Jane E. Link

Effect of Se on selenoprotein activity and thyroid hormone metabolism in beef and dairy cows and calves

Although Se is essential for antioxidant and thyroid hormone function, factors influencing its requirement are not well understood. A survey and two experiments were conducted to determine the influence of cattle breed and age on selenoprotein activity and the effect of maternal Se supplementation on cow and calf selenoprotein activity and neonatal thyroid hormone production. In our survey, four cowherds of different ages representing three breeds were bled to determine the influence of breed and age on erythrocyte glutathione peroxidase activity (RBC GPX-1). All females were nonlactating, pregnant, and consumed total mixed diets (Holstein) or grazed pasture (Angus and Hereford). In our survey of beef breeds, yearlings had greater average RBC GPX-1 activity than mature cows. In Exp. 1, neonatal Holstein heifers (n = 8) were bled daily from 0 to 6 d of age to determine thyroid hormone profile. An injection of Se and vitamin E (BO-SE) was given after the initial bleeding. Thyroxine (T4) and triiodothyronine (T3) concentrations were greatest on d 0 and decreased (P < 0.05) continuously until d 5 postpartum (156.13 to 65.88 and 6.69 to 1.95 nmol/L, d 0 to 5 for T4 and T3, respectively). Reverse T3 concentrations were 3.1 nmol/L on d 0 and decreased (P < 0.05) to 0.52 nmol/ L by d 5. In Exp. 2, multiparous Hereford cows were drenched weekly with either a placebo containing 10 mL of double-deionized H2O (n = 14) or 20 mg of Se as sodium selenite (n = 13). After 2 mo of treatment, Se-drenched cows had greater (P < 0.01) plasma concentrations than control cows (84.92 vs. 67.08 ng/mL), and at parturition, they had plasma Se concentrations twofold greater than (P < 0.05) control cows (95.51 vs. 47.14 ng Se/mL). After 4 mo, cows receiving Se had greater (P < 0.05) RBC GPX-1 activity than controls; this trend continued until parturition. Colostrum Se concentration was twofold greater (P < 0.05) in Se-drenched cows than control cows (169.97 vs. 87.00 ng/mL). Calves born to cows drenched with Se had greater (P < 0.05) plasma Se concentration, RBC GPX-1, and plasma glutathione peroxidase activity on d 0 compared with calves born to control cows. By d 7, no differences in plasma glutathione peroxidase activity in calves were observed. Maternal Se supplementation did not influence calf thyroid hormone concentrations. Selenium provided by salt and forages is not adequate for cattle in Se-deficient states.

Sensitivity of Japanese Quail (Coturnix japonica), Common Pheasant (Phasianus colchicus), and White Leghorn Chicken (Gallus gallus domesticus) Embryos to In Ovo Exposure to TCDD, PeCDF, and TCDF

Egg injection studies were performed to confirm a proposed model of relative sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this model, species are classified as belonging to one of three categories of sensitivity based on amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon receptor. Embryo lethality and relative potencies of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were compared with TCDD for Japanese quail (Coturnix japonica; least sensitive), Common pheasant (Phasianus colchicus; moderately sensitive), and White Leghorn chicken (Gallus gallus domesticus; most sensitive). Doses ranging from 0.044 to 37 pmol/g egg (0.015-12 ng/g egg) were injected into the air cell of eggs prior to incubation. LD(50) (95% confidence intervals) values, based on rate of hatching for TCDD, PeCDF, and TCDF, were 30 (25-36), 4.9 (2.3-9.2), and 15 (11-24) pmol/g egg for the quail, 3.5 (2.3-6.3), 0.61 (0.28-1.2), and 1.2 (0.62-2.2) pmol/g egg for pheasant, and 0.66 (0.47-0.90), 0.75 (0.64-0.87), and 0.33 (0.23-0.45) pmol/g egg for chicken, respectively. LD(50)-based relative potencies of PeCDF and TCDF were 6.1 and 2.0 for quail, 5.7 and 2.9 for pheasant, and 0.88 and 2.0 for chicken, respectively. TCDD was not the most potent compound among the species tested, with PeCDF and TCDF being more potent than TCDD in the quail and pheasant. TCDF was the most potent in chicken. Species sensitivity was as expected for TCDD and TCDF, whereas for PeCDF, the chicken and pheasant were similar in sensitivity and both were more sensitive than the quail. Results from companion in vitro studies are generally similar to those reported here with a few exceptions.

Transporters in the absorption and utilization of zinc and copper.

Before the discovery and elucidation of transporters, mammals were thought to cotransport Cu or Zn as an anionic complex, such as binding with an AA as a chelate or a receptor such as transferrin. In 1995, the first mammalian Zn transporter (ZnT) gene, ZnT1, was identified. However, 2 protein families are now thought to be involved in Zn transport. The ZnT family reduces intracellular Zn by aiding in efflux from the cell or promoting the influx into intracellular vesicles. The mechanism of ZnT transport against a Zn concentration gradient is unknown; however, only ZnT1 appears to be located at the plasma membrane. It has been shown to respond in tissues in a variety of ways to Zn reduction and supplementation. In our laboratory, we have found ZnT1 and metallothionein to work in concert during pharmacological Zn supplementation. The second protein family, Zip proteins, provides Zn transport from extracellular fluid or intracellular vesicles into the cytoplasm and has not been identified in a livestock species. Like Zn, no good indicator of status has been identified for Cu. However, the recent identification of Cu transporters and chaperones gives researchers the opportunity to understand the regulation of Cu trafficking where the proteins are modified by posttranslational mechanisms. Two Cu transporters, Ctr1 and Ctr3, mediate high-affinity Cu uptake. A small cytoplasmic protein, MURR1, has been identified in human hepatic tissue, but its role in Cu metabolism is unknown. The discovery of Cu chaperones that are involved in facilitating Cu absorption into proteins may provide an excellent status indicator. It has been shown that the Cu chaperone for Cu/Zn superoxide dismutase (CCS) is increased in tissue of Cu-deficient rats, induced when moderately high Zn diets are fed. We have recently found CCS in the young pig. Other Cu chaperone proteins that have been identified are COX17 and Atox1. As with CCS, they are involved in making Cu available to apo-enzymes inside the cell. It is essential that these new molecular findings be used to evaluate the bioavailability of and nutritional need for Cu and Zn in livestock.

Peptide YY3–36 and 5-Hydroxytryptamine Mediate Emesis Induction by Trichothecene Deoxynivalenol (Vomitoxin)

Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium sp. that frequently occurs in cereal grains, has been associated with human and animal food poisoning. Although a common hallmark of DON-induced toxicity is the rapid onset of emesis, the mechanisms for this adverse effect are not fully understood. Recently, our laboratory has demonstrated that the mink (Neovison vison) is a suitable small animal model for investigating trichothecene-induced emesis. The goal of this study was to use this model to determine the roles of two gut satiety hormones, peptide YY3-36 (PYY3-36) and cholecystokinin (CCK), and the neurotransmitter 5-hydroxytryptamine (5-HT) in DON-induced emesis. Following ip exposure to DON at 0.1 and 0.25mg/kg bw, emesis induction ensued within 15-30min and then persisted up to 120min. Plasma DON measurement revealed that this emesis period correlated with the rapid distribution and clearance of the toxin. Significant elevations in both plasma PYY3-36 (30-60min) and 5-HT (60min) but not CCK were observed during emesis. Pretreatment with the neuropeptide Y2 receptor antagonist JNJ-31020028 attenuated DON- and PYY-induced emesis, whereas the CCK1 receptor antagonist devezapide did not alter DONs emetic effects. The 5-HT3 receptor antagonist granisetron completely suppressed induction of vomiting by DON and the 5-HT inducer cisplatin. Granisetron pretreatment also partially blocked PYY3-36-induced emesis, suggesting a potential upstream role for this gut satiety hormone in 5-HT release. Taken together, the results suggest that both PYY3-36 and 5-HT play contributory roles in DON-induced emesis.

Dietary exposure of mink (Mustela vison) to fish from the upper Hudson River, New York, USA: Effects on reproduction and offspring growth and mortality

The effects of feeding farm-raised mink (Mustela vison) diets containing polychlorinated biphenyl (PCB)-contaminated fish from the upper Hudson River (New York, USA) on adult reproductive performance and kit growth and mortality were evaluated. Diets contained 2.5 to 20% Hudson River fish, providing 0.72 to 6.1 µg ∑PCBs/g feed (4.8-38 pg toxic equivalents [TEQWHO 2005 ]/g feed). The percentage of stillborn kits per litter was significantly increased by dietary concentrations of 4.5 µg ∑PCBs/g feed (28 pg TEQWHO 2005 /g feed) and greater. All offspring exposed to dietary concentrations of 4.5 and 6.1 µg ∑PCBs/g feed (28 and 38 pg TEQWHO 2005 /g feed) died by 10 weeks of age, and all offspring exposed to 1.5 and 2.8 µg ∑PCBs/g feed (10 and 18 pg TEQWHO 2005 /g feed) died by 31 weeks of age, leaving juveniles in the control and 0.72 µg ∑PCBs/g feed (0.41- and 4.8 pg TEQWHO 2005 /g feed) groups only. The dietary concentration predicted to result in 20% kit mortality (LC20) at six weeks of age was 0.34 µg ∑PCBs/g feed (2.6 pg TEQWHO 2005 /g feed). The corresponding maternal hepatic concentration was 0.80 µg ∑PCBs/g liver, wet weight (13 pg TEQWHO 2005 /g liver, wet wt). Mink residing in the upper Hudson River would be expected to consume species of fish that contain an average of 4.0 µg ∑PCBs/g tissue. Thus, a daily diet composed of less than 10% Hudson River fish could provide a dietary concentration of ∑PCBs that resulted in 20% kit mortality in the present study.

Comparison of Anorectic and Emetic Potencies of Deoxynivalenol (Vomitoxin) to the Plant Metabolite Deoxynivalenol-3-Glucoside and Synthetic Deoxynivalenol Derivatives EN139528 and EN139544

The mycotoxin deoxynivalenol (DON) elicits robust anorectic and emetic effects in several animal species. However, less is known about the potential for naturally occurring and synthetic congeners of this trichothecene to cause analogous responses. Here we tested the hypothesis that alterations in DON structure found in the plant metabolite deoxynivalenol-3-glucoside (D3G) and two pharmacologically active synthetic DON derivatives, EN139528 and EN139544, differentially impact their potential to evoke food refusal and emesis. In a nocturnal mouse food consumption model, oral administration with DON, D3G, EN139528, or EN139544 at doses from 2.5 to 10 mg/kg BW induced anorectic responses that lasted up to 16, 6, 6, and 3 h, respectively. Anorectic potency rank orders were EN139544>DON>EN139528>D3G from 0 to 0.5 h but DON>D3G>EN139528>EN139544 from 0 to 3 h. Oral exposure to each of the four compounds at a common dose (2.5 mg/kg BW) stimulated plasma elevations of the gut satiety peptides cholecystokinin and to a lesser extent, peptide YY3-36 that corresponded to reduced food consumption. In a mink emesis model, oral administration of increasing doses of the congeners differentially induced emesis, causing marked decreases in latency to emesis with corresponding increases in both the duration and number of emetic events. The minimum emetic doses for DON, EN139528, D3G, and EN139544 were 0.05, 0.5, 2, and 5 mg/kg BW, respectively. Taken together, the results suggest that although all three DON congeners elicited anorectic responses that mimicked DON over a narrow dose range, they were markedly less potent than the parent mycotoxin at inducing emesis.

Effect of dietary organic microminerals on starter pig performance, tissue mineral concentrations, and liver and plasma enzyme activities

Weanling pigs (n = 160) were used to evaluate dietary essential microminerals (Cu, Fe, Mn, Se, and Zn) on performance, tissue minerals, and liver and plasma enzymatic activities during a 35-d postweaning period. A randomized complete block design with 5 treatments and 8 replicates was used in this study. Organic microminerals were added to complex nursery diets at 0 (basal), 50, 100, or 150% of the requirements of microminerals listed by the 1998 NRC. A fifth treatment contained inorganic microminerals at 100% NRC and served as the positive control. Pigs were bled at intervals with hemoglobin (Hb), hematocrit (Hct), glutathione peroxidase, and ceruloplasmin activities determined. Six pigs at weaning and 1 pig per pen at d 35 were killed, and the liver, heart, loin, kidney, pancreas, and the frontal lobe of the brain were collected for micromineral analysis. The liver was frozen in liquid N for determination of enzymatic activities. The analyzed innate microminerals in the basal diet met the NRC requirement for Cu and Mn but not Fe, Se, and Zn. Performance was not affected from 0 to 10 d postweaning, but when microminerals were added to diets, ADG, ADFI, and G:F improved (P < 0.01) from 10 to 35 d and for the overall 35-d period. Pigs fed the basal diet exhibited parakeratosis-like skin lesions, whereas those fed the supplemental microminerals did not. This skin condition was corrected after a diet with the added microminerals was fed. When the basal diet was fed, Hb and Hct declined, but supplemental microminerals increased Hb and Hct values. Liver catalase activity increased (P < 0.01) when microminerals were fed. The Mn superoxide dismutase activity tended to decline quadratically (P = 0.06) when supplemental microminerals were fed above that of the basal diet. Liver plasma glutathione peroxidase activities were greater (P < 0.01) when dietary organic and inorganic micromineral were fed. Liver concentrations of microminerals increased linearly (P < 0.01) as dietary microminerals increased, indicating that the liver was the primary storage organ. Micromineral tissue concentrations were least in pigs fed the basal diet and increased (quadratic, P < 0.01) to the 50% level of organic microminerals in the various tissues collected. The results indicated that innate microminerals, Cu and Mn, from a complex nursery diet may meet the micromineral needs of the weaned pig, but the need for Fe, Se, or Zn was not met by the basal diet.

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.

Gene expression profiling in hepatic tissue of newly weaned pigs fed pharmacological zinc and phytase supplemented diets

BackgroundZinc (Zn) is an essential trace element. However, Zn bioavailability from commonly consumed plants may be reduced due to phytic acid. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Moreover, adding supplemental phytase improves Zn bioavailability. Thus, we hypothesized that benefits of pharmacological Zn supplementation result from changes in gene expression that could be further affected by supplemental phytase. The goal of this study was to investigate the effects of feeding newly weaned pigs dietary Zn (150, 1,000, or 2,000 mg Zn/kg) as Zn oxide with or without phytase [500 phytase units (FTU)/kg] for 14 d on hepatic gene expression. Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg (n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray.ResultsExpressed sequence tags for 61 putatively differentially expressed transcripts were cloned and sequenced. In addition, interrogation of a 13,297 element oligonucleotide microarray revealed 650 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation. Seven transcripts exhibiting differential expression in pigs fed pharmacological Zn with sequence similarities to genes encoding GLO1, PRDX4, ACY1, ORM1, CPB2, GSTM4, and HSP70.2 were selected for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and ACY1 (P < 0.01) mRNA abundances were confirmed to be greater in pigs fed 1,000 (n = 8) and 2,000 (n = 8) mg Zn/kg than in pigs fed 150 (n = 7) mg Zn/kg. Relative hepatic HSP70.2 (P < 0.002) mRNA abundance was confirmed to be lower in pigs fed 2,000 mg Zn/kg than in pigs fed 150 or 1,000 mg Zn/kg.ConclusionResults suggest that feeding pharmacological Zn (1,000 or 2,000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.