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.

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.

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.

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.

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.

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.

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.

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.

Increasing calcium from deficient to adequate concentration in diets for gestating sows decreases digestibility of phosphorus and reduces serum concentration of a bone resorption biomarker.

The objective of this experiment was to test the hypothesis that the concentration of Ca in diets fed to late gestating sows affects the apparent total tract digestibility (ATTD) and retention of Ca and P, serum concentrations of Ca and P, hormones, and blood biomarkers for bone formation and resorption. Thirty-six sows (average parity = 2.8) were housed in metabolism crates from day 91 to day 104 of gestation and fed 1 of 4 experimental diets containing 25, 50, 75, or 100% of the requirement for Ca. All diets met the requirement for P. The initial 5 d of each period were the adaptation period, which was followed by 4 d of quantitative collection of feces and urine. At the end of the collection period, a blood sample was collected from all sows. Results indicated that feed intake, weights of dried fecal and urine samples, and the ATTD of DM were not affected by dietary Ca, but ATTD of Ca increased (quadratic, P < 0.05) as Ca in diets increased. Urine Ca output was not affected by dietary Ca, but Ca retention increased (quadratic, P < 0.05) as Ca intake increased. Fecal P output increased (linear, P < 0.001) as dietary Ca increased, which resulted in a linear decrease (P < 0.001) in the ATTD of P. Urine P output also decreased (linear, P < 0.001) as dietary Ca increased, but P retention increased (linear, P < 0.05). Regressing the apparent total tract digestible Ca against dietary Ca intake resulted in a regression line with a slope of 0.33, indicating that true total tract digestibility of Ca in calcium carbonate was 33%. Serum concentrations of Ca and P and estrogen, calcitonin, and parathyroid hormone were not affected by dietary Ca. Serum concentration of carboxyterminal cross-linked telopeptide of type I collagen (CTX-I) decreased (linear, P < 0.05) as dietary Ca increased, which is a result of reduced bone resorption as dietary Ca increased. Serum bone-specific alkaline phosphatase tended to decrease (linear, P < 0.10) as Ca in diets increased, but the concentration of osteocalcin (OC) in serum was not affected by dietary Ca. The ratio between OC and CTX-I tended to increase (P < 0.10) as dietary Ca increased, which indicated that there was more bone formation than resorption in sows as dietary Ca increased. In conclusion, P digestibility in late gestating sows decreased, but retention of P increased, as dietary Ca increased from inadequate to adequate levels and blood biomarkers for bone resorption changed as Ca and P retention increased.

The similarity of the effect of carbohydrase or prebiotic supplementation in broilers aged 21 days, fed mixed cereal diets and challenged with coccidiosis infection.

The aim of this study was to investigate the effect on growth performance and nutrient utilisation when supplementing diets deficient in energy and protein with carbohydrase enzymes or xylo-oligosaccharide in broilers challenged with coccidia. 960 Ross 308 broilers were used in this 21-day study. The treatments were arranged into a 2×4 factorial with 2 challenge states (challenged and non-challenged) and 4 different additive types (control, xylanase alone, xylanase and ß-glucanase mixture and xylo-oligosaccharide). On day 14, the challenged group received 12× the recommended dose of coccidiosis vaccine while the non-challenged group received a sham treatment of water only. The birds and feed were weighed on days 0, 14 and 21. On day 21, two birds per pen were euthanized, the caeca were removed and the contents collected for short chain fatty acid analysis. Six more birds per pen were euthanized and ileal digesta were collected and pooled per pen for nutrient digestibility analysis. Feed intake was greater (P < 0.05) on days 14 and 21 when xylo-oligosaccharide was included in the diet compared to the xylanase and ß-glucanase mixture in birds challenged with coccidiosis. Including xylo-oligosaccharide in the diet improved (P < 0.05) the digestibility of nitrogen and supplementing diets with the xylanase and ß-glucanase mixture improved (P < 0.05) the digestibility of several amino acids. The concentration of arabinose and xylose was (P < 0.001) greater when broiler diets were supplemented with carbohydrase enzymes or xylo-oligosaccharide compared to the control. Although there was an increase in short chain fatty acid production due to the addition of carbohydrase enzymes or xylo-oligosaccharide, there was no additive effect on the %G+C profile of caecal bacteria however there was a negative effect of coccidiosis. In conclusion, the similarity in the response to carbohydrase enzymes or xylo-oligosaccharide supplementation illustrates that the hydrolysis products from carbohydrase activity may have prebiotic like effects.

Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism.

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

Effect of phytase on intestinal phytate breakdown, plasma inositol concentrations, and glucose transporter type 4 abundance in muscle membranes of weanling pigs1.

The objective of this present study was to determine the effects of phytase dosing on growth performance, mineral digestibility, phytate breakdown, and the level of glucose transporter type 4 (GLUT4) in muscle plasma membranes of weanling pigs. A total of 160 barrows were used in a randomized completely block design and assigned to 4 treatments for a 7-wk study. Depending on the feeding phase, diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8 to 6.8g/kg Ca; 7.3 to 6.3 g/kg P; negative control [NC]: 5.5 to 5.2 g/kg Ca; 5.4 to 4.7 g/kg P). NC diets were supplemented with phytase at 0 (NC); 500 (NC + 500 FTU); or 2,000 FTU/kg (NC + 2,000 FTU) phytase units/kg. Blood was collected after fasting (day 48) or feeding (day 49) for measurement of plasma inositol concentrations. On day 49, 2 pigs per pen were euthanized, and duodenal and ileal digesta samples were collected to determine inositol phosphates (InsP6-2) concentrations. High phytase supplementation increased BW on days 21, 35, and 49 (P < 0.05). Over the entire feeding period, ADG, ADFI, and feed efficiency were increased by NC + 2,000 FTU compared with the other treatments (P < 0.05). Postprandial plasma inositol concentration was increased in NC + 2,000 (P < 0.01), but there was only a tendency (P = 0.06) of a higher fasting plasma inositol concentration in this group. Inositol concentrations in the portal vein plasma (day 49) were not different among treatments. Duodenal digesta InsP5 and InsP6 concentrations were similar in PC and NC, but higher in these 2 treatments (P < 0.05) than those supplemented with phytase. Phytase supplementation decreased InsP6-4, resulting in increased InsP3-2 and myo-inositol concentrations. Similar effects were found in ileal contents. Compared with NC, phytase supplementation resulted in greater cumulative InsP6-2 disappearance (93.6% vs. 72.8% vs. 25.0%, for NC + 2,000 FTU, NC + 500 FTU and NC, respectively, P < 0.01) till the distal ileum. Longissimus dorsi muscle plasma membrane GLUT4 concentration was increased by NC + 2,000 FTU (P < 0.01) compared with NC. In summary, high phytase supplementation increased growth performance of nursery pigs. The higher myo-inositol release from phytate could contribute to the increased expression of GLUT4 in muscle plasma membranes. Further investigation is needed to determine whether this is associated with enhanced cellular glucose uptake and utilization.

The effect of carbohydrases or prebiotic oligosaccharides on growth performance, nutrient utilisation and development of small intestine and immune organs in broilers fed nutrient-adequate diets based on either wheat or barley.

BACKGROUND: Non-starch polysaccharides are large complex molecules and are found in cereal grains. This study was conducted to investigate the effect of carbohydrase enzymes or prebiotic oligosaccharides on growth performance, nutrient utilisation and weight of organs associated with the immune system in broilers fed wheat- or barley-based diets. RESULTS: In wheat-based diets, feed intake was lower (P < 0.05) following xylo-oligosaccharide supplementation, whereas in barley-based diets feed intake was greater (P < 0.05) following ß-glucanase supplementation. Gross energy digestibility was improved (P < 0.01) when either level of xylanase was added to wheat diets. Ileal digestible energy was greater (P < 0.01) in wheat diets including an additive compared with the control diet. In wheat diets, bursa weight was lower (P < 0.05) following xylo-oligosaccharide supplementation compared with the control treatment. CONCLUSIONS: The current study showed that supplemented carbohydrases or prebiotic oligosaccharides could alter the development of immune organs or small intestine without any significant effect on growth performance in broilers receiving nutrient-adequate diets. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.