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High dietary inorganic selenium has minimal effects on turkeys and selenium status biomarkers
High dietary inorganic selenium has minimal effects on turkeys and selenium status biomarkers
Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706 tions that were 5.6X, 1.7X, 1.9X, and 2.0X greater than Se-adequate levels in liver, kidney, breast, and thigh, respectively, and GPX activities in plasma, red cells, liver, kidney, and heart that were ≤2.0X Se-adequate values. In liver, kidney, heart, gizzard, breast, or thigh, no selenoprotein transcript was increased ≥2.0X, and no selenoprotein transcript was decreased ≤0.5X by 2 or 5 μg Se/g diet as compared to poults fed 0.4 μg Se/g diet. Of the 112 Se status biomarkers reported in this study, liver Se concentration was the only biomarker markedly altered by high Se status. This study provides evidence of no adverse effects in turkey poults fed up to 5 μg Se/g diet as inorganic Se. Thus, the FDA limit for Se supplementation in turkey feed can be safely raised to 0.5 μg Se/g diet. Future studies are needed to identify biomarkers for high Se status and to better understand how turkeys maintain Se homeostasis and resist Se toxicity.
ABSTRACT The current NRC turkey Se requirement is 0.2 μg Se/g diet. Recent studies evaluating tissue Se, glutathione peroxidase (GPX) activities, and selenoprotein transcript expression concluded that the dietary Se requirement of the turkey poult should be raised to 0.4 μg Se/g when supplemented with inorganic Se. The FDA currently limits Se inclusion in premixed diets for poultry and other livestock species to 0.3 μg Se/g diet. Thus, there is a need to investigate the effect of high dietary Se (>1.0 μg Se/g) in turkeys. The present study fed turkey poults 2 and 5 μg Se/g diet to characterize tissue Se accumulation in turkey poults fed high dietary inorganic Se and to evaluate the efficacy of selenoprotein activity and transcript expression as biomarkers of high Se status. Day-old male poults were fed 0.4, 2, or 5 μg Se/g for 28 d. There was no significant effect of Se supplementation on poult growth. Supplementation with 5 μg Se/g diet resulted in Se concentra-
Key words: glutathione peroxidase, selenoprotein, supplementation, transcript 2018 Poultry Science 0:1–11 http://dx.doi.org/10.3382/ps/pey413
INTRODUCTION
and Scott, 1975; Cantor et al., 1982). Only a few feeding studies have examined the effect of supernutritional (≤1.0 μg Se/g) and high (>1.0 μg Se/g) Se intake on biomarkers of Se status. An early study fed 0.1 and 1 μg Se/g diet; 1 μg Se/g diet completely prevented Se deficiency disease and reduced 6-wk mortality to from 20% to 0% (Walter and Jensen, 1963). To further refine the dietary requirement, Scott and colleagues fed up to 0.8 μg Se/g (Scott and Thompsom, 1971), and reported that tissue Se plateaued at 0.2 to 0.3 μg Se/g diet and that there was no reduction in growth in poults fed up to 0.8 μg Se/g. The FDA currently limits Se inclusion in premixed diets for poultry and other livestock species to 0.3 μg Se/g diet (United States Food and Drug Administration, 2004), regardless of differences in requirements between species (Sunde et al., 2016). Thus, there is a need to investigate the effect of high levels of dietary Se in turkeys to better characterize the impact when Se is fed at levels above the FDA limit of 0.3 μg Se/g. The activities of the selenoenzymes GPX and thioredoxin reductase (TXNRD) fall to <10% of Seadequate values when poults are fed a Se-deficient diet, and do not reach plateau levels of expression until 0.2
The current NRC turkey Se requirement for preventing overt Se deficiency disease is 0.2 μg Se/g diet, based on studies conducted over 50 yr ago (Scott et al., 1967; National Research Council, 1994). More recently, two groups supplemented turkey poults with multiple graded levels of Se up to 0.4 to 0.5 μg Se/g and found that the minimum Se requirement was 0.3 μg Se/g diet (Fischer et al., 2008; Sunde and Hadley, 2010). Our recent studies used tissue Se, glutathione peroxidase (GPX) activities, and selenoprotein transcript expression to examine the effect of supernutritional dietary Se up to 1 μg Se/g, and concluded that the dietary Se requirement of the turkey poult should be raised to 0.4 μg Se/g to achieve plateau levels of tissue Se and GPX activity in all tissues when supplemented with inorganic Se (Taylor and Sunde, 2016, 2017). Most published Se research in turkeys has focused on deficient-to-adequate levels of dietary Se (Cantor C 2018 Poultry Science Association Inc. Received April 30, 2018. Accepted August 10, 2018. 1 Corresponding author: [email protected]
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Rachel M. Taylor, Victor G. Bourget, and Roger A. Sunde1
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MATERIALS AND METHODS Reagents Molecular biology reagents were purchased from Promega (Madison, WI, USA), Invitrogen (Carlsbad, CA, USA), or Sigma (St. Louis, MO, USA). All other chemicals were of molecular biology or reagent grade.
Animals and Diets For the high-Se study reported here, day-old male Nicholas White-derived turkey poults (kindly donated by Jennie-O Turkey Store, Barron, WI) were allocated randomly to treatment and housed in battery cages (4/cage) with raised wire floors and 24-h lighting. The temperature was set at 95◦ F for the first week and lowered 5◦ F in each subsequent week. Animals were fed from stainless steel troughs ad lib, and allowed free access to deionized water in plastic waterers. The basal Se-deficient torula yeast-based diet (0.005 μg Se/g) was the same as we have used in previous turkey experiments (Taylor and Sunde, 2016, 2017) and modified from rodent diets we have used previously (Sunde and Hadley, 2010). The feed was prepared with the same lot of low-Se yeast used previously (Sunde and Hadley, 2010) to ensure Se deficiency. The diet was supplemented with 7.0% crystalline amino acids, including 0.93% methionine, to better match NRC rec-
ommendations for protein and amino acids. Vitamin E (as all-rac-α-tocopherol acetate) was supplemented at 150 mg/kg (12.5X NRC requirement), and levels of all other vitamins and minerals were supplemented to provide 150% of the requirements (National Research Council, 1994). The basal diet was supplemented with 0.4, 2, or 5 μg Se/g diet as Na2 SeO3 . Body weight was measured twice weekly and feed intake per cage was measured weekly. The animal protocol was approved by the Research Animal Resources Committee at the University of Wisconsin–Madison (protocol no. A005368).
Tissue Collection Poults from all treatments were killed at 28 d by terminal CO2 overexposure followed by exsanguination. Blood was collected in heparinized tubes and stored on ice, then spun at 1500× g for 15 min at 4◦ C (Eppendorf 5415R, F-45-24-11 rotor, Brinkmann, Westbury, NY) to separate plasma from red blood cells (RBC). RBC were reconstituted to original volume with saline phosphate buffer (76 nmol/L NaCl, 50 nmol/L sodium phosphate, pH 7.4). Breast muscle (pectoralis major), thigh muscle (iliotibialis), liver, gizzard, heart, and kidney were collected and immediately frozen at –80◦ C until analysis.
Enzyme Activity Analysis Kidney, heart, breast, and thigh were homogenized in 9 vol of sucrose buffer (20 mmol/L tris-HCl, pH 7.4, 0.25 mmol/L sucrose, 1.1 mmol/L EDTA, 0.1% peroxide-free Triton-100) and centrifuged (10,000× g, 15 min). GPX1 (glutathione peroxidase 1) activity was measured by the coupled assay procedure (Lawrence et al., 1974) using 120 μmol/L H2 O2 . GPX4 (glutathione peroxidase 4) activity was measured by the coupled assay procedure (Lei et al., 1995) using 78 μmol/L phosphatidylcholine hydroperoxide. GPX1specific activity was calculated by subtracting the activity due to GPX4, as described previously (Sunde and Hadley, 2010). In both assays, 1 enzyme unit is defined as the amount of enzyme that will oxidize 1 μmol glutathione per min under the specified conditions. TXNRD activity was measured using the goldinhibition assay with dithiobisnitrobenzoic acid (Sigma D8130) as the substrate, as described previously (Hill et al., 1997). The protein concentration was determined by the method of Lowry et al. (1951).
Tissue Se Analysis Thirty-six samples (n = 3/treatment) from liver, kidney, thigh, and breast were sent to the University of Missouri Research Reactor Center (1513 Research Park Dr, Columbia, MO 65211) for Se concentration analysis. Freeze-dried samples were analyzed for Se on a dry matter (DM) basis by standard-comparison instrumental neutron activation analysis, measured by highresolution gamma spectroscopy, as described previously
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to 0.4 μg Se/g diet (Hadley and Sunde, 1996; Fischer et al., 2008; Taylor and Sunde, 2016, 2017), making them good biomarkers of deficient-to-adequate Se status. In rodents, a few selenoprotein transcripts— notably Gpx1, Selenoh, and Selenow—also fall dramatically in Se deficiency and can be used as biomarkers of Se status to asses Se requirements (Barnes et al., 2009; Sunde et al., 2009). Turkeys and other avian species have 24 selenoproteins (Li et al., 2018; Sunde et al., 2015), of which most are preserved in Se deficiency; but in liver and kidney, 6 transcripts reliably fall to <50% of Se-adequate values in Se deficiency and are good biomarkers of deficient Se status. In these studies, however, no enzymatic or transcript biomarker was shown to be changed significantly by dietary Se up to 1 μg Se/g in the turkey (Taylor and Sunde, 2016, 2017). Sensitive and precise biomarkers of Se status across a range of intakes are necessary to further evaluate the FDA limit on Se supplementation. Thus, the present study fed day-old male turkey poults 2 and 5 μg Se/g diet with three objectives: (1) to characterize tissue Se accumulation in turkey poults fed high (≥1.0 μg Se/g) inorganic dietary Se; (2) to evaluate the efficacy of traditional selenoenzyme activity biomarkers for determining high Se status; and (3) to identify the effect of high Se status on selenoprotein transcripts as biomarkers. In addition, this study may lead to a better understanding of the susceptibility of the turkey to high Se.
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(Barnes et al., 2009; Sunde and Hadley, 2010; Taylor and Sunde, 2017). Se concentration on a wet matter basis was calculated by multiplying the DM Se concentration by the fractional DM.
Total RNA from liver, kidney, heart, gizzard, breast, and thigh (∼100 mg tissue, n = 4/treatment) was isolated with TRIzol Reagent (Invitrogen, catalog no. 15596-026) following the manufacturer’s protocol. The RNA pellet was dissolved in 300 μl diethylpyrocarbonate-treated water and quantitated using a DeNovix DS-11+ spectrophotometer (DeNovix Inc., Wilmington, DE). Relative mRNA abundance was determined by quantitative real-time PCR (qPCR). RNA (1 μg) was reverse transcribed to cDNA using the RETROscript kit (AM1710, Ambion) following the manufacturer’s protocol, and diluted 1/20 to make cDNA working stocks. Turkey gene-specific primer sets were based on the recently sequenced turkey selenoproteome (Sunde et al., 2015) and designed to span an intron–exon splice junction and amplify a 120 to 150base segment. The final 12 μl reactions contained 2 μl reverse transcribed cDNA working stock, 0.2 mmol/L turkey gene-specific forward and reverse primers, and 1X KAPA SYBR FAST qPCR Kit (KAPA Biosystems no. KK4611). Reactions were followed in a LightCycler 480 (Roche Life Science) using the manufacturer’s SYBR Green 384-II template (initial stage of 95◦ C for 5 min, followed by 45 amplification cycles of 95◦ C, 60◦ C, and 72◦ C for 10 s each). Melting curves were generated to confirm the presence of one specific product and standard curves were run for each primer set/tissue combination, and the second derivative max program was used to determine amplification efficiency following the manufacturer’s protocol. mRNA relative abundance was calculated according to Pfaffl (2001), accounting for gene-specific efficiencies, normalized to the mean of β -actin and glyceraldehyde-3-phosphate dehydrogenase expression, and expressed as a percentage of the plateau of Se-adequate (0.4 μg Se/g) levels.
Statistical Analysis Data are presented as mean ± standard error of the mean (SEM). For growth, enzyme analysis and mRNA expression, n = 4/treatment; for tissue Se concentration, n = 3/treatment. Data were analyzed by oneway ANOVA and variance equality was tested using Levene’s test for homogeneity of variances (Brown and Forsythe, 1974). When the main effect of diet was significant, differences between means were assessed by Duncan’s multiple range test (P < 0.05) with Kramer’s modification for unequal class sizes when necessary (Steel and Torrie, 1960). The curves plotted in Figures 2, 3, 4, and 6 are linear, hyperbolic or logistic regressions calculated using all individual values, similar to
Figure 1. Growth of day-old male poults supplemented with 0.4, 2, or 5 μ g Se/g diet (n = 4/treatment) for 28 d and measured biweekly. Values are means ± SEM, P = 0.17 at d 28.
Se-response curves plotted when studying Se requirements (Li and Sunde, 2016; Taylor and Sunde, 2016, 2017). For comparison, we have also plotted in Figures 2, 3, 4, 6, and S1 tissue Se and selenoenzyme activities that were published previously for poults supplemented with 0 to 1 μg Se/g diet (Taylor and Sunde, 2016, 2017). Tissue Se concentrations were measured concurrently and Se-adequate selenoenzyme activities were reanalyzed simultaneously to verify relative expression.
RESULTS Growth Day-old poults weighed an average of 58 IS ± 1.1g at the start of the experiment, and after 28 d on treatment, poults supplemented with 0.4, 2, and 5 μg Se/g diet weighed 741 ± 61, 569 ± 50, and 589 ± 70g, respectively (Figure 1), but there was no significant effect of Se supplementation on final body weight of the poults (P = 0.17). Even over the last 7 d, there was no significant difference in growth rate amongst the groups (P = 0.21). Poult growth in this experiment for poults supplemented with 2 and 5 μg Se/g diet was similar to growth of poults fed 0 to 1 μg Se/g diet in our previous experiment (Taylor and Sunde, 2016; Figure S1). These poults, supplemented with 150 mg/kg vitamin E (12.5X NRC requirement), were generally healthy and showed no gross tissue abnormalities, and there was no effect of Se treatment on tissue weights as a percentage of body weight (data not shown).
Tissue Se Concentration Se concentrations in poults fed the Se-adequate diet (0.4 μg Se/g) averaged 5.4 ± 0.5 nmol Se/g tissue wet weight in liver, 8.1 ± 0.5 nmol/g in kidney, 0.87 ± 0.04 nmol/g in breast, and 1.1 ± 0.03 nmol/g in thigh (Figure 2). Supplementation with 5 μg Se/g diet resulted in Se concentrations that averaged 5.6X, 1.7X, 1.9X, and 2.0X greater than Se-adequate levels in each tissue, respectively. Only in poult liver was the increase
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RNA Isolation and Analysis
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in tissue Se statistically significant (P < 0.05) at both 2 and 5 μg Se/g diet (Figure 2A). In kidney, breast, and thigh, tissue Se concentrations did not increase dramatically above Se-adequate values (Figure 2).
Enzyme Activity The relative rank of GPX1 activity in Se-adequate poults was kidney >> liver > heart > red cell > thigh > breast, gizzard (Figure 3). Supplementation with 5 μg Se/g diet resulted in GPX activities in plasma, red cells, liver, kidney, and heart that were ≤2X the Se-adequate values. GPX3 activity in plasma from Seadequate poults was 54.7 ± 3.2 EU/g protein; GPX3 activity in plasma in poults supplemented with 2 and 5 μg Se/g was 1.7X and 1.8X Se-adequate values (P = 0.002), respectively (Figure 3A). RBC GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 1.5X and 1.4X Se-adequate values (P = 0.008; Figure 3B). GPX1 activity in liver of Se-adequate poults was 73.4 ± 10 EU/g, and poults supplemented with 2 and 5 μg Se/g averaged 1.3X and 1.4X Se-adequate values (P = 0.27; Figure 3C). Kidney GPX1 activities in
mRNA Expression To survey selenoprotein transcript expression for potential biomarkers of Se status, we conducted qPCR of selenoprotein transcripts (Figure 5). In poult liver, no selenoprotein transcript was increased ≥2.0X and no selenoprotein transcript was decreased ≤0.5X by 2 or 5 μg Se/g diet as compared to expression in poults fed 0.4 μg Se/g diet (Figure 5A), and none of these changes were significant. Also in kidney, no selenoprotein transcript was increased ≥2.0X or decreased ≤0.5X by 2 or 5 μg Se/g; kidney GPX3 and SELENOP1 (selenoprotein P) mRNA were significantly upregulated by
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Figure 2. Effect of Se supplementation on tissue Se concentration. Se concentration in (A) liver ( r) and kidney () and (B) breast ( r) and thigh () of 28-d-old turkey poults, expressed as nmol/g wet tissue. Values are means ± SEM (n = 3/treatment). Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are Se concentrations in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
poults supplemented with 2 and 5 μg Se/g were 1.4X Se-adequate values (P = 0.001; Figure 3D), and heart GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 1.8X and 1.7X Se-adequate values (P = 0.046; Figure 3E). In gizzard, breast, and thigh, the tissues with the lowest levels of GPX1 activity, supplementation with 5 μg Se/g resulted in GPX1 activities that were 2 to 4X the Se-adequate values. Gizzard GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 2.1X and 2.7X Se-adequate values (P = 0.009; Figure 3F). Breast muscle GPX1 activities in poults were 1.8X and 2.4X Seadequate values (P = 0.004), respectively (Figure 3G), and thigh GPX1 activities were 2.4X and 3.6X Seadequate values (P = 0.001), respectively (Figure 3H). The relative rank of GPX4 activity in Se-adequate poults, as well as poults supplemented with 5 μg Se/g, was liver > heart, kidney > thigh > breast, gizzard (Figure 4). GPX4 activity in liver of Se-adequate poults was 32.7 ± 3.9 EU/g, and poults supplemented with 2 and 5 μg Se/g averaged 1.2X and 1.3X Se-adequate values (P = 0.24; Figure 4A). Kidney GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.3X and 1.2X Se-adequate values, respectively (Figure 4B), and heart GPX4 activities were 1.3X and 1.1X Se-adequate levels, respectively (Figure 4C). GPX4 activities in Se-adequate gizzard, breast, and thigh muscles were less than half the levels in liver, kidney, and heart. Gizzard GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.8X and 1.8X Se-adequate levels, respectively (Figure 4D). Breast GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.2X and 1.2X Se-adequate levels, respectively (Figure 4E), and thigh GPX4 activities were 1.3X and 1.3X Se-adequate levels, respectively (Figure 4F). Clearly, neither GPX1 nor GPX4 activity are substantially increased when turkey poults are supplemented with 5X and 12.5X the Se requirement (0.4 μg Se/g). We also determined TXNRD enzyme activity in liver, the turkey tissue with the highest level of TXNRD activity. Liver TXNRD was not significantly different between groups supplemented with 0.4, 2, or 5 μg Se/g diet, averaging 3.08 ± 0.24, 2.97 ± 0.28, and 3.48 ± 0.19 EU/g (P = 0.34; Figure 4G).
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2 μg Se/g but not by 5 μg Se/g diet (P = 0.012 and P = 0.034, respectively, Table S1). No other selenoprotein transcript was significantly altered by high dietary Se. LRP2 (megalin), the renal SELENOP1 receptor in mammals, was only expressed in kidney and was not significantly regulated by dietary Se (Figure 5B). In heart, gizzard, breast, or thigh muscle, no selenoprotein transcript was significantly up- or downregulated
by high dietary Se (Figure 5C and D). In heart, DIO1 (deiodinase 1), DIO3, GPX2, and SELENOP2 were not expressed above the level of detection; in gizzard, DIO1, GPX2, and SELENOP2 were not expressed above the level of detection (Table S1). In breast, DIO1 and GPX2 mRNA were not expressed above the level of detection; in thigh, DIO1, GPX2, and TXNRD3 were not expressed above the level of detection.
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Figure 3. Effect of Se supplementation on tissue GPX1 and GPX3 activity. GPX3 activity in (A) plasma and GPX1 activity in (B) red blood cells, (C) liver, (D) kidney, (E) heart, (F) gizzard, (G) breast, and (H) thigh of 28-d-old turkey poults. Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are activities in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
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DISCUSSION Growth and Toxicity There was no significant effect of high dietary Se on poult growth in this study. Although final weights in poults supplemented with 2 and 5 μg Se/g were 30 and 26% lower, respectively, than final weights of Seadequate poults, these differences were not statistically
significant (P = 0.17), and the final weights of all groups were also similar (P = 0.58) to average weights of poults in our previous study that used poults and diets from the same sources (Figure S1; Taylor and Sunde, 2016). Rates of growth, as determined by analysis of covariance, were also not significantly different over any period of the experiment, including the last 7 d (P = 0.21, data not shown).
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Figure 4. Effect of Se supplementation on tissue GPX4 and TXNRD activity. GPX4 activity in (A) liver, (B) kidney, (C) heart, (D) gizzard, (E) breast, (F) thigh, and (G) liver TXNRD activity of 28-d-old turkey poults. Values are means ± SEM (n = 4/treatment). Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are activities in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
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Tissue Se and Accumulation Differences
Figure 5. Effect of high dietary Se on relative mRNA expression of selenoprotein and selected Se-related transcripts. Transcript expression in (A) liver, (B) kidney, (C) breast, and (D) thigh of 28-d-old turkey poults, relative to expression in poults fed 0.4 μ g/g. Values are means ± SEM (n = 4/treatment). ∗ P < 0.05. Overall level of significance given in Table S1.
Of the 112 biomarkers of Se status (including tissue Se, GPX, and TXNRD activities, and up to 27 transcripts in seven tissues) reported in this study, liver Se was the only biomarker markedly altered by high Se status. Liver in poults supplemented with 2 μg Se/g had double the Se concentration vs. poults fed 0.4 μg Se/g, and liver in poults fed 5 μg Se/g had almost 6X Se concentration vs. Se-adequate liver. In contrast, tissue Se concentration did not markedly rise above Seadequate levels in the other three tissues studied. Kidneys of poults supplemented with 5 μg Se/g had less than 2X the Se content of kidneys from Se-adequate poults. Importantly, in this production animal, muscle Se concentration in breast and thigh also increased <2X relative to Se-adequate poults. Thus in poults fed 5 μg Se/g, liver Se was 2.3X the level in kidney and 15X the level in breast and thigh. Poults in a previous study fed 0.38 to 0.39 μg Se/g diet (starter and finishing diets, as Na2 SeO4 ) for 35 d also had liver Se at ∼8X the Se concentration as breast and thigh (Fischer et al., 2008). In mammals, however, Salbe and Levander (1990) found that liver and kidney of rats supplemented with 2.5 μg Se/g diet had 2X and 4X tissue Se, respectively, as Seadequate rats. In another experiment, liver from rats fed 5 μg Se/g diet had 4X Se-adequate levels (Raines and Sunde, 2011). Rats supplemented with 4 μg Se/g diet had liver and kidney Se concentrations of approximately 2X the level of Se found in Se-adequate samples (Whanger and Butler, 1988). Thus, at least in poults supplemented with high inorganic Se, liver is the tissue that accumulates Se, whereas muscle tissues only show modest increases. In mammals, the kidney is often the tissue that accumulates high concentrations when animals are fed elevated levels of trace minerals. Based on the lack of increase in selenoprotein transcript expression and selenoenzyme activity in turkey liver presented here, it is unlikely that the accumulated Se in liver is present
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A study focused on preventing aflatoxin toxicity reported that 10 μg Se/g diet as Na2 SeO3 had no effect on growth when fed to day-old turkey poults (Burguera et al., 1983). Previous studies with day-old chicks reported that feeding 5 μg Se/g as Na2 SeO3 for 3 wk reduced final body weights by 26% (Jensen, 1986). Other studies in chickens have shown no effects on egg weight, production or hatchability when dietary Se as Na2 SeO3 is fed to laying hens at levels up to 8 μg Se/g diet (Ort and Latshaw, 1978). In contrast, two studies in weanling pigs demonstrated that 5 μg Se/g but not 2.5 μg Se/g diet as Na2 SeO3 significantly reduced growth (Mahan and Moxon, 1984; Wahlstrom et al., 1984). Previous research in weanling rats has shown that feeding 5 μg Se/g as Na2 SeO3 for 4 wk reduced final body weights by 24% (Raines and Sunde, 2011). Thus the literature indicates that avians in general are more resistant to general Se toxicity than mammals.
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Selenoenzyme Activity as a Biomarker for High Se Status Activities of the selenoenzymes GPX1 and GPX3 are frequently used as biomarkers of Se status in humans and animals (Burk and Hill, 2015; Combs, 2015). In our previous studies focused on Se deficiency biomarkers, we found that GPX1, GPX3, GPX4, and TXNRD activities were good biomarkers that distinguished Se-deficient from Se-adequate turkey poults (Taylor and Sunde, 2016, 2017), and in other species (Sunde et al., 2016). In the present experiment, when birds were supplemented with 5 to 12.5X the dietary Se requirement, GPX4 activity did not increase in any tissue and only modest increases were seen for GPX1 activity in plasma, red cells, kidney, heart, gizzard, breast, and thigh. Liver TXNRD activity also did not rise with increasing dietary Se above the plateau. Although small increases in some biomarker levels were seen at 2 and 5 μg Se/g diet, we suggest that these minor increases do not justify raising the Se requirement for young male poults higher than 0.4 μg Se/g diet. This study demonstrates that none of the measured selenoenzyme activities can be used as biomarkers of high Se status.
Relationship Between Tissue Se and GPX Activity We have previously found that similar levels of dietary Se (0.3 to 0.4 μg Se/g) are required to raise GPX1 activity and Se concentration to plateau levels in the turkey. Plotting kidney GPX1 activities relative to tissue Se concentrations, using data from this high Se study and our previous Se-deficient-to-supernutritional study (Taylor and Sunde, 2016, 2017), reveals that these two biomarkers increase roughly in parallel in kidney over the range from Se-deficient to high Se (Figure 6A). This relationship is sigmoidal (P < 0.001), indicating that relatively less Se is incorporated into GPX1 when tissue Se is <5 nmol/g, reflecting the downregulation of GPX1 mRNA in Se deficiency. Plots of this data for breast and thigh are similar (data not shown). In liver, in contrast, there is still a near-linear increase in GPX1 activity as tissue Se increases when poults are supplemented with up to 1 μg Se/g diet (re-
sulting in liver Se <10 nmol/g). High Se supplementation (2 and 5 μg Se/g), however, had little impact on GPX1 activity (Figure 6B). This demonstrates that with high inorganic Se supplementation, turkey poults accumulate Se in liver in a form distinct from incorporation into GPX1, GPX4, and TXNRD. The lack of significant increases in any of the other selenoprotein transcripts further suggests that this Se is present in a form other than in selenoproteins.
Transcript Expression In our previous study, we found that a limited number of selenoprotein transcripts were good biomarkers for Se deficiency, including transcripts for GPX1, GPX3, and GPX4. In five out of six tissues assessed here (heart and gizzard data not shown), no selenoprotein transcript was significantly up- or downregulated by high Se. In kidney, just GPX3 and SELENOP1 transcripts were significantly upregulated, although to only <2X Se-adequate values in poults fed 2 μg Se/g diet, and nonsignificantly when fed 5 μg Se/g (Table S1). Liver GPX3 transcript expression followed a similar pattern; it was nonsignificantly upregulated to 1.8X and 1.1X of Se-adequate values by 2 and 5 μg Se/g diet, respectively (Table S1). This study, as well as our previous research, found that GPX3 transcript expression is high in many turkey tissues, including heart, gizzard, and skeletal muscle (Taylor and Sunde, 2017), indicating GPX3, the major plasma GPX, may be synthesized in a variety of tissues in the bird. In contrast, circulating plasma GPX3 in mammals is primarily synthesized in the kidney, and correspondingly Gpx3 transcript expression is biased to mammalian kidney (Avissar et al., 1994; Olson et al., 2010). The lipoprotein receptor megalin (LRP2) has been shown to be required for reuptake and recycling of SELENOP from kidney proximal tubules (Olson et al., 2008), and it is a key player in whole-body Se homeostasis (Krol et al., 2012). We measured expression of LRP2 in liver and kidney; consistent with what has been seen in other species, LRP2 was not expressed at detectable levels in turkey liver, but was moderately expressed in turkey kidney and not regulated by dietary Se level (Figure 5B, Table S1). Thus, this study found no selenoprotein transcript that could be used as a biomarker of high Se status. Given that the turkey response to high dietary Se is not mediated through alteration of selenoprotein transcript expression, a wholetranscriptome analysis might identify non-selenoprotein transcripts or pathways altered under conditions of high dietary Se.
FDA Limits on Supplemental Se in Livestock Feeds Our previous study showed that the minimum dietary Se requirement of the young poult is 0.4 ug
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in selenoproteins; the nature of this Se is yet unknown and the exact form would be of great interest. When excess Se is supplemented to animals as an organic form such as SeMet or its analogs, Se accumulates in many tissues, especially muscle, as SeMet is nonspecifically incorporated into proteins in place of Met (Pappas et al., 2011), but inorganic Se is not generally incorporated into proteins that do not contain a selenocysteine codon (Burk and Hill, 2015). It would be important to extend the length of this feeding trial to measure Se status biomarkers at finishing. This may reveal different tissue Se accumulation patterns in mature birds.
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Se/g diet when supplemented as inorganic Se, and that selenoprotein enzyme activities and tissue Se levels were little increased when poults were supplemented with up to 2.5X this level. The FDA currently limits supplemental Se, as Na2 SeO3 , Na2 SeO4 , or selenized yeast, in complete feeds to 0.3 μg Se/g diet for chickens, ducks, turkeys, sheep, swine, and cattle (United States Food and Drug Administration, 2004). The European Union uses the recommendation of the European Food Safety Authority (EFSA), which states that Se as Na2 SeO3 or SeMet may be incorporated into feed for all livestock species such that the total diet contains no more than 0.5 μg Se/g diet (EFSA Panel on Additives and Products or Substances used in Animal Feed, 2013, 2016). Collectively, our studies provide little evidence of adverse effects in turkey poults fed up to 5 μg Se/g diet as inorganic Se, and muscle tissues did not accumulate Se at greater than 2X Se-adequate levels. Raising the FDA limit for Se in complete feed, at least for turkeys, to 0.5 μg Se/g as inorganic Se, would provide the requirement of 0.4 μg Se/g, allow for a margin of safety, and would match the EFSA recommendation.
CONCLUSION The present study demonstrates that excess dietary Se does not dramatically impair growth or alter selenoenzyme activity or selenoprotein transcript expression in the young poult. Importantly, Se concentration
in extrahepatic tissues does not notably rise above Seadequate levels (0.4 μg Se/g) with dietary Se supplemented as high as 5 μg Se/g. Even turkey liver accumulates Se at less than 2X when inorganic dietary Se is supplemented at 5X the requirement. These results further indicate that the FDA limit for selenite supplementation in turkey feed can be safely raised to 0.5 μg Se/g diet. Future studies are needed to identify biomarkers for high Se status and to better understand how turkeys maintain Se homeostasis and resist Se toxicity.
SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Figure S1. Body weights of day-old male poults supplemented with 0 to 5 μg Se/g diet (n = 4 to 5/treatment) for 28 d and measured biweekly. Plot includes data for poults fed 0 to 1 μg/g as reported previously (Taylor and Sunde, 2016, 2017). Values are means ± SEM, P = 0.58 at d 28. Table S1. The effect of high dietary Se on mean fold change of selenoprotein transcripts in turkey poults, relative to poults fed 0.4 µg Se/g diet.
ACKNOWLEDGMENTS This research was supported by the National Institute of Food and Agriculture, United States
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Figure 6. GPX1 activity plotted against tissue Se concentration. Activity vs. Se in (A) kidney and (B) liver from poults supplemented with 0 to 5 μ g Se/g diet (n = 3/treatment). Logistic regression analysis (solid black line) was calculated using all data points. For reference, a dashed gray line is plotted from the origin to the mean of the 1 μ g/g treatment group. Complete data for poults fed 0 to 1 μ g/g was reported previously (Taylor and Sunde, 2016, 2017).
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Department of Agriculture (www.csrees.usda.gov), Hatch project 1013496, and by the Wisconsin Alumni Foundation (http://www.uwalumni.com) Selenium Nutrition Research Fund (No. 12046295), and by an undergraduate University of Wisconsin Cargill-Benevenga Research Stipend (to VGB).
Avissar, N., D. B. Ornt, Y. Yagil, S. Horowitz, R. H. Watkins, E. A. Kerl, K. Takahashi, I. S. Palmer, and H. J. Cohen. 1994. Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am. J. Physiol. 266:C367–C375. Barnes, K. M., J. K. Evenson, A. M. Raines, and R. A. Sunde. 2009. Transcript analysis of the selenoproteome indicates that dietary selenium requirements of rats based on selenium-regulated selenoprotein mRNA levels are uniformly less than those based on glutathione peroxidase activity. J. Nutr. 139:199–206. Brown, M. B., and A. B. Forsythe. 1974. Robust tests for the equality of variances. J. Am. Statist. Assoc. 69:364–367. Burguera, J. A., G. T. Edds, and O. Osuna. 1983. Influence of selenium on aflatoxin B1 or crotalaria toxicity in turkey poults. Am. J. Vet. Res. 44:1714–1717. Burk, R. F., and K. E. Hill. 2015. Regulation of Selenium metabolism and transport. Annu. Rev. Nutr. 35:109–134. Cantor, A. H., and M. L. Scott. 1975. Influence of dietary selenium on tissue selenium levels in turkeys. Poult. Sci. 54:262– 265. Cantor, A. H., P. D. Moorhead, and M. A. Musser. 1982. Comparative effects of sodium selenite and selenomethionine upon nutritional muscular dystrophy, selenium-dependent glutathione peroxidase, and tissue selenium concentrations of turkey poults. Poult. Sci. 61:478–484. Combs, G. F. 2015. Biomarkers of selenium status. Nutrients 7:2209– 2236. EFSA Panel on Additives and Products or Substances used in Animal Feed. 2013. Scientific opinion on the safety and efficacy of L-selenomethionine as feed additive for all animal species. EFSA J. 11:3219–3236. EFSA Panel on Additives and Products or Substances used in Animal Feed. 2016. Safety and efficacy of selenium compounds (E8) as feed additives for all animal species: sodium selenite, based on a dossier submitted by Retorte GmbH Selenium Chemicals and Metals. EFSA J. 14:4398–4423. Fischer, J., A. Bosse, E. Most, A. Mueller, and J. Pallauf. 2008. Selenium requirement of growing male turkeys. Br. Poult. Sci. 49:583–591. Hadley, K. B., and R. A. Sunde. 1996. The effect of dietary selenium on glutathione peroxidase activity in turkey poults. FASEB J. Abstr. 10:A557. Hill, K. E., G. W. McCollum, and R. F. Burk. 1997. Determination of thioredoxin reductase activity in rat liver supernatant. Anal. Biochem. 253:123–125. Jensen, L. S. 1986. Effect of Monensin on response of chicks to deficient and toxic levels of dietary selenium. Poult. Sci. 65:1163– 1166. Krol, M. B., J. Gromadzinska, and W. Wasowicz. 2012. SeP, ApoER2 and megalin as necessary factors to maintain Se homeostasis in mammals. J. Trace Elem. Med. Biol. 26:262–266. Lawrence, R. A., R. A. Sunde, G. L. Schwartz, and W. G. Hoekstra. 1974. Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake. Exp. Eye Res. 18:563– 569. Lei, X. G., J. K. Evenson, K. M. Thompson, and R. A. Sunde. 1995. Glutathione Peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium. J. Nutr. 125:1438–1446. Li, J.-L., and R. A. Sunde. 2016. Selenoprotein Transcript Level and Enzyme Activity as Biomarkers for Selenium Status and Selenium Requirements of Chickens (Gallus gallus). PLOS ONE 11, e0152392.
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REFERENCES
Li, S., F. Gao, J. Huang, Y. Wu, S. Wu, and X. G. Lei. 2018. Regulation and function of avian selenogenome. Biochim. Biophys. Acta doi: 10.1016/j.bbagen.2018.03.029. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. Mahan, D. C., and A. L. Moxon. 1984. Effect of inorganic selenium supplementation on selenosis in postweaning swine. J. Anim. Sci. 58:1216–1221. National Research Council. 1994. Nutrient Requirements of Poultry. National Academies Press, Washington, D.C. Olson, G. E., V. P. Winfrey, K. E. Hill, and R. F. Burk. 2008. Megalin mediates selenoprotein P Uptake by kidney proximal tubule epithelial cells. J. Biol. Chem. 283:6854–6860. Olson, G. E., J. C. Whitin, K. E. Hill, V. P. Winfrey, A. K. Motley, L. M. Austin, J. Deal, H. J. Cohen, and R. F. Burk. 2010. Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells. Am. J. Physiol. 298:F1244–F1253. Ort, J. F., and J. D. Latshaw. 1978. The toxic level of sodium selenite in the diet of laying chickens. J. Nutr. 108:1114–1120. Pappas, A. C., E. Zoidis, C. A. Georgiou, N. Demiris, P. F. Surai, and K. Fegeros. 2011. Influence of organic selenium supplementation on the accumulation of toxic and essential trace elements involved in the antioxidant system of chicken. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 28:446– 454. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:45e– 45. Raines, A. M, and R. A. Sunde. 2011. Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents. BMC Genomics 12:26. Salbe, A. D., and O. A. Levander. 1990. Comparative toxicity and tissue retention of selenium in methionine-deficient rats fed sodium selenate or L-selenomethionine. J. Nutr. 120:207– 212. Scott, M. L., G. Olson, L. Krook, and W. R. Brown. 1967. Seleniumresponsive Myopathies of myocardium and of smooth muscle in the young poult. J. Nutr. 91:573–583. Scott, M. L., and J. N. Thompsom. 1971. Selenium content of feedstuffs and effects of dietary selenium levels upon tissue selenium in chicks and poults. Poult. Sci. 50:1742–1748. Steel, R. G. D., and J. H. Torrie. Principles and Procedures of Statistics. New York, NY: McGraw-Hill Book; 1960. Sunde, R. A., and K. B. Hadley. 2010. Phospholipid hydroperoxide glutathione peroxidase (Gpx4) is highly regulated in male turkey poults and can be used to determine dietary selenium requirements. Exp. Biol. Med. (Maywood) 235:23–31. Sunde, R. A., A. M. Raines, K. M. Barnes, and J. K. Evenson. 2009. Selenium status highly regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome. Biosci. Rep. 29:329–338. Sunde, R. A., G. R. Sunde, C. M. Sunde, M. L. Sunde, and J. K. Evenson. 2015. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo). PLoS One 10:e0129801. Sunde, R. A., J. L. Li, and R. M. Taylor. 2016. Insights for setting of nutrient requirements, gleaned by comparison of selenium status biomarkers in turkeys and chickens versus rats, mice, and lambs. Adv Nutr. 7:1129–1138. Taylor, R. M., and R. A. Sunde. 2016. Selenoprotein transcript level and enzyme activity as biomarkers for selenium status and selenium requirements in the turkey (Meleagris gallopavo). PLoS One 11:e0151665. Taylor, R. M., and R. A. Sunde. 2017. Selenium requirements based on muscle and kidney selenoprotein enzyme activity and transcript expression in the turkey poult (Meleagris gallopavo). PLoS One 12: e0189001. United States Food and Drug Administration. 2004. Code of Federal Regulations Title 21. Food Additives Permitted in Feed and Drinking Water of Animals, sec. 573.920.
HIGH SELENIUM IN THE TURKEY Wahlstrom, R. C., T. B. Goehring, D. D. Johnson, G. W. Libal, and O. E. Olson. 1984. The relationship of hair color to selenium content of hair and selenosis in swine. Nutr. Rep. Int. 29:143– 147. Walter, E. D., and L. S. Jensen. 1963. Effectiveness of Selenium and Noneffectiveness of Sulfur Amino Acids in Prevent-
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ing Muscular Dystrophy in the Turkey. Poult. J. Nutr. 80:327– 331. Whanger, P. D., and J. A. Butler. 1988. Effects of various dietary levels of selenium as selenite or selenomethionine on tissue selenium levels and glutathione peroxidase activity in rats. J. Nutr. 118:846–852. Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey413/5101505 by Bukkyo University user on 19 September 2018
Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706 tions that were 5.6X, 1.7X, 1.9X, and 2.0X greater than Se-adequate levels in liver, kidney, breast, and thigh, respectively, and GPX activities in plasma, red cells, liver, kidney, and heart that were ≤2.0X Se-adequate values. In liver, kidney, heart, gizzard, breast, or thigh, no selenoprotein transcript was increased ≥2.0X, and no selenoprotein transcript was decreased ≤0.5X by 2 or 5 μg Se/g diet as compared to poults fed 0.4 μg Se/g diet. Of the 112 Se status biomarkers reported in this study, liver Se concentration was the only biomarker markedly altered by high Se status. This study provides evidence of no adverse effects in turkey poults fed up to 5 μg Se/g diet as inorganic Se. Thus, the FDA limit for Se supplementation in turkey feed can be safely raised to 0.5 μg Se/g diet. Future studies are needed to identify biomarkers for high Se status and to better understand how turkeys maintain Se homeostasis and resist Se toxicity.
ABSTRACT The current NRC turkey Se requirement is 0.2 μg Se/g diet. Recent studies evaluating tissue Se, glutathione peroxidase (GPX) activities, and selenoprotein transcript expression concluded that the dietary Se requirement of the turkey poult should be raised to 0.4 μg Se/g when supplemented with inorganic Se. The FDA currently limits Se inclusion in premixed diets for poultry and other livestock species to 0.3 μg Se/g diet. Thus, there is a need to investigate the effect of high dietary Se (>1.0 μg Se/g) in turkeys. The present study fed turkey poults 2 and 5 μg Se/g diet to characterize tissue Se accumulation in turkey poults fed high dietary inorganic Se and to evaluate the efficacy of selenoprotein activity and transcript expression as biomarkers of high Se status. Day-old male poults were fed 0.4, 2, or 5 μg Se/g for 28 d. There was no significant effect of Se supplementation on poult growth. Supplementation with 5 μg Se/g diet resulted in Se concentra-
Key words: glutathione peroxidase, selenoprotein, supplementation, transcript 2018 Poultry Science 0:1–11 http://dx.doi.org/10.3382/ps/pey413
INTRODUCTION
and Scott, 1975; Cantor et al., 1982). Only a few feeding studies have examined the effect of supernutritional (≤1.0 μg Se/g) and high (>1.0 μg Se/g) Se intake on biomarkers of Se status. An early study fed 0.1 and 1 μg Se/g diet; 1 μg Se/g diet completely prevented Se deficiency disease and reduced 6-wk mortality to from 20% to 0% (Walter and Jensen, 1963). To further refine the dietary requirement, Scott and colleagues fed up to 0.8 μg Se/g (Scott and Thompsom, 1971), and reported that tissue Se plateaued at 0.2 to 0.3 μg Se/g diet and that there was no reduction in growth in poults fed up to 0.8 μg Se/g. The FDA currently limits Se inclusion in premixed diets for poultry and other livestock species to 0.3 μg Se/g diet (United States Food and Drug Administration, 2004), regardless of differences in requirements between species (Sunde et al., 2016). Thus, there is a need to investigate the effect of high levels of dietary Se in turkeys to better characterize the impact when Se is fed at levels above the FDA limit of 0.3 μg Se/g. The activities of the selenoenzymes GPX and thioredoxin reductase (TXNRD) fall to <10% of Seadequate values when poults are fed a Se-deficient diet, and do not reach plateau levels of expression until 0.2
The current NRC turkey Se requirement for preventing overt Se deficiency disease is 0.2 μg Se/g diet, based on studies conducted over 50 yr ago (Scott et al., 1967; National Research Council, 1994). More recently, two groups supplemented turkey poults with multiple graded levels of Se up to 0.4 to 0.5 μg Se/g and found that the minimum Se requirement was 0.3 μg Se/g diet (Fischer et al., 2008; Sunde and Hadley, 2010). Our recent studies used tissue Se, glutathione peroxidase (GPX) activities, and selenoprotein transcript expression to examine the effect of supernutritional dietary Se up to 1 μg Se/g, and concluded that the dietary Se requirement of the turkey poult should be raised to 0.4 μg Se/g to achieve plateau levels of tissue Se and GPX activity in all tissues when supplemented with inorganic Se (Taylor and Sunde, 2016, 2017). Most published Se research in turkeys has focused on deficient-to-adequate levels of dietary Se (Cantor C 2018 Poultry Science Association Inc. Received April 30, 2018. Accepted August 10, 2018. 1 Corresponding author: [email protected]
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Rachel M. Taylor, Victor G. Bourget, and Roger A. Sunde1
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MATERIALS AND METHODS Reagents Molecular biology reagents were purchased from Promega (Madison, WI, USA), Invitrogen (Carlsbad, CA, USA), or Sigma (St. Louis, MO, USA). All other chemicals were of molecular biology or reagent grade.
Animals and Diets For the high-Se study reported here, day-old male Nicholas White-derived turkey poults (kindly donated by Jennie-O Turkey Store, Barron, WI) were allocated randomly to treatment and housed in battery cages (4/cage) with raised wire floors and 24-h lighting. The temperature was set at 95◦ F for the first week and lowered 5◦ F in each subsequent week. Animals were fed from stainless steel troughs ad lib, and allowed free access to deionized water in plastic waterers. The basal Se-deficient torula yeast-based diet (0.005 μg Se/g) was the same as we have used in previous turkey experiments (Taylor and Sunde, 2016, 2017) and modified from rodent diets we have used previously (Sunde and Hadley, 2010). The feed was prepared with the same lot of low-Se yeast used previously (Sunde and Hadley, 2010) to ensure Se deficiency. The diet was supplemented with 7.0% crystalline amino acids, including 0.93% methionine, to better match NRC rec-
ommendations for protein and amino acids. Vitamin E (as all-rac-α-tocopherol acetate) was supplemented at 150 mg/kg (12.5X NRC requirement), and levels of all other vitamins and minerals were supplemented to provide 150% of the requirements (National Research Council, 1994). The basal diet was supplemented with 0.4, 2, or 5 μg Se/g diet as Na2 SeO3 . Body weight was measured twice weekly and feed intake per cage was measured weekly. The animal protocol was approved by the Research Animal Resources Committee at the University of Wisconsin–Madison (protocol no. A005368).
Tissue Collection Poults from all treatments were killed at 28 d by terminal CO2 overexposure followed by exsanguination. Blood was collected in heparinized tubes and stored on ice, then spun at 1500× g for 15 min at 4◦ C (Eppendorf 5415R, F-45-24-11 rotor, Brinkmann, Westbury, NY) to separate plasma from red blood cells (RBC). RBC were reconstituted to original volume with saline phosphate buffer (76 nmol/L NaCl, 50 nmol/L sodium phosphate, pH 7.4). Breast muscle (pectoralis major), thigh muscle (iliotibialis), liver, gizzard, heart, and kidney were collected and immediately frozen at –80◦ C until analysis.
Enzyme Activity Analysis Kidney, heart, breast, and thigh were homogenized in 9 vol of sucrose buffer (20 mmol/L tris-HCl, pH 7.4, 0.25 mmol/L sucrose, 1.1 mmol/L EDTA, 0.1% peroxide-free Triton-100) and centrifuged (10,000× g, 15 min). GPX1 (glutathione peroxidase 1) activity was measured by the coupled assay procedure (Lawrence et al., 1974) using 120 μmol/L H2 O2 . GPX4 (glutathione peroxidase 4) activity was measured by the coupled assay procedure (Lei et al., 1995) using 78 μmol/L phosphatidylcholine hydroperoxide. GPX1specific activity was calculated by subtracting the activity due to GPX4, as described previously (Sunde and Hadley, 2010). In both assays, 1 enzyme unit is defined as the amount of enzyme that will oxidize 1 μmol glutathione per min under the specified conditions. TXNRD activity was measured using the goldinhibition assay with dithiobisnitrobenzoic acid (Sigma D8130) as the substrate, as described previously (Hill et al., 1997). The protein concentration was determined by the method of Lowry et al. (1951).
Tissue Se Analysis Thirty-six samples (n = 3/treatment) from liver, kidney, thigh, and breast were sent to the University of Missouri Research Reactor Center (1513 Research Park Dr, Columbia, MO 65211) for Se concentration analysis. Freeze-dried samples were analyzed for Se on a dry matter (DM) basis by standard-comparison instrumental neutron activation analysis, measured by highresolution gamma spectroscopy, as described previously
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to 0.4 μg Se/g diet (Hadley and Sunde, 1996; Fischer et al., 2008; Taylor and Sunde, 2016, 2017), making them good biomarkers of deficient-to-adequate Se status. In rodents, a few selenoprotein transcripts— notably Gpx1, Selenoh, and Selenow—also fall dramatically in Se deficiency and can be used as biomarkers of Se status to asses Se requirements (Barnes et al., 2009; Sunde et al., 2009). Turkeys and other avian species have 24 selenoproteins (Li et al., 2018; Sunde et al., 2015), of which most are preserved in Se deficiency; but in liver and kidney, 6 transcripts reliably fall to <50% of Se-adequate values in Se deficiency and are good biomarkers of deficient Se status. In these studies, however, no enzymatic or transcript biomarker was shown to be changed significantly by dietary Se up to 1 μg Se/g in the turkey (Taylor and Sunde, 2016, 2017). Sensitive and precise biomarkers of Se status across a range of intakes are necessary to further evaluate the FDA limit on Se supplementation. Thus, the present study fed day-old male turkey poults 2 and 5 μg Se/g diet with three objectives: (1) to characterize tissue Se accumulation in turkey poults fed high (≥1.0 μg Se/g) inorganic dietary Se; (2) to evaluate the efficacy of traditional selenoenzyme activity biomarkers for determining high Se status; and (3) to identify the effect of high Se status on selenoprotein transcripts as biomarkers. In addition, this study may lead to a better understanding of the susceptibility of the turkey to high Se.
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(Barnes et al., 2009; Sunde and Hadley, 2010; Taylor and Sunde, 2017). Se concentration on a wet matter basis was calculated by multiplying the DM Se concentration by the fractional DM.
Total RNA from liver, kidney, heart, gizzard, breast, and thigh (∼100 mg tissue, n = 4/treatment) was isolated with TRIzol Reagent (Invitrogen, catalog no. 15596-026) following the manufacturer’s protocol. The RNA pellet was dissolved in 300 μl diethylpyrocarbonate-treated water and quantitated using a DeNovix DS-11+ spectrophotometer (DeNovix Inc., Wilmington, DE). Relative mRNA abundance was determined by quantitative real-time PCR (qPCR). RNA (1 μg) was reverse transcribed to cDNA using the RETROscript kit (AM1710, Ambion) following the manufacturer’s protocol, and diluted 1/20 to make cDNA working stocks. Turkey gene-specific primer sets were based on the recently sequenced turkey selenoproteome (Sunde et al., 2015) and designed to span an intron–exon splice junction and amplify a 120 to 150base segment. The final 12 μl reactions contained 2 μl reverse transcribed cDNA working stock, 0.2 mmol/L turkey gene-specific forward and reverse primers, and 1X KAPA SYBR FAST qPCR Kit (KAPA Biosystems no. KK4611). Reactions were followed in a LightCycler 480 (Roche Life Science) using the manufacturer’s SYBR Green 384-II template (initial stage of 95◦ C for 5 min, followed by 45 amplification cycles of 95◦ C, 60◦ C, and 72◦ C for 10 s each). Melting curves were generated to confirm the presence of one specific product and standard curves were run for each primer set/tissue combination, and the second derivative max program was used to determine amplification efficiency following the manufacturer’s protocol. mRNA relative abundance was calculated according to Pfaffl (2001), accounting for gene-specific efficiencies, normalized to the mean of β -actin and glyceraldehyde-3-phosphate dehydrogenase expression, and expressed as a percentage of the plateau of Se-adequate (0.4 μg Se/g) levels.
Statistical Analysis Data are presented as mean ± standard error of the mean (SEM). For growth, enzyme analysis and mRNA expression, n = 4/treatment; for tissue Se concentration, n = 3/treatment. Data were analyzed by oneway ANOVA and variance equality was tested using Levene’s test for homogeneity of variances (Brown and Forsythe, 1974). When the main effect of diet was significant, differences between means were assessed by Duncan’s multiple range test (P < 0.05) with Kramer’s modification for unequal class sizes when necessary (Steel and Torrie, 1960). The curves plotted in Figures 2, 3, 4, and 6 are linear, hyperbolic or logistic regressions calculated using all individual values, similar to
Figure 1. Growth of day-old male poults supplemented with 0.4, 2, or 5 μ g Se/g diet (n = 4/treatment) for 28 d and measured biweekly. Values are means ± SEM, P = 0.17 at d 28.
Se-response curves plotted when studying Se requirements (Li and Sunde, 2016; Taylor and Sunde, 2016, 2017). For comparison, we have also plotted in Figures 2, 3, 4, 6, and S1 tissue Se and selenoenzyme activities that were published previously for poults supplemented with 0 to 1 μg Se/g diet (Taylor and Sunde, 2016, 2017). Tissue Se concentrations were measured concurrently and Se-adequate selenoenzyme activities were reanalyzed simultaneously to verify relative expression.
RESULTS Growth Day-old poults weighed an average of 58 IS ± 1.1g at the start of the experiment, and after 28 d on treatment, poults supplemented with 0.4, 2, and 5 μg Se/g diet weighed 741 ± 61, 569 ± 50, and 589 ± 70g, respectively (Figure 1), but there was no significant effect of Se supplementation on final body weight of the poults (P = 0.17). Even over the last 7 d, there was no significant difference in growth rate amongst the groups (P = 0.21). Poult growth in this experiment for poults supplemented with 2 and 5 μg Se/g diet was similar to growth of poults fed 0 to 1 μg Se/g diet in our previous experiment (Taylor and Sunde, 2016; Figure S1). These poults, supplemented with 150 mg/kg vitamin E (12.5X NRC requirement), were generally healthy and showed no gross tissue abnormalities, and there was no effect of Se treatment on tissue weights as a percentage of body weight (data not shown).
Tissue Se Concentration Se concentrations in poults fed the Se-adequate diet (0.4 μg Se/g) averaged 5.4 ± 0.5 nmol Se/g tissue wet weight in liver, 8.1 ± 0.5 nmol/g in kidney, 0.87 ± 0.04 nmol/g in breast, and 1.1 ± 0.03 nmol/g in thigh (Figure 2). Supplementation with 5 μg Se/g diet resulted in Se concentrations that averaged 5.6X, 1.7X, 1.9X, and 2.0X greater than Se-adequate levels in each tissue, respectively. Only in poult liver was the increase
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RNA Isolation and Analysis
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in tissue Se statistically significant (P < 0.05) at both 2 and 5 μg Se/g diet (Figure 2A). In kidney, breast, and thigh, tissue Se concentrations did not increase dramatically above Se-adequate values (Figure 2).
Enzyme Activity The relative rank of GPX1 activity in Se-adequate poults was kidney >> liver > heart > red cell > thigh > breast, gizzard (Figure 3). Supplementation with 5 μg Se/g diet resulted in GPX activities in plasma, red cells, liver, kidney, and heart that were ≤2X the Se-adequate values. GPX3 activity in plasma from Seadequate poults was 54.7 ± 3.2 EU/g protein; GPX3 activity in plasma in poults supplemented with 2 and 5 μg Se/g was 1.7X and 1.8X Se-adequate values (P = 0.002), respectively (Figure 3A). RBC GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 1.5X and 1.4X Se-adequate values (P = 0.008; Figure 3B). GPX1 activity in liver of Se-adequate poults was 73.4 ± 10 EU/g, and poults supplemented with 2 and 5 μg Se/g averaged 1.3X and 1.4X Se-adequate values (P = 0.27; Figure 3C). Kidney GPX1 activities in
mRNA Expression To survey selenoprotein transcript expression for potential biomarkers of Se status, we conducted qPCR of selenoprotein transcripts (Figure 5). In poult liver, no selenoprotein transcript was increased ≥2.0X and no selenoprotein transcript was decreased ≤0.5X by 2 or 5 μg Se/g diet as compared to expression in poults fed 0.4 μg Se/g diet (Figure 5A), and none of these changes were significant. Also in kidney, no selenoprotein transcript was increased ≥2.0X or decreased ≤0.5X by 2 or 5 μg Se/g; kidney GPX3 and SELENOP1 (selenoprotein P) mRNA were significantly upregulated by
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Figure 2. Effect of Se supplementation on tissue Se concentration. Se concentration in (A) liver ( r) and kidney () and (B) breast ( r) and thigh () of 28-d-old turkey poults, expressed as nmol/g wet tissue. Values are means ± SEM (n = 3/treatment). Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are Se concentrations in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
poults supplemented with 2 and 5 μg Se/g were 1.4X Se-adequate values (P = 0.001; Figure 3D), and heart GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 1.8X and 1.7X Se-adequate values (P = 0.046; Figure 3E). In gizzard, breast, and thigh, the tissues with the lowest levels of GPX1 activity, supplementation with 5 μg Se/g resulted in GPX1 activities that were 2 to 4X the Se-adequate values. Gizzard GPX1 activities in poults supplemented with 2 and 5 μg Se/g were 2.1X and 2.7X Se-adequate values (P = 0.009; Figure 3F). Breast muscle GPX1 activities in poults were 1.8X and 2.4X Seadequate values (P = 0.004), respectively (Figure 3G), and thigh GPX1 activities were 2.4X and 3.6X Seadequate values (P = 0.001), respectively (Figure 3H). The relative rank of GPX4 activity in Se-adequate poults, as well as poults supplemented with 5 μg Se/g, was liver > heart, kidney > thigh > breast, gizzard (Figure 4). GPX4 activity in liver of Se-adequate poults was 32.7 ± 3.9 EU/g, and poults supplemented with 2 and 5 μg Se/g averaged 1.2X and 1.3X Se-adequate values (P = 0.24; Figure 4A). Kidney GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.3X and 1.2X Se-adequate values, respectively (Figure 4B), and heart GPX4 activities were 1.3X and 1.1X Se-adequate levels, respectively (Figure 4C). GPX4 activities in Se-adequate gizzard, breast, and thigh muscles were less than half the levels in liver, kidney, and heart. Gizzard GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.8X and 1.8X Se-adequate levels, respectively (Figure 4D). Breast GPX4 activities in poults supplemented with 2 and 5 μg Se/g were 1.2X and 1.2X Se-adequate levels, respectively (Figure 4E), and thigh GPX4 activities were 1.3X and 1.3X Se-adequate levels, respectively (Figure 4F). Clearly, neither GPX1 nor GPX4 activity are substantially increased when turkey poults are supplemented with 5X and 12.5X the Se requirement (0.4 μg Se/g). We also determined TXNRD enzyme activity in liver, the turkey tissue with the highest level of TXNRD activity. Liver TXNRD was not significantly different between groups supplemented with 0.4, 2, or 5 μg Se/g diet, averaging 3.08 ± 0.24, 2.97 ± 0.28, and 3.48 ± 0.19 EU/g (P = 0.34; Figure 4G).
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2 μg Se/g but not by 5 μg Se/g diet (P = 0.012 and P = 0.034, respectively, Table S1). No other selenoprotein transcript was significantly altered by high dietary Se. LRP2 (megalin), the renal SELENOP1 receptor in mammals, was only expressed in kidney and was not significantly regulated by dietary Se (Figure 5B). In heart, gizzard, breast, or thigh muscle, no selenoprotein transcript was significantly up- or downregulated
by high dietary Se (Figure 5C and D). In heart, DIO1 (deiodinase 1), DIO3, GPX2, and SELENOP2 were not expressed above the level of detection; in gizzard, DIO1, GPX2, and SELENOP2 were not expressed above the level of detection (Table S1). In breast, DIO1 and GPX2 mRNA were not expressed above the level of detection; in thigh, DIO1, GPX2, and TXNRD3 were not expressed above the level of detection.
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Figure 3. Effect of Se supplementation on tissue GPX1 and GPX3 activity. GPX3 activity in (A) plasma and GPX1 activity in (B) red blood cells, (C) liver, (D) kidney, (E) heart, (F) gizzard, (G) breast, and (H) thigh of 28-d-old turkey poults. Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are activities in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
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DISCUSSION Growth and Toxicity There was no significant effect of high dietary Se on poult growth in this study. Although final weights in poults supplemented with 2 and 5 μg Se/g were 30 and 26% lower, respectively, than final weights of Seadequate poults, these differences were not statistically
significant (P = 0.17), and the final weights of all groups were also similar (P = 0.58) to average weights of poults in our previous study that used poults and diets from the same sources (Figure S1; Taylor and Sunde, 2016). Rates of growth, as determined by analysis of covariance, were also not significantly different over any period of the experiment, including the last 7 d (P = 0.21, data not shown).
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Figure 4. Effect of Se supplementation on tissue GPX4 and TXNRD activity. GPX4 activity in (A) liver, (B) kidney, (C) heart, (D) gizzard, (E) breast, (F) thigh, and (G) liver TXNRD activity of 28-d-old turkey poults. Values are means ± SEM (n = 4/treatment). Means without a common letter are significantly different (P < 0.05). Also shown for reference with gray symbols are activities in tissues from poults supplemented with 0 to 1.0 μ g Se/g as reported previously (Taylor and Sunde, 2016, 2017).
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Tissue Se and Accumulation Differences
Figure 5. Effect of high dietary Se on relative mRNA expression of selenoprotein and selected Se-related transcripts. Transcript expression in (A) liver, (B) kidney, (C) breast, and (D) thigh of 28-d-old turkey poults, relative to expression in poults fed 0.4 μ g/g. Values are means ± SEM (n = 4/treatment). ∗ P < 0.05. Overall level of significance given in Table S1.
Of the 112 biomarkers of Se status (including tissue Se, GPX, and TXNRD activities, and up to 27 transcripts in seven tissues) reported in this study, liver Se was the only biomarker markedly altered by high Se status. Liver in poults supplemented with 2 μg Se/g had double the Se concentration vs. poults fed 0.4 μg Se/g, and liver in poults fed 5 μg Se/g had almost 6X Se concentration vs. Se-adequate liver. In contrast, tissue Se concentration did not markedly rise above Seadequate levels in the other three tissues studied. Kidneys of poults supplemented with 5 μg Se/g had less than 2X the Se content of kidneys from Se-adequate poults. Importantly, in this production animal, muscle Se concentration in breast and thigh also increased <2X relative to Se-adequate poults. Thus in poults fed 5 μg Se/g, liver Se was 2.3X the level in kidney and 15X the level in breast and thigh. Poults in a previous study fed 0.38 to 0.39 μg Se/g diet (starter and finishing diets, as Na2 SeO4 ) for 35 d also had liver Se at ∼8X the Se concentration as breast and thigh (Fischer et al., 2008). In mammals, however, Salbe and Levander (1990) found that liver and kidney of rats supplemented with 2.5 μg Se/g diet had 2X and 4X tissue Se, respectively, as Seadequate rats. In another experiment, liver from rats fed 5 μg Se/g diet had 4X Se-adequate levels (Raines and Sunde, 2011). Rats supplemented with 4 μg Se/g diet had liver and kidney Se concentrations of approximately 2X the level of Se found in Se-adequate samples (Whanger and Butler, 1988). Thus, at least in poults supplemented with high inorganic Se, liver is the tissue that accumulates Se, whereas muscle tissues only show modest increases. In mammals, the kidney is often the tissue that accumulates high concentrations when animals are fed elevated levels of trace minerals. Based on the lack of increase in selenoprotein transcript expression and selenoenzyme activity in turkey liver presented here, it is unlikely that the accumulated Se in liver is present
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A study focused on preventing aflatoxin toxicity reported that 10 μg Se/g diet as Na2 SeO3 had no effect on growth when fed to day-old turkey poults (Burguera et al., 1983). Previous studies with day-old chicks reported that feeding 5 μg Se/g as Na2 SeO3 for 3 wk reduced final body weights by 26% (Jensen, 1986). Other studies in chickens have shown no effects on egg weight, production or hatchability when dietary Se as Na2 SeO3 is fed to laying hens at levels up to 8 μg Se/g diet (Ort and Latshaw, 1978). In contrast, two studies in weanling pigs demonstrated that 5 μg Se/g but not 2.5 μg Se/g diet as Na2 SeO3 significantly reduced growth (Mahan and Moxon, 1984; Wahlstrom et al., 1984). Previous research in weanling rats has shown that feeding 5 μg Se/g as Na2 SeO3 for 4 wk reduced final body weights by 24% (Raines and Sunde, 2011). Thus the literature indicates that avians in general are more resistant to general Se toxicity than mammals.
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Selenoenzyme Activity as a Biomarker for High Se Status Activities of the selenoenzymes GPX1 and GPX3 are frequently used as biomarkers of Se status in humans and animals (Burk and Hill, 2015; Combs, 2015). In our previous studies focused on Se deficiency biomarkers, we found that GPX1, GPX3, GPX4, and TXNRD activities were good biomarkers that distinguished Se-deficient from Se-adequate turkey poults (Taylor and Sunde, 2016, 2017), and in other species (Sunde et al., 2016). In the present experiment, when birds were supplemented with 5 to 12.5X the dietary Se requirement, GPX4 activity did not increase in any tissue and only modest increases were seen for GPX1 activity in plasma, red cells, kidney, heart, gizzard, breast, and thigh. Liver TXNRD activity also did not rise with increasing dietary Se above the plateau. Although small increases in some biomarker levels were seen at 2 and 5 μg Se/g diet, we suggest that these minor increases do not justify raising the Se requirement for young male poults higher than 0.4 μg Se/g diet. This study demonstrates that none of the measured selenoenzyme activities can be used as biomarkers of high Se status.
Relationship Between Tissue Se and GPX Activity We have previously found that similar levels of dietary Se (0.3 to 0.4 μg Se/g) are required to raise GPX1 activity and Se concentration to plateau levels in the turkey. Plotting kidney GPX1 activities relative to tissue Se concentrations, using data from this high Se study and our previous Se-deficient-to-supernutritional study (Taylor and Sunde, 2016, 2017), reveals that these two biomarkers increase roughly in parallel in kidney over the range from Se-deficient to high Se (Figure 6A). This relationship is sigmoidal (P < 0.001), indicating that relatively less Se is incorporated into GPX1 when tissue Se is <5 nmol/g, reflecting the downregulation of GPX1 mRNA in Se deficiency. Plots of this data for breast and thigh are similar (data not shown). In liver, in contrast, there is still a near-linear increase in GPX1 activity as tissue Se increases when poults are supplemented with up to 1 μg Se/g diet (re-
sulting in liver Se <10 nmol/g). High Se supplementation (2 and 5 μg Se/g), however, had little impact on GPX1 activity (Figure 6B). This demonstrates that with high inorganic Se supplementation, turkey poults accumulate Se in liver in a form distinct from incorporation into GPX1, GPX4, and TXNRD. The lack of significant increases in any of the other selenoprotein transcripts further suggests that this Se is present in a form other than in selenoproteins.
Transcript Expression In our previous study, we found that a limited number of selenoprotein transcripts were good biomarkers for Se deficiency, including transcripts for GPX1, GPX3, and GPX4. In five out of six tissues assessed here (heart and gizzard data not shown), no selenoprotein transcript was significantly up- or downregulated by high Se. In kidney, just GPX3 and SELENOP1 transcripts were significantly upregulated, although to only <2X Se-adequate values in poults fed 2 μg Se/g diet, and nonsignificantly when fed 5 μg Se/g (Table S1). Liver GPX3 transcript expression followed a similar pattern; it was nonsignificantly upregulated to 1.8X and 1.1X of Se-adequate values by 2 and 5 μg Se/g diet, respectively (Table S1). This study, as well as our previous research, found that GPX3 transcript expression is high in many turkey tissues, including heart, gizzard, and skeletal muscle (Taylor and Sunde, 2017), indicating GPX3, the major plasma GPX, may be synthesized in a variety of tissues in the bird. In contrast, circulating plasma GPX3 in mammals is primarily synthesized in the kidney, and correspondingly Gpx3 transcript expression is biased to mammalian kidney (Avissar et al., 1994; Olson et al., 2010). The lipoprotein receptor megalin (LRP2) has been shown to be required for reuptake and recycling of SELENOP from kidney proximal tubules (Olson et al., 2008), and it is a key player in whole-body Se homeostasis (Krol et al., 2012). We measured expression of LRP2 in liver and kidney; consistent with what has been seen in other species, LRP2 was not expressed at detectable levels in turkey liver, but was moderately expressed in turkey kidney and not regulated by dietary Se level (Figure 5B, Table S1). Thus, this study found no selenoprotein transcript that could be used as a biomarker of high Se status. Given that the turkey response to high dietary Se is not mediated through alteration of selenoprotein transcript expression, a wholetranscriptome analysis might identify non-selenoprotein transcripts or pathways altered under conditions of high dietary Se.
FDA Limits on Supplemental Se in Livestock Feeds Our previous study showed that the minimum dietary Se requirement of the young poult is 0.4 ug
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in selenoproteins; the nature of this Se is yet unknown and the exact form would be of great interest. When excess Se is supplemented to animals as an organic form such as SeMet or its analogs, Se accumulates in many tissues, especially muscle, as SeMet is nonspecifically incorporated into proteins in place of Met (Pappas et al., 2011), but inorganic Se is not generally incorporated into proteins that do not contain a selenocysteine codon (Burk and Hill, 2015). It would be important to extend the length of this feeding trial to measure Se status biomarkers at finishing. This may reveal different tissue Se accumulation patterns in mature birds.
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Se/g diet when supplemented as inorganic Se, and that selenoprotein enzyme activities and tissue Se levels were little increased when poults were supplemented with up to 2.5X this level. The FDA currently limits supplemental Se, as Na2 SeO3 , Na2 SeO4 , or selenized yeast, in complete feeds to 0.3 μg Se/g diet for chickens, ducks, turkeys, sheep, swine, and cattle (United States Food and Drug Administration, 2004). The European Union uses the recommendation of the European Food Safety Authority (EFSA), which states that Se as Na2 SeO3 or SeMet may be incorporated into feed for all livestock species such that the total diet contains no more than 0.5 μg Se/g diet (EFSA Panel on Additives and Products or Substances used in Animal Feed, 2013, 2016). Collectively, our studies provide little evidence of adverse effects in turkey poults fed up to 5 μg Se/g diet as inorganic Se, and muscle tissues did not accumulate Se at greater than 2X Se-adequate levels. Raising the FDA limit for Se in complete feed, at least for turkeys, to 0.5 μg Se/g as inorganic Se, would provide the requirement of 0.4 μg Se/g, allow for a margin of safety, and would match the EFSA recommendation.
CONCLUSION The present study demonstrates that excess dietary Se does not dramatically impair growth or alter selenoenzyme activity or selenoprotein transcript expression in the young poult. Importantly, Se concentration
in extrahepatic tissues does not notably rise above Seadequate levels (0.4 μg Se/g) with dietary Se supplemented as high as 5 μg Se/g. Even turkey liver accumulates Se at less than 2X when inorganic dietary Se is supplemented at 5X the requirement. These results further indicate that the FDA limit for selenite supplementation in turkey feed can be safely raised to 0.5 μg Se/g diet. Future studies are needed to identify biomarkers for high Se status and to better understand how turkeys maintain Se homeostasis and resist Se toxicity.
SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Figure S1. Body weights of day-old male poults supplemented with 0 to 5 μg Se/g diet (n = 4 to 5/treatment) for 28 d and measured biweekly. Plot includes data for poults fed 0 to 1 μg/g as reported previously (Taylor and Sunde, 2016, 2017). Values are means ± SEM, P = 0.58 at d 28. Table S1. The effect of high dietary Se on mean fold change of selenoprotein transcripts in turkey poults, relative to poults fed 0.4 µg Se/g diet.
ACKNOWLEDGMENTS This research was supported by the National Institute of Food and Agriculture, United States
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Figure 6. GPX1 activity plotted against tissue Se concentration. Activity vs. Se in (A) kidney and (B) liver from poults supplemented with 0 to 5 μ g Se/g diet (n = 3/treatment). Logistic regression analysis (solid black line) was calculated using all data points. For reference, a dashed gray line is plotted from the origin to the mean of the 1 μ g/g treatment group. Complete data for poults fed 0 to 1 μ g/g was reported previously (Taylor and Sunde, 2016, 2017).
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Department of Agriculture (www.csrees.usda.gov), Hatch project 1013496, and by the Wisconsin Alumni Foundation (http://www.uwalumni.com) Selenium Nutrition Research Fund (No. 12046295), and by an undergraduate University of Wisconsin Cargill-Benevenga Research Stipend (to VGB).
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