Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

Dietary vasodilator and vitamin C/L-arginine/choline blend improve broiler feed efficiency during finishing and reduce woody breast severity at 6 and 7 wks.

Woody breast has become a considerable economic concern to the poultry industry. This myopathy presents rigid, pale breasts characterized by replacement of lean muscle protein with connective tissue, a result of hypoxia and oxidative stress in a metabolically starved muscle with inadequate circulation. Hence, the objectives were to supplement broiler diets with ingredients specifically aimed to improve circulation and oxidative status. About 1,344 male Ross 708 broilers were assigned to 1 of 4 diets: 1) a basal diet (control), 2) basal diet plus a blend of 0.2% supplemental L-arginine, 0.17% choline bitartrate, and 0.03% vitamin C (blend), 3) 0.1% vasodilator ingredient (vasodilator), or 4) 0.02% Astaxanthin ingredient (AsX). At d 14, 28, 42, and 49, performance outcomes were collected on all birds and serum from 16 broilers/diet (n = 64) was analyzed for creatine kinase and myoglobin. Once weekly beginning on d 28, a subset of 192 broilers were measured for breast width. On d 42 and 49, breast fillets from 16 broilers/diet (n = 64) were palpated for woody breast severity, weighed, and analyzed for compression force at 1-day postmortem and water-holding capacity at 2-day postmortem. mRNA was isolated from 15 breast fillets/timepoint for qPCR quantification of myogenic gene expression. Data were analyzed using Proc Mixed (SAS Version 9.4) with the fixed effect of diet. Feed conversion ratio was improved in the blend and vasodilator-fed birds d 42 to 49, each by over 2 points (P < 0.05). Breast width was increased in the control on d 42 compared to the vasodilator and AsX-fed broilers (P < 0.05). At d 42, there were 12% greater normal fillets in blend diet-fed birds and 13% more normal scores in vasodilator-fed birds at d 49 compared to the control. At d 49, myogenin expression was upregulated in the AsX diet compared to blend and control diets (P < 0.05), and muscle regulatory factor-4 expression was increased by 6.5% in the vasodilator diet compared to the blend and AsX diets (P < 0.05). Blend and vasodilator diets simultaneously improved feed efficiency in birds approaching market weight while reducing woody breast severity.

Effects of dietary bacitracin or Bacillus subtilis on the woody breast myopathy-associated gut microbiome of Eimeria spp. challenged and unchallenged broilers.

Study suggested that dysbiosis of the gut microbiota may affect the etiology of woody breast (WB). In the current study, the cecal microbiota and WB in chickens fed three different diets were investigated. A total of 504 male chicks were used in a randomized complete block design with a 3 (Diet) × 2 (Challenge) factorial arrangement of treatments with 6 replicates per treatment, 6 treatments per block, and 14 birds per treatment. The experimental diets were a control diet (corn-soybean meal basal diet), an antibiotic diet (basal diet + 6.075 mg bacitracin/kg feed), and a probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). On d 14, birds that were assigned to the challenge treatment received a 20 × live cocci vaccine. On d 41, breast muscle hardness in live birds was palpated and grouped into normal (NB) and WB phenotypes. Cecal contents were collected and their bacterial compositions were analyzed and compared. The genomic DNA of the cecal contents was extracted and the V3 and V4 regions of 16S rRNA gene were amplified and sequenced via an Illumina MiSeq platform. There were no differences (P > 0.05) in Shannon and Chao 1 indexes between the challenges, diets, and phenotypes (NB vs. WB). However, there was a difference (P = 0.001) in the beta diversity of the samples between the challenged and nonchallenged groups. Relative bacterial abundance differed (false discovery rate, FDR < 0.05) between the challenge treatments, but there were no significant differences (FDR > 0.05) among the three diets or two phenotypes. Predicted energy metabolism, nucleotide metabolism, and amino acid and coenzyme biosynthesis activities only differed (q-value < 0.05) between challenged and nonchallenged groups. The cocci challenge altered the gut microbial composition on Butyricicoccus pullicaecorum, Sporobacter termitidis, and Subdoligranulum variabile, but the dietary antibiotic and probiotic treatments did not impact gut microbial composition. No strong association was found between WB myopathy and gut microbial composition in this study.

Effects of the in ovo injection of vitamin D3 and 25-hydroxyvitamin D3 in Ross 708 broilers subsequently fed commercial or calcium and phosphorus-restricted diets. I. Performance, carcass characteristics, and incidence of woody breast myopathy1,2,3.

Effects of the in ovo-injection of vitamin D3 (D3) and 25-hydroxyvitamin D3 (25OHD3) on broiler performance, carcass characteristics, and woody breast myopathy (WBM) incidence were investigated. Live embryonated Ross 708 broiler hatching eggs (2,880) were randomly assigned to one of the following in ovo injection treatments: (1) diluent (50 µL); diluent (50 µL) containing either (2) 2.4 µg D3; (3) 2.4 µg 25OHD3; or (4) 2.4 µg D3 + 2.4 µg 25OHD3. Eggs were injected at 18 d of incubation (doi) using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly placed in each of 6 replicate pens belonging to each in ovo injection and, dietary treatment combination. Birds were fed either a commercial diet or a diet restricted in calcium and phosphorous (ReCaP) content by 20% for the starter, grower and finisher dietary phases. Broiler performance was determined in each dietary phase and breast muscle yield was also determined at 14 and 40 d of age (doa). At 41 and 46 doa, birds were processed for determination of WBM, carcass weight, and the absolute and relative (% of carcass weight) weights of various carcass parts. Compared to birds fed the commercial diet, birds fed ReCaP diets experienced a reduction in performance from 14 to 40 doa, in breast meat yield at 41 and 46 doa, and in WBM at 41 and 46 doa. At 14 and 40 doa, breast meat yield in birds that received an in ovo injection of 25OHD3 alone was higher compared to birds that received diluent alone or a combination of D3 and 25OHD3. Lower WBM incidence in ReCaP-fed birds was associated with a lower breast weight. An increase in breast meat yield in response to 25OHD3 alone may be due to improved immunity and small intestine morphology. However, further study is needed to determine the aforementioned effects.

Research Note: Effects of supplementing cranberry and blueberry pomaces on meat quality and antioxidative capacity in broilers.

Cranberry and blueberry pomaces are rich in antimicrobial and antioxidant compounds. They have been identified as potential antibiotic alternatives in animal feed, but their antioxidative capacity for maintaining or improving the meat quality in broilers is not well documented. This study was to determine whether cranberry and wild blueberry pomaces in diets could positively influence the broiler meat quality. A total of 3,150 broilers were randomly allotted to 10 dietary treatments with bacitracin methylene disalicylate, wild cranberry pomace (CRP) (0.5 and 1% of the basal diet), and wild blueberry pomace (BLP) (0.5 and 1% of the basal diet) alone or in combination with a mixture of feed enzymes. The results showed that supplementation with the CRP or BLP did not affect meat lightness and yellowness, while the deeper red meat (higher a∗ values) was observed in the birds receiving the diet containing 0.5% BLP against those in CRP treatments (P = 0.015). In addition, inclusion of CRP or BLP in the diet did not change meat texture and proximate composition (moisture, protein, fat, ash) irrespective of pomace concentrations. Although there were no obvious effects of CRP or BLP supplementation on meat antioxidant capacity and the incidence of myopathies (P > 0.05), the upward trend of antioxidant capacity and less severity of woody breast were observed in birds fed with 0.5% CRP. Furthermore, supplementation of 0.5 or 1.0% CRP without the enzyme resulted in higher mRNA levers of Nrf, Gpx2, and HO-1 (P < 0.05). Taken together, 0.5% CRP supplementation without the enzyme could potentially maintain meat quality and attenuate the severity of woody breast.

Research Note: Dietary phytase reduces broiler woody breast severity via potential modulation of breast muscle fatty acid profiles.

Woody breast (WB) myopathy is a major concern and economic burden to the poultry industry, and for which, there is no effective solution because of its unknown etiology. In a previous study, we have shown that phytase (Quantum Blue, QB) reduces the WB severity by 5% via modulation of oxygen homeostasis-related pathways. As WB has been suggested to be associated with lipid dysmetabolism, we aimed to determine the effect of QB on WB and breast muscle fatty acid profile. Male broilers were subjected to 6 treatments (96 birds/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with QB at 500 (NC+500 FTU), and 1,000 (NC+ 1,000 FTU) or 2,000 FTU/kg of feed (NC+2,000 FTU). Woody breast and white striping scores were recorded, and fatty acid profiles were determined using gas liquid chromatography. Woody breast-affected muscles exhibited a significant higher incidence of white striping as liquid chromatography analysis reveals an imbalance of fatty acid profile in the breast of WB-affected birds with a significant higher percent of saturated fatty acids (SFA, myristic [14:0], pentadecanoic [15:0], and margaric [17:0]) and monounsaturated fatty acids (myristoleic [14:1], palmitoleic [16:1c], 10-trans-heptadecenoic [17:1t], oleic [18:1c9], and vaccenic [18:1c11]), and lower content of polyunsaturated fatty acids (PUFA) and omega-3 (P < 0.05). Quantum Blue at high doses (1,000 and 2,000 FTU) significantly reduces the percent of SFA and increases that of PUFA compared with the control group. In conclusion, WB myopathy seemed to be associated with an imbalance of fatty acid profile, and QB ameliorates the severity of WB potentially via modulation of SFA and PUFA contents.

Effect of Limestone Particle Size and Potassium Supplementation on Growth Performance, Blood Physiology, and Breast Muscle Myopathy of Male Broiler Chickens.

The experiment investigated the effects of limestone particle size and dietary potassium (K) on live performance, blood physiology, and muscle myopathies in broilers raised to 35 days of age. A total of 384 Ross male broilers were placed in 24 floor pens and fed four diets during the starter (0-16 days of age) and grower (17-33 days of age) periods containing two limestone particle sizes (fine: 0.2 mm and coarse: 0.9 mm), and amended with either 0% basal K (K-) or 0.2% added dietary K (K+) as potassium carbonate to complete the 2 × 2 factorial arrangement. Live performance was measured from 1-33 days of age. Blood physiology, woody breast (WB), and white striping (WS) scores were measured at 35 days of age. The K+ dietary treatment reduced (P < 0.05) feed intake and BWG when compared to K- during the starter and grower period. The K+ dietary treatment decreased blood Na (mmol/L), blood glucose (mg/dl), ionized blood Ca (mg/dl), TCO2 (mmol/L), blood HCO3 (mmol/L), and base excess in extracellular fluid (mmol/L) when compared to K- birds of similar body weight at 35 days of age (P ≤ 0.05). Fine limestone diets tended to reduce WB scores (3.0 vs. 2.59) when compared to coarse limestone diets at 35 days of age (P = 0.08). This study demonstrated that using 0.2% of K as potassium carbonate did not negatively affect FCR even though FI and BWG were reduced. Furthermore, fine limestone has the potential to reduce WB in breast muscle tissues; however, further research is needed to confirm these outcomes.

Quantum Blue Reduces the Severity of Woody Breast Myopathy via Modulation of Oxygen Homeostasis-Related Genes in Broiler Chickens.

The incidence of woody breast (WB) is increasing on a global scale representing a significant welfare problem and economic burden to the poultry industry and for which there is no effective treatment due to its unknown etiology. In this study, using diffuse reflectance spectroscopy (DRS) coupled with iSTAT portable clinical analyzer, we provide evidence that the circulatory- and breast muscle-oxygen homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels] in chickens with WB myopathy compared to healthy counterparts. Molecular analysis showed that blood HB subunit Mu (HBM), Zeta (HBZ), and hephaestin (HEPH) expression were significantly down regulated; however, the expression of the subunit rho of HB beta (HBBR) was upregulated in chicken with WB compared to healthy counterparts. The breast muscle HBBR, HBE, HBZ, and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) mRNA abundances were significantly down regulated in WB-affected compared to normal birds. The expression of HIF-1α at mRNA and protein levels was significantly induced in breasts of WB-affected compared to unaffected birds confirming a local hypoxic status. The phosphorylated levels of the upstream mediators AKT at Ser473 site, mTOR at Ser2481 site, and PI3K P85 at Tyr458 site, as well as their mRNA levels were significantly increased in breasts of WB-affected birds. In attempt to identify a nutritional strategy to reduce WB incidence, male broiler chicks (Cobb 500, n = 576) were randomly distributed into 48 floor pens and subjected to six treatments (12 birds/pen; 8 pens/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with quantum blue (QB) at 500 (NC + 500 FTU), 1,000 (NC + 1,000 FTU), or 2,000 FTU/kg of feed (NC + 2,000 FTU). Although QB-enriched diets did not affect growth performances (FCR and FE), it did reduce the severity of WB by 5% compared to the PC diet. This effect is mediated by reversing the expression profile of oxygen homeostasis-related genes; i.e., significant down regulation of HBBR and upregulation of HBM, HBZ, and HEPH in blood, as well as a significant upregulation of HBA1, HBBR, HBE, HBZ, and PHD2 in breast muscle compared to the positive control.

Dietary amino acids under hypoxic conditions exacerbates muscle myopathies including wooden breast and white stripping.

The occurrence of wooden breast (WB) and white striping (WS) of broiler breast myopathies may be associated with least-cost feed formulations and the inclusion of greater amounts of synthetic amino acids. Two experiments were conducted to evaluate the effects of supplemental glutamine (Gln-/+, 0 and 1%) and arginine (Arg-/+, 0 and 0.25%) in a 2 × 2 factorial arrangement. Experiment 1 consisted of 8 replicates using 32 pens and focused on live performance to 44 d of age, carcass yield, and meat quality evaluations at 45 d of age. Live performance parameters including feed intake, BW gain, mortality, and feed conversion were evaluated. Post-harvest carcass yield, breast muscle quality (including WB and WS), proximate analysis, and mineral analysis of breast muscle and tibia bone were also evaluated. Experiment 2 comprised 4 replicates using 8 pens with weekly measurements of blood physiology and muscle myopathy development from 21 to 45 d of age. Broilers fed supplemental Gln and Arg (Gln+/Arg+) produced broilers with greater BW and improved FCR. The WB and WS severity increased with Gln+ diets, while only WB increased using Arg+ diets. Weekly observations revealed the greatest increase in WB myopathies occurred between 28 to 35 d, while WS significantly increased one week later (35 to 42 d). When comparing broilers of similar BW at 45 d, Gln+ diets resulted in greater WS scores and percentage breast muscle fat. Further analysis of WB and WS scores (independent of treatment affect) revealed that increased myopathy scores were associated with increased blood CO2, H2CO3, and reduced O2 with increased meat pH, moisture content, and Ca concentration. These results indicated that increased muscle Ca is associated with increased WB, while increased dietary glutamine and lack of oxygen may have resulted in a reverse flux of the citric acid cycle and reduced electron transport chain activity resulting in increased WS.