and Diets Forty-eight male Cobb 500 broiler chickens (Baiada Hatchery, Willaston, Australia) were raised in 4 floor pens with fresh litter in a temperature-controlled room (22 to 27°C). All birds were vaccinated with Eimeriavax 4m (Bioproperties Pty Ltd., Glenorie, New South Wales, Australia) at placement and at 5 and 11 d posthatch. All procedures were approved by the Animal Ethics Committees of the University of Adelaide and the Department of Primary Industries and Resources South Australia, Adelaide. All diets were based on a standard commercial starter diet (Ridley Agriproducts Pty Ltd., Murray Bridge, Australia) without any added antibiotics or coccidiostats (Table 1). All diets met or exceeded NRC guidelines for broiler chickens (NRC, 1994). The source of n-3 PUFA was SALmate (Feedworks, Romsey, Victoria, Australia), which is composed of 42% fish oil and 58% starch. The 4 experimental diets were as follows: standard diet with no additives (control), standard diet + 50 mg/kg of zinc bacitracin (ZnB), standard diet + 2% wt/wt SALmate (2% SALmate), and standard diet + 5% wt/wt SALmate (5% SALmate) (n = 12 birds/dietary treatment; Table 1). Titanium dioxide (5 g/kg) was added to each diet as a digestibility marker for the energy metabolism phase of the experiment. Birds were fed experimental diets from day of hatch and had ad libitum access to water and experimental feed throughout the trial period.
AME Study At 13 d posthatch, 48 chickens were transferred in pairs to 24 metabolism cages located in a temperaturecontrolled room. To reduce any stress associated with isolation, birds were placed in pairs for an initial accli-
mation period. On d 15, chickens were then placed one per cage in 48 cages based on a randomized block design for the energy metabolism phase of the experiment (n = 12 birds/dietary treatment). The AME values of the experimental diets were measured in a 7-d period between 15 and 22 d posthatch as described previously (Geier et al., 2009). Birds continued to consume the experimental diets until d 25.
Sample Collection and Preparation At 25 d posthatch, all birds were killed. One bird from the ZnB group and 1 bird from the 2% SALmate group were culled during the experimental period due to poor health. Breast tissue (5 g) samples were collected for fatty acid analysis. At kill, 1 cecum and 3-cm sections of tissue and associated digesta from the midpoints of the jejunum and ileum were collected from all birds (n = 12 birds/dietary treatment) and stored at 4°C for approximately 4 h until frozen at −20°C. Samples were freeze-dried and total nucleic acid was extracted using a modification (Torok et al., 2008) of a proprietary extraction method developed by the South Australian Research and Development Institute (Stirling et al., 2004). Total nucleic acid from samples was analyzed for total gut bacterial community composition by terminal RFLP analysis (T-RFLP) or specific lactobacilli community profiles by PCR-denaturing gradient gel electrophoresis (Lac PCR-DGGE).
Microbial Profiling T-RFLP Analysis. Terminal RFLP analysis was performed on samples from all birds (n = 12 birds/dietary treatment) according to the technique outlined previously (Torok et al., 2008). The universal 16S bacterial primers 27F (Lane, 1991) and 907R (Muyzer et al., 1995) were used to amplify bacterial rDNA. All PCR reactions were run in duplicate in a MJ Research PTC225 Peltier thermal cycler (GeneWorks, Adelaide, Australia). Specificity of PCR products was analyzed by gel electrophoresis on a 1.5% agarose gel and visualized after staining with ethidium bromide. The PCR products were quantified by fluorometry and 200 ng of PCR product was digested in duplicate with 2 U of MspI (Genesearch, Arundel, Australia) in recommended enzyme buffer. Completeness of digestion was confirmed by gel electrophoresis of a known positive control. The length of the fluorescently labeled terminal restriction fragments was determined from each sample in duplicate by comparison with an internal standard (Gene Scan 1200 LIZ, Applied Biosystems, Scoresby, Victoria, Australia) after separation by capillary electrophoresis (ABI 3730 automated DNA sequencer; Applied Biosystems) and data were analyzed using GeneMapper software (Applied Biosystems). Identification of operational taxonomic units was performed as described previously (Torok et al., 2008).
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Table 1. Composition of experimental diets Ingredient (g/kg) 3
Starter crumble Zinc bacitracin (50 mg/kg of active ingredient) SALmate4 Added tallow Milled wheat CP Nutrient specifications (% as fed) Crude fat ME (MJ/kg) Calcium Total phosphorus Available phosphorus Lysine Methionine Determined values5 (g/kg of diet) Total saturated FA6 Total trans FA 18:1n-9 Total monounsaturated FA 18:2n-6 20:4n-6 Total n-6 18:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 Total polyunsaturated FA
Control
ZnB2
2% SALmate
5% SALmate
950.0 — — 21.0 29.0 20.27
949.7 0.3 — 21.0 29.0 20.27
950.0 — 20.0 12.6 17.4 20.13
950.0 — 50.0 — — 19.91
5.57 12.63 1.01 0.75 0.50 1.13 0.48
5.57 12.63 1.01 0.75 0.50 1.13 0.48
5.56 12.88 1.01 0.75 0.50 1.13 0.48
5.54 13.26 1.01 0.74 0.50 1.12 0.47
25.88 1.43 20.65 24.69 17.25 0.15 17.52 1.23 0.04 0.04 — 1.52 19.04
23.77 1.33 19.06 22.80 15.62 0.14 15.88 1.13 0.03 0.06 0.06 1.48 17.36
24.45 1.21 19.50 25.06 16.51 0.17 16.82 1.16 0.44 0.21 0.48 2.50 19.32
22.37 1.05 17.92 25.57 17.36 0.20 17.72 1.16 0.98 0.40 1.05 3.75 21.47
1
Birds were fed these experimental diets from d 0 to 15. From d 15 to 25, this diet was supplemented with 5.0 g/kg of titanium dioxide at the expense of starter crumble. 2 ZnB = zinc bacitracin. 3 Starter crumble was composed of (in g/kg): wheat (638.9), hammer-milled wheat (8.0), oats (50.0), peas (60.0), meat meal (100.0), blood meal (10.67), tallow (10.0), solvent extracted soybean meal (93.67), ground oatmeal flour (16.67), monocalcium phosphate (3.28), dicalcium phosphate dehydrate (1.09), choline chloride 75% (0.50), l-lysine HCl (2.14), Alimet (2.57, Novus International, St. Louis, MO), biotin (0.50). Vitamin and mineral mixes (2.00) exceeded NRC standards. 4 SALmate (Feedworks, Romsey, Victoria, Australia) was composed of 42% fish oil and 58% starch. 5 Determined values are means of 2 replicates. 6 FA = fatty acid.
Denaturing Gradient Gel Electrophoresis. Lactobacillus species were analyzed from all birds (n = 12 birds/dietary treatment) and on pooled samples from each diet (n = 1 sample/diet) using Lac PCR-DGGE as described previously (Walter et al., 2001). The V3 region of the 16S rDNA was amplified from the total DNA using the group-specific bacterial primers Lac1 and Lac2-GC in a Cool Gradient Palm Cycler 9600 (Corbett Research, Sydney, Australia). Individual or pooled DNA was used as template. Pooled samples were prepared by combining an equal amount of DNA from each relevant intestinal section of all 12 birds per diet. The PCR products were subjected to Lac PCR-DGGE using the Bio-Rad DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA) as described previously (Walter et al., 2001). Denaturing gradient gel electrophoresis identification ladders were prepared by combining the Lactobacillus PCR products from DNA extracted from reference Lactobacillus strains (Lactobacillus acidophilus ATCC 4356, Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, Lactobacillus johnsonii ATCC 33200, Lactobacillus reuteri ATCC 23272, and Lactobacillus salivarius ssp. sali-
varius ATCC 11741). Gels were stained with ethidium bromide and viewed by UV transillumination.
Statistical Analysis Performance data were analyzed using the SAS for Windows version 9.1 software package (SAS Institute Inc., Cary, NC). Data were compared by ANOVA using the GLM procedure with differences between diets determined by Duncan’s multiple range test. Fatty acid levels were compared by nonparametric Kruskal-Wallis ANOVA. Comparisons of intestinal microbial communities between diets and identification of bacterial species contributing to the observed differences were performed using the PRIMER-6 software package (PRIMER-E Ltd., Plymouth, UK). Individual T-RFLP and Lac PCR-DGGE data were analyzed according to the previously described procedures (Torok et al., 2008). BrayCurtis measures of similarity (Bray and Curtis, 1957) were calculated to indicate similarities between intestinal microbial communities of individual birds from the T-RFLP-generated (operational taxonomic units) data after standardization and fourth root transformation
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and the presence-absence data from Lac PCR-DGGE analysis, as scored against the Lactobacillus reference strains. One-way analysis of similarity (Clarke, 1993) was employed to determine if intestinal microbial communities were significantly different among diets for TRFLP and Lac PCR-DGGE data. Similarity percentage analyses (Clarke, 1993) were performed to indicate which Lactobacillus species had contributed to the dissimilarity among diets. Dice’s similarity coefficient based on the unweighted pair group methods using arithmetic averages clustering algorithm was calculated to compare pooled Lac PCR-DGGE profiles using the BioNumerics software package (Applied Maths, SintMartens-Latem, Belgium). For all comparisons, P < 0.05 was considered significant.
RESULTS AME Study No differences in BW gain, feed intake, feed conversion ratio, or ileal digestible energy were observed among diets during the 7-d AME study period (P > 0.05; our unpublished data). The AME values for birds fed the ZnB diet were significantly greater than all of the other diets (P < 0.05). No significant differences were observed in AME values among control, 2% SALmate, and 5% SALmate diets (P > 0.05).
PUFA Analysis of Breast Tissue As observed previously, fatty acid levels in the breast tissue were influenced significantly by diet (our unpublished data). Breast tissue levels of EPA, DHA, docosapentaenoic acid (DPA), and total n-3 PUFA were significantly greater in 2% SALmate- and 5% SALmate-fed chickens compared with birds fed the ZnB or control diet (P < 0.05). Chickens fed the 5% SALmate diet also had a greater incorporation of EPA and DHA into the breast tissue compared with chickens fed 2% SALmate (P < 0.05), with a trend toward an increase in DPA and total n-3 PUFA (P < 0.06).
Microbial Profiling T-RFLP Analysis. No significant differences were observed in the overall microbial communities of the ileum (global R = 0.012, P = 0.587) and cecum (global R = 0.027, P = 0.233) among the 4 diets (our unpublished data). The T-RFLP analysis was performed to profile the overall microbial communities in the ileum and cecum. Denaturing Gradient Gel Electrophoresis. Lactobacillus species were analyzed by comparing pooled (Figures 1a and 1b) and individual (Table 2) Lac PCRDGGE profiles. The pooled jejunal and ileal profiles of birds fed 2% SALmate and 5% SALmate and the cecal profile of birds fed 5% SALmate clustered together and consisted of L. salivarius and L. crispatus, Lactobacillus
gallinarum, and Lactobacillus amylovorus. The latter 3 species belong to the group A acidophilus taxonomic group, which cannot be distinguished using Lac PCRDGGE (Guan et al., 2003) and will be referred to here collectively as LCGA. The consensus cecal profile of birds fed 2% SALmate was characterized by a dominant band co-migrating with LCGA. The pooled profiles of all intestinal sections of birds fed ZnB and control diets contained L. johnsonii in addition to L. salivarius or LCGA, or both. When comparing individual Lactobacillus profiles, cecal profiles of birds fed the control and 2% SALmate diet were significantly different (P < 0.05; Table 2). The Lactobacillus profiles of birds fed ZnB or 5% SALmate did not differ compared with control-fed birds (P > 0.05). Similarity percentage analysis indicated that L. johnsonii contributed significantly to the overall shift in cecal Lactobacillus profiles, with L. johnsonii detected in 8 out of 12 birds fed the control diet versus 2 out of 11 birds fed the 2% SALmate diet. No significant differences in Lactobacillus profiles were observed in the jejunum (global R = 0.004, P = 0.393) or ileum (global R = 0.020, P = 0.224).
DISCUSSION The importance of the intestinal microbiota to the health of its host is well documented in numerous species including human (Guarner and Malagelada, 2003; O’Hara and Shanahan, 2006) and avian (Patterson and Burkholder, 2003). Unfavorable shifts in the intestinal microbial balance can lead to various bacteria-associated disease conditions, and importantly to the poultry industry, it has recently been reported that shifts in the microbiota can be linked with bird metabolism (Torok et al., 2008). Because diet can significantly influence the microbial composition (Owens et al., 2008; Geier et al., 2009), it was important to characterize the effects of candidate feed additives on the intestinal microbiota. Previously, a small number of studies have reported n-3 PUFA-mediated effects on the intestinal microbiota. Fish oil supplementation has been demonstrated to decrease Bacteroidaceae and increase Bifidobacteriaceae numbers in rats compared with a standard chow diet (Hekmatdoost et al., 2008). In addition, Kankaanpaa and colleagues reported that DHA decreased the in vitro growth of Lactobacillus casei Shirota and reduced the adhesion of L. casei Shirota, Lactobacillus GG, and Lactobacillus bulgaricus (Kankaanpaa et al., 2001), indicating that n-3 PUFA may have the capacity to influence the intestinal microbiota in vivo. Similar in vitro reports of the growth inhibitory properties of n-3 PUFA toward bacterial isolates have been described previously (Thompson and Spiller, 1995). The current study indicated that the inclusion of SALmate did not significantly influence the overall intestinal microbiota. Minor changes were observed in the Lactobacillus species profiles of the ceca between chickens in the control and 2% SALmate treatment groups; however, this was
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not evident in the 5% SALmate-fed birds, indicating an inconsistent or possible dose-dependent effect of n-3 PUFA. In the ileum, where Lactobacillus species are a dominant genus (Lu et al., 2003), no differences in profiles were observed. Previously, n-3 PUFA have been shown to influence fatty acid composition, adhesion characteristics, and growth of lactobacilli (Kankaanpaa et al., 2001, 2004); this may influence their capacity to colonize within the intestine. Based on T-RFLP and Lac PCR-DGGE, we can conclude that SALmate has little influence on the overall intestinal microbiota and Lactobacillus profiles. The observed shifts in the Lac-
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tobacillus species profiles did not convey any performance-enhancing effects in the current study. Although it has previously been demonstrated that shifts in the microbiota can be linked to performance differences in some instances (Torok et al., 2008), a recent study by Geier and colleagues suggested that significant diet-induced microbial shifts do not necessarily influence bird performance (Geier et al., 2009). The nature and extent of the microbial shifts that facilitate improved bird performance need to be identified, and feeding regimens to promote the colonization of an optimal microbiota need to be developed.
Figure 1. Lactobacillus PCR-denaturing gradient gel electrophoresis profiles of pooled samples for jejunum (J), ileum (I), and ceca (C) of each diet (n = 1 sample/dietary treatment; top) and analysis using Dice’s similarity (bottom). The marker lane (M) contains the identification ladder composed of Lactobacillus PCR products from the following reference strains: Lj = Lactobacillus johnsonii ATCC 33200; Lg = Lactobacillus gasseri ATCC 33323; Lac = Lactobacillus acidophilus ATCC 4356; Ls = Lactobacillus salivarius ssp. salivarius ATCC 11741; and Lr = Lactobacillus reuteri ATCC 23272. LCGA marks the migration of Lactobacillus crispatus, Lactobacillus gallinarum, and Lactobacillus amylovorus species and is represented by L. crispatus ATCC 33820. 0% = control diet; ZnB = control diet + 50 mg/kg of zinc bacitracin; 2% = control diet + 2% SALmate (Feedworks, Romsey, Victoria, Australia); and 5% = control diet + 5% SALmate.
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Table 2. One-way ANOSIM of cecal Lactobacillus species between diets2 Item Control ZnB 2% SALmate4 5% SALmate
Control
ZnB3
2% SALmate
5% SALmate
— 0.170 0.008 0.132
0.071 — 0.399 0.214
0.170 0.003 — 0.344
0.048 0.054 0.009 —
1
ANOSIM = analysis of similarity. Data are expressed as the R-statistic (above the diagonal), with significance level (P) below the diagonal. The R-statistic value indicates the extent of similarity between each pair in the ANOSIM. Values approaching unity indicate that the 2 groups are entirely separate, and a zero value indicates that there is no difference between groups. For all analyses, P < 0.05 was considered significant. For cecal microbial communities, the global R-value was 0.066 at a significance level of 0.042. n = 12 per diet. 3 ZnB = zinc bacitracin. 4 SALmate (Feedworks, Romsey, Victoria, Australia) was composed of 42% fish oil and 58% starch. 2
Terminal RFLP analysis and Lac PCR-DGGE have been used in combination in the current study to provide a high-throughput means to characterize the intestinal microbiota. These powerful, molecular-based approaches have previously been employed simultaneously to provide a snapshot of this complex environment (Geier et al., 2009). Interestingly, the current study indicated that there was little difference in the microbial populations of the intestine between birds fed diets with and without ZnB. Previously, using the same techniques, it has been reported that ZnB significantly influenced ileal bacterial communities (Geier et al., 2009), whereas numerous studies have described antibiotic-mediated shifts in intestinal bacterial profiles (Engberg et al., 2000; Knarreborg et al., 2002; Wise and Siragusa, 2007). These conflicting results may suggest significant flock variation in susceptibility of the intestinal microbial communities to antibiotics. Together, these studies indicate that further research into the intestinal microbiota of the chicken and the influence of dietary components on microbial composition is needed. In the current study, we observed no n-3 PUFAinduced effects on growth or performance. Previous studies have reported variable effects of fish oil on performance parameters (Lopez-Ferrer et al., 1999, 2001; Schreiner et al., 2005). The lack of an effect of fish oil in the current study may be attributed to differences in fish oil source and composition, dietary fat levels, or diet composition. In summary, in light of recent bans and pressure for withdrawal of antibiotics from poultry feed, a range of natural feed additives such as n-3 PUFA, prebiotics, probiotics, and plant extracts are being assessed for their capacity to influence the health status of chickens and subsequently improve growth and performance. In the current study, n-3 PUFA supplementation did not influence performance but did significantly increase the incorporation of EPA, DHA, and DPA in the breast tissue. Additionally, n-3 PUFA had little effect on the intestinal microbiota. Previous studies have indicated that n-3 PUFA may prove useful as a natural feed additive with immunomodulatory and human health-promoting effects due to increased n-3 levels of chicken meat; however, it does not appear
that n-3 PUFA alone could replicate the effects of antibiotics.
ACKNOWLEDGMENTS This project was supported by the Australian Poultry Cooperative Research Centre (Project 05-2). We thank Derek Schultz (South Australian Research and Development Institute), Evelyn Daniels (South Australian Research and Development Institute), Gabrielle Brooke (Pork Cooperative Research Centre, University of Adelaide), and Rebecca Forder (University of Adelaide) for assistance with animal handling and tissue collection. We also thank Kylee Swanson (South Australian Research and Development Institute) for tissue collection and measurement of ileal digestibility, Teresa Mammone (South Australian Research and Development Institute) for T-RFLP analysis, and Roxanne Portolesi (University of Adelaide) and David Apps (University of Adelaide) for PUFA analysis.
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Owens, B., L. Tucker, M. A. Collins, and K. J. McCracken. 2008. Effects of different feed additives alone or in combination on broiler performance, gut microflora and ileal histology. Br. Poult. Sci. 49:202–212. Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci. 82:627– 631. Schreiner, M., H. W. Hulan, R.-F. Ebrahim, J. Bohm, and R. G. Moreira. 2005. Effect of different sources of dietary omega-3 fatty acids on general performance and fatty acid profiles of thigh, breast, liver and portal blood of broilers. J. Sci. Food Agric. 85:219–226. Stirling, G. R., D. Griffin, K. Ophel-Keller, A. McKay, D. Hartley, J. Curran, A. M. Stirling, C. Monsour, J. Winch, and B. Hardie. 2004. Combining an initial risk assessment process with DNA assays to improve prediction of soilborne diseases caused by rootknot nematode (Meloidogyne spp.) and Fusarium oxysporum f. sp. lycopersici in the Queensland tomato industry. Australas. Plant Pathol. 33:285–293. Thompson, L., and R. C. Spiller. 1995. Impact of polyunsaturated fatty acids on human colonic bacterial metabolism: An in vitro and in vivo study. Br. J. Nutr. 74:733–741. Torok, V. A., K. Ophel-Keller, M. Loo, and R. J. Hughes. 2008. Gut microbiota and performance in broiler chickens: Application of methods for identifying bacterial species linked with increased energy metabolism. Appl. Environ. Microbiol. 74:783–791. Walter, J., C. Hertel, G. W. Tannock, C. M. Lis, K. Munro, and W. P. Hammes. 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using groupspecific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67:2578–2585. Wang, Y. W., C. J. Field, and J. S. Sim. 2000. Dietary polyunsaturated fatty acids alter lymphocyte subset proportion and proliferation, serum immunoglobulin G concentration, and immune tissue development in chicks. Poult. Sci. 79:1741–1748. Wise, M. G., and G. R. Siragusa. 2007. Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets. J. Appl. Microbiol. 102:1138–1149. Yang, Y., P. A. Iji, and M. Choct. 2009. Dietary modulation of gut microflora in broiler chickens: A review of the role of six kinds of alternatives to in-feed antibiotics. World’s Poult. Sci. J. 65:97–114.
Breast tissue levels of eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, and total n-3 PUFA were elevated significantly in 2% SALmate- and 5% SALmate-fed chickens compared with control and ZnB diets (P < 0.05). No significant differences in overall microbial communities were observed in the ileum or cecum as assessed by terminal RFLP (P > 0.05). Birds fed 2% SALmate had a significantly different cecal Lactobacillus species profile compared with birds fed the control diet (P < 0.05); however, no differences were observed in birds fed 5% SALmate compared with birds fed all other diets. In addition to the expected increase in breast tissue n-3 fatty acid levels, a low level of dietary n-3 PUFA also altered the intestinal Lactobacillus species profiles. However, n-3 PUFA supplementation did not alter the overall microbial communities or broiler performance.
Key words: broiler, denaturing gradient gel electrophoresis, polyunsaturated fatty acid, terminal restriction fragment length polymorphism 2009 Poultry Science 88:2399–2405 doi:10.3382/ps.2009-00126
INTRODUCTION Supplemental dietary antibiotics are used in poultry production to improve the growth and performance of broiler chickens and to reduce the incidence of infectious diseases. However, the risk of the development of antibiotic-resistant bacteria as a result of long-term antibiotic exposure led to their ban in the European Union in 2006. Other regions have not yet implement©2009 Poultry Science Association Inc. Received March 10, 2009. Accepted July 15, 2009. 1 Corresponding author: [email protected]
ed such a ban; however, consumer and environmental pressure may lead to future changes in legislation. Debate continues as to the exact mechanisms of action of antibiotics, with antimicrobial and antiinflammatory mechanisms postulated (Niewold, 2007). The need for natural antibiotic alternatives has led to the investigation of a wide range of compounds including probiotics, prebiotics, and plant extracts (Yang et al., 2009). The immunomodulatory and antiinflammatory properties of n-3 polyunsaturated fatty acids (PUFA) suggest a possible role as a replacement for antibiotics. The health-promoting effects of n-3 PUFA are well documented in humans (Calder, 2006). In poultry, the consumption of diets high in fish-derived n-3 PUFA,
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particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been demonstrated to improve BW gain (Lopez-Ferrer et al., 2001; Schreiner et al., 2005), reduce the inflammatory response (Korver and Klasing, 1997), and alter immune parameters including antibody production and lymphocyte proportion and responsiveness (Wang et al., 2000). It has also been reported that n-3 PUFA influence intestinal bacteria in vitro and in vivo (Kankaanpaa et al., 2001; Hekmatdoost et al., 2008). The aim of the current study was to compare the effects of dietary fish oil on the composition of the intestinal microbial communities, with particular focus on Lactobacillus species due to their known healthpromoting properties. Additionally, broiler growth and production traits and the fatty acid composition of breast tissue were studied.
MATERIALS AND METHODS Birds, Management, and Diets Forty-eight male Cobb 500 broiler chickens (Baiada Hatchery, Willaston, Australia) were raised in 4 floor pens with fresh litter in a temperature-controlled room (22 to 27°C). All birds were vaccinated with Eimeriavax 4m (Bioproperties Pty Ltd., Glenorie, New South Wales, Australia) at placement and at 5 and 11 d posthatch. All procedures were approved by the Animal Ethics Committees of the University of Adelaide and the Department of Primary Industries and Resources South Australia, Adelaide. All diets were based on a standard commercial starter diet (Ridley Agriproducts Pty Ltd., Murray Bridge, Australia) without any added antibiotics or coccidiostats (Table 1). All diets met or exceeded NRC guidelines for broiler chickens (NRC, 1994). The source of n-3 PUFA was SALmate (Feedworks, Romsey, Victoria, Australia), which is composed of 42% fish oil and 58% starch. The 4 experimental diets were as follows: standard diet with no additives (control), standard diet + 50 mg/kg of zinc bacitracin (ZnB), standard diet + 2% wt/wt SALmate (2% SALmate), and standard diet + 5% wt/wt SALmate (5% SALmate) (n = 12 birds/dietary treatment; Table 1). Titanium dioxide (5 g/kg) was added to each diet as a digestibility marker for the energy metabolism phase of the experiment. Birds were fed experimental diets from day of hatch and had ad libitum access to water and experimental feed throughout the trial period.
AME Study At 13 d posthatch, 48 chickens were transferred in pairs to 24 metabolism cages located in a temperaturecontrolled room. To reduce any stress associated with isolation, birds were placed in pairs for an initial accli-
mation period. On d 15, chickens were then placed one per cage in 48 cages based on a randomized block design for the energy metabolism phase of the experiment (n = 12 birds/dietary treatment). The AME values of the experimental diets were measured in a 7-d period between 15 and 22 d posthatch as described previously (Geier et al., 2009). Birds continued to consume the experimental diets until d 25.
Sample Collection and Preparation At 25 d posthatch, all birds were killed. One bird from the ZnB group and 1 bird from the 2% SALmate group were culled during the experimental period due to poor health. Breast tissue (5 g) samples were collected for fatty acid analysis. At kill, 1 cecum and 3-cm sections of tissue and associated digesta from the midpoints of the jejunum and ileum were collected from all birds (n = 12 birds/dietary treatment) and stored at 4°C for approximately 4 h until frozen at −20°C. Samples were freeze-dried and total nucleic acid was extracted using a modification (Torok et al., 2008) of a proprietary extraction method developed by the South Australian Research and Development Institute (Stirling et al., 2004). Total nucleic acid from samples was analyzed for total gut bacterial community composition by terminal RFLP analysis (T-RFLP) or specific lactobacilli community profiles by PCR-denaturing gradient gel electrophoresis (Lac PCR-DGGE).
Microbial Profiling T-RFLP Analysis. Terminal RFLP analysis was performed on samples from all birds (n = 12 birds/dietary treatment) according to the technique outlined previously (Torok et al., 2008). The universal 16S bacterial primers 27F (Lane, 1991) and 907R (Muyzer et al., 1995) were used to amplify bacterial rDNA. All PCR reactions were run in duplicate in a MJ Research PTC225 Peltier thermal cycler (GeneWorks, Adelaide, Australia). Specificity of PCR products was analyzed by gel electrophoresis on a 1.5% agarose gel and visualized after staining with ethidium bromide. The PCR products were quantified by fluorometry and 200 ng of PCR product was digested in duplicate with 2 U of MspI (Genesearch, Arundel, Australia) in recommended enzyme buffer. Completeness of digestion was confirmed by gel electrophoresis of a known positive control. The length of the fluorescently labeled terminal restriction fragments was determined from each sample in duplicate by comparison with an internal standard (Gene Scan 1200 LIZ, Applied Biosystems, Scoresby, Victoria, Australia) after separation by capillary electrophoresis (ABI 3730 automated DNA sequencer; Applied Biosystems) and data were analyzed using GeneMapper software (Applied Biosystems). Identification of operational taxonomic units was performed as described previously (Torok et al., 2008).
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Table 1. Composition of experimental diets Ingredient (g/kg) 3
Starter crumble Zinc bacitracin (50 mg/kg of active ingredient) SALmate4 Added tallow Milled wheat CP Nutrient specifications (% as fed) Crude fat ME (MJ/kg) Calcium Total phosphorus Available phosphorus Lysine Methionine Determined values5 (g/kg of diet) Total saturated FA6 Total trans FA 18:1n-9 Total monounsaturated FA 18:2n-6 20:4n-6 Total n-6 18:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 Total polyunsaturated FA
Control
ZnB2
2% SALmate
5% SALmate
950.0 — — 21.0 29.0 20.27
949.7 0.3 — 21.0 29.0 20.27
950.0 — 20.0 12.6 17.4 20.13
950.0 — 50.0 — — 19.91
5.57 12.63 1.01 0.75 0.50 1.13 0.48
5.57 12.63 1.01 0.75 0.50 1.13 0.48
5.56 12.88 1.01 0.75 0.50 1.13 0.48
5.54 13.26 1.01 0.74 0.50 1.12 0.47
25.88 1.43 20.65 24.69 17.25 0.15 17.52 1.23 0.04 0.04 — 1.52 19.04
23.77 1.33 19.06 22.80 15.62 0.14 15.88 1.13 0.03 0.06 0.06 1.48 17.36
24.45 1.21 19.50 25.06 16.51 0.17 16.82 1.16 0.44 0.21 0.48 2.50 19.32
22.37 1.05 17.92 25.57 17.36 0.20 17.72 1.16 0.98 0.40 1.05 3.75 21.47
1
Birds were fed these experimental diets from d 0 to 15. From d 15 to 25, this diet was supplemented with 5.0 g/kg of titanium dioxide at the expense of starter crumble. 2 ZnB = zinc bacitracin. 3 Starter crumble was composed of (in g/kg): wheat (638.9), hammer-milled wheat (8.0), oats (50.0), peas (60.0), meat meal (100.0), blood meal (10.67), tallow (10.0), solvent extracted soybean meal (93.67), ground oatmeal flour (16.67), monocalcium phosphate (3.28), dicalcium phosphate dehydrate (1.09), choline chloride 75% (0.50), l-lysine HCl (2.14), Alimet (2.57, Novus International, St. Louis, MO), biotin (0.50). Vitamin and mineral mixes (2.00) exceeded NRC standards. 4 SALmate (Feedworks, Romsey, Victoria, Australia) was composed of 42% fish oil and 58% starch. 5 Determined values are means of 2 replicates. 6 FA = fatty acid.
Denaturing Gradient Gel Electrophoresis. Lactobacillus species were analyzed from all birds (n = 12 birds/dietary treatment) and on pooled samples from each diet (n = 1 sample/diet) using Lac PCR-DGGE as described previously (Walter et al., 2001). The V3 region of the 16S rDNA was amplified from the total DNA using the group-specific bacterial primers Lac1 and Lac2-GC in a Cool Gradient Palm Cycler 9600 (Corbett Research, Sydney, Australia). Individual or pooled DNA was used as template. Pooled samples were prepared by combining an equal amount of DNA from each relevant intestinal section of all 12 birds per diet. The PCR products were subjected to Lac PCR-DGGE using the Bio-Rad DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA) as described previously (Walter et al., 2001). Denaturing gradient gel electrophoresis identification ladders were prepared by combining the Lactobacillus PCR products from DNA extracted from reference Lactobacillus strains (Lactobacillus acidophilus ATCC 4356, Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, Lactobacillus johnsonii ATCC 33200, Lactobacillus reuteri ATCC 23272, and Lactobacillus salivarius ssp. sali-
varius ATCC 11741). Gels were stained with ethidium bromide and viewed by UV transillumination.
Statistical Analysis Performance data were analyzed using the SAS for Windows version 9.1 software package (SAS Institute Inc., Cary, NC). Data were compared by ANOVA using the GLM procedure with differences between diets determined by Duncan’s multiple range test. Fatty acid levels were compared by nonparametric Kruskal-Wallis ANOVA. Comparisons of intestinal microbial communities between diets and identification of bacterial species contributing to the observed differences were performed using the PRIMER-6 software package (PRIMER-E Ltd., Plymouth, UK). Individual T-RFLP and Lac PCR-DGGE data were analyzed according to the previously described procedures (Torok et al., 2008). BrayCurtis measures of similarity (Bray and Curtis, 1957) were calculated to indicate similarities between intestinal microbial communities of individual birds from the T-RFLP-generated (operational taxonomic units) data after standardization and fourth root transformation
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and the presence-absence data from Lac PCR-DGGE analysis, as scored against the Lactobacillus reference strains. One-way analysis of similarity (Clarke, 1993) was employed to determine if intestinal microbial communities were significantly different among diets for TRFLP and Lac PCR-DGGE data. Similarity percentage analyses (Clarke, 1993) were performed to indicate which Lactobacillus species had contributed to the dissimilarity among diets. Dice’s similarity coefficient based on the unweighted pair group methods using arithmetic averages clustering algorithm was calculated to compare pooled Lac PCR-DGGE profiles using the BioNumerics software package (Applied Maths, SintMartens-Latem, Belgium). For all comparisons, P < 0.05 was considered significant.
RESULTS AME Study No differences in BW gain, feed intake, feed conversion ratio, or ileal digestible energy were observed among diets during the 7-d AME study period (P > 0.05; our unpublished data). The AME values for birds fed the ZnB diet were significantly greater than all of the other diets (P < 0.05). No significant differences were observed in AME values among control, 2% SALmate, and 5% SALmate diets (P > 0.05).
PUFA Analysis of Breast Tissue As observed previously, fatty acid levels in the breast tissue were influenced significantly by diet (our unpublished data). Breast tissue levels of EPA, DHA, docosapentaenoic acid (DPA), and total n-3 PUFA were significantly greater in 2% SALmate- and 5% SALmate-fed chickens compared with birds fed the ZnB or control diet (P < 0.05). Chickens fed the 5% SALmate diet also had a greater incorporation of EPA and DHA into the breast tissue compared with chickens fed 2% SALmate (P < 0.05), with a trend toward an increase in DPA and total n-3 PUFA (P < 0.06).
Microbial Profiling T-RFLP Analysis. No significant differences were observed in the overall microbial communities of the ileum (global R = 0.012, P = 0.587) and cecum (global R = 0.027, P = 0.233) among the 4 diets (our unpublished data). The T-RFLP analysis was performed to profile the overall microbial communities in the ileum and cecum. Denaturing Gradient Gel Electrophoresis. Lactobacillus species were analyzed by comparing pooled (Figures 1a and 1b) and individual (Table 2) Lac PCRDGGE profiles. The pooled jejunal and ileal profiles of birds fed 2% SALmate and 5% SALmate and the cecal profile of birds fed 5% SALmate clustered together and consisted of L. salivarius and L. crispatus, Lactobacillus
gallinarum, and Lactobacillus amylovorus. The latter 3 species belong to the group A acidophilus taxonomic group, which cannot be distinguished using Lac PCRDGGE (Guan et al., 2003) and will be referred to here collectively as LCGA. The consensus cecal profile of birds fed 2% SALmate was characterized by a dominant band co-migrating with LCGA. The pooled profiles of all intestinal sections of birds fed ZnB and control diets contained L. johnsonii in addition to L. salivarius or LCGA, or both. When comparing individual Lactobacillus profiles, cecal profiles of birds fed the control and 2% SALmate diet were significantly different (P < 0.05; Table 2). The Lactobacillus profiles of birds fed ZnB or 5% SALmate did not differ compared with control-fed birds (P > 0.05). Similarity percentage analysis indicated that L. johnsonii contributed significantly to the overall shift in cecal Lactobacillus profiles, with L. johnsonii detected in 8 out of 12 birds fed the control diet versus 2 out of 11 birds fed the 2% SALmate diet. No significant differences in Lactobacillus profiles were observed in the jejunum (global R = 0.004, P = 0.393) or ileum (global R = 0.020, P = 0.224).
DISCUSSION The importance of the intestinal microbiota to the health of its host is well documented in numerous species including human (Guarner and Malagelada, 2003; O’Hara and Shanahan, 2006) and avian (Patterson and Burkholder, 2003). Unfavorable shifts in the intestinal microbial balance can lead to various bacteria-associated disease conditions, and importantly to the poultry industry, it has recently been reported that shifts in the microbiota can be linked with bird metabolism (Torok et al., 2008). Because diet can significantly influence the microbial composition (Owens et al., 2008; Geier et al., 2009), it was important to characterize the effects of candidate feed additives on the intestinal microbiota. Previously, a small number of studies have reported n-3 PUFA-mediated effects on the intestinal microbiota. Fish oil supplementation has been demonstrated to decrease Bacteroidaceae and increase Bifidobacteriaceae numbers in rats compared with a standard chow diet (Hekmatdoost et al., 2008). In addition, Kankaanpaa and colleagues reported that DHA decreased the in vitro growth of Lactobacillus casei Shirota and reduced the adhesion of L. casei Shirota, Lactobacillus GG, and Lactobacillus bulgaricus (Kankaanpaa et al., 2001), indicating that n-3 PUFA may have the capacity to influence the intestinal microbiota in vivo. Similar in vitro reports of the growth inhibitory properties of n-3 PUFA toward bacterial isolates have been described previously (Thompson and Spiller, 1995). The current study indicated that the inclusion of SALmate did not significantly influence the overall intestinal microbiota. Minor changes were observed in the Lactobacillus species profiles of the ceca between chickens in the control and 2% SALmate treatment groups; however, this was
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not evident in the 5% SALmate-fed birds, indicating an inconsistent or possible dose-dependent effect of n-3 PUFA. In the ileum, where Lactobacillus species are a dominant genus (Lu et al., 2003), no differences in profiles were observed. Previously, n-3 PUFA have been shown to influence fatty acid composition, adhesion characteristics, and growth of lactobacilli (Kankaanpaa et al., 2001, 2004); this may influence their capacity to colonize within the intestine. Based on T-RFLP and Lac PCR-DGGE, we can conclude that SALmate has little influence on the overall intestinal microbiota and Lactobacillus profiles. The observed shifts in the Lac-
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tobacillus species profiles did not convey any performance-enhancing effects in the current study. Although it has previously been demonstrated that shifts in the microbiota can be linked to performance differences in some instances (Torok et al., 2008), a recent study by Geier and colleagues suggested that significant diet-induced microbial shifts do not necessarily influence bird performance (Geier et al., 2009). The nature and extent of the microbial shifts that facilitate improved bird performance need to be identified, and feeding regimens to promote the colonization of an optimal microbiota need to be developed.
Figure 1. Lactobacillus PCR-denaturing gradient gel electrophoresis profiles of pooled samples for jejunum (J), ileum (I), and ceca (C) of each diet (n = 1 sample/dietary treatment; top) and analysis using Dice’s similarity (bottom). The marker lane (M) contains the identification ladder composed of Lactobacillus PCR products from the following reference strains: Lj = Lactobacillus johnsonii ATCC 33200; Lg = Lactobacillus gasseri ATCC 33323; Lac = Lactobacillus acidophilus ATCC 4356; Ls = Lactobacillus salivarius ssp. salivarius ATCC 11741; and Lr = Lactobacillus reuteri ATCC 23272. LCGA marks the migration of Lactobacillus crispatus, Lactobacillus gallinarum, and Lactobacillus amylovorus species and is represented by L. crispatus ATCC 33820. 0% = control diet; ZnB = control diet + 50 mg/kg of zinc bacitracin; 2% = control diet + 2% SALmate (Feedworks, Romsey, Victoria, Australia); and 5% = control diet + 5% SALmate.
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Table 2. One-way ANOSIM of cecal Lactobacillus species between diets2 Item Control ZnB 2% SALmate4 5% SALmate
Control
ZnB3
2% SALmate
5% SALmate
— 0.170 0.008 0.132
0.071 — 0.399 0.214
0.170 0.003 — 0.344
0.048 0.054 0.009 —
1
ANOSIM = analysis of similarity. Data are expressed as the R-statistic (above the diagonal), with significance level (P) below the diagonal. The R-statistic value indicates the extent of similarity between each pair in the ANOSIM. Values approaching unity indicate that the 2 groups are entirely separate, and a zero value indicates that there is no difference between groups. For all analyses, P < 0.05 was considered significant. For cecal microbial communities, the global R-value was 0.066 at a significance level of 0.042. n = 12 per diet. 3 ZnB = zinc bacitracin. 4 SALmate (Feedworks, Romsey, Victoria, Australia) was composed of 42% fish oil and 58% starch. 2
Terminal RFLP analysis and Lac PCR-DGGE have been used in combination in the current study to provide a high-throughput means to characterize the intestinal microbiota. These powerful, molecular-based approaches have previously been employed simultaneously to provide a snapshot of this complex environment (Geier et al., 2009). Interestingly, the current study indicated that there was little difference in the microbial populations of the intestine between birds fed diets with and without ZnB. Previously, using the same techniques, it has been reported that ZnB significantly influenced ileal bacterial communities (Geier et al., 2009), whereas numerous studies have described antibiotic-mediated shifts in intestinal bacterial profiles (Engberg et al., 2000; Knarreborg et al., 2002; Wise and Siragusa, 2007). These conflicting results may suggest significant flock variation in susceptibility of the intestinal microbial communities to antibiotics. Together, these studies indicate that further research into the intestinal microbiota of the chicken and the influence of dietary components on microbial composition is needed. In the current study, we observed no n-3 PUFAinduced effects on growth or performance. Previous studies have reported variable effects of fish oil on performance parameters (Lopez-Ferrer et al., 1999, 2001; Schreiner et al., 2005). The lack of an effect of fish oil in the current study may be attributed to differences in fish oil source and composition, dietary fat levels, or diet composition. In summary, in light of recent bans and pressure for withdrawal of antibiotics from poultry feed, a range of natural feed additives such as n-3 PUFA, prebiotics, probiotics, and plant extracts are being assessed for their capacity to influence the health status of chickens and subsequently improve growth and performance. In the current study, n-3 PUFA supplementation did not influence performance but did significantly increase the incorporation of EPA, DHA, and DPA in the breast tissue. Additionally, n-3 PUFA had little effect on the intestinal microbiota. Previous studies have indicated that n-3 PUFA may prove useful as a natural feed additive with immunomodulatory and human health-promoting effects due to increased n-3 levels of chicken meat; however, it does not appear
that n-3 PUFA alone could replicate the effects of antibiotics.
ACKNOWLEDGMENTS This project was supported by the Australian Poultry Cooperative Research Centre (Project 05-2). We thank Derek Schultz (South Australian Research and Development Institute), Evelyn Daniels (South Australian Research and Development Institute), Gabrielle Brooke (Pork Cooperative Research Centre, University of Adelaide), and Rebecca Forder (University of Adelaide) for assistance with animal handling and tissue collection. We also thank Kylee Swanson (South Australian Research and Development Institute) for tissue collection and measurement of ileal digestibility, Teresa Mammone (South Australian Research and Development Institute) for T-RFLP analysis, and Roxanne Portolesi (University of Adelaide) and David Apps (University of Adelaide) for PUFA analysis.
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