Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways.

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, AKT and 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, PI3K, and AKT mRNA 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, AKT, and PKC mRNA 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-1, and mTOR mRNA 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.

Dietary Zn Deficiency Inhibits Cell Proliferation via the GPR39-Mediated Suppression of the PI3K/AKT/mTOR Signaling Pathway in the Jejunum of Broilers.

A prior investigation revealed that a lack of Zinc (Zn) could hinder intestinal cell proliferation in broiler chickens; however, the mechanisms responsible for this effect remain unclear. We aimed to investigate the possible mechanisms of dietary Zn deficiency in inhibiting the jejunal cell proliferation of broilers. For this study, a total of 112 chickens (21 days old) were randomly divided into two treatments (seven replicate cages per treatment, eight chickens per replicate cage): the control group (CON) and the Zn deficiency group. The duration of feeding was 21 d. Chickens in the control group were provided with a basal diet containing an extra addition of 40 mg Zn/kg in the form of Zn sulfate, whereas chickens in the Zn deficiency group were given the basal diet with no Zn supplementation. The results indicated that, in comparison to the CON, Zn deficiency increased (p < 0.05) the duodenal and jejunal crypt depth (CD) of broilers on d 28 and jejunal and ileal CD on d 35, and decreased (p < 0.05) the duodenal, jejunal, and ileal villus height/crypt depth (VH/CD) on d 28 and the jejunal VH, jejunal and ileal villus surface area, and VH/CD on d 35. Furthermore, Zn deficiency decreased (p < 0.0001) the number of proliferating cell nuclear antigen-positive cells and downregulated (p < 0.01) the mRNA or protein expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, phosphorylated serine-threonine kinase (AKT), phosphorylated mechanistic target of rapamycin (mTOR), G protein-coupled receptor 39 (GPR39), and extracellular-regulated protein kinase, but upregulated (p < 0.05) the mRNA or protein expression levels of P38 mitogen-activated protein kinase, c-jun N-terminal kinase (JNK) 1 and JNK2, and phosphorylated protein kinase C in the jejunum of the broilers on d 42. It was concluded that dietary Zn deficiency inhibited cell proliferation possibly via the GPR39-mediated suppression of the PI3K/AKT/mTOR signaling pathway in the jejunum of broilers.

Research progress on the biological regulatory mechanisms of selenium on skeletal muscle in broilers.

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 vivo and in vitro research, 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.

Dietary copper requirement of broilers fed a corn-soybean meal diet during 22-42 d of age.

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 CuZnSOD in liver and MAO B in 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.

Betaine supplementation alleviates corticosterone-induced hepatic cholesterol accumulation through epigenetic modulation of HMGCR and CYP7A1 genes in laying hens.

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 3 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, whereas 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), whereas 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 downregulation 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.

The chemical characteristics of different sodium iron ethylenediaminetetraacetate sources and their relative bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

BACKGROUND: Our previous studies demonstrated that divalent organic iron (Fe) proteinate sources with higher complexation or chelation strengths as expressed by the greater quotient of formation (Qf) values displayed higher Fe bioavailabilities for broilers. Sodium iron ethylenediaminetetraacetate (NaFeEDTA) is a trivalent organic Fe source with the strongest chelating ligand EDTA. However, the bioavailability of Fe when administered as NaFeEDTA in broilers and other agricultural animals remains untested. Herein, the chemical characteristics of 12 NaFeEDTA products were determined. Of these, one feed grade NaFeEDTA (Qf = 2.07 × 108), one food grade NaFeEDTA (Qf = 3.31 × 108), and one Fe proteinate with an extremely strong chelation strength (Fe-Prot ES, Qf value = 8,590) were selected. Their bioavailabilities relative to Fe sulfate (FeSO4·7H2O) for broilers fed with a conventional corn-soybean meal diet were evaluated during d 1 to 21 by investigating the effects of the above Fe sources and added Fe levels on the growth performance, hematological indices, Fe contents, activities and gene expressions of Fe-containing enzymes in various tissues of broilers. RESULTS: NaFeEDTA sources varied greatly in their chemical characteristics. Plasma Fe concentration (PI), transferrin saturation (TS), liver Fe content, succinate dehydrogenase (SDH) activities in liver, heart, and kidney, catalase (CAT) activity in liver, and SDH mRNA expressions in liver and kidney increased linearly (P < 0.05) with increasing levels of Fe supplementation. However, differences among Fe sources were detected (P < 0.05) only for PI, liver Fe content, CAT activity in liver, SDH activities in heart and kidney, and SDH mRNA expressions in liver and kidney. Based on slope ratios from multiple linear regressions of the above indices on daily dietary analyzed Fe intake, the average bioavailabilities of Fe-Prot ES, feed grade NaFeEDTA, and food grade NaFeEDTA relative to the inorganic FeSO4·7H2O (100%) for broilers were 139%, 155%, and 166%, respectively. CONCLUSIONS: The bioavailabilities of organic Fe sources relative to FeSO4·7H2O were closely related to their Qf values, and NaFeEDTA sources with higher Qf values showed higher Fe bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

Correction: Jin et al. Effects of Zinc Source and Level on the Intestinal Immunity of Xueshan Chickens under Heat Stress. Animals 2023, 13, 3025.

There were some errors in the original publication [...].

Effects of Zinc Source and Level on the Intestinal Immunity of Xueshan Chickens under Heat Stress.

Heat stress can cause intestinal inflammation, impaired barrier integrity, and decreased immunity in poultry. While zinc is known to mitigate the adverse effects of heat stress, how the dietary supplementation of different sources and levels of it can improve the heat stress capacity of Chinese landraces remains unclear. This study investigated Xueshan chickens, which are an important local breed in China. The effects of different levels of ZnS and Zn-Prot M on their intestinal immune function under heat stress were compared. We found that different levels of ZnS and Zn-Prot M could effectively reduce the secretion level of IL-6 in the serum, and 60 mg/kg was optimal. Compared with ZnS, Zn-Prot M significantly increased duodenal villus height and VH/CD ratio, thus Zn-Prot M was more effective than ZnS. Both ZnS and Zn-Prot M significantly down-regulated TNF-α, IL-1ß, and MyD88 in 102-day-old duodenum, and IL-1ß, IL-6, and NFKBIA in jejunum and ileum at 74, 88, and 102 days old, with 60 mg/kg Zn-Prot M determined as optimal. In conclusion, our study demonstrates that Zn-Prot M is superior to ZnS in improving intestinal immunity in Xueshan chickens, and 60 mg/kg is the optimal addition dose.

Dietary trace mineral pattern influences gut microbiota and intestinal health of broilers.

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 Lactobacillus in 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-1 mRNA 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.

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.

Manganese alleviates heat stress of primary cultured chick embryonic myocardial cells via enhancing manganese superoxide dismutase expression and attenuating heat shock response.

Manganese (Mn) is an essential trace element that has been shown to attenuate the adverse effects of heat stress in the heart of broiler breeders and embryos. However, the underlying molecular mechanisms involving this process remain unclear. Therefore, two experiments were conducted to investigate the possible protective mechanisms of Mn on primary cultured chick embryonic myocardial cells exposed to heat challenge. In experiment 1, the myocardial cells were exposed to 40 °C (normal temperature, NT) and 44 °C (high temperature, HT) for 1, 2, 4, 6 or 8 h. In experiment 2, the myocardial cells were preincubated with no Mn supplementation (CON), 1 mmol/L of Mn as the inorganic MnCl2 (iMn) or organic Mn proteinate (oMn) under NT for 48 h, and then continuously incubated under NT or HT for another 2 or 4 h. The results from experiment 1 showed that the myocardial cells incubated for 2 or 4 h had the highest (P < 0.0001) heat-shock protein 70 (HSP70) or HSP90 mRNA levels than those incubated for other incubation times under HT. In experiment 2, HT increased (P < 0.05) the heat-shock factor 1 (HSF1) and HSF2 mRNA levels as well as Mn superoxide dismutase (MnSOD) activity of myocardial cells compared with NT. Furthermore, supplemental iMn and oMn increased (P < 0.02) HSF2 mRNA level and MnSOD activity of myocardial cells compared with the CON. Under HT, the HSP70 and HSP90 mRNA levels were lower (P < 0.03) in iMn group than in the CON group, in oMn group than in iMn group; and the MnSOD mRNA and protein levels were higher (P < 0.05) in oMn group than in the CON and iMn groups. These results from the present study indicate that supplemental Mn, especially oMn, could enhance the MnSOD expression and attenuate heat shock response to protect against heat challenge in primary cultured chick embryonic myocardial cells.

The organic zinc with moderate chelation strength enhances the expression of related transporters in the jejunum and ileum of broilers.

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.

Zinc intake ameliorates intestinal morphology and oxidative stress of broiler chickens under heat stress.

Zinc (Zn), an essential trace element for poultry, plays a crucial role in promoting growth, improving feed conversion efficiency, enhancing antioxidant activity, and preventing disease. This study investigated the impact of different levels and sources of dietary Zn supplementation on the growth performance, intestinal morphology and antioxidant activity of broiler chickens under heat stress conditions. In this experiment, 1024 Xueshan chickens were divided into eight groups and subjected to heat stress conditions with different levels of Zn supplementation (30 mg/kg, 60 mg/kg, and 90 mg/kg) using organic or inorganic sources. Our findings indicated that dietary Zn supplementation significantly increased the feed-to-weight ratio of broilers during the experimental period under heat stress. Moreover, Zn supplementation positively increased the villus height and villus width in the jejunum and ileum at 74 and 88 days old, with the 60 and 90 mg/kg groups outperforming other groups, and organic Zn was more effective than inorganic Zn. Furthermore, Zn supplementation significantly increased serum antioxidant levels, with higher superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-px) activities, and organic Zn was more effective than inorganic Zn. This study concludes that Zn supplementation is beneficial in mitigating the detrimental impacts of heat stress on broilers. The findings suggest that employing Zn as a strategy can enhance productivity in the poultry industry by positively influencing intestinal morphology and bolstering antioxidant activity to counteract potential stress.

An optimal dietary sodium chloride supplemental level of broiler chicks fed a corn-soybean meal diet from 1 to 21 days of age.

Sodium chloride (NaCl) is usually added to diets to meet the Na and Cl requirements of broilers in the Chinese poultry industry, but the optimal dietary NaCl supplemental level was not well-established. The present study was conducted to estimate the optimal dietary NaCl supplemental level of broilers fed a corn-soybean meal diet from 1 to 21 days of age. A total of 490, 1-day-old Arbor Acres male broilers were fed a NaCl-unsupplemented corn-soybean meal basal diet (control) and the basal diet supplemented with 0.10, 0.20, 0.30, 0.40, 0.50 or 0.60% NaCl for 21 days. Regression analysis was conducted to evaluate the optimal dietary NaCl level using the best fitted broken-line or asymptotic models. As dietary supplemental NaCl levels increased, average daily gain (ADG), average daily feed intake (ADFI), blood partial pressure of CO2, total CO2, base excess and anion gap, blood concentrations of HCO3, Na and Cl, serum Na concentration, jejunal villus height (VH) and tibia ash content increased linearly and quadratically (P < 0.05), while feed/gain ratio, relative weights of heart, liver and kidney, blood K concentration, serum concentrations of K, uric acid and glucose, and osmotic pressure decreased linearly and quadratically (P < 0.05). The estimates of optimal dietary NaCl levels were 0.20-0.22% based on the best fitted broken-line or asymptotic models (P < 0.0001) of ADG, ADFI and feed/gain ratio, and 0.08-0.24% based on the best fitted broken-line or asymptotic models (P < 0.0001) of blood gas indices, serum parameters, jejunal VH, tibia ash content and organ indices. These results suggested that the optimal dietary NaCl supplemental level would be 0.24% for broilers fed the corn-soybean meal diet from 1 to 21 days of age, which is lower than the current dietary NaCl supplemental level (0.30%) in the Chinese broiler production.

Organic zinc with moderate chelation strength enhances zinc absorption in the small intestine and expression of related transporters in the duodenum of broilers.

Our previous study demonstrated that the absorption of zinc (Zn) from the organic Zn proteinate with moderate chelation strength was significantly higher than that of Zn from the inorganic Zn sulfate in the in situ ligated duodenal segment of broilers, but the underlying mechanisms are unknown. The present study aimed to determine the effect of organic Zn with moderate chelation strength and inorganic Zn on the Zn absorption in the small intestine and the expression of related transporters in the duodenum of broilers. The Zn-deficient broilers (13 days old) were fed with the Zn-unsupplemented basal diets (control) containing 25.72 and 25.64 mg Zn/kg by analysis or the basal diets supplemented with 60 mg Zn/kg as the Zn sulfate or the Zn proteinate with moderate chelation strength (Zn-Prot M) for 26 days. The results showed that the plasma Zn contents from the hepatic portal vein of broilers at 28 days and 39 days of age were increased (p < 0.05) by Zn addition and greater (p < 0.05) in the Zn-Prot M than in the Zn sulfate. On d 28, Zn addition upregulated (p < 0.05) mRNA expression of zinc transporter 1 (ZnT1), Zrt-irt-like protein 5 (ZIP5), y + L-type amino transporter 2 (y + LAT2) and b0,+-type amino acid transporter (rBAT), zinc transporter 4 (ZnT4) protein expression, and zinc transporter 9 (ZnT9) mRNA and protein expression in the duodenum. Moreover, ZnT9 mRNA expression, ZnT4, ZIP5, and rBAT protein expression, zinc transporter 7 (ZnT7), and y + LAT2 mRNA and protein expression in the duodenum of broilers on 28 days were higher (p < 0.05) in the Zn-Prot M than in the Zn sulfate. On d 39, supplemental Zn increased (p < 0.05) peptide-transporter 1 (PepT1) mRNA expression and y + LAT2 protein expression, while the mRNA expression of ZnT7 and Zrt-irt-like protein 3 (ZIP3) were higher (p < 0.05) for the Zn-Prot M than for the Zn sulfate in the duodenum. It was concluded that the Zn-Prot M enhanced the Zn absorption in the small intestine partially via upregulating the expression of ZnT4, ZnT7, ZnT9, ZIP3, ZIP5, y + LAT2, and rBAT in the duodenum of broilers.

Metabolic utilization of intravenously injected iron from different iron sources in target tissues of broiler chickens.

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-ProtM) or extremely strong (Fe-ProtES) chelation strength. 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) liver or kidney Fe concentrations and spleen FTH1 protein levels than those injected with Fe-MetW or Fe-ProtM on d 20 after injections. And they had lower (P < 0.05) liver cytochrome C oxidase mRNA levels on d 20 after injections than those injected with Fe-MetW or Fe sulphate. The results from this study indicate that intravenously injected Fe from Fe-ProtES was the least utilizable and functioned in the sensitive target tissue less effectively than Fe from Fe sulfate, Fe-MetW or Fe-ProtM.

The Effect of Bone Morphogenetic Protein 2 or Extracellular Signal-Regulated Kinase 1 Silencing on Phosphorus Utilization and Related Parameters in Primary Broiler Osteoblasts.

Two experiments were conducted to study the effect of bone morphogenetic protein 2 (BMP2) or extracellular signal-regulated kinase 1 (ERK1) silencing on phosphorus (P) utilization and related parameters in primary broiler osteoblasts. Experiment 1 was carried out to select the most efficacious siRNAs against BMP2 or ERK1 for the subsequent experiment. In experiment 2, with or without the siRNA against BMP2 or ERK1, primary broiler osteoblasts were incubated in the medium supplemented with 0.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The osteoblastic P utilization and related parameters were determined. The results showed that the si980 and si1003 were the most effective (P < 0.05) in inhibiting BMP2 and ERK1 expressions, respectively. The BMP2 silencing reduced (P < 0.004) the osteoblastic P retention rate, alkaline phosphatase (ALP) activity, BMP2 mRNA and protein expressions. Supplemental P increased (P = 0.0008) ALP activity. Significant interactions (P < 0.04) between the gene silencing and supplemental P level were observed in both mineralization formation and bone gal protein (BGP) content. The BMP2 silencing decreased (P < 0.05) mineralization formation at both 0.0 and 2.0 mmol/L of added P levels, but the decreased degree was greater at 2.0 mmol/L of added P level, while BMP2 silencing reduced (P < 0.05) BGP content at only 2.0 mmol/L of added P level. The ERK1 silencing decreased (P < 0.004) mineralization formation, ALP activity, BGP content, ERK1 mRNA, ERK1 and p-ERK1 protein expressions. Supplemental P increased (P < 0.03) mineralization formation, ALP activity, BGP content and p-ERK1 protein expression, but inhibited (P = 0.014) ERK1 protein expression. There was an interaction (P < 0.03) between the gene silencing and supplemental P level in the osteoblastic P retention rate. The ERK1 silencing decreased (P < 0.05) it regardless of 0.0 or 2.0 mmol/L of added P level, but the reduced degree was greater at 2.0 mmol/L of added P level. It was concluded that either BMP2 or ERK1 silencing suppressed P utilization, and thus either of them participated in regulating P utilization in primary broiler osteoblasts.

Effect of in ovo manganese injection on the embryonic development, antioxidation, hatchability, and performances of offspring broilers under normal and high temperatures.

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 dismutase mRNA 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.

Dentin Matrix Protein 1 Silencing Inhibits Phosphorus Utilization in Primary Cultured Tibial Osteoblasts of Broiler Chicks.

Three experiments were carried out in the present study to investigate whether dentin matrix protein 1 (DMP1) was involved in regulating phosphorus (P) metabolic utilization in primary cultured tibial osteoblasts of broiler chicks. Experiment 1 was conducted to select the optimal osteogenic inductive culture medium and the optimal induction time in primary cultured tibial osteoblasts of broiler chicks. In experiment 2, the siRNAs against DMP1 were designed, synthesized and transfected into primary cultured tibial osteoblasts of broiler chicks, and then the inhibitory efficiencies of siRNAs against DMP1 were determined, and the most efficacious siRNA was selected to be used for the DMP1 silencing. In experiment 3, with or without siRNA against DMP1, primary cultured tibial osteoblasts of broiler chicks were treated with the medium supplemented with 0.0, 1.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The P metabolic utilization-related parameters were measured. The results showed that the osteogenic induced medium 2 and 12 days of the optimal induction time were selected; Among the designed siRNAs, the si340 was the most effective (P < 0.05) in inhibiting the DMP1 expression; DMP1 silencing decreased (P < 0.05) the expressions of DMP1 mRNA and protein, P retention rate, mineralization formation, alkaline phosphatase activity and bone gla-protein content in tibial osteoblasts at all of added P levels. It is concluded that DMP1 silencing inhibited P utilization, and thus DMP1 was involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks, which provides a novel insight into the regulation of the P utilization in the bone of broilers, and will contribute to develop feasible strategies to improve the bone P utilization efficiency of broilers so as to decrease its excretion.

Research Note: Metabolic utilization of iron from different iron sources in primary cultured hepatocytes of broiler embryos.

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), CAT and 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 SDH mRNA 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.

The Effects of Inorganic Phosphorus Levels on Phosphorus Utilization, Local Bone-Derived Regulators, and BMP/MAPK Pathway in Primary Cultured Osteoblasts of Broiler Chicks.

Understanding the underlying mechanisms that regulate the bone phosphorus (P) utilization would be helpful for developing feasible strategies to improve utilization efficiency of P in poultry. We aimed to investigate the effects of inorganic P levels on P utilization, local bone-derived regulators and bone morphogenetic protein/mitogen-activated protein kinase (BMP/MAPK) pathway in primary cultured osteoblasts of broiler chicks in order to address whether local bone-derived regulators or BMP/MAPK pathway was involved in regulating the bone P utilization of broilers using an in vitro model. The primary cultured tibial osteoblasts of broiler chicks were randomly divided into one of five treatments with six replicates for each treatment. Then, cells were respectively incubated with 0.0, 0.5, 1.0, 1.5, or 2.0 mmol/L of added P as NaH2PO4 for 24 days. The results showed that as added P levels increased, tibial osteoblastic P retention rate, number and area of mineralized nodules, the mRNA expressions of endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1), bone morphogenetic protein 2 (BMP2), and the mRNA and protein expressions of matrix extracellular phosphoglycoprotein (MEPE) increased linearly (p < 0.001) or quadratically (p < 0.04), while extracellular signal-regulated kinase 1 (ERK1) mRNA expression and c-Jun N-terminal kinase 1 (JNK1) phosphorylated level decreased linearly (p < 0.02) or quadratically (p < 0.01). Correlation analyses showed that tibial osteoblastic P retention rate was positively correlated (r = 0.452-0.564, p < 0.03) with MEPE and BMP2 mRNA expressions. Furthermore, both number and area of mineralized nodules were positively correlated (r = 0.414-0.612, p < 0.03) with PHEX, DMP1, MEPE, and BMP2 mRNA expressions but negatively correlated (r = -0.566 to -0.414, p < 0.04) with the ERK1 mRNA expression and JNK1 phosphorylated level. These results suggested that P utilization in primary cultured tibial osteoblasts of broiler chicks might be partly regulated by PHEX, DMP1, MEPE, BMP2, ERK1, and JNK1.