Search

This research was to assess the dietary copper (Cu) requirement of broiler chickens fed a practical corn-soybean meal diet during 22–42 d of age. A total of 288 numbered Arbor Acres male broilers at 22 d of age were randomly allotted 6 treatments with 8 replicate cages (6 broilers per cage) per treatment. Broilers were fed a Cu-unsupplemented corn-soybean meal basal diet (control, containing 7.36 mg Cu/kg) or the basal diet added with 3, 6, 9, 12, or 15 mg Cu/kg from CuSO4·5H2O for 21 d. Quadratic, asymptotic and broken-line models were fitted and the best fitted models were selected to determine dietary Cu requirements. The results revealed that the contents of Cu in serum and liver, mRNA expression levels of Cu- and zinc-containing superoxide dismutase (CuZnSOD) in liver and monoamine oxidase b (MAO B) in heart, as well as protein expression level of CuZnSOD in liver were affected (P < 0.05) by supplemental Cu levels, and the above indices varied linearly and quadratically (P < 0.05) with increasing Cu levels. Dietary Cu requirements assessed according to the best fitted broken-line models (P < 0.05) of the above indexes were 10.45–13.81 mg/kg. It was concluded that mRNA expression levels of CuZnSODin liver and MAO Bin heart, as well as liver CuZnSOD protein expression level were new specific sensitive biomarkers for estimating dietary Cu requirements, and the dietary Cu requirement was recommended to be 14 mg/kg to support Cu metabolic needs related to key Cu-containing enzymes in broilers fed the corn-soybean meal diet during 22–42 d of age, which was higher than the dietary Cu requirement (8 mg/kg) for broilers at the corresponding stage suggested by the Chinese Feeding Standard of Chicken.

1,25-dihydroxyvitamin D3[1,25-(OH)2D3] could promote phosphorus (P) absorption in the duodenum of broilers. The vitamin D receptor (VDR) mediates the action of 1,25-(OH)2D3. However, it remains unknown whether and how VDR is involved in promoting P absorption in the duodenum of broilers by 1,25-(OH)2D3. The objective of the current study was to investigate the effect of added 1,25-(OH)2D3levels on P absorption, mRNA or protein expression levels of VDR, retinoid X receptor (RXR), type IIb sodium-phosphate cotransporter (NaPi-IIb), inorganic phosphate transporter (PiT) 1 and PiT-2, and the enrichments of VDR bound to DNA promoters of the above P transporters in the ligated duodenal loops of Arbor Acres male broilers. The duodenal loops were perfused with solutions (pH = 6.0) containing 0 (control), 12.5, or 25.0 ng/L of 1,25-(OH)2D3for up to 40 min. The results indicated that the addition of either 12.5 or 25.0 ng/L 1,25-(OH)2D3to the perfusates increased (P< 0.04) P absorption percentage and rate, as well as the mRNA expression levels of VDR, RXR, NaPi-IIb, and PiT-2, alongside protein expression levels of VDR, NaPi-IIb, and PiT-2, whereas it did not affect (P> 0.10) PiT-1 mRNA and protein expression levels. Moreover, 1,25-(OH)2D3administration increased (P< 0.01) the enrichments of VDR bound to NaPi-IIb and PiT-2 DNA promoter regions in the ligated duodenal loops of broilers, which were in line with their mRNA expression levels. These findings suggest that 1,25-(OH)2D3increased P absorption possibly through VDR-mediated transactivation of NaPi-IIb and PiT-2 genes in the duodenum of Arbor Acres male broilers.

Zinc (Zn) has been shown to attenuate the adverse effects of heat stress on broilers, but the mechanisms involving this process remain unclear. We aimed to investigate possible protective mechanisms of Zn on primary cultured hepatocytes of broiler embryos subjected to heat stress. Three experiments were conducted. In Exp. 1, hepatocytes were treated with 0, 50, 100, 200, or 400 μmol/L added Zn as inorganic Zn sulfate (iZn) for 12, 24 or 48 h. In Exp. 2, cells were exposed to 40 °C (a normal temperature [NT]) and 44 °C (a high temperature [HT]) for 1, 2, 4, 6, or 8 h. In Exp. 3, cells were preincubated with 0 or 50 μmol/L Zn as iZn or organic Zn lysine chelate (oZn) for 8 h under NT, and then incubated with the same Zn treatments under NT or HT for 4 or 6 h. The biomarkers of antioxidative status and heat stress in cells were measured. The results in Exp. 1 indicated that 50 μmol/L Zn and 12 h incubation were the optimal conditions for increasing antioxidant ability of hepatocytes. In Exp. 2, the 4 or 6 h incubation under HT was effective in inducing heat shock responses of hepatocytes. In Exp. 3, HT elevated (P < 0.01) malondialdehyde content and expressions of heat shock protein 70 (HSP70) mRNA and protein, as well as HSP90 mRNA. However, Zn supplementation increased (P < 0.05) copper zinc superoxide dismutase (CuZnSOD) activity and metallothionein mRNA expression, and effectively decreased (P < 0.05) the expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. Furthermore, oZn was more effective (P < 0.05) than iZn in enhancing CuZnSOD activity of hepatocytes under HT. It was concluded that Zn (especially oZn) could alleviate heat stress of broiler hepatocytes via enhancing their antioxidant ability and attenuating heat shock responses.

The gut microbiota play an important role in the growth and intestinal health of broilers. The present study was to investigate the gut microbiota, short-chain fatty acids, and intestinal morphology of broilers at different ages. A total of 320 one-day-old male broilers were raised in 8 replicates and fed the same corn–soybean diets for 42 D. The duodenal, jejunal, and ileal segments and their and cecal microbiota were collected on day 1, 7, 14, 21, and 42, respectively. The villous height (VH), crypt depth (CD), and their ratio of VH:CD in the duodenum, jejunum, and ileum all increased (P < 0.05) with age. Caecal acetate, propionate, butyrate, valerate, and isovalerate increased (P < 0.01), but isobutyrate decreased (P < 0.001) with age. The cecum had the greatest (P < 0.001) alpha diversity of bacterial community in broilers at different ages. Beta diversities showed distinct differences in gut microbial compositions among different ages (R = 0.55, P < 0.002) and different intestinal segments (R = 0.53, P < 0.002). Lactobacilluswas the most abundant genus in the duodenum (36∼97%), jejunum (39∼72%), and ileum (24∼96%) at all ages, and in the ileum, it was positively correlated with VH (R = 0.559, P < 0.03), VH:CD (R = 0.55, P < 0.03), and acetate contents (R = 0.541, P < 0.04) but negatively correlated (R = -0.50, P < 0.05) with isobutyrate contents. Escherichia–Shigellaand Salmonelladominated in the cecum of newly hatched broilers, and then the Bacteroidesdominated in the cecum on day 42. In the cecum, Escherichia–Shigellawas positively correlated (R = 0.577∼0.662, P < 0.05) with isobutyrate contents and Salmonellanegatively correlated (R = -0.539∼-0.843, P < 0.05) with isovalerate, butyrate, and acetate contents. These aforementioned results indicated that the most abundant Lactobacillusfrom the small intestine and the most diversity of microflora community and short-chain fatty acids in the cecum might contribute to the development of intestinal structure in the whole growing period of broilers.

An experiment was conducted to investigate the effect of dietary calcium (Ca) or phosphorus (P) deficiency on bone development and related Ca or P metabolic utilization parameters of broilers. A total of 504 one-day-old Arbor Acres male broilers were randomly assigned to 1 of 4 treatments with 7 replicates of 18 birds per replicate in a completely randomized design. A 2 (Ca levels: 1.00 and 0.35%) × 2 (nonphytate P [NPP] levels: 0.45 and 0.23%) factorial arrangement of treatments was adopted in the 21-day trial. The 4 treatments were the Ca- and P-adequate diet (1.00% Ca + 0.45% NPP), the Ca-deficient diet (0.35% Ca + 0.45% NPP), the P-deficient diet (1.00% Ca + 0.23% NPP), and the Ca- and P-deficient diet (0.35% Ca + 0.23% NPP). The greatest impact on tibia bone mineral density, bone breaking strength, and ash content was in the P-deficient diets, especially in broilers fed with the Ca-adequate diet, whereas adequate P and reduced Ca reduced (P< 0.05) these parameters compared with adequate Ca and P, but not to the same level as P deficiency. Furthermore, dietary Ca or P deficiency, especially adequate Ca and P deficiency decreased (P< 0.05) serum P, 25-hydroxyvitamin D3(25-OHD3) contents, and tibia ash Ca and P contents but increased (P< 0.05) the serum Ca content and tibia alkaline phosphatase (ALP) activity compared with adequate Ca and P. The results from this study indicated that the bone development and Ca or P metabolic utilization parameters of broilers were the most sensitive ones to dietary P deficiency, followed by dietary Ca deficiency or Ca and P deficiencies. Dietary P deficiency impaired the bone development by increasing serum Ca content and tibia ALP activity but decreasing serum P, 25-OHD3contents, and tibia ash Ca and P contents of broilers. Dietary Ca deficiency impaired bone development by increasing serum Ca content, tibia ALP activity, and tibia ash P content but decreasing serum P, 25-OHD3contents, and tibia ash Ca content of broilers.

Selenium (Se), an indispensable trace element in broiler chickens, is closely associated with the growth and development of skeletal muscles. However, the role of Se in the proliferation of broiler myoblasts and its specific biological mechanisms have not been elucidated. In the present study, an in vitro growth model of broiler pectoral myoblasts cultured with Se (Na2SeO3) for 24 h was established. Using light microscopy, Cell Counting Kit-8 (CCK-8) assay, and flow cytometry, we found that compared to the control (Con) group, Se supplemental level obviously promoted myoblast proliferation and prevented cell cycle arrest from the G1 phase to the S + G2 phase. Through intracellular reactive oxygen species (ROS) generation detection, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the study showed that the reduced ROS production caused by Se supplementation significantly decreased PTEN expression and activated the PI3K/AKT signaling pathway in myoblasts, thereby promoting the P53/P21/CyclinD1-regulated cell cycle progression, as well as the expression of proliferation-related myogenic regulatory factors (MRFs). Our findings support the potential of Se to maintain the proliferative capacity of chicken myoblasts and emphasize the importance of Se intake in regulating skeletal muscle growth and development in poultry.

This study aimed to characterize the effects of different dietary forms of supplemental manganese (Mn) on hepatic lipid deposition, gene expression, and enzyme activity in liver fat metabolism in 42-d-old broiler chickens. In total 420 one-day-old Arbor Acres (AA) broilers (rooster:hen = 1:1) were assigned randomly based on body weight and sex to 1 of 6 treatments (10 replicate cages per treatment and 7 broilers per replicate cage) in a completely randomized design using a 2 (sex) × 3 (diet) factorial arrangement. The 3 diets were basal control diets without Mn supplementation and basal diets supplemented with either Mn sulfate or Mn proteinate. No sex × diet interactions were observed in any of the measured indexes; thus, the effect of diet alone was presented in this study. Dietary Mn supplementation increased Mn content in the plasma and liver, adipose triglyceride lipase (ATGL) activity, and ATGLmRNA and its protein expression in the liver by 5.3% to 24.0% (P< 0.05), but reduced plasma triglyceride (TG), total cholesterol, and low-density lipoprotein (LDL-C) levels, liver TG content, fatty acid synthase (FAS) and malic enzyme (ME) activities, mRNA expression of sterol-regulatory element-binding protein 1 (SREBP1), FAS, stearoyl-coA desaturase (SCD), and ME, as well as the protein expression of SREBP1 and SCD in the liver by 5.5% to 22.8% (P< 0.05). No differences were observed between the 2 Mn sources in all of the determined parameters. Therefore, it was concluded that dietary Mn supplementation, regardless of Mn source, decreased hepatic lipid accumulation in broilers by inhibiting SREBP1 and SCD expression, FAS and ME activities, and enhancing ATGL expression and activity.Dietary manganese supplementation reduced hepatic lipid accumulation in broiler chickens by modulating lipid metabolism-related gene expression and enzyme activity in the liver.Dietary manganese supplementation regulates lipid deposition in broiler chickens, with the liver being a significant site of lipid metabolism. This study investigated the effects of different dietary forms of supplemental manganese on hepatic lipid deposition, gene expression, and enzyme activity in the liver fat metabolism of broiler chickens. The results showed that dietary manganese supplementation decreased the hepatic lipid accumulation of broilers by inhibiting the expression of sterol-regulatory element-binding protein 1 (SREBP1) and stearoyl-coA desaturase (SCD), as well as fatty acid synthase (FAS) and malic enzyme (ME) activities, and enhancing the expression and activity of adipose triglyceride lipase (ATGL). This reduction in excessive fat production will help improve poultry health and mitigate losses in the poultry industry.

Recent study showed that zinc (Zn) and amino acid transporters may be involved in enhancing Zn absorption from Zn proteinate with moderate chelation strength (Zn-Prot M) in the duodenum of broilers. However, the specific mechanisms by which Zn-Prot M promotes the above Zn absorption are unknown. Therefore, in this study, 3 experiments were conducted to investigate specific and direct effects of Zn-Prot M and Zn sulfate (ZnS) on Zn absorption and expression of related transporters in primary duodenal epithelial cells of broiler embryos so as to preliminarily address possible mechanisms. In experiment 1, cells were treated with 100 μmol Zn/L as ZnS or Zn-Prot M for 20, 40, 60, 80, 100, or 120 min. Experiment 2 consisted of 3 sub-experiments. In experiment 2A, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 100 or 200 μmol Zn/L as ZnS or Zn-Prot M for 60 min; in experiment 2B, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 200 μmol Zn/L of as the ZnS or Zn-Prot M for 120 min; in experiment 2C, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 or 800 μmol Zn/L as ZnS or Zn-Prot M for 120 min. In experiment 3, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 μmol Zn/L as ZnS or Zn-Prot M for 120 min. The results of experiment 1 indicated that the minimum incubation time for saturable Zn absorption was determined to be 50.83 min using the best fit line. The results in experiment 2 demonstrated that a Zn concentration of 400 μmol/L and an incubation time of 120 min were suitable to increase the absorption of Zn from Zn-Prot M compared to ZnS. In experiment 3, Zn absorption across cell monolayers was significantly increased by Zn addition (P < 0.05), and was significantly greater with Zn-Prot M than with ZnS (P < 0.05). Compared to the control, Zn addition significantly decreased Zn transporter 10 and peptide-transporter 1 mRNA expression levels and increased y + L-type amino transporter 2 (y + LAT2) protein abundance (P < 0.05). Moreover, protein expression levels of zrt/irt-like protein 3 (ZIP3), zrt–irt-like protein 5 (ZIP5), and y + LAT2 were significantly greater for Zn-Prot M than for ZnS (P < 0.05). These findings suggest that Zn-Prot M promote Zn absorption by increasing ZIP3, ZIP5 and y + LAT2 protein expression levels in primary duodenal epithelial cells.Zinc proteinate with moderate chelation strength increased the zinc absorption in primary duodenal epithelial cells of broiler embryos by promoting the protein expression levels of ZIP3, ZIP5, and y + LAT2 compared to zinc sulfate.Our previous studies demonstrated that zinc (Zn) proteinate with moderate chelation strength (Zn-Prot M) exhibited the greatest bioavailability compared to the inorganic Zn sulfate (ZnS) and other organic Zn sources with either weak or strong chelation strength in broilers. Our recent study further showed that Zn and amino acid transporters could be potentially involved in promoting the absorption of Zn as Zn-Prot M in the duodenum of broilers. Nevertheless, further in vitro experiments are required to reveal the specific mechanisms by which Zn-Prot M promotes Zn absorption, where it is necessary first to investigate the specific and direct effect of Zn-Prot M on Zn absorption and the expression of Zn and amino acid transporters compared to that of ZnS. Therefore, we performed an in vitro study and found that Zn-Prot M increased Zn absorption and protein expression levels of the zrt–irt-like protein 3 (ZIP3), zrt–irt-like protein 5 (ZIP5), and y + L-type amino transporter 2 (y + LAT2) compared to ZnS, suggesting that ZIP3, ZIP5, and y + LAT2 might be involved in promoting the absorption of Zn from Zn-Prot M in the primary cultured duodenal epithelial cells of broiler embryos.

As one of the indispensable trace elements for both humans and animals, selenium widely participates in multiple physiological processes and facilitates strong anti-inflammatory, antioxidant, and immune enhancing abilities. The biological functions of selenium are primarily driven by its presence in selenoproteins as a form of selenocysteine. Broilers are highly sensitive to selenium intake. Recent reports have demonstrated that selenium deficiency can adversely affect the quality of skeletal muscles and the economic value of broilers; the regulatory roles of several key selenoproteins (e.g., GPX1, GPX4, TXNRD1, TXNRD3, SelK, SelT, and SelW) have been identified. Starting from the selenium metabolism and its biological utilization in the skeletal muscle, the effect of the selenium antioxidant function on broiler meat quality is discussed in detail. The progress of research into the prevention of skeletal muscle injury by selenium and selenoproteins is also summarized. The findings emphasize the necessity of in vivoand in vitroresearch, and certain mechanism problems are identified, which aids their further examination. This mini-review will be helpful to provide a theoretical basis for the further study of regulatory mechanisms of selenium nutrition in edible poultry.

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1(ZO-1), PI3K, AKTand mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3Kand AKTmRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 μmol Zn/L was more effective than 100 μmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKTand PKCmRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 μmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1and mTORmRNA levels. Therefore, supplemental Zn, especially 50 μmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Excessive corticosterone (CORT) exposure could cause hepatic cholesterol accumulation in chickens and maternal betaine supplementation could decrease hepatic cholesterol deposition through epigenetic modifications in offspring chickens. Nevertheless, it remains uncertain whether providing betaine to laying hens could protect CORT-induced hepatic cholesterol accumulation via epigenetic mechanisms. This study aimed to examine the effects of dietary betaine on plasma and hepatic cholesterol contents, expression of cholesterol metabolic genes, as well as DNA methylation on their promoters in the liver of laying hens exposed to CORT. A total of 72 laying hens at 130 d of age were randomly divided into three groups: control (CON), CORT and CORT+betaine (CORT+BET) groups. The experiment lasted for 35 d. Chickens in CON and CORT groups were fed a basal diet, while the CORT+BET group chickens were fed the basal diet supplemented with 0.1% betaine for 35 d. On d 28 of the experiment, chickens in CORT and CORT+BET groups received daily subcutaneous injections of CORT (4.0 mg/kg body weight), while the CON group chickens were injected with an equal volume of solvent for 7 d. The results showed that CORT administration led to a significant increase (P<0.05) in the contents of cholesterol in plasma and liver, associated with activation (P<0.05) of sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), lecithin-cholesterol acyltransferase (LCAT) and low density lipoprotein receptor (LDLR) genes expression, and inhibition of cholesterol-7-alpha hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) genes expression in the liver compared to the CON. In contrast, CORT-induced up-regulation of HMGCR mRNA and protein abundances and down-regulation of CYP7A1 mRNA and protein abundances were completely normalized (P<0.05) by betaine supplementation. Besides, CORT injection led to significant hypomethylation (P<0.05) on HMGCR promoter and hypermethylation (P<0.05) on CYP7A1 promoter. Moreover, dietary betaine rescued (P<0.05) CORT-induced changes in methylation status of HMGCR and CYP7A1 genes promoters. These results indicate that dietary betaine addition protects laying hens from CORT-induced hepatic cholesterol accumulation via epigenetic modulation of HMGCR and CYP7A1 genes.

Two experiments were conducted to estimate standardized mineral (Ca, P, Cu, Fe, Mn, and Zn) availabilities (SMA) in commonly used feedstuffs and verify the additivity of SMA for diet formulation of broilers. In Exp. 1, after 3 d of acclimation, a total of 96 22-d-old Arbor Acres male broilers were fasted for 24 h. Then the broilers were fed a mineral-free, corn, soybean meal (SBM), or corn-soybean meal (C-SBM) diet for 4 h and the excreta samples were collected for 48 h after feed withdrawal (totally 52 h). The results showed that the endogenous losses of Ca, P, Cu, Fe, Mn, and Zn for chickens fed the mineral-free diet were 13.7, 113, 0.064, 0.593, 0.094, and 0.132 mg/52 h per bird, respectively. The standardized availability values of Ca, P, Cu, Fe, Mn, and Zn were 20.8%, 39.6%, 37.8%, 47.4%, 10.7%, and 45.2% in corn as well as 54.3%, 51.1%, 39.7%, 27.6%, 26.1%, and 51.0% in SBM, respectively. The determined values of SMA in C-SBM diet were close (P > 0.09) to the predicted summations of SMA from corn and SBM (Ca, 48.6% vs. 48.1%; P, 44.9% vs. 46.0%; Cu, 41.7% vs. 39.1%; Fe, 35.3% vs. 33.2%; Mn, 22.7% vs. 22.2%; Zn, 46.2% vs. 48.4%). In Exp. 2, a total of 144 22-d-old Arbor Acres male broilers were used to estimate the SMA values in 6 feedstuffs by using the above procedure. The results showed that the standardized availability values in wheat, wheat bran, corn distillers dried grains with solubles, cottonseed meal, rapeseed meal, and corn gluten meal diets were 24.7% to 55.8% for Ca, 35.6% to 46.6% for P, 24.5% to 45.7% for Cu, 21.6% to 43.5% for Fe, 9.3% to 34.5% for Mn, and 22.9% to 52.9% for Zn, respectively. The results from the present study indicated that the mineral-free diet could be used for estimating the endogenous losses of the above minerals and SMA values of feedstuffs for broilers, and the estimates of SMA in feedstuffs for the C-SBM diet formulation were additive.

The objective of this study was to determine the effect of organic and inorganic Fe sources on the Fe absorption and gene expression of Fe and amino acid transporters in the ligated jejunal and ileal segments of broilers. The in situ ligated jejunal and ileal loops from Fe-deficient broiler chicks (28-d-old) were perfused with Fe solutions containing 0, 3.58, or 7.16 mM Fe from one of the following Fe sources: Fe sulfate (FeSO4∙7H2O), the mixtures of FeSO4∙7H2O with either Met or Gly, Fe-Gly chelate, or three Fe-amino acid or protein chelates with weak, moderate or extremely strong chelation strengths (Fe-Met W, Fe-Pro M, or Fe-Pro ES), respectively, for up to 30 min. Iron absorption was increased (P< 0.0001) as the perfused Fe concentrations increased, and no differences (P> 0.07) were detected in the Fe absorption between the jejunum and ileum. Regardless of intestinal segments, Fe absorption was higher (P< 0.006) for Fe-Pro ES and Fe-Pro M than for FeSO4·7H2O, and for Fe-Pro ES than for Fe-Met W. Glycine but not Met supplementation increased (P< 0.03) the absorption of Fe as FeSO4. Regardless of Fe source, Fe addition inhibited (P< 0.05) the mRNA expressions of divalent metal transporter 1 (DMT1) in the jejunum and ileum, but enhanced (P< 0.05) the mRNA expressions of l-type amino transporter 1 (LAT1) and B0-type amino acid transporter 1 (B0AT1) in the jejunum and ileum. No differences (P> 0.05) among different Fe sources were observed in the mRNA expression levels of Fe and amino acid transporters in both the jejunum and the ileum. The mRNA expression levels of DMT1, ferroportin 1, B0AT1, or y+LAT1 were higher (P< 0.0001), but those of excitatory amino acid transporter 3, LAT1, or y+l-type amino transporter 2 were lower (P< 0.04) in the jejunum than in the ileum. The supplementation of inorganic or organic Fe had no effect (P> 0.14) on the protein expression levels of DMT1 and FPN1 in the jejunum and ileum. The above results indicate that organic Fe sources with stronger chelation strengths showed higher Fe absorption in the jejunum and ileum of broiler chicks. Glycine was more effective in facilitating Fe absorption than Met as a ligand. The mRNA expressions of Fe and amino acid transporters in the jejunum were different from those in the ileum. The DMT1, LAT1, and B0AT1 might be involved in the Fe absorption in the jejunum or ileum of broilers.

The particle size of selenium (Se) sources could affect Se absorption and utilization, and thus it is hypothesized that the Se bioavailability might be higher in ultrafine sodium selenite (USSe) than in sodium selenite (SSe) for broilers because of USSe’s smaller particle size. An experiment was conducted to investigate the relative bioavailability of Se as USSe relative to SSe for broiler chicks fed a conventional corn–soybean meal diet. A total of 504 one-d-old Arbor Acres commercial male broilers were randomly allotted to 1 of 7 treatments with 6 replicates per treatment in a completely randomized design involving in a 2 (Se sources) × 3 (added Se levels) factorial arrangement of treatments plus a Se-unsupplemented control diet containing 0.05 mg Se/kg by analysis for 21 d. The 2 Se sources were USSe and SSe, and the 3 added Se levels were 0.15, 0.30, or 0.45 mg Se/kg. The Se concentrations, glutathione peroxidase (GSH-Px) activities, and mRNA relative abundances in plasma, liver, or pancreas of broilers on day 14 and 21 were determined. The results showed that Se concentrations, GSH-Px activities in plasma, liver, and pancreas, and mRNA relative abundances in the liver and pancreas of broilers on day 14 and 21 increased linearly (P< 0.05) as the added Se-level increased. Furthermore, a difference (P< 0.05) between USSe and SSe was detected for GSH-PxmRNA relative abundance in the pancreas of broilers on day 14. On the basis of the slope ratios from the multiple linear regression of the pancreatic GSH-PxmRNA relative abundance of broilers at 14 d of age on daily dietary analyzed Se intake, the Se bioavailability of USSe relative to SSe (100%) was 158% (P< 0.05). The results from this study indicated that the Se from USSe was more available to broilers than the Se from SSe in enhancing the pancreatic GSH-PxmRNA expression.

Our previous study demonstrated that the zinc (Zn) proteinate with moderate chelation strength (Zn-Prot M) enhanced the Zn absorption in the small intestine partially via increasing the expression of some Zn and amino acid transporters in the duodenum of broilers. However, it remains unknown whether the Zn-Prot M could also regulate the expression of related transporters in the jejunum and ileum of broilers in the above enhancement of Zn absorption. The present study was conducted to investigate the effect of the Zn-Prot M on the expression of related transporters in the jejunum and ileum of broilers compared to the Zn sulfate (ZnS). Zinc-deficient broilers (13-d-old) were fed with the Zn-unsupplemented basal diets (control) or the basal diets supplemented with 60 mg Zn/kg as ZnS or Zn-Prot M for 26 d. The results showed that in the jejunum, compared to the control, supplementation of the organic or inorganic Zn increased (P< 0.05) mRNA and protein expression of b0,+-type amino acid transporter (rBAT), Zn transporter 10 (ZnT10), and peptide-transporter 1 (PepT1) mRNA expression and Zn transporter 7 (ZnT7) protein expression on d 28, while y+L-type amino transporter 2 (y+LAT2) mRNA and protein expression, and protein expression of ZnT7 and ZnT10 on 28 d and zrt-irt-like protein 3 (ZIP3) and zrt-irt-like protein 5 (ZIP5) on d 39 were higher (P< 0.05) for Zn-Prot M than for ZnS. In the ileum, Zn addition regardless of Zn source up-regulated (P< 0.05) mRNA expression of Zn transporter 9 (ZnT9) and ZIP3, ZIP5, and y+LAT2 protein expression on d 28, and PepT1 mRNA and protein expression, ZIP3 and y+LAT2 mRNA expression and ZnT10 protein expression on d 39. Furthermore, Zn transporter 4 (ZnT4) and ZnT9 mRNA expression and Zn transporter 1 (ZnT1) protein expression on d 28, and y+LAT2 mRNA expression and ZnT10 and PepT1 protein expression on d 39 were higher (P< 0.05) for Zn-Prot M than for ZnS. It was concluded that the Zn-Prot M enhanced the expression of the ZnT1, ZnT4, ZnT9, ZnT10, ZIP3, ZIP5, y+LAT2, and PepT1 in the jejunum or ileum of broilers compared to the ZnS.

Dietary trace minerals can impact gut flora, which can further affect intestinal health. However, the dietary balance pattern of trace minerals for the intestinal health of broilers needs to be explored. The present study was conducted to investigate the effect of the dietary pattern of Cu, Fe, Mn, Zn, and Se on the intestinal morphology, microbiota, short-chain fatty acid concentrations, antioxidant status, and the expression of tight junction proteins in broilers. A total of 240 1-d-old Arbor Acres male broilers were randomly assigned to one of five treatments with six replicate cages of eight birds per cage for each treatment. The birds were fed the corn-soybean meal basal diet supplemented with five combination patterns of trace minerals for 42 d. The dietary treatments were as follows: the inorganic sources were added to the diet based on the recommendations of the current National Research Council (NRC, T1) and Ministry of Agriculture of P.R. China (MAP) (T2) for broiler chicks, respectively; the inorganic sources were added to the diet at the levels based on our previous results of inorganic trace mineral requirements for broilers (T3); the organic sources were added to the diet at the levels considering the bioavailabilities of organic trace minerals for broilers described in our previous studies (T4); and the organic sources were added to the diet based on the recommendations of the current MAP for broiler chicks (T5). The results showed that broilers from T1 had lower (P < 0.05) crypt depth (CD), and a higher (P < 0.05) villus height: CD in duodenum on day 21 and lower CD (P < 0.05) in jejunum on day 42 than those from T3 and T4. Broilers from T1, T3, and T5 had a higher (P < 0.05) Shannon index in cecum on day 21 than those from T4. Broilers from T1 had a higher (P < 0.05) abundance of Lactobacillusin ileum on day 21 than those from T2 and T3. Broilers from T1, T2, and T5 had a higher (P< 0.05) valeric acid concentrations in cecum on day 42 than those from T3 and T4. In addition, Birds from T2 had higher (P < 0.05) Claudin-1mRNA levels in jejunum on day 42 than those from T3 and T4. And birds from T3, T4, and T5 had a higher (P < 0.05) Occludin protein expression levels in duodenum on day 42 than those from T2. These results indicate that dietary pattern of Cu, Fe, Mn, Zn, and Se influenced gut flora and intestinal health of broilers, and the appropriate pattern of Cu, Fe, Mn, Zn, and Se in the diet for intestinal health of broilers would be Cu 12 mg, Fe 229 mg, Mn 81 mg, Zn 78 mg, and Se 0.24 mg/kg (1 to 21 d of age), and Cu 11 mg, Fe 193 mg, Mn 80 mg, Zn 73 mg, and Se 0.22 mg/kg (22 to 42 d of age), when the trace minerals as inorganic sources were added to diets according to the recommendations of the current NRC.The results from the present study demonstrated that the dietary appropriate pattern of Cu, Fe, Mn, Zn, and Se for intestinal health of broilers would be Cu 12 mg, Fe 229 mg, Mn 81 mg, Zn 78 mg, and Se 0.24 mg/kg (1 to 21 d of age), and Cu 11 mg, Fe 193 mg, Mn 80 mg, Zn 73 mg, and Se 0.22 mg/kg (22 to 42 d of age), when the trace minerals as inorganic sources were added to diets according to the recommendations of the current National Research Council.Information is still scarce regarding the effect of dietary trace mineral patterns on the intestinal health of broilers. The results indicated that dietary trace mineral pattern influenced intestinal health of broilers, and the appropriate pattern of trace minerals in the diet for intestinal health of broilers would be Cu 12 mg, Fe 229 mg, Mn 81 mg, Zn 78 mg, and Se 0.24 mg/kg (1 to 21 d of age), and Cu 11 mg, Fe 193 mg, Mn 80 mg, Zn 73 mg, and Se 0.22 mg/kg (22 to 42 d of age), when the trace minerals as inorganic sources were added to diets according to the recommendations of the current National Research Council. Our results provided scientific experimental bases for improving intestinal health of broilers by nutritional strategy.

Two experiments were carried out to study the effect of in ovo manganese (Mn) injection on the embryonic development, antioxidation, hatchability, and performances of offspring broilers under normal temperature (NT) and high temperature (HT). Experiment 1 was conducted to investigate the effect of in ovo Mn injection on the embryonic hatchability of Arbor Acres broiler breeders. On D 9 of incubation, a total of 684 fertilized eggs were randomly allocated to 6 treatments: the non-injected positive control (niPC) and treatments injected with 0 (the negative control, iNC), 6.25, 12.5, 25.0, or 50.0 μg Mn/egg as Mn sulfate. Experiment 2 was conducted to investigate the effect of in ovo Mn injection on the embryonic development, antioxidation and performance of offspring broilers under NT and HT. A total of 792 fertilized eggs were randomly allocated to 6 treatments in a 1 (niPC) + 1 (iNC) + 2 (injected Mn sources: Mn sulfate and Mn proteinate) × 2 (injected Mn levels: 12.5 and 25.0 μg/egg) factorial arrangement during the embryonic stage and D1 to 28 at NT. Then, 288 birds were allotted to 12 treatments in a 6 (the above embryonic treatments) × 2 (environmental temperatures: NT-22℃ vs HT-34℃) factorial arrangement from D 29 to 42. The results showed that Mn injection affected (P <0.03) the hatchability and the maximum level of in ovo injected Mn was 25.0 μg Mn/egg. The Mn injection upregulated (P <0.05) Mn-containing superoxide dismutasemRNA expression in the embryonic heart compared to the iNC. Hyperthermia decreased (P <0.05) ADG and ADFI, breast muscle percentage, plsma alkaline phosphatase activity, and red color values of breast and thigh muscles, but increased (P <0.05) F/G, plasma aspartate aminotransferase and lactic dehydrogenase activities, total cholesterol, uric acid and triiodothyronine contents, abdominal fat, light values of breast and thigh muscles of offspring broilers. The results suggest that in ovo Mn injection can enhance antioxidant ability in the chick embryonic heart.

The current dietary Ca recommendation of broilers is primarily based on the previous studies carried out more than 30 yr ago. However, the modern commercial broilers are quite different from those more than 30 yr ago. The present experiment was conducted to evaluate an optimal dietary Ca level by bone characteristics and Ca metabolism-related gene expression of broilers fed a corn-soybean meal diet from 22 to 42 d of age. A total of 252 22-d-old Arbor Acres male broilers were randomly assigned to 1 of 7 treatments with 6 replicate cages of 6 birds per cage for each treatment. Broilers were fed the corn-soybean meal diets containing 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, or 1.10% Ca for 21 d, and each diet contained 0.31% non-phytate P. The results showed that the mineral contents in tibia and middle toe bone, mineral density in tibia and middle toe bone, middle toe ash percentage, middle toe ash Ca percentage, and tibia alkaline phosphatase mRNA expression level of broilers were influenced (P< 0.04) by dietary Ca level and increased quadratically (P< 0.05) as dietary Ca level increased. The estimates of optimal dietary Ca levels were 0.55%, 0.60%, 0.70%, 0.72%, 0.63%, 0.66%, and 0.70%, respectively, based on the best fitted broken-line, quadratic, or asymptotic models (P< 0.02) of the above sensitive indices. These results indicate that the optimal dietary Ca level would be 0.72% to support all of the Ca metabolism and bone development of broilers fed the corn-soybean meal diet from 22 to 42 d of age.The present experiment was conducted to evaluate an optimal dietary Ca level by bone characteristics and Ca metabolism-related gene expression of broilers fed a corn-soybean meal diet from 22 to 42 d of age. A total of 252 22-d-old Arbor Acres male broilers were randomly assigned to 1 of 7 treatments with 6 replicate cages of 6 birds per cage for each treatment. Broilers were fed the corn-soybean meal diets containing 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, or 1.10% Ca for 21 d, and each diet contained 0.31% non-phytate P. The results showed that the tibia and middle toe bone mineral contents, tibia and middle toe bone mineral density, middle toe ash percentage, middle toe ash Ca percentage, and tibia alkaline phosphatase mRNA expression level were sensitive criteria to estimate the optimal dietary Ca levels of broilers. The estimates of optimal dietary Ca levels were 0.55% to 0.72% based on the above sensitive criteria. These results indicate that the optimal dietary Ca level would be 0.72% to support all of the Ca metabolism and bone development of broilers fed the corn-soybean meal diet from 22 to 42 d of age.The findings from the present study are of great significance to ensure the healthy growth of broilers and reduce the interference of Ca on the palatability to broiler diets and absorption and utilization of other nutrients, as well as save Ca resources and reduce feed costs.

The present study was carried out to evaluate the effects of iron (Fe) sources and levels on the Fe concentration and expressions of iron-containing enzymes or protein in primary cultured hepatocytes of broiler embryos. The hepatocytes were incubated with 0, 0.25 and 0.50 mmol/L added Fe from either Fe sulfate, or 1 of 3 organic Fe chelates with weak (Fe-Met W), moderate (Fe-Pro M), or extremely strong (Fe-Pro ES) chelation strengths for 24 h. The results showed that all supplemental Fe treatments had higher (P< 0.05) Fe concentration, succinate dehydrogenase (SDH), CATand ferritin heavy chain 1 (FTH1) mRNA levels than those in the control group. The hepatocytes incubated with Fe-Prot ES had lower (P< 0.009) Fe concentration than those incubated with Fe sulfate, Fe-Met W or Fe-Prot M. The SDHmRNA level was lower (P< 0.05) in Fe sulfate and Fe-Prot ES groups than in Fe-Prot M group. In conclusion, the Fe from Fe-Prot ES was less utilizable than Fe from Fe sulfate, Fe-Met W or Fe-Pro M in primary cultured hepatocytes of broiler embryos.

An experiment was conducted to investigate the effect of organic and inorganic Fe sources on Fe absorption and expression of related transporters in the small intestine of broilers. Iron-deficient intact broilers (7-day-old) were fed an Fe-unsupplemented corn-soybean meal basal diet or the basal diet supplemented with 60 mg Fe/kg as Fe sulfate (FeSO4•7H2O), Fe-Met with weak chelation strength (Fe-Met W), Fe-proteinate with moderate chelation strength (Fe-Prot M) or Fe-proteinate with extremely strong chelation strength (Fe-Prot ES) for 14 d. The plasma Fe contents were enhanced (P< 0.02) by Fe addition, and greater (P< 0.0002) in Fe-Prot M and Fe-Prot ES groups than in Fe-Met W and FeSO4groups. Supplemental Fe decreased (P< 0.03) the divalent metal transporter 1 (DMT1) mRNA levels in the duodenum and jejunum, and ferroportin 1 (FPN1) mRNA levels in the duodenum on d 21, but no differences (P> 0.20) were detected among different Fe sources. Regardless of Fe source, the mRNA levels of DMT1and FPN1were higher (P< 0.02) in the duodenum than in the jejunum and ileum, and in the jejunum than in the ileum (P< 0.05). However, Fe addition did not affect (P> 0.10) the mRNA levels of amino acid transporters and protein levels of DMT1 and FPN1 in the small intestine of broilers. These results indicate that organic Fe sources with stronger chelation strength showed higher Fe absorption in broilers in vivo; the mRNA expression of Fe and amino acid transporters varied along with the extension of the small intestine; the absorption of Fe as organic Fe chelates was not mediated by the amino acid transporters in intact chicks in this study.

The current dietary copper (Cu) requirement (8 mg/kg) of broilers is mainly based on growth, hemoglobin concentration, or hematocrit, which might not be the most sensitive indices to evaluate dietary Cu requirements of broilers. The present study was carried out to estimate dietary Cu requirements of broilers fed a conventional corn-soybean meal diet from 1 to 21 d of age using biochemical or molecular biomarkers. A total of 384 one-d-old Arbor Acres male broilers were randomly allocated to 1 of 6 treatments with 8 replicates and fed a Cu-unsupplemented corn-soybean meal basal diet containing 5.17 mg Cu/kg by analysis and the basal diet supplemented with 3, 6, 9, 12 or 15 mg Cu/kg as CuSO4⋅5H2O for 21 d. Regression analysis was performed to estimate the optimal dietary Cu level using the broken-line model. Dietary supplemental Cu level affected (P< 0.05) Cu contents in serum and liver and kidney monoamine oxidase (MAO) activity, but had no effect (P> 0.05) on the growth performance, Cu contents in heart, kidney, pancreas and spleen, Cu- and zinc-containing superoxide dismutase (CuZnSOD) activity and ceruloplasmin content in serum, CuZnSOD and cytochrome c oxidase (COX) activities and ceruloplasmin, CuZnSOD, MAO A, MAO B, COX4I1 and COX1 mRNA and protein expressions in the above tissues of broilers. As dietary supplemental Cu levels increased, Cu contents in serum and liver increased linearly (P< 0.05), but kidney MAO activity decreased linearly and quadratically (P< 0.05). The estimated dietary Cu requirement based on the fitted broken-line model (P= 0.035) of kidney MAO activity was 11.30 mg/kg. In conclusion, kidney MAO activity is a new and sensitive criterion to evaluate the dietary Cu requirement of broilers, and the dietary Cu requirement was 11.30 mg/kg for broilers fed the conventional corn-soybean meal diet from 1 to 21 d of age, which is higher than the current National Research Council (NRC) Cu requirement (8 mg/kg) of broilers.

The current NRC dietary selenium (Se) requirement (0.15 mg/kg) of broilers is primarily based on growth performance data reported in 1986. Our study aimed to determine optimal dietary Se levels of broilers fed a practical corn-soybean meal diet for the full expression of selenoproteins in various tissues. A total of 384 one-d-old male broilers (n= 8 replicates/diet) were fed a basal corn-soybean meal diet or the basal diet supplemented with 0.1, 0.2, 0.3, 0.4 or 0.5 mg Se/kg in the form of Na2SeO3for 21 d. Regression analysis was conducted to evaluate the optimal dietary Se levels using broken-line, quadratic or asymptotic models. The activity of glutathione peroxidase (GPX) in the plasma, liver, kidney and pancreas, iodothyronine deiodinase (DIO) in the plasma, liver and pancreas, and thioredoxin reductase (Txnrd) in the liver and pancreas, the mRNA levels of Gpx1, Gpx4, Dio1, selenoprotein(Seleno) h, Selenopand Selenouin the liver, Gpx4, Dio1, Txnrd1, Txnrd2, Selenoh, Selenopand Selenouin the kidney, and Gpx1, Gpx4, Selenohand Selenouin the pancreas, and the protein levels of GPX4 in the liver and kidney of broilers were influenced (P< 0.05) by added Se levels, and increased quadratically (P< 0.05) with the increase of added Se levels. The estimates of optimal dietary Se levels were 0.07 to 0.36 mg/kg based on the fitted broken-line, quadratic or asymptotic models (P< 0.001) of the aforementioned selenoprotein expression in the plasma, liver and kidney, and 0.09 to 0.46 mg/kg based on the fitted broken-line models (P< 0.001) of the aforementioned selenoprotein expression in the pancreas. The results indicate that the optimal dietary Se levels would be 0.36 mg/kg to support the full expression of selenoproteins in the plasma, liver and kidney, and 0.46 mg/kg to support the full expression of selenoproteins in the pancreas of broilers fed a practical corn-soybean meal diet from 1 to 21 d of age.

No information is available regarding the utilization of iron (Fe) from different Fe sources at a target tissue level. To detect differences in Fe metabolic utilization among Fe sources, the effect of intravenously injected Fe on growth performance, hematological indices, tissue Fe concentrations and Fe-containing enzyme activities and gene expressions of Fe-containing enzymes or protein in broilers was investigated. On d 22 post-hatching, a total of 432 male chickens were randomly allotted to 1 of 9 treatments in a completely randomized design. Chickens were injected with either a 0.9% (wt/vol) NaCl solution (control) or a 0.9% NaCl solution supplemented with Fe sulphate or 1 of 3 organic Fe sources. The 3 organic Fe sources were Fe chelates with weak (Fe-MetW), moderate (Fe-ProM) or extremely strong (Fe-ProES) chelation strengths. The 2 Fe dosages were calculated according to the Fe absorbabilities of 10% and 20% every 2 d for a duration of 20 d. Iron injection did not affect (P> 0.05) ADFI, ADG or FCR during either 1 to 10 d or 11 to 20 d after injections. Hematocrit and Fe concentrations in the liver and kidney on d 10 after Fe injections, and Fe concentrations in the liver or pancreas and ferritin heavy-chain (FTH1) protein expression level in the liver or spleen on d 20 after Fe injections increased (P≤ 0.05) as injected Fe dosages increased. When the injected Fe level was high at 20% Fe absorbability, the chickens injected with Fe-ProtES had lower (P< 0.001) kidney Fe concentrations and spleen FTH1 protein levels than those injected with Fe-MetW on d 20 after injections. And they had lower (P< 0.02) liver cytochrome c oxidase mRNA levels on d 20 after injections than those injected with Fe-Prot W or Fe sulphate. The results from this study indicate that intravenously injected Fe from Fe-ProES was the least utilizable and functioned in the sensitive target tissue less effectively than Fe from Fe sulfate, Fe-MetW or Fe-ProM.

The current NRC dietary selenium (Se) requirement (0.15 mg/kg) of broilers from 22 to 42 d of age is primarily based on a previous study reported in 1986, which might not be applicable to modern classes of rapidly growing broilers. The present experiment was conducted to determine the optimal dietary Se level for meeting metabolic and functional Se requirements of broilers fed a corn-soybean meal diet from 22 to 42 d of age. A total of 336 twenty two-d-old Arbor Acres male broilers were randomly assigned to 1 of 6 treatments with 7 replicates and fed a basal corn-soybean meal diet (control, containing 0.014 mg Se/kg) and the basal diet supplemented with 0.10, 0.20, 0.30, 0.40, or 0.50 mg Se/kg from Na2SeO3for 21 d. The results showed that the Se concentrations in plasma, liver, kidney, pancreas, breast and thigh muscles, the activity of glutathione peroxidase (GPX) in plasma, liver and kidney, the mRNA expression levels of Gpx4, selenoprotein (Seleno) hand Selenouin liver, selenopand Selenohin kidney, and the protein expression levels of GPX4 in the liver and kidney of broilers were affected (P< 0.05) by supplemental Se level, and increased quadratically (P< 0.05) with the increase of supplemental Se level. The estimates of optimal dietary Se levels were 0.10 to 0.49 mg/kg based on the fitted broken-line or asymptotic models (P< 0.0001) of the above Se concentration indices, and 0.08 to 0.37 mg/kg based on the fitted broken-line, quadratic or asymptotic models (P< 0.007) of the above selenoprotein expression indices. These results indicate that the optimal dietary Se levels would be 0.49 mg/kg to support the maximum Se concentrations and 0.37 mg/kg to support the full expression of selenoproteins in plasma and various tissues of broilers fed a corn-soybean meal diet from 22 to 42 d of age.