Concentrations Of Phytase Research Articles

A New Rapid and Quantitative Assay to Determine the Phytase Activity of Feed.

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle–antibody–phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

Evaluation of the responses of broiler chickens to varying concentrations of phytate phosphorus and phytase. Ⅱ. Grower phase (day 12–23 post hatching)

A randomized complete block design study used 768 male broiler chickens to investigate the effects of phytate P (PP) and a novel consensus bacterial phytase variant (PhyG) concentration on growth performance, bone mineralization, apparent ileal digestibility (AID), and total tract retention (TTR) of nutrients in broiler chickens. Treatments were arranged in a 1 + 3 × 5 factorial with a nutrient-adequate positive control diet (PC) with 2.8 g PP/kg, 3 nutrient-reduced negative control diets (NC: PC minus 88 kcal/kg ME, 0.8 g/kg dig. Lys, 2.0 g/kg available P, 2.0 g/kg Ca and 0.5 g/kg Na) with varying PP (g/kg) levels, mainly from rice bran, at 2.3 (NC1), 2.8 (NC2), or 3.3 (NC3) and 5 PhyG doses at 0, 500, 1,000, 2,000, or 4,000 FTU/kg. All treatments had 6 replicate cages with 8 birds/cage. A commercial starter diet was fed from d 0 to 12 and the experimental diets from d 12 to 23 post hatching. Birds fed the NC2 diet without phytase had lower (P < 0.01) BW, BW gain, and feed intake (FI) as compared with birds fed the PC with the same PP level. With increasing phytate, there was a decrease (P < 0.05) in BW, BW gain, and FI. Phytase increased (P < 0.01) BW and feed efficiency of broiler chickens. An interaction (P < 0.05) between PP and phytase concentrations was observed on the AID of Met, Cys, and Thr. Linear decrease (P < 0.01) in the AID and TTR of P and Ca with increasing PP concentrations were observed. Phytase supplementation increased (P ≤ 0.05) the AID of P, Ca, and all AA. The TTR of P, Ca, and Zn was linearly increased (P < 0.01) by 112, 123, and 46%, respectively, when birds fed NC diets with 0 and 4,000 FTU/kg were compared. In conclusion, phytate reduced the growth performance and nutrient utilization of broiler chickens from d 12 to 23 post hatching while phytase ameliorated these negative effects.

Evaluation of the responses of broiler chickens to varying concentrations of phytate phosphorus and phytase. Ⅰ. Starter phase (day 1–11 post hatching)

Growth performance, tibia ash, apparent ileal digestibility (AID), and total tract retention (TTR) of nutrients responses of broiler chickens fed diets containing varying concentrations of phytate P (PP) and a novel consensus bacterial 6-phytase variant (PhyG) from d 1 to 11 post hatching were evaluated with 1,152 broiler chicks. Diets were a nutrient-adequate positive control diet (PC) with 2.8 g PP/kg or one of 15 nutrient-reduced negative control (NC: PC minus 88 kcal/kg ME, 0.8 g/kg dig. Lys, 2.0 g/kg available P, 1.8 g/kg Ca and 0.5 g/kg Na) diets with 3 PP (g/kg) levels, mainly from rice bran, at 2.3 (NC1), 2.8 (NC2), or 3.3 (NC3) and 5 PhyG supplementation at 0, 500, 1,000, 2,000, or 4,000 FTU/kg in a 1 + 3 × 5 factorial. All treatments had 6 replicate cages with 12 birds per cage. Despite comparable PP levels, birds fed the PC diet had greater (P ≤ 0.01) body weight (BW), feed intake (FI), tibia ash, AID of energy, AA, P, and Ca as compared with birds fed the NC2 without phytase. There was no interaction between PP and phytase for all responses. Increasing PP concentrations linearly decreased (P < 0.01) BW, FI, AID, and TTR of P and Ca. With phytase supplementation, there was a quadratic response (P < 0.05) in BW, FI, tibia ash, and a linear increase (P < 0.05) in the AID of energy, nitrogen, and all the measured AA. Increasing phytase dose from 0 to 4,000 FTU/kg increased (P < 0.01) AID of P and Ca by 88 and 18%, respectively. There was also a quadratic response (P ≤ 0.05) on TTR of P and Ca with increasing phytase dose. In conclusion, increasing levels of PP reduced growth performance and most nutrient utilization responses of broiler chickens while phytase supplementation positively impacted the responses of broiler chickens during d 1 to 11 post hatching.

Determining the phosphorus release of GraINzyme phytase in diets for nursery pigs.

The objective of this study was to determine the available P (aP) release curve for a new phytase source, GraINzyme Phytase (Agrivida Inc., Woburn, MA), which is expressed in corn containing an engineered Escherichia coli phytase called Phy02. Plant-expressed phytases are created by inserting phytase-encoding genes into plants resulting in their ability to produce seeds with increased concentrations of phytase. A total of 360 pigs (Line 200 × 400, DNA, Columbus, NE, initially 9.9 ± 0.19 kg) were used in a 21-d growth study. Pigs were weaned at approximately 21 d of age, randomly allotted to pens based on initial body weight (BW) and fed common starter diets. From days 18 to 21 postweaning, all pigs were fed a diet containing 0.11% aP. On day 21 postweaning, considered day 0 of the study, pens were blocked by BW and randomly allotted to one of eight dietary treatments with five pigs per pen and nine pens per treatment. Dietary treatments were formulated to include increasing aP derived from either an inorganic P source (0.11%, 0.19%, or 0.27% from monocalcium P) or increasing phytase (150, 250, 500, 1,000, or 1,500 FTU/kg). Diets were corn-soybean meal-based and contained 1.24% standardized ileal digestible Lys. On day 21 of the trial, one pig per pen (weighing closest to the mean pen BW) was euthanized and the right fibula was collected to determine bone ash using the nondefatted processing method. Overall (days 0 to 21), pigs fed increasing aP from inorganic P or phytase had increased (linear, P < 0.002) average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed (G:F; quadratic, P < 0.05). Bone ash weight (g) and percentage bone ash increased (linear, P < 0.001), with increasing inorganic P or added phytase. Based on the composition of the diets used in this study, the release equations developed for GraINzyme for ADG, G:F, bone ash weight, and percentage bone ash are as follows: aP = (0.255 × FTU)/(1299.969 + FTU), aP = (0.233 × FTU)/(1236.428 + FTU), aP = (45999.949 × FTU)/(462529200 + FTU), and aP = (0.272 × FTU)/(2576.581 + FTU), respectively.

Supplemental phytase increases phosphorus digestibility in pigs regardless of phytase source or feed pelleting

Two experiments were conducted to investigate the effects of phytase source and feed pelleting on digestibility of dry matter (DM), crude protein, organic matter (OM), and phosphorus (P) with different doses of supplemental phytase in pigs. In Exp. 1, ten barrows (31.4 ± 3.5 kg of body weight) were individually housed in metabolism crates and were allotted to a replicated 5 × 6 Latin square design with 5 diets and 6 periods. In Exp. 2, ten barrows (28.2 ± 3.2 kg of body weight) were individually housed in metabolism crates and were allotted to a replicated 5 × 5 Latin square design with 5 diets and 5 periods. Each period lasted for 10 days consisted of a 5-day adaptation period and a 5-day collection period, and the marker-to-marker method was used for fecal collection. In both experiments, a basal diet was prepared based on corn and soybean meal without phytase or inorganic P. Four experimental diets were prepared in a 2 × 2 factorial treatment arrangement with 2 phytase sources (Buttiauxella spp. or Escherichia coli) and 2 feed forms (mash or pellet). Supplemental dietary phytase concentrations were 500 and 1500 phytase units (FTU)/kg in Exp. 1 and 2, respectively. In Exp. 1, supplemental phytase at 500 FTU/kg increased apparent total tract digestibility of P (P < 0.001) with no effects of phytase source or feed pelleting. Feed pelleting increased digestibility of DM, crude protein, and OM (P < 0.001). In Exp. 2, supplemental phytase at 1500 FTU/kg increased apparent total tract digestibility of P (P < 0.001) with no effects of phytase source or feed pelleting. Feed pelleting increased digestibility of DM and OM (P < 0.001). Based on the results from the 2 experiments, supplemental phytase at 500 and 1500 FTU/kg increased P digestibility in pigs regardless of phytase source or feed pelleting, and feed pelleting increased nutrient digestibility.

Effect of high-phytase supplementation in lactation diets on sow and litter performance.

A total of 109 sows (Line 241; DNA, Columbus, NE) were used to evaluate the effect of increasing dietary phytase in lactation diets, already adequate in P, on sow and litter performance. On d 107 of gestation, sows were blocked by body weight and parity and allotted to 1 of 3 dietary treatments of increasing phytase concentration (0, 1,000, or 3,000 FYT/kg; Ronozyme HiPhos GT 2700, DSM Nutritional Products, Inc., Parsippany, NJ). The control diet contained no phytase and was formulated to contain 0.50% standardized total tract digestible phosphorus (STTD P; 0.45% available P) and 0.62% STTD calcium (0.90% total Ca). The phytase diets that contained 1,000 or 3,000 FYT/kg were also formulated to 0.50% STTD P and 0.62% STTD Ca including the release of 0.132% STTD P and 0.096% STTD Ca. Diets were balanced for net energy and fed from d 107 of gestation until weaning (d 18 ± 3). All farrowings were monitored, with farrowing duration starting at the time the first pig was born until the first dispersal of placental tissues with no subsequent pigs born. Litters were cross-fostered within treatment until 48 h postfarrowing to equalize litter size. There were no differences among treatments in sow body weight at d 107 of gestation, 24 h after farrowing, or at weaning. Sow average daily feed intake from farrowing to weaning tended to increase (linear, P = 0.093) as phytase increased. There was no evidence for difference in the number of total born pigs, as well as the percentage of stillborns, mummies, and born alive pigs at the completion of farrowing. Similarly, phytase supplementation did not influence (P > 0.05) wean-to-estrus interval or litter size after cross-fostering among dietary treatments. Although not significant (linear, P = 0.226), farrowing duration decreased as added phytase increased with a decrease of 47 min (12%) for 3,000 FYT compared with the control. There were no differences in pig weight at weaning, but as a result of increased survivability (linear, P = 0.002), litter weaning weight and overall litter weight gain increased (quadratic, P < 0.05) up to 1,000 FYT of added phytase with no further benefit observed in sows fed 3,000 FYT. In conclusion, sow feed intake tended to increase linearly with increasing added phytase. Feeding 1,000 FYT/kg maximized overall litter gain and weaning weight; however, a larger-scale study with more sows is needed to determine the addition of phytase in lactation diets to reduce farrowing duration.

269 Effects of high phytase supplementation in lactation diets on sow and litter performance

Abstract A total of 109 sows (Line 241; DNA, Columbus, NE) were used in a study to evaluate the effect of increasing phytase concentration in lactation diets on sow and litter performance. On d 107 of gestation, sows were blocked by body weight and parity and allotted to 1 of 3 dietary treatments of increasing phytase concentration (0, 1,000, or 3,000 FYT/kg; Ronozyme HiPhos 2700; DSM Nutritional Products, Inc., Parsippany, NJ). The control diet contained no phytase and was formulated to contain 0.50% standardized total tract digestible phosphorus (STTD P; 0.45% available P) and 0.62% STTD calcium (0.90% total Ca). The same STTD P and Ca concentrations were formulated for the phytase diets considering a release of 0.132 STTD P and 0.094 STTD Ca in both diets. Diets were fed from d 107 of gestation until weaning (d 18 ± 2). Litters were cross-fostered within treatment until 48 h post-farrowing to equalize litter size. Linear and quadratic response to phytase concentration was evaluated using the lmer function in R. There was no evidence for difference in sow body weight change, farrowing performance, wean-to-estrus interval, or litter size among dietary treatments. Sow average daily feed intake from farrowing to weaning tended to increase (linear, P=0.093) as phytase increased. Although not significant (linear, P =0.226), farrowing duration decreased as phytase increased. Litter weaning weight increased (quadratic, P=0.039) and overall litter gain increased (quadratic, P=0.047) with 1,000 FYT of phytase. In summary, sow feed intake tended to increase linearly with increasing phytase; however, feeding 1,000 FYT/kg maximized overall litter gain and weaning weight. This small-scale study suggests sow and litter performance benefits due to high inclusions of dietary phytase; however, a commercial trial with more sows is warranted to confirm these results.

Glicose sanguínea e taxa de excreção de amônia como indicadores da atividade de enzimas exógenas em tilápias do Nilo

A curva glicêmica e a taxa de excreção de amônia são métodos utilizados para avaliar a variação da glicose e da excreção do composto nitrogenado amônia em função de alterações na quantidade de carboidratos e proteínas, respectivamente, disponíveis nas rações. No entanto, os relatos são escassos quanto a sua utilização em pesquisas relacionadas ao uso de enzimas exógenas na alimentação de peixes. Mediante o exposto, o objetivo do trabalho foi de avaliar os níveis de glicose sanguínea e a taxa de excreção de amônia como indicadores metabólitos da eficiência da atividade de enzimas exógenas em dietas para tilápias do Nilo. Desta forma, foram realizados dois experimentos onde foram testadas a composição e concentração das enzimas fitase, alfa-amilase e protease. Os tratamentos foram compostos por dieta controle (DC), que posteriormente, foi suplementada com um blend 1 contendo 100ppm fitase + 200ppm protease, um blend 2 composto por 100ppm fitase + 400ppm protease, depois o blend 3 contendo 200ppm fitase + 200ppm protease + 133ppm alfa-amilase, e o blend 4 com 200ppm fitase + 400ppm protease + 200ppm alfa-amilase. O pico da glicemia sanguínea das tilápias do Nilo ocorreu nas primeiras 4 horas pós alimentação das tilápias em todos os tratamentos avaliados. As dietas suplementadas com fitase, protease e alfa-amilase promoveram maiores níveis de glicose sanguínea, enquanto, os picos de excreção de amônia ocorreram às 2h, 4h e 8h pós alimentação, o que pode estar relacionado ao aumento da disponibilidade de proteína pela suplementação das dietas com a protease. As concentrações de glicose sanguínea e a taxa de excreção de amônia podem ser indicadores indiretos da eficiência da atividade de enzimas digestivas em tilápias do Nilo.

Intrinsic phytase in hybrid rye increases the digestibility of phosphorus in corn and soybean meal in diets fed to growing pigs.

An experiment was conducted to test the hypothesis that inclusion of hybrid rye in diets containing corn and soybean meal (SBM) without or with microbial phytase improves the apparent total tract digestibility (ATTD) and the standardized total tract digestibility (STTD) of P because of the intrinsic phytase activity in hybrid rye. Forty-eight growing barrows (initial body weight: 39.5 ± 7.7 kg) were allotted to six diets. A basal diet containing corn and SBM; a rye-based diet; and a diet containing corn, SBM, and rye were formulated. Each diet was formulated without and with microbial phytase (500 units/kg of diet) for a total of six diets. Fecal samples were collected for 4 d following a 5-d adaptation period according to the marker-to-marker procedure. Results indicated that no interactions between diets and concentration of phytase were observed for any of the response criteria measured. The ATTD and STTD of P and the ATTD of Ca differed (P < 0.05) among diets, but regardless of diet, the concentration of P in feces was reduced (P < 0.05) by adding microbial phytase to the diets. As a consequence, microbial phytase increased (P < 0.05) ATTD and STTD of P, and the ATTD of Ca was also increased (P < 0.05) by the use of microbial phytase. Measured values for the ATTD and STTD of P in the diets containing corn, SBM, and hybrid rye without or with phytase were greater (P < 0.05) than values that were predicted based on the ATTD and STTD of P for the corn-SBM and the hybrid rye diet. The observation that STTD predicted from the individual ingredients underestimated the STTD of P in the mixed diet indicates that the intrinsic phytase in hybrid rye resulted in increased digestibility of the P in the corn and SBM included in the corn-SBM-hybrid rye diet. In conclusion, microbial phytase increased the ATTD and STTD of P and the ATTD of Ca regardless of feed ingredients used in diets fed to pigs. In addition, the intrinsic phytase from hybrid rye increased the ATTD and STTD of P in corn and SBM.

Efficacy of a Mono-Component Exogenous Protease in the Presence of a High Concentration of Exogenous Phytase on Growth Performance of Broiler Chickens

SUMMARY Increasing global population and disposable income is resulting in a dramatic rise in demand for animal-sourced food products. To meet this demand, the animal protein industry is constantly evolving to enhance efficiency and sustainability. Nutritionally, a major contributing technology to this objective is the exogenous feed enzymes. These enzymes can be included in the diet of livestock to improve digestibility, reduce nitrogen and phosphorus pollution, reduce diet cost, and improve uniformity of animal growth. However, the number of feed enzyme products on the market today is considerable and there is a lack of clarity as to how these divergent enzymes may interact in a diet. The study reported herein noted that the addition of a novel exogenous protease to a broiler chicken diet improved weight gain (+7.3%) and reduced feed conversion ratio (−4%) even though the diet already contained 2 other feed enzymes (phytase and xylanase). The results reported herein confirm the usefulness of exogenous protease and the value of such a part of a wider enzyme admixture to improve the sustainability of the broiler chicken industry.

Influence of graded concentrations of phytase in high-phytate diets on growth performance, apparent ileal amino acid digestibility, and phytate concentration in broilers from hatch to 28 D post-hatch.

An experiment was conducted to evaluate graded doses of phytase in high-phytate diets. Ross 308, male broilers (n = 600) were assigned to one of 4 diets, with 10 replicate pens/diet and 15 birds/replicate pen. Diets were a nutrient adequate positive control (PC), a negative control (NC) diet with a reduction of Ca by 0.22%, available P by 0.20%, energy by 120kcal/kg, and amino acids by 1 to 5% compared with the PC. The NC diet was supplemented with 0, 2,000, or 4,000 phytase units (FTU)/kg. Phytase increased (linear, P < 0.05) weight gain from hatch to day 18. Birds fed the NC + 4000 FTU/kg ate and gained more (P < 0.05) than birds fed the PC. The apparent ileal digestibility (AID) of all nutrients and amino acids were reduced (P < 0.05) in birds fed the NC compared with birds fed the PC. Phytase increased (linear, P < 0.10) AID of most nutrients. Digestibility was lower (P < 0.10) in birds fed the NC + 4000 FTU/kg compared with birds fed the PC. Using daily intake and AID to determine digestible nutrient intake resulted in no differences between birds fed the PC or NC + 4000 FTU/kg diets. Digestible intake of methionine or glutamate was better correlated with BW gain (P < 0.0001) than AID (P > 0.10). Phytase reduced (linear, P < 0.01) phytate concentration and increased inositol (linear, P < 0.01), phytate hydrolysis (linear, P < 0.05), and jejunal expression (linear, P < 0.05) of SNAT-1 and LAT-4 transporters. Supplementation of increasing doses of phytase in high-phytate, low-nutrient dense diets improved gain and digestibility through nearly complete phytate destruction. Digestible nutrient intake may be a better indication of broiler gain than AID alone.

Prediction models for apparent and standardized total tract digestible phosphorus in swine diets

The objectives of the present study were to develop equations for predicting apparent total tract digestible (ATTD) phosphorus (P) and standardized total tract digestible (STTD) P based on total P, phytate-P, and supplemental phytase concentrations in swine diets and to evaluate the accuracy of the prediction models using published values. A total of 500 observations from 78 experiments were employed to develop equations for predicting ATTD P and STTD P in swine diets. In the 500 observations, total P ranged from 1.05 to 7.90 g/kg, phytate-P ranged from 0.40 to 6.18 g/kg, and supplemental phytase ranged from 0 to 2.50 phytase unit/g (as-fed basis). The ATTD P was positively correlated with total P (r = 0.71; P < 0.001), phytate-P (r = 0.21; P < 0.001), and phytase concentrations (r = 0.32; P < 0.001). The STTD P was also positively correlated with total P (r = 0.71; P < 0.001), phytate-P (r = 0.21; P < 0.001), and phytase concentrations (r = 0.33; P < 0.001). Linear, quadratic, and exponential models for predicting ATTD P and STTD P were developed. Among the 3 prediction models, the exponential model which had the lowest Akaike information criterion and Bayesian information criterion was regarded the most accurate one for predicting digestible P. The accuracy of the prediction models was tested using 42 recently published STTD P values from diets with varying phytate-P and supplemental phytase concentrations that were not used in developing the prediction models. To test the accuracy of the current models, regression analysis was conducted to determine mean bias and linear bias. Predicted STTD P values were in very good agreements with measured values in all 3 equations based on nonsignificant bias in the models. In conclusion, all models were accurate in the validation test, but the exponential model was the most biologically reasonable.

Effects of a novel corn-expressed E. coli phytase on digestibility of calcium and phosphorous, growth performance, and bone ash in young growing pigs1.

Two experiments were conducted to test the hypothesis that a corn-expressed phytase increases growth performance, bone measurements, and nutrient digestibility by young growing pigs, if added to diets that are deficient in Ca and P. In Exp. 1, 60 pigs (initial BW: 10.78 ± 0.67 kg) were randomly allotted to 6 dietary treatments that included a positive control diet (PC; 0.70% total Ca and 0.60% total P) and a negative control diet (NC; 0.50% total Ca and 0.42% total P). Four additional diets were formulated by supplementing the NC diet with 250, 500, 1,000, or 1,500 phytase units (FTU)/kg. Diets were fed for 28 d and the individual BW of pigs on days 1 and 28 were recorded. Fecal samples were collected from days 25 to 27 to calculate apparent total tract digestibility (ATTD) of Ca and P. On the last day of the experiment, all pigs were euthanized, and the left femur was removed and analyzed for ash, Ca, and P. Results indicated that growth performance, ATTD of Ca and P, and bone ash measurements were reduced (P < 0.05) in NC fed pigs compared with PC fed pigs. However, growth performance, ATTD of Ca and P, and bone ash measurements were improved (linear and quadratic, P < 0.05) by including increasing concentrations of phytase to the NC diet. In Exp. 2, experimental procedures were similar to those used in Exp. 1. Forty-eight pigs (initial BW: 11.15 ± 0.85 kg) were randomly allotted to 6 dietary treatments in a 28-d experiment. Treatments included a PC diet, an NC diet, and 4 diets in which 500 or 1,000 FTU/kg of either the corn-expressed phytase or a commercial microbial phytase were added to the NC diet. Pigs fed the NC diet had reduced (P < 0.01) final BW, ADG, G:F, and bone ash concentrations compared with pigs fed the PC diet. When 500 FTU/kg phytase was fed, no differences were observed in growth performance or bone ash measurements between phytase sources, and there were no differences in growth performance among pigs fed 1,000 FTU/kg of either phytase source or the PC diet. However, regardless of concentration or source of phytase, pigs fed the PC diet had greater (P < 0.001) amount of bone ash, bone Ca, and bone P compared with pigs fed phytase diets. In conclusion, the corn-expressed phytase is effective in improving growth performance, Ca and P digestibility, and bone measurements in pigs fed diets that are deficient in Ca and P.

Influence of age and duration of feeding low-phosphorus diet on phytase efficacy in broiler chickens during the starter phase

A total of 1,408 male broiler chickens were used to evaluate the impact of age and duration of feeding low-phosphorus (P) diet on the efficacy of phytase using growth performance, nutrient utilization, tibia ash, and plasma indices. Diets were formulated with 2 non-phytate P (nPP) concentrations (i.e., 0.20 and 0.40%) and 2 phytase concentrations (i.e., 1,000 and 2,000 FTU/kg) added to the 0.20% nPP diet. Four dietary treatments with 8 replicate cages each were fed to broiler chicks at different ages and for different duration. Specifically, these were days 6 to 8 (12 birds per replicate), 12 to 14, 9 to 14, 20 to 22, or 6 to 22 (8 birds per replicate). Parameters were measured on the last day of each period. Duration of feeding effect was examined by comparing responses of birds fed for 2 or 5 D at day 14 and for 2 or 16 D at day 22; age effect was determined by comparing responses of birds fed for 2 D at age 8, 14 and 22 D post-hatching. Body weight gain and gain-to-feed ratio were increased (P < 0.01) in birds fed diets supplemented with phytase; however, an increase in duration of feeding improved (P < 0.01) feed efficiency with birds fed for 16 D performing better than birds fed for 2 D at day 22. In addition, phytase supplementation improved (P < 0.01) apparent P and calcium digestibility and retention, but the age effect on phytase efficacy was more apparent at day 14 and the duration of feeding effect was evident in birds fed for 2 D due to the increased levels of mineral utilization at that age/duration of feeding as compared with the other groups. The results of this study show that phytase efficacy was at optimum in birds fed low-P diet for 2 D at day 14. This period can be recommended for further bioefficacy studies of phytase.

Production of nitrogen fixing Azotobacter (SR-4) and phosphorus solubilizing Aspergillus niger and their evaluation on Lagenaria siceraria and Abelmoschus esculentus

AimsCurrent study was aimed to produce nitrogen fixing Azotobacter sp. (SR-4) and phosphorus solubilizers Aspergillus niger (A. niger) and to evaluate their efficiency as biofertilizers for agricultural practices. MethodsTwo biofertilizer including nitrogen fixing and phosphorus solubilizing were grown. The nitrogen fixing efficiency of Azotobacter (SR-4) was determined by Kjeldahl method. Similarly, Vanadomoybdate method was used to measure the soluble phosphorus while Heinonen method was used to analyze concentration of phytase and phosphatase in the cultures. Furthermore, both biofertilizers were tested in a field trail on Lagenaria siceraria (bottle gourd) and Abelmoschus esculentus (okra). ResultsThe Azotobacter (SR-4) strain was found efficient nitrogen fixer as 35.08 mg of nitrogen per gram of carbon was produced after 72 h of fermentation. Similarly, A. niger strain excrete extracellular phosphate solubilizing enzymes such as phytase (133UI in 48 h of fermentation) and phosphatase (170UI in 48 h of fermentation) which can solubilize the rock phosphate and make it available to plants. In field trials on selected plants (L. siceraria and A. esculentus), both biofertilizers showed significant increase in plant height, leaf length/width, fruit size and number of fruits per plant when compared with controls/untreated plants. Furthermore, plants co-inoculated with both the N fixing Azotobacter and phosphorus solubilizing A. niger have enhanced performance than those treated with each biofertilizer alone. ConclusionThe inoculation of seeds with A. niger and Azotobacter may replace costly and environment toxic chemical fertilizers with environment friendly and cost effective biofertilizers.

Increasing levels of microbial phytase increases the digestibility of energy and minerals in diets fed to pigs

The objective of this experiment was to test the hypothesis that increased dietary concentrations of a novel E. coli phytase (Smizyme) improve the apparent total tract digestibility (ATTD) of energy, dry matter (DM), calcium (Ca), phosphorus (P), and micro minerals in corn-soybean meal based diets fed to growing pigs. A total of 56 growing pigs (initial body weight: 13.6 ± 1.15 kg) were allotted to a randomized complete block design with 7 diets. Pigs were divided into 2 blocks with 28 pigs and 4 replicate pigs per diet in each block, for a total of 8 replicate pigs per diet. A positive control without phytase was formulated to contain 0.70% Ca and 0.33% standardized total tract digestible (STTD) P. A negative control diet that was similar to the positive control diet with the exception that the concentration of Ca and STTD P were reduced to 0.50 and 0.16%, respectively, was formulated by removing the dicalcium phosphate and increasing the concentration of limestone. Five additional diets that were similar to the negative control diet, but contained 250, 500, 1000, 1500, or 2500 units of microbial phytase per kg were also used. The ATTD of gross energy (GE), dry matter (DM), nitrogen (N), Ca, P, sodium (Na), potassium (K), magnesium (Mg), sulfur (S), zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn) in each diet were calculated. Data were analyzed using contrast statements. Data for ATTD and retention of P and Ca, were also analyzed using a single broken-line model. Results indicated that N retention and concentration of digestible energy (DE) and metabolizable energy (ME) of diets increased (linear, P < 0.05) as the concentration of phytase increased in the diets. The ATTD of Ca and P, and the STTD of P increased (linear and quadratic, P < 0.05) as phytase concentration increased in the diets. The concentration of phytase required for maximum retention of P and Ca was greater (P < 0.05) than the concentration required for maximum ATTD of P and Ca. The ATTD of K, Na, Mg, and Zn increased (linear, P < 0.05) as phytase increased in the diets, but that was not the case for S, Cu, Fe, and Mn. In conclusion, ATTD of GE, DM, and most macro and micro minerals increased if increased levels of phytase were added to corn-soybean meal diets that contained no inorganic P. Fecal excretion of Ca and P was reduced, and retention of Ca and P was increased by adding phytase to the diets, whereas most parameters for N balance were not affected by addition of phytase to the diets.

Effects of High Phytase Supplementation in Lactation Diets on Sow and Litter Performance

A total of 109 sows (Line 241; DNA, Columbus, NE) were used to evaluate the effect of increasing dietary phytase in lactation diets, already adequate in P, on sow and litter performance. On d 107 of gestation, sows were blocked by body weight and parity and allotted to 1 of 3 dietary treatments of increasing phytase concentration (0, 1,000, or 3,000 FYT/kg; Ronozyme HiPhos GT 2700, DSM Nutritional Products, Inc., Parsippany, NJ). The control diet contained no phytase and was formulated to contain 0.50% standardized total tract digestible phosphorus (STTD P; 0.45% available P) and 0.62% STTD calcium (0.90% total Ca). The phytase diets that contained 1,000 or 3,000 FYT/kg were also formulated to 0.50% STTD P and 0.62% STTD Ca including the release of 0.132% STTD P and 0.096% STTD Ca. Diets were balanced for net energy and fed from d 107 of gestation until weaning (d 18 ± 3). All farrowings were monitored, with farrowing duration starting at the time the first pig was born until the first dispersal of placental tissues with no subsequent pigs born. Litters were cross-fostered within treatment until 48 h postfarrowing to equalize litter size. There were no differences among treatments in sow body weight at d 107 of gestation, 24 h after farrowing, or at weaning. Sow average daily feed intake from farrowing to weaning tended to increase (linear, P = 0.093) as phytase increased. There was no evidence for difference in the number of total born pigs, as well as the percentage of stillborns, mummies, and born alive pigs at the completion of farrowing. Similarly, phytase supplementation did not influence (P > 0.05) wean-to-estrus interval or litter size after cross-fostering among dietary treatments. Although not significant (linear, P = 0.226), farrowing duration decreased as added phytase increased with a decrease of 47 min (12%) for 3,000 FYT compared with the control. There were no differences in pig weight at weaning, but as a result of increased survivability (linear, P = 0.002), litter weaning weight and overall litter weight gain increased (quadratic, P < 0.05) up to 1,000 FYT of added phytase with no further benefit observed in sows fed 3,000 FYT. In conclusion, sow feed intake tended to increase linearly with increasing added phytase. Feeding 1,000 FYT/kg maximized overall litter gain and weaning weight; however, a larger-scale study with more sows is needed to determine the addition of phytase in lactation diets to reduce farrowing duration.

Microfluidic Platform for Multimodal Analysis of Enzyme Secretion in Nanoliter Droplet Arrays.

High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multistep conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.

High doses of phytase on growth performance and apparent ileal amino acid digestibility of broilers fed diets with graded concentrations of digestible lysine.

Two experiments of the same design were conducted to determine the influence of phytase on performance and apparent ileal digestibility (AID) of amino acids in broilers fed graded concentrations of digestible lysine (dgLys). Cobb 400, male broilers were allocated to 1 of 16 diets consisting of 4 basal diets formulated at 80, 88, 96, or 104% of the Cobb 400 dgLys requirements for each feeding phase. Phytase was included in each basal diet at 0, 750, 1,500, or 3,000 phytase units (FTU)/kg. In Exp. 1, 33 birds/pen from hatch to day 42 were fed a 2-phase feeding program with 12 replicate pens/diet. In Exp. 2, there were 25 birds/pen from hatch to day 21 and 8 replicate pens/diet. Data were analyzed as a 4 × 4 factorial and means separated using orthogonal contrasts. In Exp. 1, feed intake (FI) increased (quadratic, P < 0.05) as dgLys increased in the diet. Body weight gain (BWG) increased (quadratic, P < 0.05) as dgLys concentration or phytase dose increased in the diet. As phytase dose increased in the diet, feed conversion ratio (FCR) was improved in a linear or quadratic (P < 0.05) manner depending on the dgLys concentration of the diet (dgLys × phytase, P<0.05). In Exp. 2, FI linearly (P < 0.05) increased as dgLys increased in the diet. Increasing the concentration of dgLys or phytase in the diet increased (quadratic, P < 0.05) BWG and improved (quadratic, P < 0.05) FCR. The AID of most amino acids was influenced by a dgLys × phytase interaction (P < 0.05), except threonine, valine, tryptophan, serine, cysteine, or leucine (linear or quadratic effect of phytase, P < 0.05), where phytase improved the AID in birds fed diets containing 80, 88, or 96% of the dgLys requirement, but not birds fed 104%. The predicted dgLys requirement to maximize performance, carcass, and digestible lysine intake was 97.6 to ≥ 104%. The predicted dose of phytase to maximize BWG or FCR was between 1,990 and 2,308 FTU/kg, regardless of the dgLys concentration in the diet. The predicted dose of phytase to maximize carcass weight was between 1,527 and 2,658 FTU/kg of diet and to maximize breast weight was 0 to ≥ 3,000 FTU/kg diet, depending on the dgLys concentration in the diet. In conclusion, optimal performance in the absence of phytase could be achieved at much lower levels of lysine in the presence of phytase.

The effects ofmicrobial phytases and dietary calcium and phosphorus levels on the productive performance and bone mineralization of broilers

Two trials were conducted to evaluate the effects of phytase and different levels of calcium (Ca) in an available phosphorus-limited diet (aP) on the performance, bone mineralization, and Ca and P retention of broilers. In Trial 1, 160 one-day-old male broilers were placed into 40 cages and allocated to five dietary treatments: one positive control [PC, 4.7 and 4.3 g/kg non-phytate P (NPP) and 9.0 and 8.5 g/kg Ca, from 1 to 21 days (d) and from 21 to 35 d of age, respectively] and four negative control [NC, 3.2 and 2.9 g/kg NPP and 7.0 and 6.3 g/kg Ca in starter (1–21 d) and growth (22–35 d) diet] groups comprising 4 concentrations of phytase A (FLF® 1000 FUT, 0, 250, 500 and 1000 FTU/kg). In Trial 2, 300 one-day-old broilers were placed into 60 cages and allocated to 10 dietary treatments, consisting of two NC diets (3.2 g/kg NPP), either 7.0 g/kg Ca (low Ca) or 9.0 g/kg Ca (high Ca), supplemented (0 or 500 FTU/kg) with four different phytases [OptiPhos® 5000 CT (A), Phyzyme® XP10000 TPT (B), Ronozyme® NP (C) and FLF® 1000 FUT (D)] from 1 to 14 d. In Trial 1, the PC group and the group receiving the highest dose of phytase A FLF® 1000 FUT (1000 FTU/kg) yielded significantly higher average daily gains from 1 to 21 d and higher tibia weights and ash contents than the NC, 250, and 500 FTU/kg. In Trial 2, no differences were observed in performances between animals receiving different types of phytase. Low Ca diets improved the feed efficiency from 1 to 7 d and ileal P digestibility. The interaction between Ca:aP and phytase affected (P ≤ 0.05) the feed efficiency from 1 to 7 d and ADG from 7 to 14 d. Based on this study, high levels of phytase (1000 FTU/kg) supplemented in starter (1–21 d) and growth (22–35 d) diets containing 3.2 and 2.9 g/kg NPP and 7.0 and 6.3 g/kg Ca, respectively, may be required to promote performance and bone mineralization in growing broilers. Lowering Ca levels to 7 g/kg in starting diets may exert greater effects on performance and ileal P digestibility than low levels (500 FTU/kg) of phytase.