Hatchery and Dietary Application of Synbiotics in Broilers: Performance and mRNA Abundance of Ileum Tight Junction Proteins, Nutrient Transporters, and Immune Response Markers.

This study investigated the effects of a synbiotic consisting of inulin, Enterococcus faecium, Pediococcus acidilactici, Bifidobacterium animalis, and Lactobacillus reuteri given orally to day (d)-of-hatch (DOH) broiler chicks at the hatchery and in the feed for a 21 d period. A total of 480 Cobb male broilers were randomly divided into one of four treatments using a 2 × 2 factorial design as follows: (1) control (CTRL) group receiving a gel-only oral application on DOH at the hatchery prior to transport and a non-medicated basal corn/soybean meal starter diet; (2) hatchery synbiotic (HS) receiving an oral gel containing the synbiotic (0.5 mL/bird) at the hatchery and the basal diet; (3) CTRL + dietary synbiotic at 0.5 kg/MT (DS); and (4) HS + dietary synbiotic at 0.5 kg/MT (HSDS). On d 7 and d 21, one bird per pen (eight replicate pens/group) was euthanized, and the ileum was immediately removed for qPCR analysis. Data were subjected to a 2-way ANOVA using GLM procedure (JMP Pro17). A significant diet × hatchery interaction was observed in feed conversion ratio (FCR) from d 14 to d 21 (p = 0.013) where the HS, DS, and HSDS treatments had a significantly lower FCR compared to the CTRL. However, no significant interaction effect was observed for body weight gain (BWG) or FCR during the overall experimental period. No significant interaction was observed in mRNA abundance of the evaluated genes in the ileum on d 7 and d 21. Gel application with the synbiotic significantly reduced sodium-dependent glucose cotransporter 1 (SGLT1) mRNA abundance on d 7 (p = 0.035) in comparison to birds receiving gel alone. Regardless of hatchery application, dietary synbiotic supplementation significantly reduced Toll-like receptor (TLR)2, TLR4, and interleukin (IL)-10 mRNA abundance on d 7 (p = 0.013). In conclusion, these findings showed that hatchery and dietary synbiotic application could have a potential beneficial impact on broiler intestinal immunity by regulating the TLR response, a key element of innate immunity. FCR was improved from d 14 to d 21 after synbiotic application. Future research involving extended grow-out studies with a disease challenge would expand on the implications of an early application of synbiotics.

Fate of transgenic soybean DNA and immune response of broilers fed genetically modified DP-3Ø5423-1 soybean.

Increased use of genetically modified (GM) plants in the food and feed industry has raised several concerns about the presence of unwanted genes in the food chain and potential associated health risks. In recent years, several studies have compared the nutrient contents of GM crops to conventional counterparts, and some have also tracked the fate of novel DNA fragments and proteins in the gastrointestinal (GIT) and their presence in several tissues. This study was conducted to investigate the fate of transgenic PHP19340A DNA fragment containing gm-fad2-1 (Soybean Event DP-3Ø5423-1) gene in digestive tract contents, blood, internal organs, and muscle tissues. The effects of feeding DP-3Ø5423-1 full-fat soybean meal (FFSBM) to broiler chickens on immune response and blood profiles were also evaluated on d 35. Day-old Ross 308 birds (n = 480) were randomly allocated to 24 floor pens in a 2 × 2 factorial arrangement with diet and gender as main factors. Birds were fed diets containing 20% of either DP-3Ø5423-1 or non-GM FFSBM for 35 d. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (Pro13). Based on PCR analysis, transgenic PHP19340A DNA fragment containing gm-fad2-1 gene was degraded throughout the digestive system to reach undetectable level in the cecal digesta. Moreover, there was no transgenic gene translocation to blood, organs, or muscle tissue. Feeding DP-3Ø5423-1 FFSBM to broilers had no effect on mRNA abundance of IL-1ß, IL-2, IL-6, IL-12B, IL-17A, IFNγ, TNFα, and NF-κB in the spleen or on blood profile. In conclusion, these findings indicate that the examined transgenic fragment in DP-3Ø5423-1 FFSBM progressively degraded in the GIT and did not translocate into blood or tissues. Along with the immune response and blood profile findings, it can be assumed that DP-3Ø5423-1 soybean is safe and unlikely to pose any health risks to broilers or consumers.

Performance, fatty acid composition, and liver fatty acid metabolism markers of broilers fed genetically modified soybean DP-3Ø5423-1.

Several genetically modified (GM) plants have been produced and approved by regulatory agencies worldwide for cultivation and commercialization. Soybean and its by-products are major components of poultry diets and approximately 74% of world production is obtained from GM soybean events. The aim of this study was to evaluate the nutrient composition of DP-3Ø5423-1 extruded full-fat soybean meal (FFSBM) and near isoline non-GM control FFSBM included in broiler diets. Also assessed were their effects on bird performance, body composition, intestinal morphology, tissue fatty acid profile, and mRNA abundance of fatty acid metabolism markers. A total of 480 Ross 308 d of hatch birds were randomly allocated to 24 floor pens in a 2 × 2 factorial arrangement with diet and gender as main factors. Birds were fed diets containing 20% of either DP-3Ø5423-1 or control FFSBM for 35 d. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (Pro13). No significant interaction (P > 0.05) was observed between treatment groups in terms of performance and carcass composition. Morphological measurements of the jejunum and ileum were not influenced by the SBM treatments. Dietary addition of the DP-3Ø5423-1 FFSBM resulted in higher monounsaturated fatty acid composition of the thigh muscle and abdominal fat. Moreover, dietary treatment had no significant impact on the mRNA abundance of metabolic markers ACCα, FAS, MTTP, SREBP1, PPARα, PPARγ, AMPK-α1, SOD, CAT, and GPx in the liver. In conclusion, our results showed that DP-3Ø5423-1 extruded FFSBM is nutritionally equivalent to non-GM near-isoline counterpart with a comparable genetic background as evidenced by feed analyses except for fatty acid composition. Furthermore, the findings of this study clearly indicate that the examined DP-3Ø5423-1 FFSBM yields similar bird performance as conventional FFSBM.

Inclusion of Quillaja Saponin Clarity Q Manages Growth Performance, Immune Response, and Nutrient Transport of Broilers during Subclinical Necrotic Enteritis.

Necrotic enteritis (NE) is an intestinal disease that results in poor performance, inefficient nutrient absorption, and has a devastating economic impact on poultry production. This study evaluated the effects of a saponin-based product (Clarity Q, CQ) during an NE challenge. A total of 1200 male chicks were randomly assigned to four dietary treatments (10 pens/treatment; 30 birds/pen): treatment 1 (NC), a non-medicated corn-soybean basal diet; treatment 2 (PC), NC + 50 g/metric ton (MT) of bacitracin methylene disalicylate (BMD); and treatments 3 (CQ15) and 4 (CQ30), NC + 15 and 30 g/MT, respectively. On the day (d) of placement, birds were challenged by a coccidia vaccine to induce NE. On d 8, 14, 28, and 42, performance parameters were measured. On d 8, three birds/pen were necropsied for NE lesions. On d 8 and d 14, jejunum samples from one bird/pen were collected for mRNA abundance of tight junction proteins and nutrient transporter genes. Data were analyzed in JMP (JMP Pro, 16), and significance (p ≤ 0.05) between treatments was identified by Fisher's least significant difference (LSD) test. Compared to PC and NC, CQ15 had higher average daily gain (ADG), while CQ30 had lower average daily feed intake (ADFI) and feed conversion ratio (FCR). NE lesions in the duodenum were lower in CQ15 compared to all other treatments. On d 8, mRNA abundance of CLDN1, CLDN5, AMPK, PepT2, GLUT2, and EAAT3 were significantly greater in CQ30 (p < 0.05) compared to both PC and NC. On d 14, mRNA abundance of ZO2 and PepT2 was significantly lower in PC when compared to all treatments, while that of ANXA1, JAM3, and GLUT5 was comparable to CQ15. In summary, adding Clarity Q to broiler diets has the potential to alleviate adverse effects caused by this enteric disease by improving performance, reducing intestinal lesions, and positively modulating the mRNA abundance of various tight junction proteins and key nutrient transporters during peak NE infection.

Influence of dietary vitamin E and selenium supplementation on broilers subjected to heat stress, Part II: oxidative stress, immune response, gut integrity, and intestinal microbiota.

This study evaluated the effects of vitamin E (Vit E) and selenium (Se) supplementation on mRNA abundance of antioxidant, immune response, and tight junction genes, as well as taxonomic and functional profiles of ileal microbiota of broilers exposed to daily 4-h elevated temperature during d 28 to 35. A total of 640-day-old Cobb male broiler chicks were randomly allocated to 32 floor pens in a 2 × 2 factorial arrangement that included ambient temperature (thermoneutral [TN] or heat stress [HS]) and dietary treatments (basal diet or Vit E + Se). Vit E and organic Se were added to the basal diet at the rate of 250 mg/kg and 1 mg/kg, respectively. Liver and jejunum tissue samples were taken on d 27 (1 bird/pen), d 28 and d 35 (2 birds/pen) from birds for qPCR analysis. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP. Ileal contents were taken on d 27 and d 35 for microbial profiling. Microbiota data were analyzed in QIIME 2 and significance between treatments identified linear discriminant analysis effect size (LEfSe, P < 0.05). Dietary Vit E/Se significantly downregulated the mRNA levels of HSPs in liver and jejunal tissues of the HS-challenged birds both on d 28 and d 35. Moreover, mRNA abundance of TLR2, TNFα, IFNγ, IL-1ß, IL-10, and iNOS in the liver were significantly downregulated in birds fed the Vit E/Se diet on d 35. However, dietary treatment had no significant impact on oxidative stress, immunity, and gut integrity related genes analyzed in jejunal tissues on d 28 and d 35, except downregulation of IFNγ on d 35 (P = 0.052). LEfSe analysis revealed that Lachnospiraceae FE2018 and Ruminococcaceae NK4A214 groups was enriched in the Vit E/Se birds on d 35. Moreover, PICRUSt analysis predicted significant functional differences among the treatment groups. In conclusion, dietary supplementation of Vit E/Se mitigated the negative effects of HS potentially via improving antioxidant status, regulating cytokine responses and modifying ileal microbiota and its function.

Influence of dietary vitamin E and selenium supplementation on broilers subjected to heat stress, Part I: Growth performance, body composition and intestinal nutrient transporters.

High ambient temperature is one of the most common stressors in modern poultry production, resulting in reduced feed intake, weight gain, and increased mortality. This study evaluated the effects of vitamin E (Vit E) and organic selenium (Se) supplementation on performance, body composition, core body temperatures, and mRNA abundance of nutrient transporters in the jejunum of broilers exposed to daily 4-h elevated temperature during d 28 to 35. A total of 640 Cobb male birds were randomly allocated to 32 floor pens in a 2 × 2 factorial arrangement that included ambient temperature (thermoneutral, [TN]; or heat stress, [HS]) and dietary treatments (basal diet or Vit E + Se). Four rooms were used (2 TN and 2 HS) each housing half of the 8 replicate pens per group. Vit E and organic Se were added to the basal diet at the rate of 250 mg/kg and 1 mg/kg diet, respectively. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (SAS). During the HS period, birds fed the Vit E/Se diet had significantly lower mortality compared to nonsupplemented group (1.92% vs. 7.01%). Moreover, dietary Vit E/Se supplementation had a significant effect on performance by increasing BWG, FI, and European production efficiency factor (EPEF) during the entire experimental period (d 0-35). Dietary Vit E and Se supplementation significantly increased carcass, tissue, lean, and fat weights as well as bone mineral content (BMC) and bone mineral density (BMD) on d 35. Birds fed Vit E/Se supplemented diet had significantly lower (P = 0.010) core body temperature compared to birds fed the basal diet on d 30. Dietary treatment did not influence mRNA abundance of PepT1, SGLT1, or NaPi-IIb on d 28 or d 35. However, HS significantly upregulated levels of PepT1 and NaPi-IIb (P < 0.001) and downregulated that of SGLT1 (P = 0.017) on d 28. In conclusion, dietary Vit E and Se supplementation significantly improved broiler growth performance and carcass composition, and reduced heat-related mortality and core body temperature (on d 30) without influencing the mRNA abundance of intestinal nutrient transporters.

Managing broilers gut health with antibiotic-free diets during subclinical necrotic enteritis.

Necrotic enteritis (NE) caused by Clostridium perfringens is among the most important enteric diseases in poultry production. This study examined the effects of 2 probiotics (Prob) and a synbiotic (Synb) during a naturally occurring NE challenge. On the day of hatch, 1200 Cobb male broilers were randomly allocated to 5 groups (8 pens/treatment, 30 birds/pen) including 1) negative control (NC): corn-soybean meal diet; 2) positive control (PC): NC + 453 g Stafac20/907 kg feed; 3) Prob 1: NC + 453 g Prob 1/907 kg feed; 4) Prob 2: NC + 453 g Prob 2/907 kg feed; and 5) Synb: NC + 453 g Synb/907 kg feed. One day after placement, birds were challenged by a coccidia vaccine to induce NE. Feed intake and body weights were measured on day 8 (NE onset) and end of starter (day 14) and grower (28) periods. On day 8, the small intestines of 3 birds/pen were examined for NE lesions. Ileal mucosal scrapings from one bird/pen were collected on day 8 and day 28 to profile the microbiota using 16S rRNA sequencing. Data were analyzed in JMP or QIIME 2 and significance between treatments identified by LSD or linear discriminant analysis effect size (P < 0.05). The Synb group significantly lowered NE lesion scores on day 8 and reduced day 0-14 mortality by 50% compared with NC. FCR was significantly better in all the groups, whereas ADG was higher in PC, Synb, and Prob 2 groups compared with NC from day 0 to day 28. Lower lesion scores in the Synb group were accompanied by lower relative abundance of Alistipes, ASF356, Faecalibaculum, Lachnospiraceae UCG-001, Muribaculum, Oscillibacter, Parabacteroides, Rikenellaceae RC9 gut group, Ruminococcaceae UCG-014, and Ruminiclostridium 9 compared with NC on day 8. On day 28, relative abundance of Lactobacillus was lower, whereas abundance of Bacteroides, Barnesiella, Butyricicoccus, CHKCI001, Eisenbergiella, Eubacterium hallii group, Helicobacter, Ruminococcaceae UCG-005, Ruminococcus torques group, and Sellimonas was significantly higher in the NC birds than in the Synb and Prob 2 groups. Collectively, these data indicate that during a subclinical naturally occurring NE, supplementation of specific additives could be effective in reducing intestinal lesions and mortality, and improving performance potentially through developing a signature microbial profile in the intestinal mucosal layer.

The Modulating Effect of Dietary Beta-Glucan Supplementation on Expression of Immune Response Genes of Broilers during a Coccidiosis Challenge.

This study investigated the effects of a yeast-derived ß-glucan (Auxoferm YGT) supplementation on mRNA expression of immune response genes in the spleen, thymus, and bursa of broiler chickens during a mixed Eimeria infection. Day (d)-old chicks (n = 1440) were fed diets containing 0% or 0.1% YGT. On d 8 post-hatch, half the replicate pens (n = 8) were challenged with a mixed inoculum of E. acervulina, E. maxima, and E. tenella. On d 10 and d 14 post-hatch, the spleen, thymus, and bursa were collected to evaluate mRNA abundance by quantitative real-time PCR. Data were analyzed using PROC GLIMMIX model (2-way interaction) and differences were established by LS-MEANS with significance reported at p ≤ 0.05. In spleen tissues at d 10, expression of interleukin (IL)-10 and inducible nitric oxide synthase (iNOS) were elevated in both 0.1% YGT-fed challenged and non-challenged birds. In thymus tissues at d 14, expression of IL-10, IL-17F, interferon (IFN)-γ, iNOS, and macrophage migration inhibitory factor (MIF) were elevated in challenged birds fed 0.1% YGT. In bursal tissues at d 10 and d 14, expression of IL-10, IFN-γ, iNOS (d 10 only), and MIF were elevated in 0.1% YGT-fed challenged and non-challenged birds. Dietary ß-glucan supplementation to chicken diets modulated their immune response to the Eimeria challenge.

Effect of Probiotics and Multi-Component Feed Additives on Microbiota, Gut Barrier and Immune Responses in Broiler Chickens During Subclinical Necrotic Enteritis.

The withdrawal of antibiotic growth promoters from poultry feed has increased the risk of necrotic enteritis (NE) outbreaks. This study examined the effects of a probiotic (PROB) or probiotic/prebiotic/essential oil supplement (PPEO) during a subclinical NE challenge. On day (d) of hatch, 960 male broilers were randomized to four groups (8 pens/treatment, 30 birds/pen) including (1) negative control (NC): corn-soybean meal diet; (2) positive control (PC): NC + 20 g Virginiamycin/ton diet; (3) NC + 227 g PROB/ton diet; and (4) NC + 453 g PPEO/ton diet. One d after placement, birds were challenged by a coccidia vaccine to induce NE. Feed intake and body weights were measured on d 8 (NE onset) and end of each feeding period. On d 8, the small intestines of three birds/pen were examined for NE lesions. Jejunum samples and ileal mucosal scrapings from one bird/pen were respectively collected to measure mRNA abundance (d 8 and d 14) and profile the microbiota (d 8 and d 42). Data were analyzed in JMP or QIIME 2 and significance between treatments identified by LSD (P < 0.05). PROB and PPEO had significantly lower mortality (d 0-14) and NE lesion scores compared to NC. Feed conversion ratio was significantly lower in PC, PROB, and PPEO, while average daily gain was higher in PPEO and PC groups compared to NC from d 0-42. On d 8 and d 14, mRNA abundance of claudin-3 was higher in PPEO compared to NC. On d 14, compared to NC, mRNA abundance of sIgA and PGC-1α in PROB and PPEO were lower and higher, respectively. Compared to NC, PPEO increased mTOR abundance on d 14. On d 8, relative abundance of Clostridium sensu stricto 1, Ruminiclostridium9, Prevotellaceae, Prevotellaceae UCG-014, ASF356, and Muribaculaceae was higher in NC compared to PPEO and PROB, while Lactobacillus was lower in NC. Escherichia-Shigella had higher abundance in PC compared to PPEO and PROB. Collectively, these data indicate that during a subclinical naturally occurring NE, supplementation of PROB or PPEO supports performance and reduces intestinal lesions, potentially through modifying tight junction proteins, gut microbiota, immune responses, and cell metabolism.

Necrotic Enteritis in Broiler Chickens: The Role of Tight Junctions and Mucosal Immune Responses in Alleviating the Effect of the Disease.

Necrotic enteritis (NE) continues to present major challenges to the poultry industry, and the etiologic agent Clostridium perfringens is the fourth leading cause of bacterially-induced food- borne illnesses in the US. This study was designed to evaluate the effects of a probiotic during naturally occurring NE. On day of hatch, 1080 Cobb 500 male broilers were randomly allocated to three groups (12 replicate pens/treatment, 30 birds/pen) including 1) negative control (NC): corn-soybean meal diet; 2) positive control (PC): NC + 20 mg virginiamycin/kg diet (0.450 kg Stafac®20/ton); and 3) NC + PrimaLac (1.36 and 0.91 kg/ton from 1-21 and 22-42 days, respectively). One day (d) post placement, all birds were challenged by a commercial live oocyst coccidia vaccine as a predisposing factor to NE. Body weight and feed intake were measured at the onset of NE (d 8) and end of each feeding phase. On d 8, small intestines of two birds/pen were examined for NE lesions, and jejunum samples from one bird were collected for mRNA gene expression analysis of tight junction proteins, cytokines, and nutrient transporters. Data were analyzed using the Jump (JMP) software and significance between treatments identified by LSD (P < 0.05). Compared to NC, supplementation of probiotic reduced d 1-42 mortality; however, PC was the only group with significantly lower mortality. Despite significantly improved feed conversion ratio (FCR) in PC and probiotic groups during d 1-42, average daily gain was only higher in PC (77.69 g/bird) compared with NC (74.99 g/bird). Furthermore, probiotic and PC groups had significantly reduced lesion scores in the duodenum and jejunum compared to NC. Expression of claudin-3 was higher, while expression of zonula occluden-2 tended (P = 0.06) to be higher in probiotic-supplemented birds compared to NC. Moreover, birds fed the probiotic diet had significantly higher expression of IL-10, IL-17, AMPK-α1, and SGLT1 mRNA compared to NC birds. The expression of PepT1 was higher for the probiotic-supplemented group compared to PC. IFN-γ expression was lower in PC compared to NC, while there was no difference between probiotic and NC. There were no differences in gene expression of sIgA, TNF-α, IL-1ß, and IL-22 among treatments. Collectively, these data indicate that in a naturally occurring NE model, supplementation of a probiotic helps to improve FCR and reduce lesions, potentially due to the improvements in mRNA expression of tight junctions, cytokines, and nutrient transporters.

Dietary Non-Drug Feed Additive as an Alternative for Antibiotic Growth Promoters for Broilers During a Necrotic Enteritis Challenge.

Necrotic enteritis, caused by Clostridium perfringens, is an enteric disease that leads to poor performance and increased mortality, resulting in significant economic losses in poultry production. This study evaluated the effects of a proprietary prebiotic, probiotic, and plant extract blend on performance of broilers during coccidiosis challenge leading to necrotic enteritis (NE). In total, 744 Cobb500 male broilers were randomly allocated to 3 treatments (8 replicates, 31 birds/pen) including, the negative control (NC) fed a basal diet; the positive control (PC) fed a basal diet with Virginiamycin; and the additive group fed basal diet with a blend of prebiotic, probiotic, and plant extract (BSN). A unique, naturally occurring NE model developed to mimic field conditions was implemented to challenge the birds. This model consists of spraying a concentrated commercial coccidiosis vaccine on litter and feed upon bird placement, which, in conjunction with the presence of C. perfringens spores in the environment, leads to the development of a NE outbreak one week post vaccine application. At the onset of NE on d7, three birds/pen were selected for scoring NE lesions. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were recorded on days 7, 14, 28, and 42. Carcass composition was assessed by dual energy X-ray absorptiometry (DXA) analysis on day 42. Dietary supplementation of BSN significantly (p < 0.05) improved FCR during starter and grower periods. Dietary treatments had no effect on NE lesions in the small intestine. DXA analysis revealed slightly higher lean content in BSN birds compared to NC. These results showed that dietary supplementation of the BSN blend significantly improved broilers performance during the early NE challenge phase, as well as in the grower period.

Effects of Dietary Direct Fed Microbial Supplementation on Performance, Intestinal Morphology and Immune Response of Broiler Chickens Challenged With Coccidiosis.

Poultry coccidiosis is a costly intestinal disease that leads to considerable tissue damage, inefficient nutrient absorption, increased mortality, and predisposition to secondary infections. This study evaluated the effects of a direct feed microbial (DFM) dietary additive on performance, intestinal morphology, and immune response of broilers during a mixed coccidiosis challenge. In total, 840 Cobb500 male broilers were randomly allocated to 3 treatments (7 replicates, 40 birds/pen) including negative control (NC) fed basal diet; positive control (PC) fed basal diet with coccidiosis challenge; and DFM supplemented diet, with coccidiosis challenge. At 15 days of age, all birds except for the NC treatment were orally gavaged with live oocysts of a commercial vaccine. On d 21 (6 days post challenge), 4 birds/pen were randomly selected and euthanized for scoring of coccidia-caused lesions in the duodenum, jejunum, and ceca. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were recorded on d 7, 14, 28, and 42. Jejunal and ileal tissue samples were taken for histomorphological assessment from 2 birds/pen on d 21. Ileal samples were also taken for mRNA expression analysis on d 14 and d 21. The DFM birds had significantly greater BWG than PC birds during d 0-21 (P < 0.05). No differences were observed among the treatment groups in terms of FI and FCR. Dietary DFM supplementation significantly reduced lesion scores in the duodenum and jejunum when compared with PC group (P < 0.05). The coccidia challenge significantly reduced (P < 0.05) ileal villus height when compared to the non-challenged group on d 21. Conversely, dietary DFM supplementation alleviated the negative effects of coccidiosis by increasing ileal villus area on d 21 (P < 0.05). The challenged birds had significantly greater expression of IFN-γ and IL-1ß in the ileum on d 21. Based on these findings, dietary DFM supplementation may help restore broiler performance during the starter and early grower periods during coccidiosis, likely by maintaining gut integrity via improving intestinal morphology and also by reducing disease severity as manifested by lower lesion scores.