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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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

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 b 0,+ -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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of in ovo zinc (Zn) injection in improving the embryonic development in eggs from Zn-deficient hens, via epigenetic and antioxidant mechanisms, was examined. A completely randomized design involving a 1 (the non-injected control) + 1 (the injected control with sterilized water) + 2 (Zn source) × 2 (Zn level) factorial arrangement of treatments was used. The two injected Zn sources were inorganic Zn sulfate and organic Zn-lysine chelate with a moderate chelation strength, and the two injected Zn levels were 50 and 100 μg Zn/egg. In ovo Zn injection decreased (P < 0.05) embryonic mortality, and increased (P < 0.05) hatchability and healthy chick ratio. In ovo Zn injection increased (P < 0.05) embryonic tibia Zn content, but had no effect (P > 0.05) on copper (Cu)- and Zn-containing superoxide dismutase (CuZnSOD) activities and metallothionein IV (MT4) levels or their mRNA expression levels and malondialdehyde (MDA) levels in the embryonic liver. In ovo Zn injection had no effect (P > 0.05) on the global level of DNA methylation or DNA methylation and histone 3 lysine 9 (H3K9) acetylation levels of the MT4 promoter in the embryonic liver. However, the organic Zn had higher (P < 0.05) levels of DNA methylation and H3K9 acetylation than inorganic Zn. These data demonstrate that in ovo Zn injection improved the embryonic development, and the organic Zn was more effective than inorganic Zn in enhancing DNA methylation and H3K9 acetylation in the liver MT4 promoter, but the precise mechanisms require further investigations.

To investigate the effects of environmental temperature and dietary Zn on egg production performance, egg quality and antioxidant status, as well as expression of heat-shock proteins (HSP) in tissues, of laying broiler breeders, we used a completely randomised design with a 2×3 factorial arrangement of treatments. The two environmental temperatures were normal (21±1°C, NT) and high (32±1°C, HT). The three dietary Zn sources were a Zn-unsupplemented basal diet (CON), and the basal diet supplemented with 110 mg Zn/kg as either the inorganic Zn sulphate (iZn) or the organic Zn proteinate with a moderate chelation strength (oZn). HT decreased (P<0·002) egg weight, laying rate, eggshell strength, thickness and weight, but increased (P≤0·05) rectal temperature, broken egg rate, misshapen egg rate, feed:egg ratio, Cu Zn superoxide dismutase activities in liver and pancreas, as well as metallothionein (MT) level in pancreas, and HSP70 mRNA levels in liver and pancreas of laying broiler breeders. Broiler breeders fed the oZn diet had higher (P<0·04) Zn content in the liver, as well as MT levels in the liver and pancreas, compared with those fed the CON diet. Under HT, broiler breeders fed the oZn diet had higher (P<0·05) Zn content in the pancreas compared with those fed the iZn and CON diets. The results from this study indicated that HT impaired egg production performance and eggshell quality possibly because of the disturbed redox balance and HSP homoeostasis, whereas the oZn is more available than the iZn for pancreatic Zn of heat-stressed laying broiler breeders.

An experiment was conducted to estimate the optimal dietary zinc (Zn) level of brown-egg laying hens fed a corn-soybean meal diet from 20 to 40 weeks of age. A total of 120 20-week-old Beijing Red commercial laying hens were randomly allotted by bodyweight to one of five treatments with six replicates of four birds each in a completely randomized design, and fed a Zn-unsupplemented corn-soybean meal basal diet containing 27.95 mg Zn/kg by analysis and the basal diets supplemented with 30, 60, 90, or 120 mg Zn/kg as Zn sulfate (reagent grade ZnSO 4 ·7H 2 O) for a duration of 20 weeks. Laying performance, egg quality, tissue Zn concentrations, and activities of serum alkaline phosphatase (AKP), and liver copper-Zn superoxide dismutase (CuZnSOD) were measured. Regression analyses were performed to estimate an optimal dietary Zn level whenever a significant quadratic response (P < 0.05) was observed. Tibia Zn concentration (P = 0.002) and serum AKP activity (P = 0.010) showed significant quadratic responses to dietary supplemental Zn levels. The estimates of dietary Zn requirements for brown-egg laying hens from 20 to 40 weeks of age were 71.95 and 64.63 mg/kg for tibia Zn concentration and serum AKP activity, respectively. The results from this study indicate that the tibia Zn might be a more suitable and reliable parameter for Zn requirement estimation, and the optimal dietary Zn level would be about 72 mg/kg for brown-egg laying hens fed a corn-soybean meal diet from 20 to 40 weeks of age.

The role of maternal dietary zinc supplementation in protecting the embryos from maternal hyperthermia-induced negative effects via epigenetic mechanisms was examined using an avian model (Gallus gallus). Broiler breeder hens were exposed to two maternal temperatures (21°C and 32°C) × three maternal dietary zinc treatments (zinc-unsupplemented control diet, the control diet + 110 mg zinc/kg inorganic or organic zinc) for 8 weeks. Maternal hyperthermia increased the embryonic mortality and induced oxidative damage evidenced by the elevated mRNA expressions of heat shock protein genes. Maternal dietary zinc deficiency damaged the embryonic development associated with the global DNA hypomethylation and histone 3 lysine 9 hyperacetylation in the embryonic liver. Supplementation of zinc in maternal diets effectively eliminated the embryonic mortality induced by maternal hyperthermia and enhanced antioxidant ability with the increased mRNA and protein expressions of metallothionein IV in the embryonic liver. The increased metallothionein IV mRNA expression was due to the reduced DNA methylation and increased histone 3 lysine 9 acetylation of the metallothionein IV promoter regardless of zinc source. These data demonstrate that maternal dietary zinc addition as an epigenetic modifier could protect the offspring embryonic development against maternal heat stress via enhancing the epigenetic-activated antioxidant ability.

An experiment was carried out to determine the bioavailability of the organic Zn-methionine chelate relative to inorganic Zn source (ZnSO4•7H2O) for broiler chicks fed a conventional corn-soybean meal diet. A total of 504 1-day-old Arbor Acres commercial male broiler chicks were randomly allotted to one of seven treatments in a completely randomized design involving a 2 × 3 factorial arrangement with three levels of added Zn (30, 60, or 90 mg of Zn/kg) and two Zn sources (Zn-methionine chelate and Zn sulfate) plus a Zn-unsupplemented control diet containing 29.2 mg of Zn/kg by analysis for an experimental phase of 21 days. Bone and pancreas were collected for testing Zn concentrations and pancreas metallothionein (MT) messenger RNA (mRNA) level at 7 or 21 days of age. The results showed that bone and pancreas Zn concentrations and MT mRNA level in pancreas increased linearly (P < 0.0001) at 7 or 21 days of age as added Zn level increased. Based on slope ratios from multiple linear regressions of the pancreas, MT mRNA level at 7 days and pancreas Zn concentration at 21 days on added Zn level and the bioavailability values of the Zn-methionine chelate relative to ZnSO4•7H2O (100%) were 120 and 115%, respectively (P > 0.22). The results indicated that the Zn from the Zn-methionine chelate was just as bioavailable as the Zn from Zn sulfate for broilers.

Heat stress (HS) is a significant concern in broiler chickens, which is vital for global meat supply in the dynamic field of poultry farming. The impact of heat stress on the ileum and its influence on the redox homeostatic genes in chickens remains unclear. We hypothesized that adding zinc to the feed of heat-stressed broilers would improve their resilience to heat stress. However, this study aimed to explore the effects of organic zinc supplementation under HS conditions on broiler chickens' intestinal histology and regulation of HS index genes. In this study, 512 Xueshan chickens were divided into four groups: vehicle, HS, 60 mg/kg zinc, and HS + 60 mg/kg zinc groups. Findings revealed that zinc supply positively increased the VH and VH: CD in the ileum of the broilers compared to the HS group, while CD and VW decreased in Zn and HS+Zn supplemented broilers. Zn administration significantly increased superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and decreased the enzymatic activities of reactive oxygen species (ROS) and malondialdehyde (MDA) compared to the HS group. In addition, Zn administration significantly increased relative ATP, complex I, III, and V enzyme activity compared to the HS group. Furthermore, the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), lactate transporter 3 (LPCAT3), peroxiredoxin (PRX), and transferrin receptor (TFRC) in the protein levels was extremely downregulated in HS+Zn compared to the HS group. Zn supply significantly decreased the enrichment of RORγ, P300, and SRC1 at target loci of ACSL4, LPCAT3, and PRX compared to the HS group. The occupancies of histone active marks H3K9ac, H3K18ac, H3K27ac, H3K4me1, and H3K18bhb at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. Moreover, H3K9la and H3K18la at the locus of ACSL4 and LPCAT3 were significantly decreased in HS+Zn compared to the HS group. This study emphasizes that organic Zn is a potential strategy for modulating the oxidative genes ACSL4, LPCAT3, PRX, and TFRC in the ileum of chickens via nuclear receptor RORγ regulation and histone modifications. [ABSTRACT FROM AUTHOR]

Simple Summary: The intestinal tract is an important part of the immune system and forms a congenital barrier against food antigens and pathogenic microorganisms. In poultry, heat stress can induce intestinal mucosal injury, damage intestinal tight junctions and microvillous structures, and trigger an inflammatory response and enterogenic infection. As an essential trace element, zinc has been shown to mitigate the adverse effects of heat stress on broilers. However, how the dietary supplementation of different sources and levels of zinc can improve the heat stress capacity of Chinese landraces remains unclear. Here, we investigated the effects of different levels of zinc sulfate (ZnS) and zinc proteinate with moderate chelation strength (Zn-Prot M) on the intestinal immune function under heat stress in Xueshan chickens, which comprise an important local breed in China. 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. ZnS and Zn-Prot M effectively 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 Zn-Prot M was more effective than ZnS. Both ZnS and Zn-Prot M significantly down-regulated TNF-α, IL-1β, and MyD88 in 102-day-old duodenum, and IL-1β, 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. [ABSTRACT FROM AUTHOR]