Dietary inclusion of phytase and stimbiotic decreases mortality and lameness in a wire ramp challenge model in broilers.

RESEARCH HIGHLIGHTS: Wire ramp model reproducibly induced lameness/BCO in broilers.Treatments did not affect growth, but phytase with stimbiotic significantly reduced BCO.Phytase increased circulating inositol, and wire flooring decreased bone inositol.

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems.

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%-92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry. IMPORTANCE: Clostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

Amino acid and mineral digestibility, bone ash, and plasma inositol is increased by including microbial phytase in diets for growing pigs.

BACKGROUND: The effect of microbial phytase on amino acid and energy digestibility is not consistent in pigs, which may be related to the phytase dosage or the adaptation length to the diet. Therefore, an experiment was conducted to test the hypotheses that increasing dietary phytase after an 18-day adaptation period: 1) increases nutrient and energy digestibility; 2) increases plasma P, plasma inositol, and bone ash of young pigs; and 3) demonstrates that maximum phytate degradation requires more phytase than maximum P digestibility. RESULTS: Data indicated that increasing inclusion of phytase [0, 250, 500, 1,000, 2,000, and 4,000 phytase units (FTU)/kg feed] in corn-soybean meal-based diets increased apparent ileal digestibility (AID) of Trp (quadratic; P < 0.05), and of Lys and Thr (linear; P < 0.05), and tended to increase AID of Met (linear; P < 0.10). Increasing dietary phytase also increased AID and apparent total tract digestibility (ATTD) of Ca and P (quadratic; P < 0.05) and increased ATTD of K and Na (linear; P < 0.05), but phytase did not influence the ATTD of Mg or gross energy. Concentrations of plasma P and bone ash increased (quadratic; P < 0.05), and plasma inositol also increased (linear; P < 0.05) with increasing inclusion of phytase. Reduced concentrations of inositol phosphate (IP)6 and IP5 (quadratic; P < 0.05), reduced IP4 and IP3 (linear; P < 0.05), but increased inositol concentrations (linear; P < 0.05) were observed in ileal digesta as dietary phytase increased. The ATTD of P was maximized if at least 1,200 FTU/kg were used, whereas more than 4,000 FTU/kg were needed to maximize inositol release. CONCLUSIONS: Increasing dietary levels of phytase after an 18-day adaptation period increased phytate and IP ester degradation and inositol release in the small intestine. Consequently, increasing dietary phytase resulted in improved digestibility of Ca, P, K, Na, and the first 4 limiting amino acids, and in increased concentrations of bone ash and plasma P and inositol. In a corn-soybean meal diet, maximum inositol release requires approximately 3,200 FTU/kg more phytase than that required for maximum P digestibility.

Effects of bacteriophage on Salmonella Enteritidis infection in broilers.

Bacteriophages (BP) are viruses that can infect bacteria. The present study evaluated the effect of BP on Salmonella infected broilers. A number of 150 day-old broilers were used in a completely randomized design with five treatments that included: (1) basal diet from day 0 to 28; (2) basal diet + 0.3 g/kg of colistin from day 0 to 28; (3) basal diet from day 1 to 13, and basal diet + 0.4 g/kg of colistin from day 14 to 28; (4) basal diet + 1 g/kg of BP from day 0 to 28; (5) basal diet + 1.5 g/kg of BP from day 0 to 28. On day 13, 15 chickens from each treatment were challenged by Salmonella Enteritidis (SE), while fifteen from each treatment were not; instead, they were kept in the same cage with the challenged chickens (exposed chickens). At 7 and 14 days post-challenge, the number of SE and coliform bacteria in the cecum and liver of colistin and BP-fed birds was lower than the control treatment. In exposed and challenged chickens, the height and surface area of villus were greater in the BP and colistin-supplemented groups. Serum concentrations of aspartate aminotransferase and alanine transaminase were greater, while serum albumin and triglycerides concentrations were lower in the control treatment. The liver of the challenged chickens had more pathological lesions than exposed birds. BP significantly decreased PPARγ gene expression in exposed chickens. In the challenged and exposed chickens, TLR4 gene expression was lower in BP and colistin-treated birds as compared to the control. In conclusion, adding BP to the diet from the day of age prevents the spread of Salmonella.

Impact of Phytase Supplementation on Meat Quality of Heat-Stressed Broilers.

Heat stress (HS) is one of the most challenging stressors to poultry production sustainability. The adverse effects of HS range from feed intake and growth depression to alteration of meat quality and safety. As phytase supplementation is known to improve nutrient utilization and consequently growth, we undertook the present study to evaluate the effects of dietary phytase on growth and meat quality in heat-stressed broilers. A total of 720 day-old hatch Cobb 500 chicks were assigned to 24 pens within controlled environmental chambers and fed three diets: Negative Control (NC), Positive Control (PC), and NC diet supplemented with 2000 phytase units (FTU)/kg) of quantum blue (QB). On day 29, birds were exposed to two environmental conditions: thermoneutral (TN, 25 °C) or cyclic heat stress (HS, 35 °C, 8 h/d from 9 a.m. to 5 p.m.) in a 3 × 2 factorial design. Feed intake (FI), water consumption (WI), body weight (BW), and mortality were recorded. On day 42, birds were processed, carcass parts were weighed, and meat quality was assessed. Breast tissues were collected for determining the expression of target genes by real-time quantitative PCR using the 2-ΔΔCt method. HS significantly increased core body temperature, reduced feed intake and BW, increased water intake (WI), elevated blood parameters (pH, SO2, and iCa), and decreased blood pCO2. HS reduced the incidence of woody breast (WB) and white striping (WS), significantly decreased drip loss, and increased both 4- and 24-h postmortem pH. Instrumental L* and b* values were reduced (p < 0.05) by the environmental temperature at both 4- and 24-h postmortem. QB supplementation reduced birds' core body temperature induced by HS and improved the FCR and water conversion ratio (WCR) by 1- and 0.5-point, respectively, compared to PC under HS. QB increased blood SO2 and reduced the severity of WB and WS under TN conditions, but it increased it under an HS environment. The abovementioned effects were probably mediated through the modulation of monocarboxylate transporter 1, heat shock protein 70, mitogen-activated protein kinase, and/or glutathione peroxidase 1 gene expression, however, further mechanistic studies are warranted. In summary, QB supplementation improved growth performance and reduced muscle myopathy incidence under TN conditions. Under HS conditions, however, QB improved growth performance but increased the incidence of muscle myopathies. Therefore, further QB titration studies are needed.

A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens.

Nontyphoidal Salmonella spp. are a leading cause of human gastrointestinal infections and are commonly transmitted via the consumption of contaminated meat. To limit the spread of Salmonella and other food-borne pathogens in the food chain, bacteriophage (phage) therapy could be used during rearing or pre-harvest stages of animal production. This study was conducted to determine if a phage cocktail delivered in feed is capable of reducing Salmonella colonization in experimentally challenged chickens and to determine the optimal phage dose. A total of 672 broilers were divided into six treatment groups T1 (no phage diet and unchallenged); T2 (phage diet 106 PFU/day); T3 (challenged group); T4 (phage diet 105 PFU/day and challenged); T5 (phage diet 106 PFU/day and challenged); and T6 (phage diet 107 PFU/day and challenged). The liquid phage cocktail was added to mash diet with ad libitum access available throughout the study. By day 42 (the concluding day of the study), no Salmonella was detected in faecal samples collected from group T4. Salmonella was isolated from a small number of pens in groups T5 (3/16) and T6 (2/16) at ∼4 × 102 CFU/g. In comparison, Salmonella was isolated from 7/16 pens in T3 at ∼3 × 104 CFU/g. Phage treatment at all three doses had a positive impact on growth performance in challenged birds with increased weight gains in comparison to challenged birds with no phage diet. We showed delivering phages via feed was effective at reducing Salmonella colonization in chickens and our study highlights phages offer a promising tool to target bacterial infections in poultry.

Can the broiler industry rely on results of existing life cycle assessment and environmental assessments studies to inform broilers' nutritional strategies?

The goal of this systematic review is to investigate the applicability of the results from existing life cycle analysis (LCA) and environmental assessments studies in informing nutritional strategies for environmentally sustainable poultry meat production. This paper reports on a Rapid Evidence Assessment (REA) of articles published between 2000 and 2020. The studies reviewed were conducted in developed countries including UK, France, Germany, Sweden, Norway, The Netherlands, Denmark, Belgium, Canada, and USA. All articles were written in English. The REA includes studies on LCA of differing strains of meat poultry and production systems, studies on poultry manure emission and studies on environmental assessments of plant-based feed ingredients. The review covered studies on soil carbon dynamics associated with plant-based ingredients. Web of Science, Scopus, and PubMed were used to obtain the 6,142 population articles. The multistage screening process resulted in 29 studies from which 15 studies included LCA while the rest 14 studies analyzed NH3 emission of broilers. All studies based on LCA were descriptive and did not include replications. Only 12 studies assessed the effect of interventions to reduce NH3 emission of broiler litter using replicated layout designs. It is concluded that the broiler industry in UK, EU, and North America cannot rely on results of existing LCA and environmental assessments studies to inform their nutritional strategy and poultry meat production due to a shortage of reliable in vivo data assessing interventions in controlled studies.

Potential role of endoplasmic reticulum stress in broiler woody breast myopathy.

Although broiler (meat-type) chickens are one of the most efficient protein sources that supports the livelihoods and food security of billions of people worldwide, they are facing several challenges. Due to its unknown etiology and heavy economic impact, woody breast (WB) myopathy is one of the most challenging problems facing the poultry industry, and for which there is no effective solution. Here, using a primary chicken myotube culture model, we show that hypoxia and endoplasmic reticulum (ER) stress are an integral component of the etiology of the myopathy. Multiple components of the ER stress response are significantly upregulated in WB as compared with normal muscle, and this response was mimicked by hypoxic conditions in chicken primary myotube culture. In addition, apoptotic pathways were activated as indicated by increases in active caspase 3 protein levels in both WB-affected tissues and hypoxic myotube culture, and caspase 3 activity and apoptosis in hypoxic myotube culture. Finally, as a phenotypic hallmark of WB is enhanced fibrosis and increased collagen aggregation, here, we show that hypoxic conditions increase collagen 1A1 and 1A2 gene expression, as well as collagen 1 protein levels in primary myotubes. These effects were partially reversed by tauroursodeoxycholic acid (TUDCA), an ER-stress inhibitor, in myotube culture. Taken together, these findings indicate that hypoxia and ER stress are present in WB, hypoxia can upregulate the cell death arm of the unfolded protein response (UPR) and lead to collagen production in a culture model of WB. This opens new vistas for potential mechanistic targets for future effective interventions to mitigate this myopathy.

Apparent digestibility of energy and nutrients and efficiency of microbial phytase is influenced by body weight of pigs.

An experiment was conducted to test the hypothesis that regardless of pig body weight (BW), increasing dietary phytase results in increased phytate degradation and improved digestibility of minerals, amino acids (AA), and gross energy (GE). Eighteen pigs were equipped with a T-cannula in the distal ileum and allotted to a triplicated 6 × 3 Youden square design with six diets and three collection periods of 7 d, for a total of nine replicate pigs per diet. This design was repeated four times to simulate four production phases, and there was a 7-d resting period before each collection phase started (BW at start of collections: 29.3, 53.6, 85.1, and 114.4 kg for phases 1, 2, 3, and 4, respectively). Six corn-soybean meal diets were formulated by including 0, 250, 500, 1,000, 2,000, or 4,000 phytase units/kg feed (FTU). The six diets were used throughout the experiment. Samples of feces and ileal digesta were collected in each period. Results indicated that regardless of pig BW, increasing inclusion of phytase increased (quadratic; P < 0.05) apparent ileal digestibility (AID) of crude protein (CP) and most AA, increased apparent total tract digestibility (ATTD) of Ca, P, K, Mg (linear and quadratic; P < 0.05), and Na (linear; P < 0.05), but decreased (linear and quadratic; P < 0.05) AID and ATTD of GE. In all phases, ileal concentrations of inositol phosphate (IP) 6, IP5, IP4, and IP3 decreased (linear and quadratic; P < 0.05), whereas ileal inositol increased (linear and quadratic; P < 0.05) with increasing dietary phytase. However, as pig BW increased, AID of GE, CP, and AA increased (linear, P < 0.05), and the AID of a few AA (Met, Phe, Thr, Trp, Ala, Asp, Gly, and Ser) also increased quadratically (P < 0.05). The ATTD of GE, K, and Mg increased (linear and quadratic; P < 0.05), but ATTD of Ca and Na (linear; P < 0.05) and of P (linear and quadratic; P < 0.05) decreased as pig BW increased. Ileal IP6 and IP3 (linear and quadratic; P < 0.05) and ileal IP5 and IP4 (linear; P < 0.05) increased, whereas ileal inositol decreased (linear; P < 0.05) as pig BW increased. In conclusion, regardless of pig BW, increasing dietary phytase increased phytate degradation and inositol release in the small intestine, and consequently increased mineral and AA digestibility. Older pigs have reduced Ca, P, and Na digestibility, but increased K, Mg, AA, and GE digestibility compared with younger pigs. The efficiency of dietary phytase to degrade phytate appears to decrease as pigs get older.

The influence of dietary phytase in pig nutrition is often investigated using pigs from 20 to 40 kg, but there are limited data to demonstrate that data obtained in young pigs can be extrapolated to pigs above 40 kg. Therefore, an experiment was conducted to determine effects of increasing phytase levels (0, 250, 500, 1,000, 2,000, and 4,000 phytase units) on phytate breakdown and nutrient digestibility of pigs throughout four productive phases (25 to 50 kg, 50 to 75 kg, 75 to 100 kg, and 100 to 125 kg). Results indicated that regardless of pig body weight, the digestibility of macro-minerals and most amino acids increased with increasing dietary phytase because of increased phytate breakdown.

The Growth Performance, Nutrient Digestibility, Gut Bacteria and Bone Strength of Broilers Offered Alternative, Sustainable Diets Varying in Nutrient Specification and Phytase Dose.

This study assessed the use of locally sourced sustainable feed ingredients, rapeseed meal (RSM) and maize dried distiller grains with solubles (DDGS) in diets over traditional ingredients on the growth performance, bone strength and nutrient digestibility of broilers. This work also investigated the effects of supplementing exogenous phytase in two doses (500 vs. 1500 FTU/kg). Using male Ross 308 chicks (n = 320) assigned to receive one of four experimental diets: (1) Positive control diet 1 (PC1), a wheat, soya-based diet + 500 FTU/kg phytase. (2) Positive control diet 2, RSM/DDGS diet + 500 FTU/kg phytase (PC2). (3) Negative control (NC) reduced nutrient RSM/DDGS diet, no phytase. (4) The NC diet plus 1500 FTU/kg phytase (NC+). PC1 birds displayed higher feed intake and body weight gain consistently throughout the trial (p < 0.001) as well as increased body weight by 28 d and 42 d (p < 0.001). Whole-body dual emission X-ray absorptiometry (DXA) analysis revealed PC1 birds also had higher bone mineral density (BMD), bone mineral content (BMC), total bone mass, total lean mass and total fat mass than birds offered other treatments (p < 0.01). Diet had no significant effect on bone strength. Phytase superdosing improved the digestibility of dry matter (DM), neutral detergent fibre (NDF), gross energy (GE), calcium (Ca), potassium (K) and magnesium (Mg) compared to birds in other treatment groups. The phytase superdose also improved performance in comparison to birds offered the NC diet. Phytase superdosing increased the IP6 and IP5 degradation and increased the ileal inositol concentration of the birds. N excretion was lower for birds offered the traditional wheat−soya diet and highest for those offered the high-specification RSM/DDGS diet with a commercial dose of phytase. The addition of a phytase superdose to the negative control diet (NC+) reduced P excretion of birds by 15% compared to birds offered NC.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.

Increased microbial phytase increased phytate destruction, plasma inositol, and feed efficiency of weanling pigs, but reduced dietary calcium and phosphorus did not affect gastric pH or fecal score and reduced growth performance and bone ash.

An experiment was conducted to test two hypotheses: 1) reducing dietary Ca and P reduces gastric pH and diarrhea in weanling pigs; 2) negative effects of low Ca and P on pig growth performance may be overcome if phytase is added to the diets. A total of 320 weanling pigs (6.35 ± 0.87 kg) were allotted to eight corn-soybean meal-based diets in a randomized complete block design with five pigs per pen. Two phase 1 (days 1 to 14) control diets containing 100 or 50% of total Ca and digestible P relative to the requirement, and six diets in which 500, 2,000, or 16,000 units of phytase/kg feed (FTU) were added to each control diet were formulated. Phytase was assumed to release 0.16% total Ca and 0.11% digestible P. Common diets were fed in phases 2 (days 15 to 27) and 3 (days 28 to 42). Growth performance data were recorded within each phase. Data for fecal scores and gastrointestinal pH were recorded for phase 1. Colon content (day 14), the right femur (days 14 and 42), and blood samples (days -1, 14, 27, and 42) were collected from one pig per pen. In phase 1, reducing Ca and P did not reduce gastric pH or fecal score, but pigs fed the 50% diets had reduced (P < 0.05) average daily gain (ADG) and average daily feed intake (ADFI) compared with pigs fed the 100% diets. In both 50% and 100% diets, phytase above 500 FTU increased (P < 0.05) gain:feed ratio (G:F) and tended (P < 0.10) to reduce gastric pH of pigs. From days 1 to 42, pigs fed the 50% diets tended (P < 0.10) to have reduced ADG and ADFI compared with pigs fed the 100% diets, but among the 100% diets, pigs tended (P < 0.10) to have a linear increase in G:F as phytase level increased. Pigs fed the 50% diets had reduced (P < 0.05) concentrations of inositol phosphate esters (IP) in the colon and reduced bone ash (days 14 and 42) compared with pigs fed the 100% diets. Phytase did not affect bone ash or most blood metabolites. Concentrations of IP in the colon decreased, whereas plasma inositol increased (d 14; P < 0.05) in pigs fed diets with phytase (≥ 500 FTU). In pigs fed the 100% diets, IP in the colon linearly decreased (P < 0.05), but plasma inositol linearly increased (P < 0.05) with increasing levels of phytase. In conclusion, reducing Ca and P in diets for weanling pigs did not influence gastric pH or fecal score, but compromised growth performance and bone ash. However, regardless of dietary Ca and P, high doses of phytase increased phytate degradation and inositol absorption, which consequently increased G:F of pigs.

Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid.

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI's evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

Reduced concentrations of limestone and monocalcium phosphate in diets without or with microbial phytase did not influence gastric pH, fecal score, or growth performance, but reduced bone ash and serum albumin in weanling pigs.

An experiment was conducted to test the hypothesis that reducing limestone and monocalcium phosphate in diets for weanling pigs by lowering the concentration of Ca and P or by including microbial phytase in the diet will reduce stomach pH and fecal score and will improve growth performance of pigs. A total of 160 weanling pigs (5.75 ± 1.04 kg) were allotted to four corn-soybean meal-based diets in a completely randomized design with five pigs per pen. Diets for phase 1 (d 1 to 15) were formulated using a 2 × 2 factorial design with 2 concentrations of Ca and P (adequate or deficient levels of total Ca and digestible P) and 2 inclusion levels of phytase (0 or 2,000 units/kg feed). Phytase was assumed to release 0.16% total Ca and 0.11% digestible P. Common diets were fed in phases 2 (d 16 to 21) and 3 (d 22 to 35). Fecal scores were recorded in phase 1 and on d 15, gastric pH was measured and a blood sample and the right femur were collected from one pig per pen. Growth performance data were recorded within each phase. Results indicated that in phase 1, at deficient dietary Ca and P, pigs fed the diet with phytase had greater (P < 0.05) average daily gain (ADG) and gain to feed (G:F) compared with pigs fed the diet without phytase, but in diets with adequate levels of Ca and P, no effect of phytase inclusion was observed (interaction, P < 0.05). Without phytase, pigs fed the diet with deficient Ca and P had reduced (P < 0.05) G:F compared with pigs fed the diet with adequate Ca and P, but if phytase was included, there was no effect of Ca and P on G:F (interaction, P < 0.05). For phases 2 and 3, and from d 1 to 35, no differences among dietary treatments were observed for ADG or G:F. Bone ash was greater (P < 0.05) in pigs fed diets with adequate Ca and P than in pigs fed diets with deficient Ca and P, but no effect of phytase inclusion was observed on bone ash. Concentrations of Ca and P did not affect stomach pH or fecal score, but pigs fed diets with phytase tended (P < 0.10) to have reduced stomach pH and fecal score compared with pigs fed diets without phytase. Pigs fed diets with adequate Ca and P had greater (P < 0.05) albumin in serum than pigs fed the Ca- and P-deficient diets. In conclusion, phytase inclusion in phase 1 diets may reduce diarrhea, but lowering Ca and P does not reduce stomach pH or fecal score and decreases bone ash, although growth performance during the entire weanling period is not affected.

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.

Quantities of ash, Ca, and P in metacarpals, metatarsals, and tibia are better correlated with total body bone ash in growing pigs than ash, Ca, and P in other bones.

The objective was to determine correlations between individual bones and total body bone ash to identify the bone that is most representative of total body bone ash in growing pigs. Twenty growing pigs were allotted to 1 of 2 diets that were formulated to contain 60% or 100% of the requirement for standardized total tract digestible (STTD) P. Both diets had an STTD Ca to STTD P ratio of 1.90:1. Growth performance and carcass weights were determined. Metacarpals, metatarsals, femur, tibia, fibula, 3rd and 4th ribs, and 10th and 11th ribs, and all other bones from the left half of the carcass were collected separately. Each bone was defatted and ashed. Pigs fed the diet containing 100% of required Ca and P had greater (P < 0.05) average daily gain, gain to feed, and ash concentration (%) in total and all individual bones except femur and fibula compared with pigs fed the diet containing 60% of required Ca and P. Calcium and P concentrations in bone ash were not affected by dietary treatments. Weights (g) of bone ash, bone Ca, and bone P were greater (P < 0.05) or tended to be greater (P < 0.10) for pigs fed the diet containing 100% of required Ca and P. Correlation coefficients between the weight of ashed metacarpals, metatarsals, and tibia and the weight of total bone ash were >0.95. In conclusion, metacarpals, metatarsals, and tibia were more representative of total body bone ash compared with other bones.

Formulation of diets for pigs based on a ratio between digestible calcium and digestible phosphorus results in reduced excretion of calcium in urine without affecting retention of calcium and phosphorus compared with formulation based on values for total calcium.

An experiment was conducted to test the hypothesis that formulating diets for pigs based on a ratio between standardized total tract digestible (STTD) Ca and STTD P instead of total Ca and STTD P does not decrease Ca retention, but increases P utilization. Forty barrows (59.4 ± 3.8 kg) were individually housed in metabolism crates and allotted to four corn-soybean meal-based diets in a randomized complete block design with two blocks and five pigs per diet in each block. Diets were formulated using a 2 × 2 factorial design with two diet formulation principles (total Ca or STTD Ca) and two inclusion levels of microbial phytase (0 or 500 units per kg of feed). Phytase was assumed to release 0.11% STTD P and 0.16% total Ca. Diets were formulated based on requirements for total Ca and STTD P or a ratio between STTD Ca and STTD P of 1.25:1. Diets were fed for 11 d and fecal and urine samples were collected from feed provided from day 6 to day 10. Interactions (P < 0.05) between diet formulation principle and phytase level were observed for Ca intake, Ca in feces, Ca absorbed, Ca retained, P digestibility, P absorbed, and P in urine. Phytase increased (P < 0.05) the digestibility of Ca in both total Ca and STTD Ca diets. Without phytase, Ca intake, Ca in feces, and Ca absorbed was greater (P < 0.05) from pigs fed total Ca diets than from pigs fed STTD Ca diets, but P absorbed, P digestibility, and P in urine was greater (P < 0.05) from pigs fed STTD Ca diets than from pigs fed total Ca diets. However, in the presence of phytase, no differences between diet formulation principles were observed in these variables. Regardless of phytase, Ca in urine was lower (P < 0.05) from pigs fed STTD Ca diets than from pigs fed total Ca diets. There were no differences in Ca retention between pigs fed STTD Ca diets and total Ca diets, but pigs fed total Ca diets retained less (P < 0.05) Ca if diets contained phytase. No differences in P retention were observed between diet formulation principles, but pigs fed non-phytase diets retained more (P < 0.05) P than pigs fed diets with phytase. In conclusion, because diets formulated based on STTD Ca contain less Ca than total Ca diets, pigs fed STTD Ca diets excreted less Ca in urine, but retention of Ca was not affected. Formulating non-phytase diets based on STTD Ca instead of total Ca increased P absorption, which confirms the detrimental effect of excess Ca on P digestibility. However, P retention was not improved if pigs were fed STTD Ca diets.

Formulating diets based on digestible calcium instead of total calcium does not affect growth performance or carcass characteristics, but microbial phytase ameliorates bone resorption caused by low calcium in diets fed to pigs from 11 to 130 kg.

An experiment was conducted to test the hypothesis that the requirement for Ca expressed as a ratio between standardized total tract digestible (STTD) Ca and STTD P obtained in short-term experiments may be applied to pigs fed diets without or with microbial phytase from 11 to 130 kg. In a 5-phase program, 160 pigs (body weight: 11.2 ± 1.8 kg) were randomly allotted to 32 pens and 4 corn-soybean meal-based diets in a 2 × 2 factorial design with 2 diet formulation principles (total Ca or STTD Ca), and 2 phytase inclusion levels (0 or 500 units/kg of feed) assuming phytase released 0.11% STTD P and 0.16% total Ca. The STTD Ca:STTD P ratios were 1.40:1, 1.35:1, 1.25:1, 1.18:1, and 1.10:1 for phases 1 to 5, and STTD P was at the requirement. Weights of pigs and feed left in feeders were recorded at the end of each phase. At the conclusion of phase 1 (day 24), 1 pig per pen was euthanized and a blood sample and the right femur were collected. At the end of phases 2 to 5, a blood sample was collected from the same pig in each pen. At the conclusion of the experiment (day 126), the right femur of 1 pig per pen was collected and carcass characteristics from this pig were measured. No interactions were observed between diet formulation principle and phytase inclusion for growth performance in any phase and no differences among treatments were observed for overall growth performance. Plasma Ca and P and bone ash at the end of phase 1 were also not influenced by dietary treatments. However, on day 126, pigs fed nonphytase diets formulated based on total Ca had greater bone ash than pigs fed STTD Ca-based diets, but if phytase was used, no differences were observed between the 2 formulation principles (interaction P < 0.05). At the end of phases 2 and 3, pigs fed diets without phytase had greater (P < 0.05) plasma P than pigs fed diets with phytase, but no differences were observed at the end of phases 4 and 5. A negative quadratic effect (P < 0.05) of phase (2 to 5) on the concentration of plasma Ca was observed, whereas plasma P increased (quadratic; P < 0.05) from phases 2 to 5. However, there was no interaction or effect of diet formulation principle or phytase inclusion on any carcass characteristics measured. In conclusion, STTD Ca to STTD P ratios can be used in diet formulation for growing-finishing pigs without affecting growth performance or carcass characteristics and phytase inclusion ameliorates bone resorption caused by low dietary Ca and P.

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.

Muscle Metabolome Profiles in Woody Breast-(un)Affected Broilers: Effects of Quantum Blue Phytase-Enriched Diet.

Woody breast (WB) myopathy is significantly impacting modern broilers and is imposing a huge economic burden on the poultry industry worldwide. Yet, its etiology is not fully defined. In a previous study, we have shown that hypoxia and the activation of its upstream mediators (AKT/PI3K/mTOR) played a key role in WB myopathy, and supplementation of quantum blue (QB) can help to reduce WB severity via modulation of hypoxia-related pathways. To gain further insights, we undertook here a metabolomics approach to identify key metabolite signatures and outline their most enriched biological functions. Ultra performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS) identified a total of 108 known metabolites. Of these, mean intensity differences at P < 0.05 were found in 60 metabolites with 42 higher and 18 lower in WB-affected compared to unaffected muscles. Multivariate analysis and Partial Least Squares Discriminant analysis (PLS-DA) scores plot displayed different clusters when comparing metabolites profile from affected and unaffected tissues and from moderate (MOD) and severe (SEV) WB muscles indicating that unique metabolite profiles are present for the WB-affected and unaffected muscles. To gain biologically related molecule networks, a stringent pathway analyses was conducted using IPA knowledge-base. The top 10 canonical pathways generated, using a fold-change -1.5 and 1.5 cutoff, with the 50 differentially abundant-metabolites were purine nucleotide degradation and de novo biosynthesis, sirtuin signaling pathway, citrulline-nitric oxide cycle, salvage pathways of pyrimidine DNA, IL-1 signaling, iNOS, Angiogenesis, PI3K/AKT signaling, and oxidative phosphorylation. The top altered bio-functions in term of molecular and cellular functions in WB-affected tissues included cellular development, cellular growth and proliferation, cellular death and survival, small molecular biochemistry, inflammatory response, free radical scavenging, cell signaling and cell-to-cell interaction, cell cycles, and lipid, carbohydrate, amino acid, and nucleic acid metabolisms. The top disorder functions identified were organismal injury and abnormalities, cancer, skeletal and muscular disorders, connective tissue disorders, and inflammatory diseases. Breast tissues from birds fed with high dose (2,000 FTU) of QB phytase exhibited 22 metabolites with significantly different levels compared to the control group with a clear cluster using PLS-DA analysis. Of these 22 metabolites, 9 were differentially abundant between WB-affected and unaffected muscles. Taken together, this study determined many metabolic signatures and disordered pathways, which could be regarded as new routes for discovering potential mechanisms of WB myopathy.