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1.
Poult Sci ; 103(11): 104114, 2024 Jul 25.
Article in English | MEDLINE | ID: mdl-39214056

ABSTRACT

Various types of dietary fats undergo distinct fermentation processes by gut microbes, potentially leading to the production of neurotransmitters that can influence the gut. Serotonin and dopamine are recognized neurotransmitters with positive effects on gut function. A broiler chicken trial was conducted to evaluate the influence of dietary fat types on protein expression of 2 neurotransmitter transporters, dopamine (DAT) and serotonin (5-HTT). A total of 560 day-old (Ross 708) male broiler chicks were randomly assigned to 7 dietary treatments. The experimental treatments included a basal diet of corn-soybean meal (SBM), supplemented with 3% of various fats: poultry fat (CON), olive oil (OLIV), fish oil (FISH), canola oil (CANO), lard (LARD), coconut oil (COCO), or flaxseed oil (FLAX). Bodyweight (BW) and feed conversion ratio (FCR) were recorded. Ileal tissues were aseptically collected to determine the expression levels of DAT and 5-HTT through western blot analysis. In addition, plasma samples were analyzed for reactive oxygen metabolites (d-ROM) tests on d 55. Results showed that dietary fat type inclusion did not have any detrimental effect on growth performance parameters. The expression levels of DAT were higher (P < 0.05) in FLAX treatments compared to CON treatments on d 20 and d 55, respectively. Similarly, with 5-HTT levels, FLAX, CANO, and LARD treatments were higher (P < 0.05) than CON treatments on d 20 and d 55. However, higher levels of oxidative stress (d-ROM values) were recorded in COCO (32.75 Carr U), CANO (29 Carr U), and CON treatments (25.5 Carr U) compared to FLAX (18.5 Carr U; P < 0.05) treatment. These findings suggest that incorporating dietary flaxseed oil at a 3% level in the diet has significant potential to elevate the expression levels of intestinal DAT and 5-HTT without inducing oxidative stress.

2.
BMC Genomics ; 25(1): 580, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38858615

ABSTRACT

BACKGROUND: Understanding growth regulatory pathways is important in aquaculture, fisheries, and vertebrate physiology generally. Machine learning pattern recognition and sensitivity analysis were employed to examine metabolomic small molecule profiles and transcriptomic gene expression data generated from liver and white skeletal muscle of hybrid striped bass (white bass Morone chrysops x striped bass M. saxatilis) representative of the top and bottom 10 % by body size of a production cohort. RESULTS: Larger fish (good-growth) had significantly greater weight, total length, hepatosomatic index, and specific growth rate compared to smaller fish (poor-growth) and also had significantly more muscle fibers of smaller diameter (≤ 20 µm diameter), indicating active hyperplasia. Differences in metabolomic pathways included enhanced energetics (glycolysis, citric acid cycle) and amino acid metabolism in good-growth fish, and enhanced stress, muscle inflammation (cortisol, eicosanoids) and dysfunctional liver cholesterol metabolism in poor-growth fish. The majority of gene transcripts identified as differentially expressed between groups were down-regulated in good-growth fish. Several molecules associated with important growth-regulatory pathways were up-regulated in muscle of fish that grew poorly: growth factors including agt and agtr2 (angiotensins), nicotinic acid (which stimulates growth hormone production), gadd45b, rgl1, zfp36, cebpb, and hmgb1; insulin-like growth factor signaling (igfbp1 and igf1); cytokine signaling (socs3, cxcr4); cell signaling (rgs13, rundc3a), and differentiation (rhou, mmp17, cd22, msi1); mitochondrial uncoupling proteins (ucp3, ucp2); and regulators of lipid metabolism (apoa1, ldlr). Growth factors pttg1, egfr, myc, notch1, and sirt1 were notably up-regulated in muscle of good-growing fish. CONCLUSION: A combinatorial pathway analysis using metabolomic and transcriptomic data collectively suggested promotion of cell signaling, proliferation, and differentiation in muscle of good-growth fish, whereas muscle inflammation and apoptosis was observed in poor-growth fish, along with elevated cortisol (an anti-inflammatory hormone), perhaps related to muscle wasting, hypertrophy, and inferior growth. These findings provide important biomarkers and mechanisms by which growth is regulated in fishes and other vertebrates as well.


Subject(s)
Bass , Gene Expression Profiling , Animals , Bass/genetics , Bass/growth & development , Bass/metabolism , Female , Male , Metabolomics , Muscle Development/genetics , Transcriptome , Muscle, Skeletal/metabolism , Muscle, Skeletal/growth & development , Metabolome , Liver/metabolism
3.
Poult Sci ; 102(10): 102897, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37562125

ABSTRACT

Ginger contains bioactive compounds that possess anti-inflammatory and antimicrobial properties. In this study, 432-day-old Ross 708 broiler male chicks were randomly allocated to 6 dietary treatments to investigate the effect of ginger root extract (GRE) on immunocompetence and growth performance to 6 wk of age. Treatment 1 (CON) consisted of chicks fed a corn-soybean meal (SBM), a base diet without GRE. Treatment 2 (MX) chicks were given basal diets containing bacitracin methylene disalicylate (BMD) at 0.055 g/kg. Treatments 3 (GRE-0.375%), 4 (GRE-0.75%), 5 (GRE-1.5%), and 6 (GRE-3%) were fed similar diet to control with GRE supplemented at 0.375%, 0.75%, 1.5%, and 3%, respectively. Moreover, HPLC analysis of GRE was carried out to determine the concentration of bioactive compounds found in GRE. Each treatment consisted of 6 replicate pens with 12 chicks/pen. Bodyweight (BW) and feed conversion ratio (FCR) were recorded. Results show that the concentration of bioactive compounds increased with increasing GRE supplementation. Likewise, dietary GRE supplementation did not have any detrimental effect on growth performance parameters up to 1.5%, as values for BWG was not different from CON and MX; however, 3% GRE had the poorest FCR and a lower BWG as compared to other treatments. On d 27 and d 41, fecal and cecal concentrations of total bacteria count (TBC), Escherichia coli, Lactobacillus spp., and Bifidobacterium spp enumerated using selective plating media showed that GRE supplementation significantly reduced (P < 0.05) the amount of TBC and E. coli but increased the number of beneficial microorganisms such as Lactobacillus spp. and Bifidobacterium spp. On d 20, no significant differences were observed (P > 0.05) among all treatments for antibody titer against Newcastle disease virus and total IgY antibodies; however, on d 27, GRE-0.75% had the highest value for both immune indicators and was not different from MX. Dietary supplementation of GRE up to 1.5% enhanced the immune system and suppressed E. coli while promoting the growth of healthy bacteria, without any detrimental effect on growth performance.


Subject(s)
Chickens , Zingiber officinale , Animals , Male , Escherichia coli , Diet/veterinary , Dietary Supplements/analysis , Plant Extracts/pharmacology , Immunocompetence , Animal Feed/analysis , Animal Nutritional Physiological Phenomena
4.
Animals (Basel) ; 13(4)2023 Feb 16.
Article in English | MEDLINE | ID: mdl-36830484

ABSTRACT

Consumer demand for retail cage-free eggs is driving the layer industry towards greater use of extensive housing environments. However, there is limited research on how these environments affect egg production characteristics of brown egg layers, as was the focus of this study. Five housing environments were evaluated under typical industry conditions, including conventional cages, enrichable colony cages, enriched colony cages, cage-free and free-range. Three different brown egg laying strains were housed in the different housing environments and managed according to standard husbandry practices and stocking densities. Data collection for the strains began at 17 weeks of age, with a base period of 28 days for feed weigh backs and egg quality assessments. Housing environment had a highly significant (p < 0.0001) effect on all egg production characteristics measured, including egg production rates (% hen-day and % hen-housed), feed consumption (g/bird/day), feed conversion (egg g/feed g), and mortality rate (%) as well as percent grade A, B, and loss. Previous research revealed better egg production metrics for white egg layers in caged environments than extensive environments. In contrast, we observed brown egg layers had optimum production results for the free-range housing environments, and the poorest performance in enrichable colony cages.

5.
Poult Sci ; 100(7): 101134, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34089939

ABSTRACT

Spray-dried plasma (SDP) contains immunoglobulins and glycoproteins that possess antibacterial properties. Two floor-pen trials were conducted to determine the efficacy of dietary SDP and bacitracin methylene disalicylate (BMD) antibiotic in reducing intestinal colonization by Salmonella Enteritidis (SE) in broiler chickens. Experiment 1 was a 2-wk, 3 × 2 factorial design consisting of 6 treatments. Treatment CON consisted of chicks fed unmedicated corn-soybean meal (SBM) basal without SDP. Treatment BMD consisted of chicks given unmedicated corn-SBM basal into which BMD was added at 0.055g/kg diet. Treatment SDP consisted of chicks given unmedicated corn-SBM basal into which SDP was added at 30g/kg diet. Treatments CON-SE, BMD-SE, and SDP-SE consisted of chicks that were given diets similar to CON, BMD, and SDP, respectively, and were each inoculated with 7.46 × 108 CFU SE /mL at 1 day of age. Experiment 2 was a 42-day trial that was similar to Experiment 1 in design, except that chicks were placed on fresh clean litter. On d 3, 7, 14, and 28 post-challenge (PC), ceca SE concentration was enumerated on xylose lysine tergitol-4 (XLT4) agar. Body weight gain (BWG) and feed conversion ratio (FCR) were also recorded. Results for d 3 showed that BMD- and SDP-fed chicks had similar (P > 0.05) cecal SE (3.39 log 10 CFU / g and 3.58 log 10 CFU / g, respectively), but these levels were lower (P < 0.05) than that of CON-fed chicks (5.68 log 10 CFU / g). A similar trend was observed on d 7 and 14 PC. The BMD- and SDP-fed chicks also had higher BWG and FCR (P < 0.05) when compared with CON-fed chicks up to d 14. Thereafter, only BMD treatment sustained this growth-promoting effect till d 42 in SE-challenged birds. In conclusion, BMD and SDP showed similar efficacy in reducing cecal Salmonella and in mitigating consequent growth-depressing effect(s) in broiler chicks up to 2 wk of age.


Subject(s)
Bacitracin , Chickens , Animal Feed/analysis , Animals , Cecum , Diet , Salmonella enteritidis
7.
Poult Sci ; 99(12): 6774-6782, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33248593

ABSTRACT

Broiler embryonic development depends on the nutrients that are available in the egg, which includes mostly water, lipids, and proteins. Carbohydrates represent less than 1%, and free glucose only 0.3%, of the total nutrients. Considering that energy requirements increase during incubation and metabolism is shifted toward the use of glycogen stores and gluconeogenesis from amino acids, extensive muscle protein degradation in the end of incubation can compromise chick development in the initial days after hatch. Significant prehatch changes occur in embryonic metabolism to parallel the rapid embryonic development. Oral consumption of the amniotic fluid begins around 17 d of incubation and promotes rapid development of the intestinal mucosa, which is characterized by morphological changes and increased expression and activity of enzymes and transporters. Furthermore, ingested substrates are stored as nutritional reserves to be used during hatching and in the first week after hatch. At hatch, this limited-nutrient store is directed to the functional development of the gastrointestinal tract to enable assimilation of exogenous nutrients. In ovo feeding is an alternative to deliver essential nutrients to chick embryos at this critical and challenging phase. The improved nutritional status and physiological changes triggered by in ovo feeding can resonate throughout the entire rearing period with significant health and economic gains. The present review addresses the main changes in metabolism and intestinal development throughout incubation, and also addresses scientific advances, limitations and future perspectives associated with the use of in ovo feeding that has been regarded as an important technology by the poultry industry.


Subject(s)
Animal Husbandry , Chickens , Energy Metabolism , Animals , Chick Embryo , Chickens/anatomy & histology , Chickens/growth & development , Chickens/metabolism , Gluconeogenesis , Glycogen/metabolism , Technology
8.
Transl Anim Sci ; 4(3): txaa137, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32832857

ABSTRACT

Locally grown feed ingredients of high energy and protein content, such as peanuts, maybe economically feasible alternatives to corn and soybean meal in broiler diets. Even though normal-oleic peanuts have been demonstrated to be a viable feed ingredient for poultry, few studies to date have examined the use of high-oleic peanuts (HO PN) as an alternative feed ingredient for broiler chickens. Thus, we aimed to determine the effect of feeding HO PN on broiler performance, nutrient digestibility, and intestinal morphology. Three isocaloric, isonitrogenous experimental diets were formulated with 1) dietary inclusion of ~10% coarse-ground whole HO PN; 2) a corn-soybean meal control diet with 5.5% added poultry fat; and 3) a control diet supplemented with 5.5% oleic fatty acid oil. Three-hundred Ross 708 broilers were randomly placed in 10 replicate pens per treatment with 10 chicks per pen and raised until 42 d. Body weights (BW) and feed intake were determined weekly, and feed conversion ratio (FCR) was calculated. Jejunum samples were collected at 42 d for histomorphometric analysis. Analysis of variance was performed on all variables using a general linear mixed model in JMP Pro14. Broilers in the HO PN group had lower (P < 0.05) BW and higher FCR than other treatment groups at weeks 2 and 6. There were no significant differences in the jejunum villi surface area between the treatment groups. However, broilers fed the HO PN diet had greater (P = 0.019) apparent metabolizable energy relative to the other treatment groups, suggesting improved nutrient uptake of dietary fats and/or carbohydrates in the HO PN treatment group. However, additional studies are warranted to further define the nutritional value of HO PN as an alternative poultry feed ingredient.

9.
Poult Sci ; 99(4): 2236-2245, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32241509

ABSTRACT

Early feeding trials using peanut meal prepared from normal-oleic peanuts helped to identify peanuts as a suitable alternative feed ingredient for poultry. Yet no studies to date have examined the use of high-oleic peanuts (HO-PN) as a feed ingredient for meat type chickens. Therefore, this study aimed to determine the effect of feeding whole unblanched HO-PN on the fatty acid profile of the meat produced from broilers. At hatch male chicks were randomly placed in raised wire cages, in 10 replicate pens per treatment with 10 chicks per pen, and fed with one of the 3 isocaloric, isonitrogenous diets ad libitum for 42 days: (1) conventional control of soybean meal + corn, (2) 10 to 12% HO-PN and corn diet, or (3) control diet spiked with ≈6.0% oleic acid oil. All body weights (BW) were collected, and broiler selection for processing was determined by individual BW within one-half a standard deviation of the experiment 42-D mean BW, with one bird selected per pen (10 replicate pens per treatment, 3 treatments, 10 birds selected per treatment, yielding a total sample size of 30 birds). Performance was determined weekly and breast samples were analyzed for fatty acid and amino acid profile. All data was analyzed using analysis of variance, with t-test mean comparisons at P < 0.05. BW were similar between broilers fed the HO-PN and control diet, while feed conversion ratio of broilers fed the HO-PN diet was significantly higher at weeks 2, 4, and 6 in comparison to the other treatments (P ≤ 0.03). Broilers fed with HO-PN diet had reduced carcass and pectoralis major weights in comparison to the other treatments. Chicken breast from broilers fed the HO-PN diet had significantly reduced saturated and trans fatty acid content in comparison to the controls (P ≤ 0.0002). Although additional studies must be conducted, this study suggests that feeding whole unblanched HO-PN to broiler chickens may serve as a means to enrich the meat produced with unsaturated fatty acids.


Subject(s)
Arachis/chemistry , Chickens/metabolism , Fatty Acids/metabolism , Oleic Acid/metabolism , Animal Feed/analysis , Animals , Chickens/growth & development , Diet/veterinary , Dietary Supplements/analysis , Male , Oleic Acid/administration & dosage , Random Allocation
10.
ACS Omega ; 5(2): 1080-1085, 2020 Jan 21.
Article in English | MEDLINE | ID: mdl-31984264

ABSTRACT

Previous studies have demonstrated that allergenic feed proteins from peanuts in the diets of layer hens are not detected in the eggs produced. Hence, in this study, we aimed to determine if soy and/or peanut proteins in poultry feed rations of broiler chickens or layer hens would be transferred or detectable in the meat or eggs produced. To meet this objective, 99 layer hens and 300 broiler chickens were equally divided into treatment groups and fed one of three experimental diets: control soybean meal and corn diet, whole unblanched high-oleic peanut and corn diet (HO PN), or a control diet spiked supplemented with oleic acid (OA) oil. At termination, broiler chickens were processed, and chicken breast samples of the left pectoralis muscle were collected, and eggs were collected from layers. Total protein extracts from pooled egg samples and chicken breast samples were subjected to enzyme-linked immunosorbent assay (ELISA) methods and immunoblotting analysis with rabbit antipeanut agglutinin antibodies and rabbit antisoy antibodies for the detection of peanut and soy proteins. Peanut and soy proteins were undetected in all pooled egg samples and individual chicken breast meat samples using immunoblotting techniques with rabbit antipeanut agglutinin and rabbit antisoy antibodies. Moreover, quantitative ELISA allergen detection methods determined all pooled egg samples and individual meat samples as "not containing" peanut or soy allergens. Therefore, this study helps to evaluate the risk associated with the potential transfer of allergenic proteins from animal feed to the products produced for human consumption.

11.
Poult Sci ; 98(10): 5188-5197, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31111917

ABSTRACT

Previous studies have identified peanut meal prepared from normal-oleic peanuts as a suitable and economical ingredient for broiler feed. However, to date, no studies have examined the use of new, high-oleic peanut (HO-PN) cultivars as a feed ingredient for poultry. This project aimed to determine the effect of HO-PNs as a feed ingredient for broiler chickens on the quality and sensory attributes of the meat produced. To test 3 experimental diets, male broiler chicks were randomly placed, at hatch, in raised-wire cages, in 10 replicate pens per treatment with 10 chicks per cage. For 6 wk, chicks were fed, ad libitum, one of the three isocaloric, isonitrogenous diets: (1) a conventional soybean meal plus corn control diet, (2) 10 to 12% HO-PN + corn diet, or (3) a control corn diet spiked with ≈6.0% oleic fatty acid oil (OA). At 42 D, 3 broilers per pen (30 per treatment) were processed to determine meat quality and for consumer evaluation. Carcass weights and breast yields were reduced in broilers fed HO-PN, while leg carcass yields were greater in broilers fed HO-PN in comparison to the other groups. Chicken breast from broilers fed HO-PN had reduced meat-pH, reduced L* color values, and increased cooked loss compared to other treatments. Nevertheless, a group of 100-consumer panelists scored all 3-treatment groups similar in terms of sensory attributes for cooked chicken. While additional studies must be performed, this study suggest that HO-PN may be a suitable broiler feed ingredient.


Subject(s)
Animal Feed/analysis , Arachis/chemistry , Meat/analysis , Nuts/chemistry , Oleic Acid/analysis , Animals , Chickens , Diet/veterinary , Male , Random Allocation
12.
Front Vet Sci ; 2: 45, 2015.
Article in English | MEDLINE | ID: mdl-26664972

ABSTRACT

Anti-nutritional factors (ANFs) in feed ingredients can challenge gut health and reduce nutrient utilization. Birds typically activate their innate immune system as a protective response against the adverse effects of ANF, which often involves the secretion of mucin. Although dietary supplementation of exogenous enzymes are commonly used to alleviate the adverse effects of ANF on apparent nutrient digestibility, little is known about how they affect gut health, particularly in relation to the morphological development and mucin secretion of enteric mucosa. We carried out two trials to examine the effect of dietary supplementation of different types of exogenous enzymes on gut health of by accessing the effect of jejunum morphological development and ileal enteric adherent mucin thickness layer in turkeys. Dietary ß-mannanase supplementation reduced ileal adherent mucin thickness layer (804 vs 823 µg/g; p < 0.05), while a commercial blend of xylanase, amylase, and protease (XAP) reduced ileal adherent mucin layer thickness (589 vs 740 µg/g; p < 0.05); thus reducing the apparent endogenous loss of nutrients. Both enzyme supplements also affected gut morphological characteristics. In comparison to the control treatment, dietary ß-mannanase supplementation improved the jejunum tip width (219 vs 161; p < 0.05), base width (367 vs 300; p < 0.05), surface area (509,870 vs 380, 157; p < 0.05) and villi height/crypt depth ratio (7.49 vs 5.70; p < 0.05), and XAP improved the crypt depth (p < 0.05). In conclusion, dietary supplementation of exogenous enzymes may help alleviate the adverse effects of ANF on nutrient utilization by directly or indirectly removing the mucosal irritation that stimulates enteric mucin secretion.

13.
Zoo Biol ; 27(3): 188-99, 2008 May.
Article in English | MEDLINE | ID: mdl-19360617

ABSTRACT

Woolly monkeys (Lagothrix sp.) are threatened species and numerous zoos have failed to sustain successful populations. The most common causes of death in captive woolly monkeys are related to pregnancy and hypertension. The objective of this retrospective study was to evaluate serum concentrations of a large number of captive woolly monkeys to establish baseline means and compare these concentrations with their closest related species to determine potential abnormalities. Serum analyses from 30 woolly monkeys housed at two institutions (Apenheul, The Netherlands and The Louisville Zoo, KY, USA) over 12 yr were collected. The statistical model included gender, age group (young, 0-4 yr of age; middle, 5-9 yr; and old, 10+ yr), and zoological institution. All panel result means were similar to previously reported concentrations for howler (Alouatta sp.) and spider monkeys (Ateles sp.) with the possible exception of alanine aminotransferase and gamma-glutamyl-transferase being higher, whereas creatinine and phosphorus were lower. The serum glucose mean of 6.7 mmol/L is above the baseline range for humans and spider monkeys. Alkaline phosphatase (ALP), alanine aminotransferase, and sodium (Na) were higher in females and magnesium (Mg) was higher in males (P<0.05). ALP, Mg, and phosphorus were highest (P<0.05) and calcium and sodium tended to be highest (P<0.10) in the oldest animals. Ferritin tended to be highest (P<0.10) in the oldest animals. Albumin, ALP, chloride, Na, and total bilirubin were higher for Zoo A, whereas gamma-glutamyl-transferase, glucose, and lactate dehydrogenase were lower for Zoo A (P<0.05). Areas of potential woolly monkey health risk were noted and discussed. Future studies are needed to determine free-ranging serum concentrations to elucidate parameters that contain aberrant concentrations and decrease health status.

14.
J Nutr Biochem ; 16(6): 339-46, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15936645

ABSTRACT

A 303-bp cDNA of intestinal zinc exporter (ZnT1) was isolated from chicken jejunum by reverse transcriptase-polymerase chain reaction and sequenced, and showed 42% homology to Homo sapiens and Rattus novergicus intestinal ZnT1 genes. This specific probe was used to examine the effect of zinc-methionine (ZnMet) administration on the mRNA expression of ZnT1 and on small intestinal development and functionality. In this study, ZnMet was injected into the naturally consumed amniotic fluid of 17-day-old chicken embryos. The ZnT1 gene showed an approximately 200% increase in its mRNA levels from 48 h post-ZnMet injection, as compared to the control. An analysis of the gene expression of the brush-border enzymes and transporters showed increased mRNA expression of sucrase isomaltase, leucine-aminopeptidase, sodium-glucose cotransporter and Na+K+ATPase transporter (Na+K+ATPase) from 48 h post-ZnMet injection, in comparison to controls. Significant increases (P<.05) in the biochemical activity of the brush-border enzymes and transporters, and in jejunal villus surface area were detected from day of hatch (96 h post-ZnMet injection) as compared to controls. These results suggest that ZnMet administration into prenatal intestine via injection into the amniotic fluid enhances intestinal development and improves its functionality.


Subject(s)
Chickens/metabolism , Intestine, Small/growth & development , Membrane Proteins/physiology , Zinc/physiology , Amino Acid Sequence , Amnion , Animals , Cation Transport Proteins , Chick Embryo/growth & development , Chick Embryo/metabolism , Chickens/growth & development , DNA, Complementary/analysis , Gene Expression Profiling , Injections , Intestine, Small/embryology , Intestine, Small/enzymology , Intestine, Small/metabolism , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Methionine/administration & dosage , Methionine/analogs & derivatives , Methionine/pharmacokinetics , Microvilli/enzymology , Microvilli/metabolism , Molecular Sequence Data , Organometallic Compounds/administration & dosage , Organometallic Compounds/pharmacokinetics , RNA, Messenger/biosynthesis , Rats , Zinc/metabolism
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