Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Effects of Dietary Colostrum Basic Protein on Bone Growth and Calcium Absorption in Mice.

Colostrum basic protein (CBP) is a trace protein extracted from bovine colostrum. Previous studies have shown that CBP can promote bone cell differentiation and increase bone density. However, the mechanism by which CBP promotes bone activity remains unclear. This study investigated the mechanism of the effect of CBP on bone growth in mice following dietary supplementation of CBP at doses that included 0.015%, 0.15%, 1.5%, and 5%. Compared with mice fed a normal diet, feeding 5% CBP significantly enhanced bone rigidity and improved the microstructure of bone trabeculae. Five-percent CBP intake triggered significant positive regulation of calcium metabolism in the direction of bone calcium accumulation. The expression levels of paracellular calcium transport proteins CLDN2 and CLDN12 were upregulated nearly 1.5-fold by 5% CBP. We conclude that CBP promotes calcium absorption in mice by upregulating the expression of the calcium-transporting paracellular proteins CLND2 and CLND12, thereby increasing bone density and promoting bone growth. Overall, CBP contributes to bone growth by affecting calcium metabolism.

Structural characterisation of deer sinew peptides as calcium carriers, their promotion of MC3T3-E1 cell proliferation and their effect on bone deposition in mice.

Deer sinew as a by-product has high collagen and nutritional value. This study focuses on its hydrolysate being used as a calcium carrier to develop functional foods. The chelation mechanism was analyzed by SEM, EDS, UV-vis, FTIR, and fluorescence spectroscopy and zeta potential analysis after using peptide-sequenced deer sinew peptides for chelation with calcium ions. The results showed that the chelation of deer sinew peptides with calcium ions occurs mainly at the O and N atoms of carboxyl, amino and amide bonds. In vitro and in vivo studies revealed that deer sinew peptide-calcium chelate (DSPs-Ca) promoted the proliferation of MC3T3-E1 cells without toxic side effects and increased the alkaline phosphatase activity. The DSPs-Ca group improved the bone microstructure induced by low calcium, as well as up-regulated the expression of genes responsible for calcium uptake in the kidneys, as evidenced by serum markers, bone sections, bone parameters, and gene expression analyses in low-calcium-fed mice. From the above, it can be concluded that DSPs-Ca is expected to be a calcium supplement food for promoting bone health.

Effects of phytase supplementation on broilers fed with calcium and phosphorus-reduced diets, challenged with Eimeria maxima and Eimeria acervulina: influence on growth performance, body composition, bone health, and intestinal integrity.

An experiment was conducted to evaluate the effects of phytase in calcium (Ca) and available phosphorous (avP)-reduced diet on growth performance, body composition, bone health, and intestinal integrity of broilers challenged with Eimeria maxima and Eimeria acervulina. A total of 672 14-day-old male broilers were allocated to a 2 × 4 factorial arrangement with 6 replicates per treatment and 14 birds per replicate. Two factors were Eimeria challenge and 4 dietary treatments: 1) a positive control (PC; 0.84% Ca and 0.42% avP); 2) a negative control (NC; 0.74% Ca and 0.27% avP); 3) NC + 500 FTU/Kg of phytase (NC + 500PHY); and 4) NC + 1,500 FTU/Kg of phytase (NC + 1500PHY). On d 14, birds in the Eimeria-challenged groups received a solution containing 15,000 sporulated oocysts of E. maxima and 75,000 sporulated oocysts of E. acervulina via oral gavage. At 5 d postinoculation (DPI), the challenged birds showed a higher (P < 0.01) FITC-d level than the unchallenged birds. While the permeability of the NC group did not differ from the PC group, the phytase supplementation groups (NC + 500PHY and NC + 1500PHY) showed lower (P < 0.05) serum FITC-d levels compared to the NC group. Interaction effects (P < 0.05) of Eimeria challenge and dietary treatments on feed intake (FI), mucin-2 (MUC2) gene expression, bone ash concentration, and mineral apposition rate (MAR) were observed. On 0 to 6 and 0 to 9 DPI, Eimeria challenge decreased (P < 0.01) body weight (BW), body weight gain (BWG), FI, bone mineral density (BMD), bone mineral content (BMC), bone area, fat free bone weight (FFBW), bone ash weight, bone ash percentage and bone ash concentration; and it showed a higher FCR (P < 0.01) compared to the unchallenged group. The reduction Ca and avP in the diet (NC) did not exert adverse effects on all parameters in birds, and supplementing phytase at levels of 500 or 1,500 FTU/Kg improved body composition, bone mineralization, and intestinal permeability, with the higher dose of 1,500 FTU/Kg showing more pronounced enhancements. There was an observed increase in FI (P < 0.01) when phytase was supplemented at 1,500 FTU/Kg during 0 to 6 DPI. In conclusion, results from the current study suggest that dietary nutrients, such as Ca and avP, can be moderately reduced with the supplementation of phytase, particularly in birds infected with Eimeria spp., which has the potential to save feed cost without compromising growth performance, bone health, and intestinal integrity of broilers.

Effect of day-old chick weight on the response of broilers to high doses of exogenous phytase.

A total of 360 Ross 708 male broiler chicks were used in an 8 treatment and 9 replicate cage study to explore the influence of day-old chick weight on the efficacy of exogenous phytase. Treatments were arranged as a 2 × 4 factorial with the factors being diet (a positive (PC) and negative control (NC) varying in nutrient density fed without or with 2 concentrations of exogenous phytase) and chick weight (light; <38.5 g or heavy; >42 g). Chicks were sourced from the same breeder flock, with light and heavy chicks being selected from the naturally occurring heterogeneity in the population. The diets were corn-soybean meal based and the PC was formulated to meet the nutrient requirements of male broiler chicks. The NC was formulated to contain 120 kcal/kg, 0.5, 0.18, and 0.18% less apparent metabolizable energy (AME), crude protein (CP), calcium (Ca), and phosphorus (P), respectively, than the PC. Amino acid (AA) density in the NC was also reduced in line with the reduction in CP and the manufacturers' recommendations for the effect of phytase on amino acid digestibility. Phytase at either 1,000 FYT/kg or 3,000 FYT/kg was added only to the NC to create a total of 4 diets. Water and mash feed were available ad libitum and were offered to 8 replicate cages, each containing 5 chicks. The experiment was conducted over a period of 3 wk and diets were offered in 2 phases (starter from d 1 to 10 and grower from d 11 to 21). Growth performance was monitored at the end of each diet phase and on d 21 ileal digesta were collected for estimation of apparent digestibility of energy (DE), nitrogen (N), Ca, P, dry matter (DM), and AA. There were no statistically significant interactions between diet and day-old chick weight for any of the measured parameters. Light chicks had significantly lower weight gain (approx. 5%) at both d 10 and d 21 compared with heavy chicks. This effect was principally associated with reduced feed intake and there was no significant effect of chick weight on feed conversion ratio (FCR). Chick weight had no effect on ileal nutrient digestibility. The reduction in nutrient density from the PC to the NC generated a significant reduction in weight gain (around 12%) and a significant increase in FCR (1.68 vs. 1.83). This effect was associated with a significant reduction in ileal DE (approximately 150 kcal/kg) and in the digestibility of several AA. Exogenous phytase significantly increased weight gain, reduced FCR and generated a significant increase in the ileal digestibility of energy, N, P, and several AA. Although chick weight and diet did not interact statistically, heavy chicks benefited more than light chicks from high doses of exogenous phytase across almost all measured end points which was confirmed by regression analysis. In conclusion, light chicks have inferior performance outcomes than heavy chicks principally because of reduced feed intake, but putatively not in digestive capacity per se. Exogenous phytase is effective in improving performance and nutrient digestibility in nutrient deficient diets. The effect of chick weight per se, and also breeder flock age, on the utility of supra-nutritional inclusion concentrations of exogenous phytase warrants further study.

Circadian regulation of calcium and phosphorus homeostasis during the oviposition cycle in laying hens.

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (PiT-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens.

Effects of different dietary vitamin D combinations during the grower phase and the feed restriction phase on growth performance and sternal morphology, mineralization, and related genes expression of bone metabolism in Pekin ducks.

Our study aimed to investigate the effects of different dietary vitamin D (VD) combinations during the grower (1-32 d of age) and feed restriction (33-52 d of age) phases on growth performance. We also evaluated sternal morphology, mineralization, and related genes expression of bone metabolism as well as absorption of calcium and phosphorous in duodenal mucosa and kidney in Pekin ducks. During the grower phase, we used 2 VD regimes (Group A: 3,160 IU/kg VD3; Group B: 400 IU/kg VD3 + 69 µg/kg 25-OH-D3). Each dietary treatment had 50 replicate pens of 10 ducks per pen. During the feed restriction phase, 30 replicate pens selected from Group A and Group B, repetitively, were redivided into 5 different dietary VD regimes to form a 2 × 5 experimental design. Each group consisted of 6 replicates, each with 10 ducks. During the feed restriction phase, we evaluated 5 different dietary VD combinations were as follows: T1: 2,000 IU/kg VD3 ; T2: 5,000 IU/kg VD3; T3: 3,620 IU/kg VD3 + 34.5 µg/kg 25-OH-D3; T4: 2,240 IU/kg VD3 + 69 µg/kg 25-OH-D3; T5: 1,800 IU/kg VD3 + 80 µg/kg 25-OH-D3). Results showed that Group B combinations with T5 had a better growth performance and breast meat deposition (P < 0.1). Regardless of 5 dietary VD regimes during the feed restriction phase, Group B significantly increased (P < 0.05) 52 d sternal depth and tended to increase (P < 0.1) 52 d sternal defatted weight, ash content, and phosphate (P) content of ducks. A significant interactive effect (P < 0.05) was observed on the mRNA abundance of DMP1 and Sost1 as well as RANKL/OPG in sternum and of VDR in duodenal mucosa of ducks at 52 d of age between dietary VD combinations during 2 phases. These results indicated that dietary VD regimes during the grower phase could affect the effectiveness of dietary VD regimes during the feed restriction phases; Dietary VD combinations of both phases could affect the genes expression of bone formation and the absorption as well as reabsorption of calcium and phosphorus in duodenum and kidney.

Oral calcium and vitamin D supplements differentially alter exploratory, anxiety-like behaviors and memory in male rats.

Oral calcium and calcium plus vitamin D supplements are commonly prescribed to several groups of patients, e.g., osteoporosis, fracture, and calcium deficiency. Adequate and steady extracellular calcium levels are essential for neuronal activity, whereas certain forms of calcium supplement (e.g., CaCO3) probably interfere with memory function. However, it was unclear whether a long-term use of ionized calcium (calcium chloride in drinking water ad libitum), vitamin D supplement (oral gavage) or the combination of both affected anxiety and memory, the latter of which was probably dependent on the hippocampal neurogenesis. Here, we aimed to determine the effects of calcium and/or vitamin D supplement on the anxiety- and memory-related behaviors and the expression of doublecortin (DCX), an indirect proxy indicator of hippocampal neurogenesis. Eight-week-old male Wistar rats were divided into 4 groups, i.e., control, calcium chloride-, 400 UI/kg vitamin D3-, and calcium chloride plus vitamin D-treated groups. After 4 weeks of treatment, anxiety-, exploration- and recognition memory-related behaviors were evaluated by elevated pulse-maze (EPM), open field test (OFT), and novel object recognition (NOR), respectively. The hippocampi were investigated for the expression of DCX protein by Western blot analysis. We found that oral calcium supplement increased exploratory behavior as evaluated by OFT and the recognition index in NOR test without any effect on anxiety behavior in EPM. On the other hand, vitamin D supplement was found to reduce anxiety-like behaviors. Significant upregulation of DCX protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats, suggesting their positive effects on neurogenesis. In conclusion, oral calcium and vitamin D supplements positively affected exploratory, anxiety-like behaviors and/or memory in male rats. Thus, they potentially benefit on mood and memory in osteoporotic patients beyond bone metabolism.

Effect of dietary calcium concentration and exogenous phytase on inositol phosphate degradation, mineral digestibility, and gut microbiota in growing pigs.

Variations in the dietary Ca concentration may affect inositol phosphate (InsP) degradation, and thereby, P digestibility in pigs. This study assessed the effects of dietary Ca concentration and exogenous phytase on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs with ileal cannulation. In a completely randomized row-column design with four periods, eight ileal-cannulated barrows (initial body weight 27 kg) were fed four corn-soybean- and rapeseed meal-based diets containing 5.5 or 8.5 g Ca/kg dry matter (DM), with or without 1,500 FTU of an exogenous hybrid-6-phytase/kg diet. No mineral P was added and the P concentration in the feed was 4.8 g P/kg DM. Prececal InsP6 disappearance in pigs fed diets containing exogenous phytase was lower (P = 0.022) with additional Ca than without. Concentrations of InsP2-4 isomers and myo-inositol in the distal ileal digesta and prececal P digestibility were greater (P < 0.001) with exogenous phytase than without exogenous phytase. In feces, InsP6 disappearance was lower (P < 0.002) and concentration of InsP5 and InsP4 isomers was higher (P ≤ 0.031) with additional Ca compared to without additional Ca. The prececal amino acid digestibility, energy digestibility, and hindgut disappearance of energy did not differ. The Shannon diversity index of the microbiota in the distal ileal digesta and feces was similar among the diets but was lower in the distal ileal digesta than in the feces (P < 0.001). Permutation analysis of variance revealed no dietary differences between the bacterial groups within the ileal digesta and fecal samples (P > 0.05). In conclusion, additional Ca reduced the effect of exogenous phytase on prececal InsP6 degradation. Endogenous InsP degradation was impaired by additional Ca only in the hindgut but the abundance of bacterial genera in feces was not affected.

The dietary calcium concentration can influence the release of phosphorus from phytate in growing pigs. This study assessed the effects of dietary calcium and exogenous phytase on inositol phosphate (InsP) degradation and nutrient digestibility in ileal-cannulated, growing pigs. The phosphorus, calcium, and myo-inositol concentrations in the blood, microbiota composition in the ileal digesta and feces, and volatile fatty acid concentrations in the feces were also evaluated. Additional dietary calcium decreased prececal inositol hexakisphosphate (InsP6) disappearance, but only with exogenous phytase. Concentrations of InsP2-4 isomers and myo-inositol in the ileal digesta and prececal phosphorus digestibility were greater with exogenous phytase, but not affected by dietary calcium concentration. In contrast, fecal InsP6 disappearance was lower and the concentration of InsP4-5 isomers in feces was greater with additional dietary calcium. Regarding microbiota, the Shannon diversity index was lower in ileal digesta than in feces but was unaffected by dietary calcium concentration or exogenous phytase. In conclusion, dietary calcium concentration is relevant for phytate disappearance in feces, but not in the ileal digesta. However, when exogenous phytase is used, the dietary calcium concentration is important because prececal phytate degradation is changed.

Purification, Identification, Chelation Mechanism, and Calcium Absorption Activity of a Novel Calcium-Binding Peptide from Peanut (Arachis hypogaea) Protein Hydrolysate.

A novel calcium-binding peptide was purified from peanut protein hydrolysate using gel filtration chromatography and identified using HPLC-MS/MS. Its amino acid sequence was determined as Phe-Pro-Pro-Asp-Val-Ala (FPPDVA, named as FA6) with the calcium-binding capacity of 15.67 ± 0.39 mg/g. Then, the calcium chelating characteristics of FPPDVA were investigated using ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, particle size, and zeta potential. The results showed that FPPDVA interacted with calcium ions, the chelation of calcium ions induced FPPDVA to fold and form a denser structure, the calcium-binding sites may mainly involve oxygen atoms from the carboxyl residues of Asp and Ala, and Phe possessed contact energy and carbonyl residues of Val. Microstructure analysis showed that FPPDVA-calcium chelate exhibited a regularly ordered and tightly aggregated sheets or block structures. Additionally, FPPDVA-calcium chelate had good gastrointestinal digestive stability and thermal stability. The results of everted rat intestinal sac and Caco-2 cell monolayer experiments showed that FPPDVA-calcium chelate could promote calcium absorption and transport through the Cav1.3 and TRPV6 calcium channels. These data suggest that FPPDVA-calcium chelate possesses the potential to be developed and applied as calcium supplement.

A first model of the fate of dietary calcium and phosphorus in broiler chickens.

To reduce P excretion and increase the sustainability of poultry farms, one needs to understand the mechanisms surrounding P metabolism and its close link with Ca metabolism to precisely predict the fate of dietary P and Ca and related requirements for birds. This study describes and evaluates a model developed to estimate the fate of Ca and P consumed by broilers. The Ca and P model relies on three modules: (1) digestion of Ca and P; (2) dynamics of Ca and P in soft tissue and feathers; and (3) dynamics of body ash. Exogenous phytase affects the availability of Ca and P; thus, to predict the absorption of those minerals, the model also accounts for the effect of phytase on Ca and P digestibility. We used a database to estimate the consequences of dietary Ca, P, and phytase over feed intake response. This study followed a four-step process: (1) Ca and P model development and its coupling with a growth broiler model; (2) model behavior assessment; (3) sensitivity analysis to identify the most influential parameters; and (4) external evaluation based on three databases. The proportion of P in body protein and the Ca to P ratio in bone are the most sensitive parameters of P deposition in soft tissue and bone, representing 91 and 99% of the total variation. The external evaluation results indicated that body water and protein had an overall mean square prediction error (rMSPE) of 7.22 and 12.3%, respectively. The prediction of body ash, Ca, and P had an rMSPE of 7.74, 11.0, and 6.56%, respectively, mostly errors of disturbances (72.5, 51.6, and 90.7%, respectively). The rMSPE for P balance was 13.3, 18.4, and 22.8%, respectively, for P retention, excretion, and retention coefficient, with respective errors due to disturbances of 69.1, 99.9, and 51.3%. We demonstrated a mechanistic model approach to predict the dietary effects of Ca and P on broiler chicken responses with low error, including detailed simulations to show the confidence level expected from the model outputs. Overall, this model predicts broilers' response to dietary Ca and P. The model could aid calculations to minimize P excretion and reduce the impact of broiler production on the environment. A model inversion is ongoing that will enable the calculation of Ca and P dietary quantities for a specific objective. This will simplify the use of the model and the feed formulation process.

Dietary calcium and nonphosphate phosphorus interaction influences tibiotarsus development and related gene expression of broilers from 1 to 21 days of age.

The dietary needs of calcium (Ca) and phosphorus (P) are interdependent, thus accurate evaluation of Ca and P requirements of broilers to support skeleton health and optimal growth is critical. The present study was carried out to investigate the effects of dietary Ca and nonphytate P (NPP) levels and their interactions on growth performance, tibiotarsus characteristics, tibiotarsus metabolism-related enzyme and proteins, and their gene expression of broilers, so as to provide a rational recommendation for Ca and NPP levels in diet. A total of 540 one-day-old Arbor Acres male broilers were randomly allotted to 1 of 15 treatments with 6 replicate cages of 6 birds per cage for each treatment in a completely randomized design involving a 5 × 3 factorial arrangement of treatments (5 levels of Ca × 3 levels of NPP). The birds were fed the corn-soybean meal diet containing 0.60%, 0.70%, 0.80%, 0.90%, or 1.00% Ca and 0.35%, 0.40%, or 0.45% NPP for 21 d. Dietary Ca level affected (P < 0.03) the bone mineral density, bone mineral content (BMC), breaking strength, ash percentage and ash Ca contents in tibia, which showed linear (P < 0.006) responses to dietary Ca levels. Dietary NPP level affected (P < 0.05) tibia BMC, ash percentage, and FGF23 mRNA level. Broilers that received 0.40% and 0.45% NPP had higher (P < 0.04) tibia BMC and ash percentage than those that received 0.35% NPP, but no differences (P > 0.05) were found between 0.40% and 0.45% NPP. Broilers that received 0.40% NPP had higher (P = 0.02) tibia FGF23 mRNA level than those that received 0.35% NPP, but no differences (P > 0.05) were detected between 0.40% and 0.45% NPP or 0.45% and 0.35% NPP. The interactions between dietary Ca and NPP affected (P < 0.05) ADG, ALP activity, bone gal protein, FGF23 contents, and the mRNA expression levels ALP and bone gal protein in tibia of broilers. Results from the present study indicate that dietary Ca and NPP interaction influences growth, tibiotarsus development, and related gene expression of broiler chickens. Considering all the criteria, the dietary levels of 0.90% Ca and 0.45% NPP would be optimal for both growth and tibiotarsus development of broilers fed a conventional corn-soybean meal diet from 1 to 21 d of age.

Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium.

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D3 and Ca (HS+D3/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D3 and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D3/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D3/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D3/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D3/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D3/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D3/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D3/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D3 and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.

Effects of preharvest sprays of iodine, selenium and calcium on apple biofortification and their quality and storability.

The low dietary intake of iodine (I) and selenium (Se) by humans leads to serious health and socioeconomic problems. Therefore, enrichment of plants with I and Se using fertilisers containing these micronutrients is commonly recommended. In this study, we examined the impacts of combined spraying of I as iodide or iodate, Se as selenite or selenate, and calcium (Ca) as Ca-chloride on the enrichment of 'Red Jonaprince' (Malus domestica Borth.) apples, as well as fruit quality and their storability. Sprays were applied 2 weeks before harvest at rates of 0.5 kg I, 0.25 kg Se and 7 kg Ca per ha. Trees not sprayed with these nutrients served as controls. The tested sprays caused leaf burn, but they did not affect the cold injury of buds and shoots. Those sprays had no effect on yield, fruit size and russeting or skin colouring. At harvest, sprayed apples contained about 50 times more I and Se and 30% more Ca than the control fruit. After storage, compared to the control fruit, sprayed apples were firmer, had more organic acids and were less susceptible to disorders, such as bitter pit, internal breakdown and decay caused by Neofabraea spp. The results indicate that preharvest spraying with I, Se and Ca at high rates can be recommended to effectively enrich apples with I and Se and to simultaneously improve their storability.

A novel calcium-binding peptide from bovine bone collagen hydrolysate and chelation mechanism and calcium absorption activity of peptide-calcium chelate.

A novel calcium-binding peptide from bovine bone collagen hydrolysate was screened based on a new target-the calcium-sensing receptor (CaSR), and its chelation mechanism and calcium absorption activity were investigated. Glu-Tyr-Gly exhibited superior binding affinities to CaSR because of its interaction with the active sites of the CaSR Venus Flytrap (VFT) domain. Glu-Tyr-Gly-Ca may exist in five potential chelation modes and its potential chelation mechanism was that calcium ions were located in the center and surrounded by ionic bonds (carboxyl group) and coordination bonds (carbonyl, amino, and carboxyl group). Glu-Tyr-Gly-Ca was slightly damaged in the intestinal fluid and at different temperatures, whereas it was severely damaged in the gastric fluid and acidic conditions. The results of the calcium dialysis percentage and Caco-2 cells experiments showed that Glu-Tyr-Gly-Ca possessed good calcium transport activity and bioavailability. The findings provided theoretical basis for Glu-Tyr-Gly-Ca as potential calcium supplement to improve intestinal calcium absorption.

Dietary calcium and non-phytate phosphorus levels affect the performance, serum biochemical indices, and lipid metabolism in growing pullets.

This experiment aimed to study the effects of dietary calcium (Ca) and non-phytate phosphorus (NPP) levels on performance, serum biochemical indices, and lipid metabolism in Beijing You Chicken (BYC), a local chicken. A 3 × 3 factorial design was adopted, dietary Ca levels were 0.66, 0.71, and 0.76%, NPP levels were 0.25, 0.30, and 0.35%. A total of 648 ten-wk-old BYC growing pullets were randomly divided into 9 groups with 6 replicates per group, and 12 birds per replicate. Growth performance, serum biochemical indices, and lipid metabolism indicators from 10 to 16 wk were measured. The results showed as follows: 1) Dietary Ca and NPP alone did not affect growth performance, but the interaction of dietary Ca and NPP affected average feed intake (AFI) of growing pullets (P < 0.05). The AFI was the lowest for the group with 0.71% Ca and 0.25% NPP (3,550.0 g, P = 0.036). 2) Dietary Ca level significantly affected serum P content (P < 0.05); dietary NPP had an influence trend on serum Ca content (P= 0.054). Dietary NPP levels and the interaction of Ca and NPP significantly affected alkaline phosphatase (AKP) activity. 3) Dietary Ca levels significantly affected TC content and HDL-C content (P < 0.05). Dietary NPP level significantly affected TG content (P < 0.05), the TG content in 0.25% and 0.30% NPP groups was significantly lower than that in 0.35% NPP group (P < 0.05). The interaction of dietary Ca and NPP significantly affected TG, TC and HDL-C contents (P < 0.05). TG, TC, and LDL-C levels were lower and HDL-C levels were the highest in the group with 0.66% Ca and 0.25% NPP. In summary, appropriate dietary Ca level can regulate serum TG, TC, and HDL-C content. Dietary Ca and NPP levels can be adjusted in pullet phase to avoid excessive obesity during the egg-laying period. This study recommended that dietary 0.66% Ca and 0.25% NPP benefit for the lipid metabolism of BYC growing pullets without affecting the performance.

Evaluating a novel consensus bacterial 6-phytase variant on growth performance of broilers fed U.S. commercial diets deficient in nutrients and energy through 63 days of age.

An experiment was conducted to evaluate the efficacy of a novel consensus bacterial 6-phytase variant expressed in Trichoderma reesei (PhyG) in broilers fed corn-soybean meal-based diets with application of dose-specific full nutrient and energy matrix values. Ross 708, straight-run broilers (n = 2,016) were assigned to one of 7 dietary treatments, with 12 replicate pens/diet and 24 birds/pen. Diets were a nutrient adequate control (PC), nutrient reduced negative controls 1, 2, and 3 (NC1, NC2, and NC3) with reductions in available phosphorus (avP) by 0.15%, 0.18%, and 0.19%, calcium (Ca) by 0.17%, 0.20%, and 0.21%, dig amino acids (AA) by 0.02%-0.05%, sodium (Na) by 0.03%-0.05%, and metabolizable energy (ME) by 62.8, 68.8, and 69.5 kcal/kg, respectively. Other diets were the NC1, NC2, and NC3 respectively supplemented with 500 (PhyG500), 1,000 (PhyG1000), and 2,000 (PhyG2000) FTU/kg. Over the 63-day feeding period, decreasing nutrient specifications lowered body weights (P < 0.05) in broilers from 4,518 g in PC to 4,256 g and 4,191 g and increased body weight-corrected feed conversion ratio (FCR, P < 0.05) from 1.92 in PC to 2.06 and 2.08 in the NC2 and NC3, respectively. Compared with PC, PhyG maintained (P > 0.05) BW in broilers fed PhyG500 (4,474 g), PhyG1000 (4,417 g), and PhyG2000 (4,449 g). Moreover, PhyG at all dose-levels maintained (P > 0.05) overall FCR vs. PC. The NC1, NC2, and NC3 diets decreased (P < 0.05) tibia ash vs. PC, and each PhyG500, PhyG1000, and PhyG2000 completely restored tibia ash to the similar levels (P > 0.05) as the PC. Carcass yield was decreased (P < 0.05) by NC1 (80.63%), NC2 (80.51%), and NC3 (80.31%) vs. PC (81.96%) with complete alleviation by PhyG500 (82.11%), PhyG1000 (81.80%), and PhyG2000 (81.54%). In conclusion, the novel consensus phytase variant completely compensated for the reduction in dietary avP, Ca, dig AA, and ME at each dose-level and maintained growth performance, bone quality, carcass characteristics, and nutrient digestibility in a typical corn-soybean mean based diet fed to broilers through 63 days of age.

As any reduction in dietary nutrients can negatively impact broiler productivity mitigating these effects is imperative. Dietary reductions in minerals, amino acids, and energy can all result in decreased growth and performance. Previous studies have demonstrated that exogenous phytase added to poultry diets can mitigate decreased growth caused by diets deficient in minerals, amino acids, and energy. In this current study, the addition of dose-dependent digestible AA and ME matrix values in addition to a mineral (P and Ca) down-specification in novel consensus bacterial 6-phytase variant supplemented diets resulted in maintained growth performance, bone quality, carcass characteristics, and nutrient digestibility equivalent to those of a nutritionally adequate diet. This study demonstrated the extra-phosphoric efficacy of phytase, at on top of the phosphoric efficacy, in a typical corn-soybean meal based diet fed to broilers through 63 days of age. The results of this current study confirmed that it is possible to account for phytase contributions for increased AA and energy availability, in addition to P and Ca.

Fish feeds supplemented with calcium-based buffering minerals decrease stomach acidity, increase the blood alkaline tide and cost more to digest.

Predatory fish in the wild consume whole prey including hard skeletal parts like shell and bone. Shell and bone are made up of the buffering minerals calcium carbonate (CaCO3) and calcium phosphate (Ca3(PO4)2). These minerals resist changes in pH, meaning they could have physiological consequences for gastric acidity, digestion and metabolism in fish. Using isocaloric diets supplemented with either CaCO3, Ca3(PO4)2 or CaCl2 as non-buffering control, we investigated the impacts of dietary buffering on the energetic cost of digestion (i.e. specific dynamic action or SDA), gastric pH, the postprandial blood alkalosis (the "alkaline tide") and growth in juvenile rainbow trout (Oncorhynchus mykiss). Increases in dietary buffering were significantly associated with increased stomach chyme pH, postprandial blood HCO3-, net base excretion, the total SDA and peak SDA but did not influence growth efficiency in a 21 day trial. This result shows that aspects of a meal that have no nutritional value can influence the physiological and energetic costs associated with digestion in fish, but that a reduction in the SDA will not always lead to improvements in growth efficiency. We discuss the broader implications of these findings for the gastrointestinal physiology of fishes, trade-offs in prey choice in the wild, anthropogenic warming and feed formulation in aquaculture.

Anti-hyperalgesic effects of photobiomodulation therapy (904 nm) on streptozotocin-induced diabetic neuropathy imply MAPK pathway and calcium dynamics modulation.

Several recent studies have established the efficacy of photobiomodulation therapy (PBMT) in painful clinical conditions. Diabetic neuropathy (DN) can be related to activating mitogen-activated protein kinases (MAPK), such as p38, in the peripheral nerve. MAPK pathway is activated in response to extracellular stimuli, including interleukins TNF-α and IL-1ß. We verified the pain relief potential of PBMT in streptozotocin (STZ)-induced diabetic neuropathic rats and its influence on the MAPK pathway regulation and calcium (Ca2+) dynamics. We then observed that PBMT applied to the L4-L5 dorsal root ganglion (DRG) region reduced the intensity of hyperalgesia, decreased TNF-α and IL-1ß levels, and p38-MAPK mRNA expression in DRG of diabetic neuropathic rats. DN induced the activation of phosphorylated p38 (p-38) MAPK co-localized with TRPV1+ neurons; PBMT partially prevented p-38 activation. DN was related to an increase of p38-MAPK expression due to proinflammatory interleukins, and the PBMT (904 nm) treatment counteracted this condition. Also, the sensitization of DRG neurons by the hyperglycemic condition demonstrated during the Ca2+ dynamics was reduced by PBMT, contributing to its anti-hyperalgesic effects.

Effects of 25-hydroxycholecalciferol (25-OH-D3) and 1-hydroxycholecalciferol (1-OH-D3) on serum bone biomarkers and calcium and phosphorus balance and concentrations of energy in diets without or with microbial phytase fed to sows in late gestation.

The objective was to test the hypothesis that supplementation of diets for gestating sows with 25-hydroxycholecalciferol (25-OH-D3) or 1-hydroxycholecalciferol (1-OH-D3) affects serum biomarkers for bone and increases Ca and P balance and the apparent total tract digestibility (ATTD) of gross energy (GE), and the concentrations of digestible energy (DE) and metabolizable energy (ME) in diets without or with microbial phytase. Sixty multiparous sows were allotted to 1 of 6 diets. Diets were formulated using a 3 × 2 factorial with 3 inclusions of supplemental vitamin D metabolite (no metabolite, 25-OH-D3, or 1-OH-D3) and 2 inclusion levels of microbial phytase (0 or 1,000 units). Sows were housed individually in metabolism crates and feces and urine were collected quantitatively. Results indicated that there was no difference in the ATTD of dry matter (DM) and GE and concentration of DE among the 3 diets containing microbial phytase, but the ATTD of DM and GE and concentration of DE was greater (P < 0.05) in diets containing 1-OH-D3 compared with the diet without a vitamin D metabolite if phytase was not used (interaction; P < 0.05). In diets without microbial phytase, ME was greater in diets containing either one of the 2 vitamin D metabolites than in the diet without a vitamin D metabolite, but among diets with microbial phytase, the ME of the 1-OH-D3 diet was less than of the 25-OH-D3 diet (interaction; P < 0.05). No effect of microbial phytase on concentrations of DE and ME was observed. There was no interaction between supplementation of microbial phytase and vitamin D metabolites for Ca and P balances, and regardless of metabolite supplementation, use of microbial phytase increased (P < 0.05) the ATTD and retention of Ca and P. Regardless of dietary phytase, the ATTD and retention of Ca and P increased (P < 0.05) for sows fed a diet containing one of the vitamin D metabolites compared with sows fed the diet without a vitamin D metabolite. Serum biomarkers for bone resorption or bone tissue synthesis were not affected by experimental diets. In conclusion, the ATTD of DM and GE, concentrations of DE and ME, and Ca and P balance in phytase-free diets fed to sows in late gestation were increased by supplementation with 1-OH-D3 or 25-OH-D3, but no differences between the 2 vitamin D metabolites were observed. Supplementation of diets with microbial phytase increased Ca and P balance, but did not affect DE and ME of diets.

The role of vitamin D is to increase absorption of calcium and phosphorus in the gastrointestinal tract and maintain serum concentrations of calcium, but dietary vitamin D needs to be converted to an active form by 2-hydroxylation steps that take place in the liver and the kidneys. The conversion efficiency to active vitamin D may be increased if pre-hydroxylated metabolites rather than vitamin D are provided, which also increases calcium and phosphorus utilization. In a previous experiment it was also demonstrated that a vitamin D metabolite increases energy absorption in gestating sows. It is possible that use of a vitamin D metabolite and phytase have additive effects and the hypothesis, therefore, was that supplementation of a vitamin D metabolite increases calcium and phosphorus balance and energy digestibility in diets fed to gestating sows without or with microbial phytase. Results indicated that in diets without phytase, the 2 vitamin D metabolites increased energy concentration in diets by increasing apparent energy digestibility. There was no interaction between supplementation of phytase and vitamin D metabolites for calcium and phosphorus balances. Use of phytase and vitamin D metabolites increased calcium and phosphorus digestibility and retention.