animal
Animal, page 1 of 11 © The Animal Consortium 2014 doi:10.1017/S1751731114000536
Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract D. Józefiak1†, B. Kierończyk1, M. Rawski1, M. Hejdysz1, A. Rutkowski1, R. M. Engberg2 and O. Højberg2 1 Department of Animal Nutrition and Feed Management, Poznań University of Life Science, Poznań, Poland; 2Department of Animal Science, Aarhus University, Tiele, Denmark
(Received 1 October 2013; Accepted 4 February 2014)
The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract. Keywords: broiler chicken, Clostridium perfringens, fat, digestibility, fermentation
Implication The present study elucidates how different dietary fat sources influence digestion and fermentation activity of the endogenous microbiota in broiler chickens. The study demonstrates the secondary effects of Clostridium perfringens challenge and fat type in broiler chickens, indicating that both factors affect fermentation in the broiler gastrointestinal tract.
Introduction
Clostridium perfringens is ubiquitous in the environment and may thus be ingested with soil, feces, dust, feed or even †
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drinking water (Van Immerseel et al., 2009; Schocken-Iturrino et al., 2010). Avirulent C. perfringens strains are often found in the gastrointestinal tract (GIT) of healthy chickens where they may reside without harming the host bird. However, simultaneous blooming of virulent C. perfringens strains and colonization of the small intestine by Eimeria spp. may trigger the development of necrotic enteritis (NE). There are many dietary factors influencing C. perfringens colonization of the chicken GIT. In this connection, the role of dietary carbohydrates and protein has received much attention. The inclusion of certain grains containing high amounts of viscous carbohydrates, for example rye, wheat or barley in poultry diets, leads to higher colonization of C. perfringens, which may impair chicken performance (Choct, 2009). Further, it has been reported that the growth of C. perfringens is greatly 1
Józefiak, Kierończyk, Rawski, Hejdysz, Rutkowsk, Engberg and Højberg supported by dietary protein – in particular, protein of animal origin, like fish meal (Dahiya et al., 2006). However, the influence of lipids on the GIT microbiota is still not completely clarified. Lipids are known to influence the intestinal microbiota indirectly through their impact on digesta viscosity, intestinal transit time and digestion in the small intestine. Apart from the microbial population, the absorption of fats is influenced by many other factors – for example fat source and type, carbon chain length and degree of fatty acid saturation, bile salt concentration in the digesta, and finally chicken age (Engberg et al., 1996; Knarreborg et al., 2002). However, in the literature there is only limited information about possible interactions between dietary fat source and C. perfringens challenge. It is well documented that a highly digestible and unsaturated fat source, for example soy oil, decreases small intestinal numbers of C. perfringens compared with poorly digestible lard and tallow (Knarreborg et al., 2002). Moreover, in our earlier studies, we observed that a C. perfringens challenge resulted in significantly decreased pH of crop, gizzard and ileum contents and reduced the numbers of lactic acid bacteria in the ceca (Józefiak et al., 2012); however, the manner in which it affects microbial fermentation activity remains unclear. Therefore, the aim of the present study was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the GIT of broilers, challenged with virulent C. perfringens strains. Material and methods Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. The birds were kept in floor pens (1.2 × 0.8 m) over a production period of 42 days. The lightning regime provided 23 h of light and 1 h of dark during the 1st week, 19 h of light and 5 h of dark from 7 to 21 days, and 23 h of light and 1 h of dark from 22 to 42 days of age. In Experiment 1, birds were fed with diets containing soybean oil (SO), palm kernel fatty acid distillers (PKFDs), rendered pork fat (RPF) and lard (L). In Experiment 2, birds were fed diets containing rapeseed oil (RO), coconut oil (CO), beef tallow (BT) and palm oil (PO). In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains: 73.70560-e (PFGE type S11), strain 200302–1-1-Ba (PFGE type S48) and strain 97.73338–3 (PFGE type T7). Isolated from birds with dignosed necrotic enteritis and all producing α-toxin and NetB as described in detail by Józefiak et al. (2012).
Diets and feeding program The composition of the experimental diets is shown in Tables 1 and 2. The fatty acid composition of the applied fats is shown in Table 3. The diets were provided in the form of mash; all raw materials were ground in a disc mill (Skiold A/S, Denmark) at 2.5-disc distance, mixed without any heat treatment and fed ad libitum to all birds. In the last 5 days (37 to 42 days) of the experiment, 0.2% of the wheat was 2
replaced by titanium oxide as an internal marker for the calculation of nutrient digestibility. The experiment complied with the guidelines of the Local Ethic Commission (Poznań University of Life Sciences, Poland) with respect to animal experimentation and care of animals under study.
Data collection The feed intake (FI) and BW of the chickens were measured on d 14, 28 and 42. Mortality was registered throughout the experimental period. At the end of the trial (42 days) from each experimental group 18 randomly picked chickens (three chickens from six pens) were killed by cervical dislocation. For digesta analyses (pH and organic acid concentrations), the contents of crop, ileum and ceca from three birds per pen were pooled (n = 6, replicate digesta samples per segment of ~10 g each). Approximately 5 g of the samples was used immediately for the pH analyses. The rest of the samples was immediately frozen and stored at −20°C for later analysis of organic acids. For the evaluation of apparent metabolizable energy (AMEn) and total tract fat digestibility, 100 g of the excreta from each pen was collected at 42 days (n = 12). The excreta samples were immediately frozen, freeze-dried and ground before further analyses. Chemical analyses Feed samples were analyzed in duplicate for CP, crude fat and crude fiber using AOAC (2005) methods 976.05, 920.39 and 2002.04, respectively. The iodine value in all fats was analyzed according to the AOAC (2005) 993.20 method and the fatty acid profile by methods described in detail by Edwards (1967). The concentration of titanium dioxide was determined according to the method described by Short et al. (1996), and the samples were prepared according to the procedure reported by Myers et al. (2004). Gross energy was determined using an adiabatic bomb calorimeter (KL 12Mn, Precyzja-Bit PPHU, Poland) standardized with benzoic acid. The nitrogen content was analyzed using the Kjel Foss Automatic 16210 (A/S N. Foss Electric, Hillerød, Denmark) and the CP content was calculated using a multiplication factor of 6.25. Fat content was determined using Soxtex System HT 1043, Extraction Unit (Foss Tecator, Hillerød, Denmark). Digestibility calculations The apparent digestibility coefficients of fat was analyzed as described in detail by Józefiak et al. (2011). The AMEn content of the experimental diets was calculated according to Hill and Anderson (1958) using titanium dioxide as an indigestible marker. Organic acids and pH analyses The pH of pooled digesta samples from crop, ileum and ceca was measured immediately after slaughter using a combined glass and reference electrode (Elmetron, Zabrze, Poland). The concentration of short-chain fatty acids and lactic acid in the contents of the different gastrointestinal segments was determined by gas chromatography, as described earlier by Canibe et al. (2007), using a Hewlett Packard gas
Fat and C. perfringens challenge in chickens Table 1 Composition of the basal diets: Experiment 1 Diet 1 to 14 days
15 to 42 days
2
3
4
1
2
3
4
57.19 36.9 2.2 – – – 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
56.99 36.9 – 2.4 – – 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
56.20 37.1 – – 3.0 – 1.0 1.5 0.42 0.38 0.01 0.12 0.27 –
56.99 36.9 – – – 2.4 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
66.82 26.5 4.0 – – – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
66.42 26.6 – 4.3 – – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
65.12 26.8 – – 5.4 – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
66.32 26.6 – – – 4.4 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
12.3 22.00 5.03 2.70 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 4.86 2.70 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 5.07 2.69 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 5.58 2.69 0.85 1.28 0.58 0.94 0.84 0.39
13.3 18.00 6.85 2.50 0.60 1.04 0.49 0.80 0.70 0.25
13.3 18.00 7.14 2.50 0.60 1.04 0.49 0.80 0.70 0.25
13.3 18.00 8.14 2.48 0.60 1.04 0.49 0.79 0.70 0.25
13.3 18.00 7.23 2.50 0.60 1.04 0.49 0.79 0.70 0.25
1 Ingredient (%) Corn Soybean meal 46.8% Soybean oil Palm kernel fatty acids distillers Rendered pork fat Lard Vitamin-mineral premix2 Monocalcium phosphate Limestone NaCl NaHCO3 L-lysine HCl 98 DL-Methionine L-Threonine Calculated composition (%) ME (MJ/kg) CP Crude fat Crude fiber Calcium Lysine Methionine Methionine + cystine Treonine Non-phytate P
1
ME = metabolizable energy. 1 1, soybean oil; 2, palm kernel fatty acids distillers; 3, rendered pork fat; 4, Lard. 2 Provided the following per kilogram of diet: vitamin A, 11.166 IU; cholecalciferol, 2.500 IU; vitamin E, 80 mg; menadione, 2.50 mg; B12, 0.02 mg; folic acid, 1.17 mg; choline, 379 mg; D-pantothenic acid, 12.50 mg; riboflavin, 7.0 mg; niacin, 41.67 mg; thiamine, 2.17 mg; D-biotin, 0.18 mg; pyridoxine, 4.0 mg; ethoxyquin, 0.09 mg; Mn (MnO2), 73 mg; Zn (ZnO), 55 mg; Fe (FeSO4), 45 mg; Cu (CuSO4), 20 mg; I (CaI2O6), 0.62 mg; Se (Na2SeO3), 0.3 mg.
chromatograph (model 6890; Hewlett Packard, Agilent Technologies, Naerum, Denmark) equipped with a flame ionization detector and a 30-m ZB-5 column with an internal diameter of 0.32 mm and coated with 5%-phenyl 95%-di-methylpolysiloxane with a film thickness of 0.25 μm. The data are presented as total organic acids (acetate + propionate + butyrate + isobutyrate, valerate, iso-valerate and lactic acid).
Statistical analysis Statistical analysis of the results was performed using the GLM of SAS, (1990) according to the following general model: Yij ¼ μ + αi + βj + ðαβÞij + δij ; where Yij was the observed dependent variable; μ was the overall mean; αi was the effect of fat source; βj was the effect of C. perfringens challenge; (αβ)ij was the interaction between fat and C. perfringens challenge; and δij was the random error. In cases in which the overall effect was significant (P < 0.05), means were compared pairwise (pdiff).
Results are presented as least square mean values with pooled standard error of the mean (s.e.m.). Results
Experiment 1 The performance results are summarized in Table 4. During the period from 1 to 14 days and from 14 to 28 days, none of the dietary fats affected the BWG of broiler chickens. From 28 to 42 days (P = 0.015) and during the entire experiment (P = 0.004), a significant response to dietary fat source was observed. From 28 to 42 days, the highest BWG was observed in birds fed L, being significantly higher than that of birds fed diets with PKFDs and SO. The birds fed diets with RPF did not differ significantly from the rest of the treatments. FI was significantly affected by dietary fat type throughout the experiment. Irrespective of the experimental period, birds receiving SO had the lowest FI, being significantly lower than that of other treatments from 28 to 42 days and in the entire experimental period. In the period from 3
Józefiak, Kierończyk, Rawski, Hejdysz, Rutkowsk, Engberg and Højberg Table 2 Composition of the basal diets: Experiment 2 Diet 1 to 14 days
Ingredient (%) Corn Soybean meal 46.8% Rapeseed oil Coconut oil Beef tallow Palm oil Vitamin-mineral premix2 Monocalcium phosphate Limestone NaCl NaHCO3 L-Lysine HCl 98 DL-Methionine L-Threonine Calculated composition (%) ME (MJ/kg) CP Crude fat Crude fiber Calcium Lysine Methionine Methionine + cystine Treonine Non-phytate P
15–42d
11
2
3
4
1
2
3
4
57.19 36.9 2.2 – – – 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
56.99 36.9 – 2.4 – – 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
56.20 37.1 – – 3.0 – 1.0 1.5 0.42 0.38 0.01 0.12 0.27 –
56.99 36.9 – – – 2.4 1.0 1.5 0.42 0.38 0.01 0.13 0.27 –
66.82 26.5 4.0 – – – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
66.42 26.6 – 4.3 – – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
65.12 26.8 – – 5.4 – 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
66.32 26.6 – – – 4.4 1.0 0.8 0.11 0.33 0.01 0.17 0.23 0.03
12.3 22.00 4.86 2.70 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 5.03 2.70 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 5.58 2.69 0.85 1.28 0.58 0.94 0.84 0.39
12.3 22.00 5.07 2.70 0.85 1.28 0.58 0.94 0.84 0.39
13.3 18.00 6.85 2.50 0.60 1.04 0.50 0.80 0.70 0.25
13.3 18.00 7.15 2.50 0.60 1.04 0.50 0.80 0.70 0.25
13.3 18.00 8.14 2.48 0.60 1.04 0.49 0.79 0.70 0.25
13.3 18.00 7.23 2.50 0.60 1.04 0.50 0.80 0.70 0.25
ME = metabolizable energy. 1 1, rapeseed oil; 2, coconut oil; 3, beef tallow; 4, palm oil. 2 Provided the following per kilogram of diet: vitamin A, 11.166 IU; cholecalciferol, 2.500 IU; vitamin E, 80 mg; menadione, 2.50 mg; B12, 0.02 mg; folic acid, 1.17 mg; choline, 379 mg; D-pantothenic acid, 12.50 mg; riboflavin, 7.0 mg; niacin, 41.67 mg; thiamine, 2.17 mg; D-biotin, 0.18 mg; pyridoxine, 4.0 mg; ethoxyquin, 0.09 mg; Mn (MnO2), 73 mg; Zn (ZnO), 55 mg; Fe (FeSO4), 45 mg; Cu (CuSO4), 20 mg; I (CaI2O6), 0.62 mg; Se (Na2SeO3), 0.3 mg.
Table 3 Fatty acid composition and iodine value of the fat sources used in Experiments 1 and 2 Fatty acid composition (%) C10:0 C12:0 C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:1 Iodine value (g/100g) nd = not detectable.
4
Soybean oil
Palm kernel fatty acids distillers
Rendered pork fat
Lard
Rapeseed oil
Coconut oil
Beef tallow
Palm oil
nd nd 0.1 10.5 0.2 nd 3.8 21.7 53.1 7.4 0.3 0.2 0.3 131.0
5.4 50.6 19.2 9.4 nd nd 3.0 6.4 1.4 nd nd nd nd 3.8
nd 0.1 2.1 26.8 3.8 0.7 16.1 43.5 3.9 0.3 0.2 0.6 nd 47.0
nd nd 1.3 26.3 2.1 0.6 15.8 42.8 8.1 0.5 0.2 0.7 nd 50.5
nd 0.2 0.1 4.2 0.4 nd 1.8 58 20.5 9.8 nd nd 0.4 111.0
2.9 54.0 17.7 10.9 nd nd 2.2 8.7 1.3 nd nd nd nd 7.2
nd 0.1 3.3 25.3 3.4 nd 19.2 37.5 2.8 1.6 1.0 0.7 nd 40.0
nd nd 0.8 42.7 0.1 0.1 5.1 39.8 10.7 0.2 0.3 0.1 nd 49.5
Table 4 The effect of fat type and Clostridium perfringens challenge on the performance of broiler chickens: Experiment 1 Significance (P-value)
Treatment Fat Source Challenge1
Palm kernel fatty acids distillers
Rendered pork fat
Lard
Effect of treatments
Interaction
−
+
−
+
−
+
−
+
Model RMSE
Fat
Challenge
Fat × challenge
362 893 1293 2549bc
355 891 1289 2535c
367 911 1325 2603abc
366 884 1320 2570bc
358 885 1352 2595abc
358 946 1317 2621abc
374 924 1376 2675ab
380 949 1402 2730a
5 7 8 10
0.29 0.19 0.02 0.01
0.98 0.39 0.81 0.78
0.96 0.25 0.78 0.73
488 1381c 1948d 3817c
486 1406bc 2045cd 3937bc
499 1441abc 2151abc 4091ab
508 1446abc 2076bcd 4030bc
489 1429bc 2177abc 4095ab
498 1485ab 2178abc 4160ab
538 1487ab 2270a 4295a
539 1525a 2235ab 4299a
6 8 11 14
0.01 0.002
