Soy Content of Basal Diets Determines the Effects of Supplemental Selenium in Male Mice

Abstract

The effects of supplemental Se in rodent models may depend upon composition of the basal diet to which it is added. Wild-type male littermates of Transgenic Adenocarcinoma of Mouse Prostate mice were fed until 18 wk of age 1 of 2 Se-adequate stock diets high in soy (HS) or low in phytoestrogens (LP) or the same diets supplemented with 3.0 mg Se/kg diet as seleno-methylselenocysteine. Body and abdominal fat pad weights were lower (P < 0.01) in mice fed the HS diet. Supplemental Se reduced fat pad weights in mice receiving the LP diet but increased body and fat pad weights in mice consuming the HS formulation (P-interaction < 0.005). Serum free triiodothyronine concentrations were unaffected by supplemental Se in mice fed the LP diet but were decreased by Se supplementation of mice given the HS feed (P-interaction < 0.02). Free thyroxine concentrations were higher in mice consuming the HS diet regardless of Se intake (P < 0.001). Hepatic mRNA for iodothyronine deiodinase I was lower (P < 0.001) in mice fed the HS diet. Supplementation of Se increased this mRNA (P < 0.001) in both diet groups. Results from this study show a significant interaction between the composition of basal diets and the effects of supplemental Se with respect to body composition. These findings have important implications for future studies in rodent models of the effects of supplemental Se on heart disease, cancer, diabetes, and other conditions related to body weight and composition.

Introduction

The Selenium and Vitamin E Cancer Prevention Trial, involving over 35,500 participants, was terminated prematurely in 2008 due the failure of supplements of vitamin E (all rac-α-tocopheryl acetate) and Se (as selenomethionine) to reduce the risk of prostate cancer and to an unexpected slight increase in risk of prostate cancer and type 2 diabetes in men receiving vitamin E and Se, respectively (1). This failure of supplemental Se and vitamin E to provide the expected protective effects has led not only to a critical reevaluation of previous epidemiologic and intervention studies showing benefits of supplementation with these nutrients (2–4), but also to a reconsideration of the adequacy of current animal models and feeding protocols to accurately mimic the effects of dietary components in humans (5).

Numerous studies have been conducted to examine the effects of Se on development and progression of prostate cancer in rodent models (Table 1). In all studies in which the basal diet was free of phytoestrogens (semipurified diets and others labeled “free of phytoestrogens”) (6–11), Se supplementation was efficacious regardless of the chemical form given, its mode of delivery, or the rodent model used. In contrast, in those studies in which the reported basal diet included soy (12–14), a rich source of phytoestrogens, Se supplements had no significant effects, regardless of their chemical form or the animal model in which they were tested. In the reports of five other studies, the basal diet was described only as “commercial rodent chow” (15, 16) or was not stated (17–19). These observations strongly suggest that effects of supplemental Se may be determined by the composition of the basal diet to which it is added.

TABLE 1.

Previous studies of prostate cancer prevention by Se in rodents¹

Reference	Species	Inducing agent	Se form(s)	Se concentration	Delivery mode	Basal diet2	Results
(11)	TRAMP2 mouse	Transgenic	MSA	3.0 mg/kg BW	Daily oral gavage	AIN-93M (Harlan-Teklad)	Decreased neuro-endocrine tumors
(14)	Noble rat	T + E2	SeMet	1.5, 3.0 mg/kg diet	Diet	NIH-07 (Harlan-Teklad)	No effect
(12)	Wistar-Unilever rat	MNU + T	SeMet, Se yeast	1.3, 3.0, 9.0 mg/kg diet	Diet	Teklad 4%, Purina 5001	No effect
(7)	Copenhagen rat	Dunning R3327 tumors	MSC	1 mg/kg diet	Diet	AIN-93G	Decreased tumor area, weight
(10)	TRAMP mouse	Transgenic	MSA, MSC	3 mg/kg BW	Daily oral gavage	AIN-93M (Harlan-Teklad)	Decreased tumor weight; increased survival
(9)	Lady mouse	Transgenic	SeMet (in combination with vitamin E + lycopene)	“Human equivalent of 200 μg/d”	Diet	Purina Mills Test Diet “free of phyto-estrogens”	Increased survival; decreased PCa, metastases
(15)	Nude mouse	DU145 xenografts	MSA	4 mg/kg BW	Single oral dose daily	“commercial rodent chow”	Increased apoptosis
(16)	Nude mouse	DU145, PC3 xenografts	MSA, MSC, Se-Met, selenite	4 mg/kg BW	Daily single oral dose	“commercial rodent chow” (Harlan-Teklad)	Decreased tumor volume, weight, angiogenesis; increased apoptosis
(17)	Nude mouse	LAPC-4 xenografts	Selenite	2 mg/kg BW	Intraperitoneal injection 3X/wk	Not stated	Decreased tumor volume, PSA
(18)	Nude mouse	LNCaP xenografts	MSC	100 μg/mouse	Intraperitoneal daily injection	Not stated	Decreased tumor growth, PSA, AR expression
(8)	Lady mouse	Transgenic	SeMet (in combination with vitamin E + lycopene)	“Human equivalent of 200 μg/d”	Diet	Purina Mills Test Diet“free of phyto-estrogens”	75% decrease in PCa incidence, PCNA; increased p27
(6)	Nude mouse	PC3 orthotopic xenografts	Selenate, SeMet,MSC, Se yeast	0.3, 3.0 mg/L	Drinking water	Research Diets Inc. D19101(semipurified)	Selenite (3.0 mg/L) decreased tumor growth
(19)	Wistar-Unilever rat	MNU + T	SeMet	1.5, 3.0 mg/kg diet	Diet	Not stated	No effect
(13)	F344 Rat	DMAB	Not stated	2 mg/kg diet	Diet	Oriental MF, Oriental YeastCo, Ltd.	No effect

¹AR, androgen receptor; BW, body weight; DMAB, 3,2′-dimethyl-4-aminobiphenyl; E2, 17β-estradiol; MNU, N-methyl-N-nitrosourea; MSA, methylseleninic acid; MSC, seleno-methylselenocysteine; p27, cyclin-dependent kinase inhibitor 1B; PCa, prostate cancer; PCNA, proliferating cell nuclear antigen; PSA, prostate specific antigen; SeMet, L-selenomethionine; T, testosterone; TRAMP, Transgenic Adenocarcinoma of Mouse Prostate; Vitamin E, D-α-tocopherol succinate.

²For each basal diet the complete description of the diet and its source, as included by the authors in the cited reference, is given.

Evidence that a high intake of soy constituents by humans is associated with lower prostate cancer incidence has been extensively reviewed (20–24) and results from preclinical experiments are generally in agreement (25–28). Several commercially available rodent nonpurified diets include high concentrations of dehulled soybean meal (29). Chemopreventive dietary components, such as Se, when added as supplements to a basal diet high in soy may show limited additional protection beyond that provided by the high concentration of preventive substances already in the basal diet.

The possible effects of high soy intake from commercial rodent diets on various experimental endpoints have been reviewed (30, 31). The potential for producing spurious results due to unsuspected interactions of supplements with soy in basal diets may be quite high. For example, Hsu et al. (25) showed that soy in combination with green tea produced significant protective effects in a rat model of prostate cancer not seen with either component individually. Thus, the interaction of soy and green tea could exaggerate the apparent effects of green tea tested individually if experimental animals in such a study are fed basal diets high in soy.

To test the hypothesis that the soy content of a basal diet may determine effects of Se added to that diet, we examined mice exposed from conception to basal stock diets devoid of or high in soy, unsupplemented or supplemented with a naturally occurring food form of Se. Our previous work in healthy rats showed a potential interaction of soy and Se (32, 33). In this study, we chose to examine aspects of thyroid hormone metabolism, body weight, and body fat as indicators of possible interactions. Se plays a role in thyroid hormone metabolism by means of its incorporation into and activation of Se-dependent deiodinases (34, 35). These selenoenzymes catalyze interconversions among thyroid hormones, which are major regulators of metabolic rate. In addition, previous work from our laboratory and others has repeatedly demonstrated an antiobesogenic effect in rodents of high soy consumption (33, 36–39). Thus, we reasoned that examination of thyroid hormone metabolism and body composition could reveal interactions of Se and soy and suggest possible mechanisms for their combined effects. This work provides evidence that basal diet composition may significantly alter the apparent effects of nutrients, other dietary components, or plant chemicals that are added to those diets as supplements.

Materials and Methods

Animals and housing.

All procedures involving animals were approved by the Brigham Young University Institutional Animal Care and Use Committee. Mice were housed in shoebox cages with wood chip bedding in a temperature- and light-controlled room (12 h, 0600–1800, light; 12 h, 1800–0600, dark) and were given free access to food (as described below) and tap water.

Male and female hemizygous C57BL/6 transgenic TRAMP⁷ mice (40), and male and female wild-type FVB mice were purchased from Jackson Laboratory and used as breeders. Dams were fed one of the four experimental diets (described below) for at least 30 d before mating (32, 33). Hemizygous C57BL/6 TRAMP breeders were crossed with wild-type FVB mice to produce F1 wild-type C57BL/6 X FVB mice. Dams continued consuming their respective diets through pregnancy and lactation. Male pups were weaned at 21 d to the diets consumed by their dams and genotyped using standard techniques (41, 42).

Diets.

Mice were fed one of two pelleted basal stock diets, with or without a supplement of Se, in a 2 × 2 factorial design. The first was a modification of the NIH-07 open formula diet from which soy and alfalfa were eliminated to produce a low-phytoestrogen (LP) diet (Zeigler Bros; Phytoestrogen Reduced Rodent Diet I). The second was a closed formula, high-soy (HS) diet in which dehulled soybean meal was the most abundant ingredient (8604, Harlan Teklad) (Table 2). These diets have been compared to one another for their effects on various endpoints such as energy expenditure and adiposity (43), AMP kinase activation (44), fetal programming of glucose homeostasis (45), fertility (46), depression (47), gene expression regulation (32), and neuronal apoptosis (48). Previous analysis showed that the LP diet contained ~10 mg isoflavones (aglycone genistein + daidzein)/kg diet (49). In contrast, according to the manufacturer’s quarterly testing of the dehulled soybean meal included in the HS diet, this formulation would have provided a mean concentration of 620 mg isoflavones (range 420–700 mg)/kg diet during this study.

TABLE 2.

Composition of basal diets fed to mice with or without supplemental Se¹

	HS	LP
Top 10 ingredients by weight
1	Dehulled soybean meal	Ground corn
2	Wheat middlings	Ground wheat
3	Flaked corn	Fish meal
4	Ground corn	Wheat middlings
5	Fish meal	Lactic casein
6	Cane molasses	Dried skim milk
7	Ground wheat	Corn gluten meal
8	Dried whey	Corn oil
9	Soybean oil	Brewers dried yeast
10	Brewers dried yeast	Dry molasses
Proximate composition
Protein, g/kg	243	235
Fat, g/kg	47	53
Carbohydrate, g/kg	402	513
Crude fiber, g/kg	40	25
Neutral detergent fiber, g/kg	124	67
Ash, g/kg	74	64.5
Genistein + daidzein, g/kg	0.62	0.01
Energy density, kJ/kg	12,560	14,528
Energy from protein, %	32	27
Energy from fat, %	14	14
Energy from carbohydrate, %	54	59
Amino acids, g/kg
Arginine	15.3	11.3
Cystine	3.7	2.7
Histidine	5.8	5.9
Isoleucine	10.0	11.7
Leucine	19.0	21.6
Lysine	14.0	14.2
Methionine	4.2	5.9
Phenylalanine+tyrosine	20.0	20.7
Threonine	9.4	9.4
Tryptophan	2.9	2.5
Valine	11.0	13.6
Minerals, mg/kg
Calcium	14,000	12,000
Chloride	5000	4300
Cobalt	0.71	0.52
Copper	25	13
Iodine	2	1.84
Iron	300	245
Magnesium	3000	1500
Manganese	100	96
Phosphorus	11,000	9500
Potassium	10,000	5500
Se	0.34	0.45
Sodium	3000	3000
Zinc	80	60
Vitamins, mg/kg
Biotin	0.38	0.37
Cholecalciferol	0.060	0.125
Choline	2530	1646
Folate	3	3
Menadione	40	3.1
Niacin	63	76.5
Pantothenic acid	21	31.7
Riboflavin	8	7
Thiamin	27	17.3
Vitamin A	3.78	1.98
Vitamin B-6	13	10
Vitamin B-12	0.05	0.048
Vitamin E	54	22
Linoleic acid, mg/kg	18.7	22.8

¹HS, high-soy; LP, low-phytoestrogen.

Basal diets contained 0.34–0.45 mg Se/kg diet, which is higher than the minimum concentration (0.15 mg Se/kg diet) recommended by the AIN (50). All other nutrients were provided in both diets at concentrations that met or exceeded the minimum recommendations of the AIN (50). One-half of the diets were supplemented with 3.0 mg Se/kg diet as MSC, a naturally occurring food form of this mineral, whose tissue uptake in rats was reported to be more efficient than that of other Se forms (51). This high but nontoxic concentration was used in previous studies of supplemental Se in rodent models (6, 12, 14, 19). The MSC used in this study was a generous gift of Kelatron Corporation (product no. BISEMC0501, Se-methylselenocysteine trituration 0.5%) and was incorporated into the diets by the manufacturers during formulation prior to pelleting.

In this work, 13 mice received the unsupplemented LP diet, 17 were given the unsupplemented HS diet, 7 were fed the Se-supplemented LP mixture, and 9 received the Se-supplemented HS formulation. The unequal numbers reflect the breeding efficiency of the pups’ dams and sires, which were fed the respective diets 1 mo before mating. When subsets of mice from each group, rather than the whole group, were analyzed (e.g., due to limiting serum volume for multiple replicates of hormone assays), mice were chosen at random for inclusion in those subsets.

Body composition and food intake.

Pups continued to receive their respective diets until 18 wk, when they were weighed then killed by decapitation. Blood was collected for serum analysis (33). Abdominal fat pads were dissected and weighed. Livers were harvested and flash frozen for subsequent analysis of Se status.

Food intake was measured during the 3 d before mice were killed. Mice were transferred into individual shoebox cages the morning of the third day preceding killing, with a measured amount of food. Each morning for the next 3 d, food remaining in the tray was weighed and uneaten food on the cage bottom was recovered and weighed to correct the estimate of food consumption. Fresh food was then added and the total amount available at the beginning of the next 24-h period was weighed. This procedure was repeated up to and including the morning of the day mice were killed.

Se status and serum hormone concentrations.

The Se status of mice was determined by assay of hepatic cytosolic GPX1 activity (52) and liver Se concentration (53), as we previously reported. Serum hormone concentrations were assayed using commercially available ELISA kits (Free T3 and Free T4 ELISA kits, no. F3106T and no. F4107T, respectively, Calbiotech.

Steady-state mRNA analysis.

Hepatic expression of the gene for Dio1 and for the invariant genes Hprt and Tbp (54) were quantified by real-time qPCR. Total RNA was isolated from mouse livers as previously described (32, 55, 56). Equal quantities from each of three individual pure and intact RNA samples, chosen at random within each dietary group, were combined to form a total RNA pool for that group. Total RNA pools were reverse transcribed using random hexamers. First-strand cDNA was used in real-time qPCR analysis (LightCycler, Roche Applied Science). The PCR primers used for genotyping and qPCR were designed using sequences in GenBank and the National Center for Biotechnology Information’s Primer-BLAST program (57) (Supplemental Table 1).

For each gene, at least three quantitative PCR analyses were performed. Each analysis included three replicates for each diet. The concentration of each sample was determined based on an internal standard curve using the second derivative maximum of the amplification curve as calculated by LightCycler software (version 2.0). Calculated concentrations for Dio1 replicates were normalized to the concentrations of the invariant genes Hprt and Tbp (32, 55, 56).

Statistical analysis.

Pearson’s correlational analysis was used to examine associations among body weight, body fat pads, liver Se concentrations, and serum hormone concentrations. ANOVA using the General Linear Model (Minitab) was used to determine main effects of Se and soy and their interactions. This was followed by Fisher’s pairwise comparisons to determine significance of differences between dietary groups, as we previously reported (33). Differences for which P values were 0.05 in 2-sided tests were considered significant.

For each gene, ANOVA followed by Fisher’s tests was performed on all Hprt/Tbp-normalized replicates for each dietary treatment. In addition, for each gene, a normalized mean value was calculated for each dietary group. To determine relative expression among the four dietary groups for each gene, the Hprt/Tbp-normalized mean for each of the other dietary groups was divided by the Hprt/Tbp-normalized mean of the LP-0 Se group (32).

Results

Food consumption and Se status.

Food intake and liver cytosol GPX1 activity were not significantly affected by the diet treatments (data not shown). Supplemental Se increased the liver Se concentration (P < 0.001), but neither soy nor the interaction had an effect (Fig. 1A).

FIGURE 1.

Liver Se concentrations (A), body (B) and abdominal fat pad (C) weights, serum free T3 (D), and free T4 (E) concentrations, and hepatic Dio1 mRNA concentrations (F) of male mice exposed to a LP or HS diet with or without supplemental Se from conception through 18 wk of age. Values are mean ± SEM, n = 4–5 (A), 7–17 (B), 4–8 (C), 4–12 (D), 4–10 (E), and 8–9 (F). Significant effects (P < 0.05) of D, Se, and D × Se are shown. Means without a common letter differ, P < 0.05. D, diet; HS, high-soy; LP, low-phytoestrogen; T3, triiodothyronine; T4, thyroxine.

Body composition.

A high intake of soy was associated with a lower body weight (P < 0.01) (Fig. 1B) and there was an interaction between dietary soy and supplemental Se (P-interaction < 0.005). In mice fed the LP diet, body weight was not affected by supplemental Se, but in those receiving the HS diet, supplemental Se increased body weight (P < 0.05). Relative abdominal fat weight also was reduced by high soy intake (P < 0.001) and the interaction was significant. Supplemental Se reduced fat pad weights in mice fed the LP diet (P < 0.05) and increased them in mice fed the HS diet (P-interaction < 0.001).

Serum hormone concentrations.

Serum T3 concentrations were inversely correlated with body weight (r = −0.69; P = 0.006) and abdominal fat (r = −0.71; P = 0.005) (Fig. 1D). In mice fed the HS diet, serum T3 concentrations were higher (P < 0.001) than in mice given the LP formulation. Mice receiving Se supplements had slightly lower T3 concentrations overall than those that did not (P < 0.05). In mice fed the LP diet, supplemental Se did not affect T3 concentrations, whereas in mice given the HS diet, the Se supplement reduced serum T3 (P-interaction < 0.02).

Intake of the HS diet was associated with higher serum T4 concentrations (P < 0.001), but neither Se intake nor the interaction had a significant effect (Fig. 1E).

Steady-state iodothyronine deiodinase mRNA concentrations.

Supplemental Se increased mRNA for Dio1 (P < 0.001) regardless of the basal diet consumed, whereas intake of the HS diet was associated with lower concentrations (P < 0.001) of this mRNA, independent of dietary Se concentration (Fig. 1F). The interaction of Se and basal diet (P-interaction < 0.001) produced a greater effect of supplemental Se in mice fed the HS diet than in those receiving the LP formulation.

Discussion

Results from this study are consistent with previous work showing that: 1) provision of dietary Se to rodents in excess of the nutritionally adequate concentration increases tissue Se concentration but not activity of GPX1 (35, 50, 58); 2) high soy intake is associated with lower body weight and adiposity in animals and humans (30, 31, 33, 36–39, 59–61), whereas the main effect of Se on these measurements is not significant (33); and 3) high soy intake increases T3 concentrations, which leads to lower body and fat pad weights (60). This report shows a significant Se-soy interaction with respect to body weight and composition in mice.

In this study, one-half of the mice received a LP stock diet devoid of soy, whereas one-half was fed a HS stock diet, similar to those used in previous prostate cancer prevention studies. The HS diet provided soy components in their naturally occurring dietary blend. The Se supplement was also supplied in a naturally occurring, nutritionally relevant form of the mineral (7, 10, 16). Results from this approach are likely more relevant to human supplementation than other animal feeding protocols. Effects shown in animals consuming semipurified diets supplemented with a purified, isolated dietary component are not always seen when the same supplement is given to humans consuming self-selected mixed diets of ordinary foods (62–64). This report is consistent with others showing that effects of supplements may be diet-dependent (65).

To determine which component or components in the basal diets may account for the differential effects of supplemental Se, we focused first on those compounds whose concentration differed most between the two formulations. Six ingredients (ground corn, ground wheat, fish meal, wheat middlings, molasses, Brewers yeast) were among the top 10 in both diets (Table 2). The largest difference in composition was in isoflavones, for which the concentration in the HS diet was >60 times greater than in the LP formulation. However, soy contains other biologically active compounds that confer health benefits (66–68). In addition, stock diets made from natural ingredients can vary in both macro- and micronutrient composition. It is possible that other nutrients whose concentrations differed between diets may have contributed to the observed effects. This possibility was considered in the work of Bu and Lephart (69), who fed rats the two diets used in this study, plus a third diet (NIH-07) containing an intermediate concentration of soy and isoflavones (~0.20 g/kg). They observed an inverse association between body weight and soy content of the diets, with the highest body weights observed in rats fed the LP diet, intermediate weights in rats given the moderate-soy diet, and lowest weights in rats fed the HS diet. These results do not preclude the possibility that other components played a role in the observed dietary effects. However, although there was no consistent pattern among the diets in other nutrients, there was a clear inverse relationship and dose response between soy intake and body weight and fat. The lack of an association of body weight and fat with any other component of the diets except soy strongly argues that the effects of diet on body weight and composition seen in this work and many previous studies are due primarily to components found in soy.

The results of this study showed that the formulation of rodent basal diets may affect results and conclusions drawn in studies of Se supplements added to those diets. If the basal diet is low in soy, supplemental Se added to that diet may show antiobesogenic effects. In contrast, if the basal diet is high in soy, supplemental Se may promote adiposity. It should also be noted that the observation of a significant interaction between two dietary compounds does not by itself specify which component is altering the metabolism and effects of the other. An alternative interpretation of these data is that Se supplementation reduces the beneficial effects of high soy consumption. In this work, the antiobesogenic effects of soy were seen only in mice not given supplemental Se. In mice given a high-Se supplement, high soy intake had no effect on body weight or fat pad mass.

The iodothyronine deiodinases (DIO1, 2, and 3) are selenoenzymes that catalyze deiodinations of T4 to the active T3, to reverse T3, and also T3 to the inactive diiodothyronine (34, 35). Active T3 is generated for local use in specific tissues by DIO2, whereas DIO1 is primarily responsible for producing T3 for export into blood and maintaining systemic T3 concentrations. To the extent that higher Dio1 mRNA concentrations translate to higher DIO1 activity, it would be expected that a higher Dio1 mRNA concentration would be associated with lower T4 and higher T3 (because DIO1 converts T4 to T3), along with lower body weight and fat pads.

This pattern was seen in mice receiving the LP diet. In these mice, supplemental Se significantly increased Dio1, lowered T4, and decreased fat pads. In contrast, in mice fed the HS basal diet, the pattern of decreased T3 and increased body weight and fat pads due to Se supplementation, although internally consistent, would be expected to result from decreased Dio1 mRNA and activity rather than the increase seen (Fig. 1F). This may be due in part to the unexpectedly low Dio1 mRNA concentration in mice consuming the unsupplemented HS diet. Our observation of lower Dio1 mRNA concentrations and higher T4 concentrations in mice receiving the HS diet is internally consistent and agrees with results from both in vitro (70) and in vivo (71) studies showing that soy components can inhibit DIO1 activity and, in animals, produce corresponding increases in serum T4.

Other factors besides those examined in this study can play a role in determining each of the outcomes measured. The contributions of different deiodinases to tissue T3 concentrations, and their modulation by diet, remain to be explored. In addition, several other factors may affect activity of deiodinases and blood concentrations of T3 and T4 (34, 35). For example, estrogens stimulate thyroid hormone synthesis and release (72), which may explain in part the higher T3 and T4 concentrations observed in mice fed high concentrations of estrogen-like soy isoflavones. It is also reasonable to speculate that different molecules in soy may have variable affects on hormone metabolism that make it difficult to account for the overall influence of a complex mixture of such compounds.

In summary, this study provides evidence that the effects of Se supplementation can depend on the soy content of basal diets to which it is added. Supplemental Se increased Dio1 mRNA and reduced body fad pads in mice fed a zero-soy formulation representative of the average North American diet(37, 60). In contrast, in mice consuming a HS basal diet representative of typical Asian diets (37, 60), supplemental Se had the opposite effects of reducing serum T3 and increasing body weight and fat pads. These findings have important implications for researchers studying the effects of supplemental Se in rodent models of diseases related to body weight and composition. The antiobesogenic effects of high soy intake may include modulation of brown adipose tissue uncoupling protein-1, core body temperature, brain neuropeptide Y concentrations, or other metabolic regulators (60). The potential effects of supplemental Se on these processes in animals fed zero- and HS diets merit further investigation.