- Email: [email protected]
Impact of a Macleaya cordata-derived alkaloid extract on necrotic enteritis in broilers
Impact of a Macleaya cordata-derived alkaloid extract on necrotic enteritis in broilers G. D. Xue,∗ S. B. Wu,∗ M. Choct,∗ A. Pastor,† T. Steiner,† and R. A. Swick∗,1 ∗
Department of Animal Science, School of Environmental and Rural Sciences, University of New England, Armidale, NSW 2351, Australia; and † Phytobiotics Futterzusatzstoffe GmbH, Wallufer St 10 A, 65343 Eltville, Germany 9 and 108 to 109 Clostridium perfringens on d 14. Each treatment had 7 replicate floor pens with 17 birds each. NE challenge negatively affected growth performance, livability, and carcass traits. Regardless of challenge, IQA increased feed intake and gain on d 24 (P < 0.05) and 35 (P < 0.01) and improved FCR (P < 0.05), flock uniformity (P < 0.01) and breast meat yield (P < 0.001) on d 35. Supplementation of IQA also reduced lesions in the duodenum (P < 0.05), jejunum (P < 0.001), and ileum (P < 0.001). This study suggests that IQA may protect broilers from NE indicating its role as a promising antibiotic alternative.
ABSTRACT Necrotic enteritis (NE) causes significant economic losses in the broiler chicken industry, especially in birds raised without in-feed antibiotics. A standardized blend of plant-derived isoquinoline alkaloids (IQA) derived from Macleaya cordata has shown to have anti-inflammatory potency and promoted animal productivity. This study investigated the effects of IQA supplementation on broiler chickens under NE challenge. A 2 × 2 factorial arrangement of treatments was employed with factors: NE challenge (no or yes), and additives (no additive or IQA at 0.15 g/kg) in starter, grower, and finisher diets. Birds were challenged with Eimeria spp. on d
Key words: necrotic enteritis, isoquinoline alkaloids, anti-inflammation, performance, broiler 2017 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pex164
INTRODUCTION
Isoquinoline alkaloids (IQA) derived from Papaveraceae plants consist of a mixture of quarternary benzo phenanthridine and protopine alkaloids. The active ingredient of IQA, sanguinarine, was found to be anti-inflammatory (Niu et al., 2012) and inhibits the activation of NF-κB, the key regulator of inflammatory response (Chaturvedi et al., 1997; Wullaert et al., 2011). Niewold and Backer (2010) reported that sanguinarine inhibited lipopolysaccharide induced nitric oxide response of macrophages in vitro. Macrophages are involve in the up-regulation of pro-inflammatory cytokines observed in chickens during Eimeria infection, an important factor that predisposes chickens to NE (Lillehoj and Li, 2004; Hong et al., 2006). The hypothesis of the current study was that IQA might provide protection to broiler chickens against NE. Thus, an experiment was conducted to examine whether IQA supplementation improves growth performance and reduces the prevalence of intestinal NE lesions in broilers subjected to NE challenge.
Poultry necrotic enteritis (NE) is caused by Clostridium perfringens (Cp). The ban of in-feed antibiotics in the European Union has led to elevated incidence of NE (Shojadoost et al., 2012). Thus, currently, there is a heightened interest in finding antibiotic alternatives to control NE. Inflammation is a vital consequence of NE that has not been investigated extensively. Proinflammatory cytokines and chemokines increase dramatically in chickens during NE infection (Lee et al., 2011). As inflammation is an energy-consuming process, it has been estimated that vertebrates increase their resting metabolic rate by 8 to 27% during immunologic challenge (Martin et al., 2003). Inflammation reduces feed consumption, impairs intestinal morphology, compromises nutrient absorption, and alters anabolic processes in skeletal muscle in order to divert nutrients toward immune function (Klasing et al., 1987; Klasing, 2005; Willing and Van Kessel, 2007). This at least partially explains the impaired performance observed in broilers subjected to NE infection. However, the effect of inflammation on performance reduction during NE infection may have been underestimated.
MATERIALS AND METHODS Experimental Design and Bird Husbandry
C
2017 Poultry Science Association Inc. Received March 9, 2017. Accepted June 19, 2017. 1 Corresponding author: [email protected]
All experimental procedures involved in this study were approved by the Animal Ethics Committee of 1
2
XUE ET AL.
University of New England (Australia, AEC 15–064). Day-old Ross 308 male chicks (N = 476) were obtained from a local hatchery (Baiada Country Road Hatchery, Tamworth, NSW, Australia) and vaccinated against infectious bronchitis, Marek’s disease, and Newcastle disease. Birds were reared in an environmentally controlled floor pen facility with fresh softwood shavings used as litter (The Rob Cumming Poultry Innovation Centre at Kirby, University of New England, Australia). The facility was disinfected prior to bird arrival. Birds were weighed and placed to ensure uniform starting pen weights. The temperature and lighting program followed Ross 308 recommendations (Aviagen, 2014). The study used a 2 × 2 factorial arrangement of treatments with 7 replicate pens per treatment, each with 17 birds. Factors were: NE challenge (no or yes), and additives (none: no additive, IQA: 0.15 g/kg blend of R plant-derived isoquinoline alkaloids (Sangrovit Extra, Germany) on top of basal diets of starter, grower and finisher). Diets were based on wheat (20, 20, 20%), sorghum (25.2, 25.0, 29.3%) barley (16, 16, 20%), soybean meal (29.3, 24.9, 20.3%), meat and bone meal (6.2, 5.4, 4.8) in starter, grower and finisher respectively (Table S1). Diets were formulated to meet Ross 308 nutrient recommendations for standard ileal digestible amino acids and other nutrients. Birds had ad libitum access to water and feed for the entire experiment period. On d 35, birds were weighed individually to calculate the coefficient of variation (CV) as a measure of flock uniformity. Cumulative pen feed intake and pen live weight were recorded on d 0, 24 and 35 and used to calculate mean bird feed intake (FI), weight gain (BWG), and feed conversion ratio (FCR, corrected for mortality). Mortality was recorded as it occurred.
NE Challenge On d 9, each bird in the challenge group was inoculated with 1 mL per os of unattenuated field strains of Eimeria (Bioproperties Pty Ltd., Sydney, Australia) in phosphate-buffered saline (PBS) suspension containing 5,000 oocysts each of E. maxima and E. acervulina, and 2,500 oocysts of E. brunetti. Unchallenged controls received 1 mL of sterile PBS. On d 14, each bird in the challenge group was inoculated with 1 mL of Cp suspension (108 to 109 CFU/mL). A primary poultry isolate of Cp type A strain EHE-NE18 (CSIRO Livestock Industries, Geelong, Australia) was used and incubated as previously reported (M’Sadeq et al., 2015). A physical partition between unchallenged and challenged groups was applied to prevent cross contamination.
Sampling, Lesion Scoring and Carcass Traits Sampled birds throughout the experimental period were selected randomly and euthanized by cervical dislocation. On d 16, the entire length of the section of
small intestine (duodenum, jejunum, and ileum) underwent a lesion scoring process, based on a previously reported lesion scoring system ranging from 0 to 4 (Prescott et al., 1978; Broussard et al., 1986). On d 35, de-boned, skinless breast muscle, thigh with drumstick portions, and abdominal fat were excised and weighed from 3 birds from each replicate pen. Results of carcass traits were calculated from the average and expressed as a percentage of whole bird live weight.
Statistical Analysis R Statistical analysis was conducted using the IBM R Statistics package version 22 (IBM CorporaSPSS tion, Armonk, NY, United States). The main effects of NE challenge, additives and interactions were examined by analysis of variance, using the General Linear Model. Intestinal lesion scores and mortality data were analyzed by the nonparametric Kruskal-Wallis test, as the data were not normally distributed. Treatment means were separated using Tukey HSD multiple range test where appropriate. Statistical significance was declared at P < 0.05.
RESULTS AND DISCUSSION Table 1 shows BWG, FI, FCR, and mortality results for the d 0 to d 24 period. Overall, it was shown that the NE challenge negatively affected performance. Challenged birds had reduced FI and BWG and increased mortality and FCR (P < 0.001) compared to unchallenged controls. Regardless of NE challenge, inclusion of IQA led to higher FI (P < 0.01) and BWG (P < 0.05). No challenge by additive interactions were detected (P > 0.05). For the d 0 to d 35 period, NE challenged birds had lower FI, BWG and higher FCR and mortality (P < 0.001) as compared to unchallenged controls (Table 2). Regardless of NE challenge, the IQA supplemented groups had higher FI (P < 0.01), BWG (P < 0.01) and lower FCR (P < 0.05) and birds were more uniform as detected by lower BWG CV % (P < 0.01) as compared to those fed diets with no additive. During the experimental period, no challenge by additive interactions were detected for any of the measured performance parameters (P > 0.05). Dietary treatment did not affect mortality (P > 0.05). As illustrated in Table 3, NE challenge reduced (P < 0.001) the relative breast and thigh yields. Regardless of NE challenge, IQA supplementation increased breast meat yield compared to control diets (P < 0.001). Treatments had no significant effect on relative abdominal fat (P > 0.05) and no challenge by additive interactions in carcass traits were detected (P > 0.05). Intestinal lesion scores measured on d 16 are shown in Table 4. Challenge by additive interactions were observed for lesions occurring in the duodenum (P < 0.05), jejunum (P < 0.001) and ileum (P < 0.001). Additives did not affect lesion scores in any intestinal section in unchallenged groups.
MACLEAYA CORDATA ALKALOID FOR NECROTIC ENTERITIS IN BROILERS
3
Table 1. Performance of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge from d 0 to 24. Interaction
Feed Intake g/bird
Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Weight gain g/bird
FCR
Mortality %
1637 1708 1366 1393 30
1321 1380 1019 1025 33
1.239 1.238 1.340 1.360 0.011
0.0 3.4 13.4 14.3 1.5
1673a 1379b
1350a 1022b
1.239b 1.350a
1.7b 13.9a
1501b 1551a
1170b 1202a
1.290 1.299
6.7 8.8
< 0.001 0.011 0.227
< 0.001 0.039 0.082
< 0.001 0.140 0.106
< 0.001 0.427 0.528
Means not sharing the same superscripts are significantly different (P < 0.05). R None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoline alkaloids (Sangrovit Extra) in starter, grower and finisher. a-b 1
Table 2. Performance of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge from d 0 to 35. Interaction Additives Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Feed Intake g/bird
Weight gain g/bird
FCR
Mortality %
BW Uniformity (CV %)
3340 3502 3123 3216 34
2398 2549 2175 2253 31
1.393 1.374 1.436 1.427 0.006
0.8 4.3 14.6 17.1 1.6
9.7 6.7 11.0 6.7 0.6
3421a 3169b
2473a 2214b
1.384b 1.432a
2.6b 15.9a
8.2 8.8
3231b 3359a
2286b 2401a
1.415a 1.401b
7.7 10.7
10.3a 6.7b
< 0.001 0.007 0.429
< 0.001 < 0.001 0.218
< 0.001 0.037 0.416
< 0.001 0.329 0.824
0.535 0.001 0.513
Means not sharing the same superscripts are significantly different (P < 0.05). R None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoline alkaloids (Sangrovit Extra) in starter, grower and finisher. a-b 1
Challenge with NE only elevated duodenal (P < 0.05) and ileal (P < 0.001) lesion scores in un-supplemented birds but not in IQA-fed birds (P > 0.05). In the jejunum, NE challenge resulted in higher lesion scores compared to unchallenged groups and lower lesion scores were found in IQA-fed birds (P < 0.001). Jeroch et al. (2009) reported IQA improved the performance of turkeys fed wheat-based diets, similar to the barley-wheat (up to 40%) based diet used in the current study. Vieira et al. (2008) also found that IQA supplementation increased body weight and feed efficiency in broiler chickens. The current study demonstrated for the first time, the positive role of IQA supplementation in controlling the impacts of NE in broiler chickens. Firstly, IQA controlled the development of
intestinal NE lesion of broilers under NE challenge. Olkowski et al. (2006) found that early stages of NE showed strong inflammatory responses. The lamina propria was hyperemic and infiltrated with numerous inflammatory cells, mainly heterophilic granulocytes with most significant early changes seen at the interface of the basal domain of enterocytes and lamina propria. Activation of NF-κB was found to result in disorganization of tight junction protein at intestinal barrier in vitro (Ye et al., 2006). This indicates that intestinal inflammation may initiate NE lesion development and IQA supplementation may suppress this inflammatory response, leading to a reduction in NE lesions as observed in the current study. Secondly, IQA may provide protection to the performance of NE challenged chicken by
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XUE ET AL.
Table 3. Carcass traits (g/100 g live weight) of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge on d 35. Treatment1 Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Breast yield
Thigh yield
Abdominal fat
20.5 21.6 19.3 20.0 0.19
18.9 18.6 18.1 18.0 0.10
0.8 0.9 0.8 0.9 0.02
21.1a 19.7b
18.7a 18.0b
0.9 0.8
18.5 18.3
0.8 0.9
b
19.9 20.8a
< 0.001 < 0.001 0.269
< 0.001 0.301 0.574
SUPPLEMENTARY DATA Supplementary data are available at PSCIEN online. Table S1. Ingredient and nutrient composition of experimental basal diets.
0.389 0.247 0.467
ACKNOWLEDGMENTS
a-b
Means not sharing the same superscripts are significantly different (Tukey test; P < 0.05). 1 None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoR Extra) in starter, grower and finisher. line alkaloids (Sangrovit
Table 4. Duodenum, jejunum, and ileum NE lesion score of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge on d 16. Treatment Challenge No No Yes Yes SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Treatment
Duodenum Additives None IQA None IQA
Jejunum
explain the greater flock uniformity of the IQA group who might be more tolerant to the stimulus with uniformed FI and metabolism among the flock. However, the mechanism of IQA in controlling NE in broiler chicken is pending on further investigation. In future work with IQA, measurement and direct monitoring of inflammatory status would be warranted.
Ileum
b
0.21 0.43b 2.14a 0.50b 0.19
c
b
0.00 0.07c 2.64a 1.29b 0.24
0.00 0.14b 1.79a 0.79b 0.17
0.32b 1.32a
0.04b 1.96a
0.07b 1.29a
1.18 0.46
1.32 0.68
0.89 0.46
0.039 0.329 0.014
< 0.001 0.454 < 0.001
< 0.001 0.769 < 0.001
a-d Means not sharing the same superscripts are significantly different (P < 0.05). 1 None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoR Extra) in starter, grower and finisher. line alkaloids (Sangrovit
controlling inflammation and its physiologic consequences. Inflammation triggered by NE can lead to gluconeogenesis in order to retain circulating glucose level, particularly during the anorexia condition. In the present study, NE challenge lowered the cumulative FI by 17% at d 24. To compensate glucose production from the impaired FI, skeletal muscle would need to be catabolized into amino acids such as glutamine to provide the key substrate of gluconeogenesis resulting in a large loss of lean meat (Lacey and Wilmore, 1990; Wu et al., 1991). This was reflected in the present study as demonstrated by decreased BWG, breast and thigh meat yields, and poorer FCR as a result of NE challenge, whereas these have been alleviated when IQA was included in the diet. In addition, it may further
Phytobiotics Futterzusatzstoffe GmbH (Germany) is gratefully acknowledged for funding this project. The first author (G. D. Xue) was a recipient of a postgraduate scholarship from Phytobiotics Futterzusatzstoffe GmbH (Germany) and a fee waiver scholarship from the University of New England (Australia).
REFERENCES Aviagen. 2014. Broiler Nutrition Specification Ross 308. Huntsville, Alabama, USA. Broussard, C. T., C. L. Hofacre, R. K. Page, and O. J. Fletcher. 1986. Necrotic enteritis in cage-reared commercial layer pullets. Avian Dis. 30:617–619. Chaturvedi, M. M., A. Kumar, B. G. Darnay, G. B. N. Chainy, S. Agarwal, and B. B. Aggarwal. 1997. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-κB activation, IκBα phosphorylation, and degradation. J. Biol. Chem. 272:30129–30134. Hong, Y. H., H. S. Lillehoj, S. H. Lee, R. A. Dalloul, and E. P. Lillehoj. 2006. Analysis of chicken cytokine and chemokine gene expression following Eimeria acervulina and Eimeria tenella infections. Vet. Immunol. Immunopathol. 114:209–223. Jeroch, H., K. Kozlowski, J. Jeroch, K. Lipinski, Z. Zdunczyk, and J. Jankowski. 2009. Efficacy of the phytogenic (PapaverR in growing monogastric animals. aceae) additive Sangrovit Z¨ uchtungskunde 81:279–293. Klasing, K. C. 2005. Interplay between diet, microbes, and immune defenses of the gastrointestinal tract. Pages 229–254 In: Physiological and Ecological Adaptations to Feeding in Vertebrates. J. M. Starck, and T. Wang eds. Science, Enfield, New Hampshire. Klasing, K. C., D. E. Laurin, R. K. Peng, and D. M. Fry. 1987. Immunologically mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin-1. J. Nutr. 117:1629– 1637. Lacey, J. M., and D. W. Wilmore. 1990. Is glutamine a conditionally essential amino acid? Nutr. Rev. 48:297–309. Lee, K. W., H. S. Lillehoj, W. Jeong, H. Y. Jeoung, and D. J. An. 2011. Avian necrotic enteritis: Experimental models, host immunity, pathogenesis, risk factors, and vaccine development. Poult. Sci. 90:1381–1390. Lillehoj, H. S., and G. Li. 2004. Nitric oxide production by macrophages stimulated with coccidia sporozoites, lipopolysaccharide, or interferon-γ , and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella. Avian Dis. 48:244–253. M’Sadeq, S. A., S. B. Wu, R. A. Swick, and M. Choct. 2015. Dietary acylated starch improves performance and gut health in necrotic enteritis challenged broilers. Poult. Sci. 94:2434–2444. Martin, L. B., A. Scheuerlein, and M. Wikelski. 2003. Immune activity elevates energy expenditure of house sparrows: a link between direct and indirect costs? Proc. Biol. Sci. 270:153–158.
MACLEAYA CORDATA ALKALOID FOR NECROTIC ENTERITIS IN BROILERS Niewold, T. A., and K. D. Backer. 2010. The in vitro antiinflammatory property of sanguinarine containing extracts is consistent with enhanced in vivo performance in production animals. Proc. 13th European Poultry Conference, France. Niu, X. F., T. Fan, W. F. Li, W. Xing, and H. M. Huang. 2012. The anti-inflammatory effects of sanguinarine and its modulation of inflammatory mediators from peritoneal macrophages. Eur. J. Pharmacol. 689:262–269. Olkowski, A. A., C. Wojnarowicz, M. Chirino-Trejo, and M. D. Drew. 2006. Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis. Res. Vet. Sci. 81:99–108. Prescott, J. F., R. Sivendra, and D. A. Barnum. 1978. The use of bacitracin in the prevention and treatment of experimentally-induced necrotic enteritis in the chicken. Can. Vet. J. 19:181–183. Shojadoost, B., A. R. Vince, and J. F. Prescott. 2012. The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review. Vet. Res. 43:74.
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Vieira, S. L., O. A. Oyarzabal, D. M. Freitas, J. Berres, J. E. M. Pe˜ na, C. A. Torres, and J. L. B. Coneglian. 2008. Performance of broilers fed diets supplemented with sanguinarine-like alkaloids and organic acids. J. Appl. Poult. Res. 17:128–133. Willing, B. P., and A. G. Van Kessel. 2007. Enterocyte proliferation and apoptosis in the caudal small intestine is influenced by the composition of colonizing commensal bacteria in the neonatal gnotobiotic pig. J. Anim. Sci. 85:3256–3266. Wu, G., J. R. Thompson, and V. E. Baracos. 1991. Glutamine metabolism in skeletal muscles from the broiler chick (Gallus domesticus) and the laboratory rat (Rattus norvegicus). Biochem. J. 274:769–774. Wullaert, A., M. C. Bonnet, and M. Pasparakis. 2011. NF-κB in the regulation of epithelial homeostasis and inflammation. Cell Res. 21:146–158. Ye, D., I. Ma, and T. Y. Ma. 2006. Molecular mechanism of tumor necrosis factor-α modulation of intestinal epithelial tight junction barrier. Am. J. Physiol. Gastrointest. Liver Physiol. 290:G496– G504.
Department of Animal Science, School of Environmental and Rural Sciences, University of New England, Armidale, NSW 2351, Australia; and † Phytobiotics Futterzusatzstoffe GmbH, Wallufer St 10 A, 65343 Eltville, Germany 9 and 108 to 109 Clostridium perfringens on d 14. Each treatment had 7 replicate floor pens with 17 birds each. NE challenge negatively affected growth performance, livability, and carcass traits. Regardless of challenge, IQA increased feed intake and gain on d 24 (P < 0.05) and 35 (P < 0.01) and improved FCR (P < 0.05), flock uniformity (P < 0.01) and breast meat yield (P < 0.001) on d 35. Supplementation of IQA also reduced lesions in the duodenum (P < 0.05), jejunum (P < 0.001), and ileum (P < 0.001). This study suggests that IQA may protect broilers from NE indicating its role as a promising antibiotic alternative.
ABSTRACT Necrotic enteritis (NE) causes significant economic losses in the broiler chicken industry, especially in birds raised without in-feed antibiotics. A standardized blend of plant-derived isoquinoline alkaloids (IQA) derived from Macleaya cordata has shown to have anti-inflammatory potency and promoted animal productivity. This study investigated the effects of IQA supplementation on broiler chickens under NE challenge. A 2 × 2 factorial arrangement of treatments was employed with factors: NE challenge (no or yes), and additives (no additive or IQA at 0.15 g/kg) in starter, grower, and finisher diets. Birds were challenged with Eimeria spp. on d
Key words: necrotic enteritis, isoquinoline alkaloids, anti-inflammation, performance, broiler 2017 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pex164
INTRODUCTION
Isoquinoline alkaloids (IQA) derived from Papaveraceae plants consist of a mixture of quarternary benzo phenanthridine and protopine alkaloids. The active ingredient of IQA, sanguinarine, was found to be anti-inflammatory (Niu et al., 2012) and inhibits the activation of NF-κB, the key regulator of inflammatory response (Chaturvedi et al., 1997; Wullaert et al., 2011). Niewold and Backer (2010) reported that sanguinarine inhibited lipopolysaccharide induced nitric oxide response of macrophages in vitro. Macrophages are involve in the up-regulation of pro-inflammatory cytokines observed in chickens during Eimeria infection, an important factor that predisposes chickens to NE (Lillehoj and Li, 2004; Hong et al., 2006). The hypothesis of the current study was that IQA might provide protection to broiler chickens against NE. Thus, an experiment was conducted to examine whether IQA supplementation improves growth performance and reduces the prevalence of intestinal NE lesions in broilers subjected to NE challenge.
Poultry necrotic enteritis (NE) is caused by Clostridium perfringens (Cp). The ban of in-feed antibiotics in the European Union has led to elevated incidence of NE (Shojadoost et al., 2012). Thus, currently, there is a heightened interest in finding antibiotic alternatives to control NE. Inflammation is a vital consequence of NE that has not been investigated extensively. Proinflammatory cytokines and chemokines increase dramatically in chickens during NE infection (Lee et al., 2011). As inflammation is an energy-consuming process, it has been estimated that vertebrates increase their resting metabolic rate by 8 to 27% during immunologic challenge (Martin et al., 2003). Inflammation reduces feed consumption, impairs intestinal morphology, compromises nutrient absorption, and alters anabolic processes in skeletal muscle in order to divert nutrients toward immune function (Klasing et al., 1987; Klasing, 2005; Willing and Van Kessel, 2007). This at least partially explains the impaired performance observed in broilers subjected to NE infection. However, the effect of inflammation on performance reduction during NE infection may have been underestimated.
MATERIALS AND METHODS Experimental Design and Bird Husbandry
C
2017 Poultry Science Association Inc. Received March 9, 2017. Accepted June 19, 2017. 1 Corresponding author: [email protected]
All experimental procedures involved in this study were approved by the Animal Ethics Committee of 1
2
XUE ET AL.
University of New England (Australia, AEC 15–064). Day-old Ross 308 male chicks (N = 476) were obtained from a local hatchery (Baiada Country Road Hatchery, Tamworth, NSW, Australia) and vaccinated against infectious bronchitis, Marek’s disease, and Newcastle disease. Birds were reared in an environmentally controlled floor pen facility with fresh softwood shavings used as litter (The Rob Cumming Poultry Innovation Centre at Kirby, University of New England, Australia). The facility was disinfected prior to bird arrival. Birds were weighed and placed to ensure uniform starting pen weights. The temperature and lighting program followed Ross 308 recommendations (Aviagen, 2014). The study used a 2 × 2 factorial arrangement of treatments with 7 replicate pens per treatment, each with 17 birds. Factors were: NE challenge (no or yes), and additives (none: no additive, IQA: 0.15 g/kg blend of R plant-derived isoquinoline alkaloids (Sangrovit Extra, Germany) on top of basal diets of starter, grower and finisher). Diets were based on wheat (20, 20, 20%), sorghum (25.2, 25.0, 29.3%) barley (16, 16, 20%), soybean meal (29.3, 24.9, 20.3%), meat and bone meal (6.2, 5.4, 4.8) in starter, grower and finisher respectively (Table S1). Diets were formulated to meet Ross 308 nutrient recommendations for standard ileal digestible amino acids and other nutrients. Birds had ad libitum access to water and feed for the entire experiment period. On d 35, birds were weighed individually to calculate the coefficient of variation (CV) as a measure of flock uniformity. Cumulative pen feed intake and pen live weight were recorded on d 0, 24 and 35 and used to calculate mean bird feed intake (FI), weight gain (BWG), and feed conversion ratio (FCR, corrected for mortality). Mortality was recorded as it occurred.
NE Challenge On d 9, each bird in the challenge group was inoculated with 1 mL per os of unattenuated field strains of Eimeria (Bioproperties Pty Ltd., Sydney, Australia) in phosphate-buffered saline (PBS) suspension containing 5,000 oocysts each of E. maxima and E. acervulina, and 2,500 oocysts of E. brunetti. Unchallenged controls received 1 mL of sterile PBS. On d 14, each bird in the challenge group was inoculated with 1 mL of Cp suspension (108 to 109 CFU/mL). A primary poultry isolate of Cp type A strain EHE-NE18 (CSIRO Livestock Industries, Geelong, Australia) was used and incubated as previously reported (M’Sadeq et al., 2015). A physical partition between unchallenged and challenged groups was applied to prevent cross contamination.
Sampling, Lesion Scoring and Carcass Traits Sampled birds throughout the experimental period were selected randomly and euthanized by cervical dislocation. On d 16, the entire length of the section of
small intestine (duodenum, jejunum, and ileum) underwent a lesion scoring process, based on a previously reported lesion scoring system ranging from 0 to 4 (Prescott et al., 1978; Broussard et al., 1986). On d 35, de-boned, skinless breast muscle, thigh with drumstick portions, and abdominal fat were excised and weighed from 3 birds from each replicate pen. Results of carcass traits were calculated from the average and expressed as a percentage of whole bird live weight.
Statistical Analysis R Statistical analysis was conducted using the IBM R Statistics package version 22 (IBM CorporaSPSS tion, Armonk, NY, United States). The main effects of NE challenge, additives and interactions were examined by analysis of variance, using the General Linear Model. Intestinal lesion scores and mortality data were analyzed by the nonparametric Kruskal-Wallis test, as the data were not normally distributed. Treatment means were separated using Tukey HSD multiple range test where appropriate. Statistical significance was declared at P < 0.05.
RESULTS AND DISCUSSION Table 1 shows BWG, FI, FCR, and mortality results for the d 0 to d 24 period. Overall, it was shown that the NE challenge negatively affected performance. Challenged birds had reduced FI and BWG and increased mortality and FCR (P < 0.001) compared to unchallenged controls. Regardless of NE challenge, inclusion of IQA led to higher FI (P < 0.01) and BWG (P < 0.05). No challenge by additive interactions were detected (P > 0.05). For the d 0 to d 35 period, NE challenged birds had lower FI, BWG and higher FCR and mortality (P < 0.001) as compared to unchallenged controls (Table 2). Regardless of NE challenge, the IQA supplemented groups had higher FI (P < 0.01), BWG (P < 0.01) and lower FCR (P < 0.05) and birds were more uniform as detected by lower BWG CV % (P < 0.01) as compared to those fed diets with no additive. During the experimental period, no challenge by additive interactions were detected for any of the measured performance parameters (P > 0.05). Dietary treatment did not affect mortality (P > 0.05). As illustrated in Table 3, NE challenge reduced (P < 0.001) the relative breast and thigh yields. Regardless of NE challenge, IQA supplementation increased breast meat yield compared to control diets (P < 0.001). Treatments had no significant effect on relative abdominal fat (P > 0.05) and no challenge by additive interactions in carcass traits were detected (P > 0.05). Intestinal lesion scores measured on d 16 are shown in Table 4. Challenge by additive interactions were observed for lesions occurring in the duodenum (P < 0.05), jejunum (P < 0.001) and ileum (P < 0.001). Additives did not affect lesion scores in any intestinal section in unchallenged groups.
MACLEAYA CORDATA ALKALOID FOR NECROTIC ENTERITIS IN BROILERS
3
Table 1. Performance of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge from d 0 to 24. Interaction
Feed Intake g/bird
Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Weight gain g/bird
FCR
Mortality %
1637 1708 1366 1393 30
1321 1380 1019 1025 33
1.239 1.238 1.340 1.360 0.011
0.0 3.4 13.4 14.3 1.5
1673a 1379b
1350a 1022b
1.239b 1.350a
1.7b 13.9a
1501b 1551a
1170b 1202a
1.290 1.299
6.7 8.8
< 0.001 0.011 0.227
< 0.001 0.039 0.082
< 0.001 0.140 0.106
< 0.001 0.427 0.528
Means not sharing the same superscripts are significantly different (P < 0.05). R None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoline alkaloids (Sangrovit Extra) in starter, grower and finisher. a-b 1
Table 2. Performance of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge from d 0 to 35. Interaction Additives Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Feed Intake g/bird
Weight gain g/bird
FCR
Mortality %
BW Uniformity (CV %)
3340 3502 3123 3216 34
2398 2549 2175 2253 31
1.393 1.374 1.436 1.427 0.006
0.8 4.3 14.6 17.1 1.6
9.7 6.7 11.0 6.7 0.6
3421a 3169b
2473a 2214b
1.384b 1.432a
2.6b 15.9a
8.2 8.8
3231b 3359a
2286b 2401a
1.415a 1.401b
7.7 10.7
10.3a 6.7b
< 0.001 0.007 0.429
< 0.001 < 0.001 0.218
< 0.001 0.037 0.416
< 0.001 0.329 0.824
0.535 0.001 0.513
Means not sharing the same superscripts are significantly different (P < 0.05). R None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoline alkaloids (Sangrovit Extra) in starter, grower and finisher. a-b 1
Challenge with NE only elevated duodenal (P < 0.05) and ileal (P < 0.001) lesion scores in un-supplemented birds but not in IQA-fed birds (P > 0.05). In the jejunum, NE challenge resulted in higher lesion scores compared to unchallenged groups and lower lesion scores were found in IQA-fed birds (P < 0.001). Jeroch et al. (2009) reported IQA improved the performance of turkeys fed wheat-based diets, similar to the barley-wheat (up to 40%) based diet used in the current study. Vieira et al. (2008) also found that IQA supplementation increased body weight and feed efficiency in broiler chickens. The current study demonstrated for the first time, the positive role of IQA supplementation in controlling the impacts of NE in broiler chickens. Firstly, IQA controlled the development of
intestinal NE lesion of broilers under NE challenge. Olkowski et al. (2006) found that early stages of NE showed strong inflammatory responses. The lamina propria was hyperemic and infiltrated with numerous inflammatory cells, mainly heterophilic granulocytes with most significant early changes seen at the interface of the basal domain of enterocytes and lamina propria. Activation of NF-κB was found to result in disorganization of tight junction protein at intestinal barrier in vitro (Ye et al., 2006). This indicates that intestinal inflammation may initiate NE lesion development and IQA supplementation may suppress this inflammatory response, leading to a reduction in NE lesions as observed in the current study. Secondly, IQA may provide protection to the performance of NE challenged chicken by
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XUE ET AL.
Table 3. Carcass traits (g/100 g live weight) of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge on d 35. Treatment1 Challenge Additives No None No IQA Yes None Yes IQA SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Challenge × Additives
Breast yield
Thigh yield
Abdominal fat
20.5 21.6 19.3 20.0 0.19
18.9 18.6 18.1 18.0 0.10
0.8 0.9 0.8 0.9 0.02
21.1a 19.7b
18.7a 18.0b
0.9 0.8
18.5 18.3
0.8 0.9
b
19.9 20.8a
< 0.001 < 0.001 0.269
< 0.001 0.301 0.574
SUPPLEMENTARY DATA Supplementary data are available at PSCIEN online. Table S1. Ingredient and nutrient composition of experimental basal diets.
0.389 0.247 0.467
ACKNOWLEDGMENTS
a-b
Means not sharing the same superscripts are significantly different (Tukey test; P < 0.05). 1 None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoR Extra) in starter, grower and finisher. line alkaloids (Sangrovit
Table 4. Duodenum, jejunum, and ileum NE lesion score of broiler chickens supplemented with isoquinoline alkaloids (IQA) under necrotic enteritis challenge on d 16. Treatment Challenge No No Yes Yes SEM Main Effects Challenge No Yes Additives1 None IQA P value Challenge Additives Treatment
Duodenum Additives None IQA None IQA
Jejunum
explain the greater flock uniformity of the IQA group who might be more tolerant to the stimulus with uniformed FI and metabolism among the flock. However, the mechanism of IQA in controlling NE in broiler chicken is pending on further investigation. In future work with IQA, measurement and direct monitoring of inflammatory status would be warranted.
Ileum
b
0.21 0.43b 2.14a 0.50b 0.19
c
b
0.00 0.07c 2.64a 1.29b 0.24
0.00 0.14b 1.79a 0.79b 0.17
0.32b 1.32a
0.04b 1.96a
0.07b 1.29a
1.18 0.46
1.32 0.68
0.89 0.46
0.039 0.329 0.014
< 0.001 0.454 < 0.001
< 0.001 0.769 < 0.001
a-d Means not sharing the same superscripts are significantly different (P < 0.05). 1 None = un-supplemented; IQA = 0.15 g/kg plant-derived isoquinoR Extra) in starter, grower and finisher. line alkaloids (Sangrovit
controlling inflammation and its physiologic consequences. Inflammation triggered by NE can lead to gluconeogenesis in order to retain circulating glucose level, particularly during the anorexia condition. In the present study, NE challenge lowered the cumulative FI by 17% at d 24. To compensate glucose production from the impaired FI, skeletal muscle would need to be catabolized into amino acids such as glutamine to provide the key substrate of gluconeogenesis resulting in a large loss of lean meat (Lacey and Wilmore, 1990; Wu et al., 1991). This was reflected in the present study as demonstrated by decreased BWG, breast and thigh meat yields, and poorer FCR as a result of NE challenge, whereas these have been alleviated when IQA was included in the diet. In addition, it may further
Phytobiotics Futterzusatzstoffe GmbH (Germany) is gratefully acknowledged for funding this project. The first author (G. D. Xue) was a recipient of a postgraduate scholarship from Phytobiotics Futterzusatzstoffe GmbH (Germany) and a fee waiver scholarship from the University of New England (Australia).
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