Does dietary supplementation with phytases affect the thermoregulatory and behavioral responses of pullets in a tropical environment?

Dietary supplementation of two types of phytases (fungal and bacterial) with different dosages (300 and 900 FTUs) was evaluated in the thermoregulatory and behavioral responses of replacement pullets in a tropical environment. 288 Hy-Line White laying birds with a mean weight of 639.60 ± 6.05 g, clinically healthy, and eight weeks old were used in the study. Respiratory rate (RR, breaths. min⁻¹), Cloacal temperature (CT, °C), Surface temperature with feathers (STWF, °C), and Surface temperature featherless (STF, °C) were measured in the morning and afternoon. Behavioral data were observed through the following activities: sitting, eating, drinking, exploring feathers (EF), non-aggressive pecking (NAP), and object pecking (OP) recorded every 10 min from 6 a.m. to 5 p.m. Environmental variables were measured along with thermoregulatory and behavioral responses. There was an interaction for RR between phytase and period of the day (P < 0.05). The lowest RR (morning) was observed in fungal phytase. STF and STWF were higher (P < 0.05) in the afternoon. Birds supplemented with fungal phytase showed lower STWF (P < 0.05). The variables that contributed to explain physiological and behavioral responses are shown in order of importance for (i) periods of day: morning (sitting, STWF, drinking, eating, and CT) and afternoon (STF, STWF, OP, drinking, eating, RR and sitting); (ii) phytases: fungal (STF, STWF, RR, sitting, eating and drinking); and bacterial (RR, STF, STWF, CT and sitting). Thermoregulatory and behavioral responses were similar between dosages, but different between types of phytases. Birds supplemented with fungal phytase used sensible heat dissipation mechanisms and exhibited thermal comfort behaviors. The 300 and 900 FTUs phytase doses did not influence the thermoregulatory and behavioral responses of birds, while they showed natural heat dissipation and heat stress behaviors in the afternoon. We recommend a dietary supplementation of 300 FTUs fungal phytases.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Effects of phytase supplementation on production performance,egg and bone quality,plasma biochemistry and mineral excretion of layers fed varying levels of phosphorus

Excessive fecal excretion of phosphorus (P) has increasingly become an environmental issue due to oversupply of P in layer rations, and thus it is imperative to minimize safety margins for P to ensure the sustainability of the egg industry. In this study, a 12-week feeding trial (22 to 34 weeks of age) was conducted to evaluate the effects of phytase supplementation on production performance, plasma biochemistry, egg and bone quality and P excretion of laying hens fed various levels of non-phytate P (NPP). Forty-eight Lohmann white laying hens were randomly allocated to one of six corn–soybean meal–oat-based diets: diets containing 2.0, 2.5 or 3.0 g/kg NPP without phytase, and diets containing 1.0, 1.5 or 2.0 g/kg NPP with phytase (1 000 U/kg diet) where phytase inclusion was expected to provide 1.0 g/kg NPP to laying hens, thus making the phytase-unsupplemented treatment served as a control for the phytase-supplemented treatment accordingly. Productive performance was recorded during the experimental period. Blood and egg samples were collected, and digestibility studies were conducted at weeks 6 and 12 of the experiment. Bone mineralization was evaluated at the end of the experiment. Egg weight and egg production, feed consumption, BW and feed conversion ratio of laying hens fed lower NPP diets supplemented with phytase were comparable to those of hens fed high NPP phytase-unsupplemented controls. Eggshell thickness, specific gravity, Haugh units, tibia bone mineral density, tibia ash percent, plasma P and other biochemical parameters were not significantly different among dietary treatments. Total P intake, excretion and retention were affected by diet (P < 0.001), but its deposition in eggs was not significantly different. Contrast analysis further showed that total P excretion of phytase present vs phytase absent was averagely reduced by 40.4 mg/hen per day (P < 0.01). Moreover, total P excretion was linearly (P < 0.01) reduced with lowering dietary NPP, and this relationship was similar regardless of whether phytase was supplemented or not. The results from this study indicated that NPP levels in laying hen diets could be reduced to 1.0 g/kg (excluding the portion of NPP released by phytase) with the inclusion of phytase, without negative effects on production performance and health of the hens, thereby diminishing P excretion into environment.     

Interactive effects of vitamins A and K3 on laying performance,egg quality,tibia attributes and antioxidative status of aged Roman Pink laying hens

Extending laying cycle is a tendency in hen breeding, but egg quality declines as laying hens age. The present study was conducted to investigate the interactive effects of vitamins A and K₃ on laying performance, egg and tibia quality, and antioxidative status of aged Roman Pink laying hens. In a 3 × 3 factorial arrangement, 1 080 87-week-old laying hens were allocated to nine groups with eight replicates in each group. Deficient, adequate and excess vitamins A (0, 7 000 and 14 000 IU/kg) and K₃ (0, 2.0 and 4.0 mg/kg) were supplemented into a basal diet with 1 320 IU/kg of vitamin A and 0.5 mg/kg of vitamin K₃. After 2 weeks of adaption to basal diet, hens were fed corresponding diets for 8 weeks. Vitamins A and K₃ did not significantly affect the laying performance. However, they showed interactive effects on yolk ratio at week 93 as well as tibia weight and diameter (P < 0.05), and hens fed deficient vitamins A and K₃ had the highest yolk ratio and tibia weight, but the lowest tibia diameter. Compared with deficient addition, adequate or excess vitamins A and K₃ increased yolk color at weeks 93 and 97 (P < 0.05). Compared with hens fed deficient or excess vitamins, hens fed adequate vitamins A and K₃ had higher eggshell strength at week 93 or 97 (P < 0.05). Increasing vitamin A elevated plasma total superoxide dismutase (T-SOD) activity and decreased hepatic glutathione peroxidase (GSH-Px) activity (P < 0.05). Excess vitamin K₃ increased hepatic T-SOD activity (P < 0.05). Vitamins A and K₃ exhibited interaction on the activities of antioxidative enzymes in eggshell gland (P < 0.05), and adequate or excess vitamins A and K₃ increased the activities of GSH-Px, T-SOD and catalase (CAT). Adequate and excess vitamin A up-regulated the mRNA expression of GSH-Px1, GSH-Px3 and SOD1 in eggshell gland (P < 0.05). Vitamins A and K₃ showed interactive effects on CAT mRNA expression in eggshell gland (P < 0.05) and hens fed adequate vitamins A and K₃ had the highest CAT mRNA levels. In conclusion, dietary addition of vitamins A and K₃ improved the eggshell quality and yolk color as well as antioxidative status in eggshell gland of aged laying hens. Adequate vitamins A and K₃ showed beneficial effects and excess levels did not exhibit superior effects.     

Daily energy expenditure and water turnover in two breeds of laying hens kept in floor housing

Laying hens are increasingly kept in barn or free-range systems, which not only allows birds to move freely but also potentially entails higher energy expenditures due to higher locomotor activity. Therefore, the aim of our study was to quantify the daily energy expenditure (DEE) and water turnover in freely moving laying hens. For that purpose, 10 Lohmann Selected Leghorn (LSL) and 10 Lohmann Brown (LB) hens were obtained from a conventional breeding company at 17 weeks of age. The trial started when birds reached an age of 34 weeks. All 20 birds were kept together in the same littered floor pen (12.1 m²). The pen was equipped with perches, a nest box, feeding and nipple drinkers. The DEE was determined individually for all experimental birds (n = 20) for a total of nine days using the doubly labelled water (DLW) method. Lohmann Brown hens were heavier than LSL hens, but laying rate did not differ between the two breeds, that is, one egg per hen and day during the study period. Average egg mass was 63.1 ± 0.20 g in LB and 61.7 ± 0.12 g in LSL hens, which converted to an egg energy content of 420 and 410 kJ/egg, respectively. Dilution spaces for oxygen and hydrogen differed between the breeds but not the respective turnover rates. Total body water as a percentage of body mass (LB: 54.4%, LSL: 53.8%; SEM = 0.7, F_1,18 = 0.41, P = 0.513) and total water intake (TWI) per day (LB: 275 ml/day, LSL: 276 ml/day; SEM = 20, F_1,17 = 0, P = 0.994) did not differ between LB and LSL hens. Individual DEE increased with body mass in LB but not in LSL hens. Average DEE did not differ between the two breeds (LB: 1 501 kJ/day; LSL: 1 520 kJ/day; SEM = 32.1, F_1,17 = 2.54, P = 0.131). However, when comparing the DEE on a metabolic mass basis, LSL hens expended with 984 kJ/kg^0.75 on average significantly more energy per day than LB hens (895 kJ/kg^0.75; SEM = 20.3, F_1,18 = 10.1, P = 0.005). Our results suggest that the DLW technique is a viable method to measure the energy expenditure and water turnover over several days in laying hens. Furthermore, we show that laying hens kept in floor pens fit into the general pattern of DEE among wild birds.     

Effect of inorganic phosphate supplementation on egg production in Hy-Line Brown layers fed 2000 FTU/kg phytase

Phytase has long been used to decrease the inorganic phosphorus (Pi) input in poultry diet. The current study was conducted to investigate the effects of Pi supplementation on laying performance, egg quality and phosphate–calcium metabolism in Hy-Line Brown laying hens fed phytase. Layers (n = 504, 29 weeks old) were randomly assigned to seven treatments with six replicates of 12 birds. The corn–soybean meal-based diet contained 0.12% non-phytate phosphorus (nPP), 3.8% calcium, 2415 IU/kg vitamin D₃ and 2000 FTU/kg phytase. Inorganic phosphorus (in the form of mono-dicalcium phosphate) was added into the basal diet to construct seven experimental diets; the final dietary nPP levels were 0.12%, 0.17%, 0.22%, 0.27%, 0.32%, 0.37% and 0.42%. The feeding trial lasted 12 weeks (hens from 29 to 40 weeks of age). Laying performance (housed laying rate, egg weight, egg mass, daily feed intake and feed conversion ratio) was weekly calculated. Egg quality (egg shape index, shell strength, shell thickness, albumen height, yolk colour and Haugh units), serum parameters (calcium, phosphorus, parathyroid hormone, calcitonin and 1,25-dihydroxyvitamin D), tibia quality (breaking strength, and calcium, phosphorus and ash contents), intestinal gene expression (type IIb sodium-dependent phosphate cotransporter, NaPi-IIb) and phosphorus excretion were determined at the end of the trial. No differences were observed on laying performance, egg quality, serum parameters and tibia quality. Hens fed 0.17% nPP had increased (P < 0.01) duodenum NaPi-IIb expression compared to all other treatments. Phosphorus excretion linearly increased with an increase in dietary nPP (phosphorus excretion = 1.7916 × nPP + 0.2157; R² = 0.9609, P = 0.001). In conclusion, corn–soybean meal-based diets containing 0.12% nPP, 3.8% calcium, 2415 IU/kg vitamin D₃ and 2000 FTU/kg phytase would meet the requirements for egg production in Hy-Line Brown laying hens (29 to 40 weeks of age).     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Effects of ambient temperature and methionine supplementation of a low protein diet on the performance of laying hens

The effects of ambient temperature and the supplementation of methionine to a low protein diet on egg production, egg quality, blood constituents and nitrogen excretion of laying hens were studied. The objective was to derive an environmental friendly feed formulation for warm climate. Seventy-two 29-week-old commercial White Leghorn hens of Babcock ISA white strain were used in this trial. The design is a completely randomized design with a 2×3 factors arrangement of treatments. Two constant ambient temperatures were 24±1°C and 34±1°C with 85% relative humidity. The three dietary treatments were 170 g kg⁻¹ crude protein, 140 g kg⁻¹ crude protein and 140 g kg⁻¹ crude protein supplemented with methionine 1.4 g kg⁻¹. Hens were allotted into six groups according to egg production and body weight. Birds were raised in individual wire cages for the experimental feeding period of five weeks. At the end of the feeding trial, one replicate of laying hens (four birds) from each treatment were selected for a four-day metabolic study for the daily collection of the excreta. The blood samples were withdrawn from the wing vein for analysis of hematocrit, blood glucose, sodium, potassium, magnesium, calcium, phosphorus and uric acid. Experimental results indicate that increases in ambient temperature significantly depress feed intake, egg production, egg weight and live weight of laying hens. High ambient temperature also caused inferior egg quality, including shell weight, shell thickness, shell breaking strength and specific gravity. Ambient temperature also changes the egg components with heavier egg albumin and yolk in the low-temperature group. Increasing ambient temperature also caused an increase in pH value in the plasma. This increase revealed a trend of depressed glucose (P<0.05) in the plasma of the laying hens. The dietary treatments, however, did not significantly influence feed intakes. Except egg weight, laying hens that were fed with the low protein (140 g kg⁻¹) with methionine supplemented diet produced similar numbers of egg and feed conversion as the layers fed with the high protein diet. The low protein with methionine supplemented diet produced significantly lighter eggs than the high dietary protein diet under the high ambient temperature, but produced heavier egg under the low ambient temperature. The concentration of uric acid in the plasma and nitrogen in the excreta of the high protein group was significantly higher than the other two low protein dietary groups (P<0.05).     

Detection of Transgenic and Endogenous Plant DNA Fragments and Proteins in the Digesta,Blood, Tissues,and Eggs of Laying Hens Fed with Phytase Transgenic Corn

The trials were conducted to assess the effects of long-term feeding with phytase transgenic corn (PTC) to hens on laying performance and egg quality, and investigate the fate of transgenic DNA and protein in digesta, blood, tissues, and eggs. Fifty-week old laying hens (n = 144) were fed with a diet containing 62.4% PTC or non-transgenic isogenic control corn (CC) for 16 weeks. We observed that feeding PTC to laying hens had no adverse effect on laying performance or egg quality (P>0.05) except on yolk color (P<0.05). Transgenic phyA2 gene and protein were rapidly degraded in the digestive tract and were not detected in blood, heart, liver, spleen, kidney, breast muscle, and eggs of laying hens fed with diet containing PTC. It was concluded that performance of hens fed diets containing PTC, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with CC. There was no evidence of phyA2 gene or protein translocation to the blood, tissues, and eggs of laying hens.     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Thermoneutral zone for laying hens based on environmental conditions,enthalpy and thermal comfort indexes

Controlling environmental conditions inside laying hens facilities systems and their effects on physiology and performance is essential in defining management strategies to alleviate the adverse effects of thermal stress in laying hens. Thus, we estimated thermoneutral zones for laying hens exposed to different heat-challenging conditions based on environmental conditions, enthalpy, and thermal comfort indexes being evaluated out in four thermal environment-controlled wind tunnels equipped with heating and air moistening function, housed in an experimental room with an area of 31.92 m². Clustering analysis and empirical models were used to estimate thermoneutral zones for laying hens based on environmental conditions, enthalpy and thermal comfort indexes, and compare them with data available in the literature through graphics. The thermoneutral zones characterizing homeostasis for laying hens based on respiration rate (RR) are as follows: from 25.9 to 29.9 °C for air dry-bulb temperature (t_db), from 67 to 75 for temperature-humidity index (THI), from 68 to 73 for black globe-humidity index (BGHI), from 45 to 56 kJ kg dry air⁻¹ for enthalpy (H) and 441.7–465.6 W for radiant heat load (RHL). Comfort limits for physiological responses cloacal temperature (t_clo), surface temperature (t_sur) and RR found in this study are 39.4–39.9 °C, 26.5 to 29.9 °C and 30 to 67 mov. min⁻¹, respectively. The number of repetitions and the use of mathematical modeling to be worked on, may directly impact the amplitude of each limit to be established for each variable of interest.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Dietary supplementation of pyrroloquinoline quinone disodium protects against oxidative stress and liver damage in laying hens fed an oxidized sunflower oil-added diet

The protective effects of dietary pyrroloquinoline quinone disodium (PQQ.Na₂) supplementation against oxidized sunflower oil-induced oxidative stress and liver injury in laying hens were examined. Three hundred and sixty 53-week-old Hy-Line Gray laying hens were randomly allocated into one of the five dietary treatments. The treatments included: (1) a diet containing 2% fresh sunflower oil; (2) a diet containing 2% thermally oxidized sunflower oil; (3) an oxidized sunflower oil diet with 100 mg/kg of added vitamin E; (4) an oxidized sunflower oil diet with 0.08 mg/kg of PQQ.Na₂; and (5) an oxidized sunflower oil diet with 0.12 mg/kg of PQQ.Na₂. Birds fed the oxidized sunflower oil diet showed a lower feed intake compared to birds fed the fresh oil diet or oxidized oil diet supplemented with vitamin E (P=0.009). Exposure to oxidized sunflower oil increased plasma malondialdehyde (P<0.001), hepatic reactive oxygen species (P<0.05) and carbonyl group levels (P<0.001), but decreased plasma glutathione levels (P=0.006) in laying hens. These unfavorable changes induced by the oxidized sunflower oil diet were modulated by dietary vitamin E or PQQ.Na₂ supplementation to levels comparable to the fresh oil group. Dietary supplementation with PQQ.Na₂ or vitamin E increased the activities of total superoxide dismutase and glutathione peroxidase in plasma and the liver, when compared with the oxidized sunflower oil group (P<0.05). PQQ.Na₂ or vitamin E diminished the oxidized sunflower oil diet induced elevation of liver weight (P=0.026), liver to BW ratio (P=0.001) and plasma activities of alanine aminotransferase (P=0.001) and aspartate aminotransferase (P<0.001) and maintained these indices at the similar levels to the fresh oil diet. Furthermore, oxidized sunflower oil increased hepatic DNA tail length (P<0.05) and tail moment (P<0.05) compared with the fresh oil group. Dietary supplementation of PQQ.Na₂ or vitamin E decreased the oxidized oil diet induced DNA tail length and tail moment to the basal levels in fresh oil diet. These results indicate that PQQ.Na₂ is a potential antioxidant and is as effective against oxidized oil-related liver injury in laying hens as vitamin E. The protective effects of PQQ.Na₂ against liver damage induced by oxidized oil may be partially due to its role in the scavenging of free radicals, inhibiting of lipid peroxidation and enhancing of antioxidant defense systems.     

Bioavailability of zinc sources and their interaction with phytates in broilers and piglets

Zinc (Zn) is essential for swine and poultry and native Zn concentrations in feedstuffs are too low to meet their Zn requirement. Dietary Zn bioavailability is affected by phytate, phytase and Zn supplemented in organic form is considered as more bioavailable than inorganic sources. A meta-analysis using GLM procedures was processed using broiler and piglet databases to investigate, within the physiological response of Zn, (1) the bioavailability of inorganic and organic Zn sources (Analysis I); (2) the bioavailability of native and inorganic Zn dependent from dietary phytates, vegetal and supplemental phytase activity (Analysis II). Analysis I: the bioavailability of organic Zn relative to inorganic Zn sources ranged, depending on the variable, from 85 to 117 never different from 100 (P > 0.05). The coefficients of determination of the regressions were 0.91 in broilers and above 0.89 in piglets. Analysis II: in broilers, bone Zn was explained by supplemental Zn (linear and quadratic, P < 0.001) and by supplemental phytase (linear, P < 0.001). In piglets, the interaction between dietary Zn and phytates/phytases was investigated by means of a new variable combining dietary phytic phosphorus (PP) and phytase activity. This new variable represents the remaining dietary PP after its hydrolysis in the digestive tract, mainly due to phytase and is called non-hydrolyzed phytic phosphorus (PP_NH). Bone Zn was increased with native Zn (P < 0.001), but to a lower extent in high PP or low phytase diets (ZN_N × PP_NH, P < 0.001). In contrast, the increase in bone zinc in response to supplemental Zn (P < 0.001) was not modulated by PP_NH (P > 0.05). The coefficients of determination of the regressions were 0.92 in broilers and above 0.92 in piglets. The results from the two meta-analyses suggest that (1) broilers and piglets use supplemented Zn, independent from Zn source; (2) broiler use native Zn and the use is slightly enhanced with supplemental phytase; (3) however, piglets are limited in the use of native Zn because of the antagonism of non-hydrolyzed dietary phytate. This explains the higher efficacy of phytase in improving Zn availability in this specie.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

An Optimal Dietary Zinc Level of Brown-Egg Laying Hens Fed a Corn–Soybean Meal Diet

An experiment was conducted to estimate the optimal dietary zinc (Zn) level of brown-egg laying hens fed a corn–soybean meal diet from 20 to 40 weeks of age. A total of 120 20-week-old Beijing Red commercial laying hens were randomly allotted by bodyweight to one of five treatments with six replicates of four birds each in a completely randomized design, and fed a Zn-unsupplemented corn–soybean meal basal diet containing 27.95 mg Zn/kg by analysis and the basal diets supplemented with 30, 60, 90, or 120 mg Zn/kg as Zn sulfate (reagent grade ZnSO₄·7H₂O) for a duration of 20 weeks. Laying performance, egg quality, tissue Zn concentrations, and activities of serum alkaline phosphatase (AKP), and liver copper-Zn superoxide dismutase (CuZnSOD) were measured. Regression analyses were performed to estimate an optimal dietary Zn level whenever a significant quadratic response (P < 0.05) was observed. Tibia Zn concentration (P = 0.002) and serum AKP activity (P = 0.010) showed significant quadratic responses to dietary supplemental Zn levels. The estimates of dietary Zn requirements for brown-egg laying hens from 20 to 40 weeks of age were 71.95 and 64.63 mg/kg for tibia Zn concentration and serum AKP activity, respectively. The results from this study indicate that the tibia Zn might be a more suitable and reliable parameter for Zn requirement estimation, and the optimal dietary Zn level would be about 72 mg/kg for brown-egg laying hens fed a corn–soybean meal diet from 20 to 40 weeks of age.     

Effect of rearing density on pecking behaviour and plumage condition of laying hens in two types of aviary

The objective of this experiment was to investigate the effect of rearing density on pecking behaviour and plumage during rearing and throughout the laying period in aviaries. Chicks were reared on sand at high (H; 13 m⁻²) or low (L; 6.5 m⁻²) density, in four rearing pens of 390 chicks and eight pens of 195 chicks, respectively, each pen measuring 30 m². Proportions of chicks per pen performing various types of pecking behaviour were recorded by scan sampling during 16 observation bouts in each rearing pen at 6 weeks of age and during 24 observation bouts at 12 weeks. Individual body weights and plumage condition were recorded. Later, these pullets were housed at 17 hens m⁻² in Tiered Wire Floor (TWF; 3 H and 3 L pens of 275 hens) and Laco-Volétage (2 H and 2 L pens of 275 hens) aviaries. At 35 weeks, two samples of eight hens from each aviary pen were observed for pecking behaviour in a test pen. Throughout the laying period, additional records were collected on pecking behaviour, body weight, plumage condition, egg production, and mortality. The L birds had better plumage condition at 6 weeks of age and throughout the laying period. These birds also ground pecked more frequently than H birds during rearing and the laying period. At 12 weeks, L birds feather pecked less than H birds, but no relationship was found between rearing density and feather-pecking behaviour during the laying period. Although TWF hens feather pecked more frequently than Volétage hens, there was no interaction between rearing density and type of aviary for the various pecking behaviours.     

Low levels of organic compound trace elements improve the eggshell quality,antioxidant capacity,immune function,and mineral deposition of aged laying hens

In the egg production industry, trace elements are required as additional dietary supplements to play vital roles in performance and egg quality. Compared to inorganic microelements (ITs), appropriate dose of organic trace microelements (OTs) are environmentally friendly and sufficient to satisfy the needs of hens. In order to evaluate the extent to which low-dose OTs replace whole ITs, the effects of organic copper, zinc, manganese, and iron compound on the performance, eggshell quality, antioxidant capacity, immune function, and mineral deposition of old laying hens were investigated. A total of 1 080 57-week-old Jing Hong laying hens were assigned to five groups with six replicates of 36 layers each for an 8-week experimental period. The birds were fed either a basal diet (control treatment (CT)) or the basal diet supplemented with commercial levels of inorganic trace elements (IT 100%) or the equivalent organic trace elements at 20%, 30%, and 50% of the inorganic elements (OT 20%, OT 30%, and OT 50%, respectively). Results showed that compared with those in the CT treatment, feeding hens with inorganic or organic microelement diet had significant effects on the eggshell quality, antioxidant capacity, immune function, and mineral deposition of old laying hens (P < 0.05). The eggshell strength and ratio between OT 30%, OT 50%, and IT 100% were similar at weeks 4 and 8, and the eggshell thickness of these groups was also similar at weeks 6 and 8. At week 8, the eggshell colour in OT 50% was darker than that in IT 100%. The mineral content in the eggshells of OT 50% and IT 100% significantly increased (P < 0.001), with no significant difference in effective thickness, mammillary thickness, and mammillary knob width between groups. There were no differences in the malondialdehyde content, total antioxidant capacity, and total superoxide dismutase activity in serum between OT 30%, OT 50%, and IT100%. While the catalase activities, the interleukin-1β, interleukin-10, immunoglobulin G, and immunoglobulin M concentrations in serum were not significantly different between OT 50% and IT 100%. The mineral contents in the faeces of the organic groups were considerably reduced compared with those in IT 100% (P < 0.001). In conclusion, dietary supplementation with 30–50% organic compound microelements has the potential to replace 100% inorganic microelements in the hen industry for improving eggshell quality, mineral deposition in the eggshell, antioxidant capacity, and immune function, and reducing emissions to the environment without negative effects on laying performance.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg⁻¹) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg⁻¹) were less than that of the wild-type phytase (3,524 U mg⁻¹), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.