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.

The Probiotic Bacillus subtilis MB40 Improves Immunity in a Porcine Model of Listeriosis.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Research note: Lateral transmission of Histomonas meleagridis in turkey poults raised on floor pens.

Histomoniasis is caused by the protozoa Histomonas meleagridis (HM) that are laterally transmitted among birds leading to high mortality in commercial flocks. This study tested an HM infection model assessing the lateral transmission of HM in turkey poults raised on floor pens. Day (d)-old female turkey poults (n = 320) were individually wing-tagged and allocated to one of four treatment groups (4 floor pens/group and 20 poults/pen) based on the percentage of poults inoculated with HM: 1) 10% (HM10); 2) 20% (HM20); 3) 30% (HM30); and 4) 40% (HM40). On d 9, seeder poults intracloacally received a 1 mL inoculum/bird containing ∼80,000 histomonads. Poults were individually weighed on d 0, 9, and 25 and feed intake recorded on per pen basis. On d 25, all birds were euthanized by cervical dislocation and ceca and liver were evaluated for HM lesions. Data were analyzed using JMP (Pro16) and significance (P ≤ 0.05) between treatments were determined by LSD test. Mortality was 7.63%, 12.5%, 21.58%, and 20.59%, while transmission rates from inoculated to non-inoculated birds were 62.5%, 57.5%, 92.43%, and 78.75% in HM10, HM20, HM30, and HM40 groups, respectively. Average daily feed intake was proportionally reduced with the increasing number of inoculated poults from HM10 to HM40. Average daily gain was significantly lower in HM30 and HM40 poults compared to those in HM10 and HM20 during the postchallenge period (d 10-25). Therefore, we herein report the successful lateral transmission of HM among turkey poults raised on floor pens. This research model closely resembles commercial field conditions and affords a much-needed platform for conducting relevant basic and applied research on histomoniasis in poultry.

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.

Ileal microbial composition in genetically distinct chicken lines reared under normal or high ambient temperatures.

BACKGROUND: Heat stress (HS) has negative effects on poultry productivity, health and welfare resulting in economic losses. Broiler chickens are particularly susceptible to HS due to their high metabolic rate and rapid growth. The commensal intestinal bacterial populations have an important physiological role in the host and could ameliorate the negative effect of HS on the host. Thus, the aim of this study was to compare changes in the ileal (IL) microbiota in four different broiler lines during HS. RESULTS: Day-old broiler chicks from Giant Jungle Fowl (JF), Athens Canadian Random Bred (ACRB), 1995 Random Bred (L1995), and Modern Random Bred (L2015) lines were raised under thermoneutral (TN) conditions until day (d) 28. On d 29 birds were subjected to TN (24 °C) or chronic cyclic HS (8 h/d, 36 °C) condition till d 56. On d 56 two birds per pen were euthanized, and IL luminal content (IL-L) and mucosal scrapings (IL-M) were collected for bacterial DNA isolation. Libraries were constructed using V3-V4 16S rRNA primers and sequenced using MiSeq. DNA sequences were analyzed using QIIME2 platform and SILVA 132 database for alpha and beta diversity, and taxonomic composition, respectively. Functional property of microbiota was predicted using the PICRUSt 2 pipeline and illustrated with STAMP software. Shannon index was significantly elevated in IL-M under HS. ß-diversity PCoA plots revealed separation of microbial community of L2015-TN from JF-TN, JF-HS, ACRB-TN, and ACRB-HS in the IL-M. PERMANOVA analysis showed a significant difference between microbial community of L1995-HS compared to ACRB-HS and JF-TN, L1995-TN compared to ACRB-HS and JF-TN, L2015-HS compared to ACRB-HS and ACRB-TN, L2015-HS compared to JF-TN, L2015-TN compared to ACRB-HS and JF-TN, and ACRB-HS compared to JF-TN in the IL-L. The impact of HS on microbial composition of IL-M was more prominent compared to IL-L with 12 and 2 taxa showing significantly different relative abundance, respectively. Furthermore, differences in microbiota due to the genetic line were more prominent in IL-M than IL-L with 18 and 8 taxa showing significantly different relative abundance, respectively. Unlike taxonomy, predicted function of microbiota was not affected by HS. Comparison of L2015 with JF or ACRB showed significant changes in predicted function of microbiota in both, IL-M and IL-L. Differences were most prominent between L2015 and JF; while there was no difference between L2015 and L1995. CONCLUSIONS: These data indicate the genetic line × temperature effect on the diversity and composition of IL microbiota. Moreover, the data showcase the effect of host genetics on the composition of IL microbiota and their predicted function. These data are of critical importance for devising nutritional strategies to maintain GIT microbial balance and alleviate the negative effects of HS on broiler chickens' performance and health.

Heat Stress and Feed Restriction Distinctly Affect Performance, Carcass and Meat Yield, Intestinal Integrity, and Inflammatory (Chemo)Cytokines in Broiler Chickens.

This study was conducted to distinguish the effects of heat stress (HS) and feed intake (FI) on broiler chicken's physiological responses. Day-old male Cobb 500 broilers (n = 672) were allocated to three treatments: (1) control (CTL): birds raised under normal temperature (23°C) from day 29 to 42; (2) cyclic heat stress (CHS): birds exposed to high temperatures (8 h/day at 35°C; from 9:30 am to 5:30 pm) from day 29 to 42; (3) pair-fed (PF): birds raised under thermoneutral condition but fed the same amount of feed as CHS from day 29 to 42. On day 42, 15 birds/pen were processed, to measure carcass and meat yields. To measure blood parameters and gut integrity (using fluorescein isothiocyanate-dextran), on day 42, CHS birds were sampled before (Pre-CHS) and 2 h after (Post-CHS) the temperature increased. Furthermore, after sampling CTL birds, they were exposed to 2h heat and sampled (acute heat stress, AHS). Data were analyzed using one-way ANOVA (JMP Pro15) and significance between treatments identified by LSD (P < 0.05). BW and relative carcass yield were significantly higher in CTL compared to CHS and PF. Compared to CHS, PF had significantly higher BW and lower relative carcass yield. Breast yield was significantly higher for CTL and PF, while leg quarters and wings yield were significantly lower compared to CHS. Gut barrier integrity was significantly altered in Post-CHS and AHS compared to CTL. mRNA abundances of tumor necrosis factor-α, C-C motif chemokine ligand-20, heat shock protein (HSP)-27, and HSP70 were significantly higher in Post-CHS and AHS compared to CTL. AHS had significantly higher mRNA abundances of CARD domain containing (NLRC)-3 and NLRC5 inflammasomes, and lower superoxide dismutase (SOD)-1 and SOD2 abundance compared with CTL. PF had significantly higher liver weight (% BW) compared to all other groups; while abdominal fat was significantly higher in Pre-CHS compared to CTL, PF, and AHS. Together, these data indicate that the negative effects of HS are partially due to reduced FI. However, the negative effect of HS on gut integrity, average daily gain, feed conversion ratio, and meat yield are direct and independent of the reduced FI during the HS. Thus, warrant investigating the underlying mechanisms in future research.

Centennial Review: Recent developments in host-pathogen interactions during necrotic enteritis in poultry.

Necrotic enteritis (NE) is a significant enteric disease in commercial poultry with considerable economic effect on profitability manifested by an estimated $6 billion in annual losses to the global industry. NE presents a unique challenge, being a complex enteric disease that often leads to either clinical (acute) or subclinical (chronic) form. The latter typically results in poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio [FCR]) with low mortality rates, and represents the greatest economic impact on poultry production. The use of antibiotic growth promoters (AGPs) has been an effective tool in protecting birds from enteric diseases by maintaining enteric health and modifying gut microbiota, thus improving broilers' production efficiency and overall health. The removal of AGPs presented the poultry industry with several challenges, including reduced bird health and immunity as well as questioning the safety of poultry products. Consequently, research on antibiotic alternatives that can support gut health was intensified. Probiotics, prebiotics, essential oils, and organic acids were among various additives that have been tested for their efficacy against NE with some being effective but not to the level of AGPs. The focus of this review is on the relationship between NE pathogenesis, microbiome, and host immune responses, along with references to recent reviews addressing production aspects of NE. With a comprehensive understanding of these dynamic changes, new and programmed strategies could be developed to make use of the current products more effectively or build a stepping stone toward the development of a new generation of supplements.

Hypoxia further exacerbates woody breast myopathy in broilers via alteration of satellite cell fate.

Woody breast (WB) condition has created a variety of challenges for the global poultry industry. To date, there are no effective treatments or preventative measures due to its unknown (undefined) etiology. Several potential mechanisms including oxidative stress, fiber-type switching, cellular damage, and altered intracellular calcium levels have been proposed to play a key role in the progression of the WB myopathy. In a previous study, we have shown that WB is associated with hypoxia-like status and dysregulated oxygen homeostasis. As satellite cells (SC) play a pivotal role in muscle fiber repair and remodeling under stress conditions, we undertook the present study to determine satellite cell fate in WB-affected birds when reared in either normoxic or hypoxic conditions. Modern random bred broilers from 2015 (n = 200) were wing banded and reared under standard brooding practices for the first 2 wk post-hatch. At 15 d, chicks were divided in 2 body weight-matched groups and reared to 6 wk in either control local altitude or hypobaric chambers with simulated altitude of 6,000 ft. Birds were provided ad libitum access to water and feed, according to the Cobb recommendations. At 6 wk of age, birds were processed and scored for WB, and breast samples were collected from WB-affected and unaffected birds for molecular analyses (n = 10/group). SCs were isolated from normal breast muscle, cultured in vitro, and exposed to normoxia or hypoxia for 2 h. The expression of target genes was determined by qPCR using 2-∆∆Ct method. Protein distribution and expression were determined by immunofluorescence staining and immunoblot, respectively. Data were analyzed by the Student's t test with significance set at P < 0.05. Multiple satellite cell markers, myogenic factor (Myf)-5 and paired box (PAX)-7 were significantly decreased at the mRNA and protein levels in the breast muscle from WB-affected birds compared to their unaffected counterparts. Lipogenic-and adipogenic-associated factors (acetyl-CoA carboxylase, ACCα; fatty acid synthase, FASN, malic enzyme, ME; and ATP citrate lyase, ACLY) were activated in WB-affected birds. These data were supported by an in vitro study where hypoxia decreased the expression of Myf5 and Pax7, and increased that of ACCα, FASN, ME, and ACLY. Together, these data indicate that under hypoxic condition, SC change fate by switching from a myogenic to an adipogenic program, which explains at least partly, the etiology of the WB myopathy.

Spirulina platensis Inclusion Reverses Circulating Pro-inflammatory (Chemo)cytokine Profiles in Broilers Fed Low-Protein Diets.

Proteins are considered the most expensive nutrients in commercial modern broiler production, and their dietary inclusion at low levels is pivotal to minimize feed costs and reduce nitrogen waste. The quest for an environmentally friendly source of proteins that favor the formulation of low protein diets without compromising broiler health, welfare, and growth performance has become a hotspot in nutrition research. Due to its high protein content, the naturally growing Spirulina microalgae is considered a promising nutrient source. The purpose of the present study was, therefore, to determine the effects of Spirulina supplementation on liver bacterial translocation, hematological profile, and circulating inflammatory and redox markers in broilers fed a low-protein diet. One-day-old Ross 708 male broilers (n = 180) were randomly assigned into one of three experimental treatments: standard diet as a control, low protein diet, and low protein diet supplemented with 100 g/kg of Spirulina. Target molecular markers were measured in the peripheral blood circulation using real-time quantitative PCR. Reducing dietary proteins increased bacterial translocation and systemic inflammation as indicated by proportions of basophils among blood leukocytes. The expression levels of circulating pro-inflammatory cytokines [interleukin (IL)-3, IL-6, IL-4, IL-18, and tumor necrosis factor-α], chemokines (CCL-20), and NOD-like receptor family pyrin domain containing 3 inflammasome were significantly upregulated in birds fed the low protein diet compared with the control. The inclusion of Spirulina reversed these effects, which indicates that Spirulina reduces systemic inflammation- and bacterial translocation-induced by a low protein diet and could be a promising alternative protein source in poultry diets.

Phytogenic Water Additives Improve Broiler Growth Performance via Modulation of Intermediary Metabolism-Related Signaling Pathways.

A ban on the use of antibiotic growth promoters (AGPs) has fueled and promoted scientific research towards the identification of reliable and effective alternatives. The supplementation of phytogenics AV/SSL12 (AVSSL) and Superliv Gold (SG) in water has been shown to improve broiler feed efficiency (FE) via modulation of hypothalamic neuropeptides. However, their effects on peripheral metabolic pathways are still unknown. The present study was undertaken to determine the effects of AVSSL and SG on lipid and protein metabolism-associated pathways in various tissues. Day-old male Cobb 500 chicks (n = 288) were randomly assigned to 3 treatment groups, with 8 replicates of 12 birds each. The treatment groups were fed a basal diet and supplemented with AVSSL or SG in the drinking water at a rate of 2, 4, and 7 mL/100 birds/d during the starter, grower, and finisher phases, respectively. The control group were fed a basal diet with no additive supplementation. On d 35, liver, adipose, and muscle tissue were collected from one bird per pen (8 birds/group). Data were analyzed using Student's T-test to compare one treatment group to the control using Graph Pad Prism version 6.0 for Windows. In the liver, the levels of phosphorylated acetyl-CoA carboxylase alpha (ACCα) were significantly increased in both the AVSSL and SG groups compared to the control. The hepatic expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP) was significantly downregulated in both treated groups compared to the control. AVSSL supplementation downregulated the hepatic expression of SREBP-2 and adiponectin (AdipoQ), while SG administration upregulated hepatic AdipoR1/R2 mRNA abundances compared to the untreated group. Both AVSSL and SG treatments upregulated hepatic stearoyl-CoA desaturase-1 (SCD-1) gene expression compared to their untreated counterparts. In the adipose tissue, the levels of phosphorylated hormone-sensitive lipase (HSL) at Ser855/554 site were increased in both the AVSSL and SG groups compared to the control. However, ATGL protein expression was decreased in SG compared to the untreated group. In the muscle, the levels of phosphorylated mechanistic target of rapamycin (mTOR) were increased in the AVSSL, but decreased in the SG group compared to the control. Collectively, these data indicate that supplementation of the phytogenics AVSSL and SG in water reduced hepatic lipogenesis-related proteins and increased adipose tissue lipolysis- and muscle protein synthesis-associated targets, which might explain, at least partially, the improvement in FE observed in previous research.

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.

Research Note: Phytobiotics modulate the expression profile of circulating inflammasome and cyto(chemo)kine in whole blood of broilers exposed to cyclic heat stress.

Heat stress (HS) is a critical concern to the poultry industry as it affects both productivity and well-being. Various managerial and nutritional strategies have been proposed to mitigate the negative effects of HS in chickens, with plant-based additives showing promise. Recently, we reported the positive effect of a phytogenic feed additive (PFA) on growth performance in HS birds. Owing to the antioxidant nature of these compounds, we sought to further explore the effect of PFA on whole blood circulating chemokines, cytokines, and inflammasomes in HS broilers. Broilers (600 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to 2 environmental conditions (12 h cyclic heat stress, HS, 35°C vs. thermoneutral condition [TN], 24°C) and fed 3 diets (control, PFA-C 250 ppm, PFA-C 400 ppm) in a 2 × 3 factorial design. After 21 d of cyclic HS, blood samples were collected for target gene expression analysis. HS upregulated the expression of superoxide dismutase 1 (SOD1) and downregulated glutathione peroxidase-3 (GPX-3), and there was diet × temperature interaction for SOD2, GPX-1, and GPX-3, where gene expression was increased by PFA-C250 during HS but was unchanged for PFA-C400. Plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) content were increased by HS. Gene expression of interleukin-18 (IL-18) was decreased by HS, without further effect of PFA. HS increased tumor necrosis factor α (TNFα), but this effect was mitigated by PFA-C400. C-C motif chemokine ligands 4 and 20 (CCL4 and CCL20) showed a similar pattern to TNFα, with PFA-C400 ameliorating the negative effect of HS. The nucleotide-binding, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome was decreased by HS and further lowered by PFA-C400, but the nucleotide-binding oligomerization domain, leucine-rich repeat, and CARD domain containing 3 (NLRC3) and nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) inflammasomes were increased by PFA under TN conditions, with no effects of HS. Heat shock proteins (HSP) and heat shock factors (HSF) were unaffected by PFA or HS. Together these data indicate that gene expression of circulating inflammatory factors are dysregulated during HS, and supplemental dietary PFA may be protective.

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.

Radical Response: Effects of Heat Stress-Induced Oxidative Stress on Lipid Metabolism in the Avian Liver.

Lipid metabolism in avian species places unique demands on the liver in comparison to most mammals. The avian liver synthesizes the vast majority of fatty acids that provide energy and support cell membrane synthesis throughout the bird. Egg production intensifies demands to the liver as hepatic lipids are needed to create the yolk. The enzymatic reactions that underlie de novo lipogenesis are energetically demanding and require a precise balance of vitamins and cofactors to proceed efficiently. External stressors such as overnutrition or nutrient deficiency can disrupt this balance and compromise the liver's ability to support metabolic needs. Heat stress is an increasingly prevalent environmental factor that impairs lipid metabolism in the avian liver. The effects of heat stress-induced oxidative stress on hepatic lipid metabolism are of particular concern in modern commercial chickens due to the threat to global poultry production. Chickens are highly vulnerable to heat stress because of their limited capacity to dissipate heat, high metabolic activity, high internal body temperature, and narrow zone of thermal tolerance. Modern lines of both broiler (meat-type) and layer (egg-type) chickens are especially sensitive to heat stress because of the high rates of mitochondrial metabolism. While this oxidative metabolism supports growth and egg production, it also yields oxidative stress that can damage mitochondria, cellular membranes and proteins, making the birds more vulnerable to other stressors in the environment. Studies to date indicate that oxidative and heat stress interact to disrupt hepatic lipid metabolism and compromise performance and well-being in both broilers and layers. The purpose of this review is to summarize the impact of heat stress-induced oxidative stress on lipid metabolism in the avian liver. Recent advances that shed light on molecular mechanisms and potential nutritional/managerial strategies to counteract the negative effects of heat stress-induced oxidative stress to the avian liver are also integrated.

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.

Chondronecrosis with osteomyelitis in broilers: Further defining a bacterial challenge model using the wire flooring model.

Lameness in broiler chickens is a significant animal welfare and financial issue. Bacterial chondronecrosis with osteomyelitis (BCO) leading to lameness can be enhanced by rearing young broilers on wire flooring. Using the wire floor system, we identified Staphylococcus agnetis as the predominant isolate in BCO of the proximal tibiae and femora, and blood of lame broilers. Administration of S. agnetis isolates in water can induce lameness. We now report that the wire floor system increases bacterial translocation into the blood stream. We have also determined that approximately 105 CFU/mL is the minimum effective dose in the drinking water and that challenge at 10, 20, or 30 days of age produces similar incidences of lameness. BCO isolates of S. agnetis are much more effective than other Staphylococcus species and can overwhelm the protective effects of some commercial probiotics. Finally, we also demonstrated that the BCO lameness induced by administration of S. agnetis in the drinking water is transmissible to unchallenged broilers in the same pen.