Introduction

Vitamin E (VE) and selenium (Se) have been recognized for several years as nutrients critical for optimal livestock growth, productivity, and health [1]. In animal physiology, VE and Se work together. VE is recognized as the first line of defense against lipid peroxidation, protecting polyunsaturated fatty acids (PUFA) in cell membranes by quenching free radical activity in biomembranes at the early stage of free radical attack [2, 3]. The first line of antioxidant defense, which is composed of VE, other fat-soluble antioxidants and metal-binding proteins in the cell, is not sufficient to completely prevent free radical formation [4]. Therefore, the second level of defense consisting of chain-breaking antioxidants, e.g., glutathione (GSH) and selenoproteins, plays an important role [5, 6].

However, the exact interrelationship between VE and Se is unknown and may vary between species. Many researchers have focused on the beneficial effects of VE and Se on the immune responses of domestic animals. Ebeid et al. [7] reported that supplemental dietary VE and Se enhanced growth performance, serum antioxidative status, and immune responsiveness in growing rabbits. In chickens, VE and Se deficiencies cause multiple alterations in the antioxidant system and adversely affect the redox state of the superficial pectoralis muscle [8]. Supplementation with Se at 3 and 30 days of life increased dairy calve neutrophil function and glutathione peroxidase activity and reduced the incidence of health disorders, indicating that dietary Se at adequate levels supports the antioxidant system of the body [9]. Comparing the effects of different dietary Se sources, Falk et al. [10] found that organic Se positively influences the immune response and antioxidant capacity of pigs.

Because of the special physiological characteristics of fur-bearing animals, the feed resources contain high levels of PUFA [11]. However, when fox or mink diets contain rancid fats or are high in PUFA, the animals are subjected to yellow fat disease [11]. Minks fed unsaturated fatty acid diets require an adequate supply of VE, which effectively protects the growing mink and prevents yellow fat disease [12, 13]. VE deficiency may also lead to yellow fat disease of foxes [14]. Previous studies have indicated that dietary Se supplementation may prevent lesions caused by VE deficiency in mink [15] and in other carnivorous animals, especially during growth [16]. Müller et al. [17] found an essential role of Se in the prevention of lipid and protein oxidation in rabbits, especially during periods of VE deficiency.

Several recent studies have shown that VE or Se may have an adverse effect on the body. Miller et al. [18] performed a meta-analysis and found that high dosage VE supplementation may increase all-cause mortality. Aburto and Britton [19] found that high levels of VE in chicken diets negatively affect the utilization of vitamin D3 (VD3) when VD3 is present at a marginal level. Moreover, Kim and Mahan [20] suggested that dietary Se from inorganic or organic sources is toxic at ≥ 5 ppm Se in growing finishing pigs. Nolan et al. [21] indicated that high doses of VE and Se may be deleterious to liver function, especially in the presence of high-fat diets in rats. In vitro studies on lymphocytes from sheep [22], cattle [23], and mice [24] showed that high levels of Se and VE added to these cells inhibit their responses and have a toxic effect.

In conclusion, VE and Se are essential for domestic animals, including fur-bearing animals, but excessive addition of VE and Se might be harmful to these organisms. There are no reports on the requirement of VE and Se in blue fox diets. Hence, it is necessary to explore the appropriate concentrations of VE and Se in blue fox diets. The objectives of the present research were to study the effects of VE and Se on the growth performance, blood antioxidant capacity, and blood metabolism abilities of blue foxes as well as to elucidate the VE and Se nutritional requirements of blue foxes.

Materials and Methods

Animal Diets, Management, and Experimental Design

In the present study, there were 160 blue foxes at 120 ± 5 days of age randomly assigned to eight groups with an equal number of males and females in each group. A 4 × 2 factorial experiment with four supplemental levels of VE (0, 100, 200, or 400 mg/kg dry matter) from α-tocopherol acetate (effective substance concentration of 50%) and two supplemental levels of Se (0 or 0.2 mg/kg) from glycine selenium with 1% Se concentrations (measured concentration of 0.86 ± 0.07 g/kg) was conducted. All diets met the nutrient requirements of growing foxes [11]. The composition and chemical analysis of the basal and the other trial diets are shown in Table 1. The experimental period was from mid-September to pelting in December. The first 7 days comprised the adaptation period, during which animals were allowed to be accustomed to the experimental diets based on dry and powdery components mixed with water. All foxes were individually housed in conventional cages (1 m × 0.8 m × 0.8 m) in two row sheds. The diets were provided twice a day namely at 7:00 and 15:00, and water was provided ad libitum. The use of animals in the present study followed guidelines similar to those provided by the Canadian Council on Animal Care [25].

Table 1 Ingredient and chemical composition of the basal diet (g/kg as dry matter)
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Sample Collection and Preparation

The sample of the basal diet was randomly sampled by dietary sampler and thoroughly mixed. This sample and supplementations were shrunk by the quarter method. Three replicates were measured for each sample. The amounts of feed given and that of leftover feed collected daily during the experimental period were weighed. The body weight (BW) of foxes was recorded in the morning before feeding on the 1st, 30th, and 60th day. After weighing on day 30 of the trial, 14 healthy blue foxes (7 females and 7 males) were randomly weighed before feeding and selected for digestibility measurements. Feces, feed refusals, and urine from the four consecutive days were collected daily. To feces, 10% sulfuric acid was added in accordance with 5% of the fresh weight and stored at − 20 °C. At the end of the experiment, all feces from one animal were mixed. Thereafter, 10% feces of the gross weight as well as feed refusals were dried to a constant weight at 60 ± 5 °C and then ground through a 40-mesh screen. The weight data of the whole feces and feed refusals were collected before and after drying. Urine was preserved with 10% sulfuric acid and 4 drops of toluene, and the urine volume of each animal was recorded. The mixed urine samples were filtered through filter paper, and 10 mL of each filtrate were stored at − 20 °C.

For final blood sampling, twelve healthy blue foxes (6 females and 6 males) were randomly selected after weighing on the day of pelting. After the blue fox was paralyzed with chlorinated succinylcholine, 5 mL blood was collected by heart puncture using serum separation tubes (BD-Pharmingen, USA). The blue foxes were then killed by an overdose of succinylcholine and pelted by skilled workers. Blood samples were centrifuged at 3500 r/min for 10 min, placed into 1.5-mL centrifuge tubes and stored at − 20 °C until analysis.

Analysis of Diets, Feces, and Urine Samples

The air-dried diets and feces were analyzed for dry matter (DM) and nitrogen (N) according to the procedures of AOAC [26]. Crude fat (CF) of feed and feces was determined by a diethyl ether extraction-submersion method [26]. The dietary calcium (Ca) and total phosphorus (total P) were estimated by the AOAC method [26].

The analysis of dietary and fecal Se was performed according to AOAC [27]. The Se standard solution was a selenite standard solution (1000 μg Se/mL in 1% HNO3; commercially available from the National Standard Research Center of China), which was diluted to 10 μg Se/L with 0.1 M HCl. The selenite solution was further diluted to 0, 1, 2, 4, and 8 μg Se/L with 0.1 M HCl. Dietary and fecal samples (0.2 g each) were transferred into a sealed polytetrafluoroethylene beaker, and 8 mL of concentrated (99.99%) nitric acid and several glass beads were carefully added. The samples were digested using a microwave-assisted (Anton Paar, Austria) high-pressure digestion system until the samples were in solution and nitric oxide fumes subsided according to the instructions. The solutions were cooled at room temperature, and a constant volume of 50 mL was obtained by adding 1% nitric acid. The urine samples (5 mL each) were treated according to methods published by Błażewicz et al. [28]. Briefly, the liquid sample, 9.4 mL of deionized water and 0.1 mL of concentrated (99.99%) nitric acid were placed in a polytetrafluoroethylene beaker and subjected to the same procedures described above for the solid samples. The processed diet, feces, serum, and urine samples (10 mL) were analyzed using an atomic fluorescence spectrometer (AFS-9x, Jitian Instruments, Beijing, People’s Republic of China) according to AOAC [27] using the supporting atomic fluorescence spectrometer software.

The concentrations of VE (α-tocopherol) in diets were measured according to the China Standard Press method (GB17812-2008). In detail, 10 g of the sample (accurately to 0.0001 g) and 50 mL of ascorbic acid ethanol solution were placed in a 250-mL round bottom flask, heated in a water bath until boiling point, washed with nitrogen and cooled slightly. Next, 10 mL of potassium hydroxide solution was added to the sample, mixed evenly, and boiled to obtain saponification under dinitrogen (N2) flow for 30 min. The solution was washed with 5 mL of ethanol and 5 mL of water from the top of the condenser tube and then cooled to approximately 40 °C. The saponification liquids were transferred into a 500-mL funnel that contained 100 mL of anhydrous ether. The flask was rinsed 2 to 3 times with distilled water, shaken for 2 min, and allowed to rest in a static state. The water phase was transferred into a new separating funnel and extracted twice with ether. The ether phases were combined, dehydrated by adding anhydrous sodium sulfate, and transferred to a 250-mL brown volumetric flask. After dissolving, butylated hydroxytoluene (100 mg) was then added into the volumetric flask and fixed with ether to the scale. A certain volume of liquid was placed in a rotating evaporator and evaporated to dry in a water bath at approximately 50 °C. The residue was dissolved in methanol to 10 mL and filtered with a 0.45-μm membrane before analysis.

Analysis of Serum Samples

Prior to the measurement of the Se concentration, serum samples (0.5 mL each) were treated as described for urine samples. The serum VE concentrations were determined according to Alvarez and De [29]. In brief, a plasma sample (0.5 mL) and 400 μL of ethanol were placed in 15-mL polypropylene tubes and vortex mixed for 10 s. The mixture was extracted with 2 mL of n-hexane/dichloromethane (90/10, v/v) by vortex mixing for 60 s and centrifugation at 3000×g for 5 min. The n-hexane/dichloromethane was carefully transferred to a glass test tube and evaporated to dryness under a stream of N2. The sample was dissolved in 250 μL of methanol and transferred to a micro-vial for the auto-sampler (Supelco, USA).

The α-tocopherol standard solution (1 mL of 1.0 mg/mL) was added into a 10-mL brown volumetric flask, evaporated to dryness under a steam of N2 and then diluted to 1, 2, 4, 8, and 10 mg/L with methanol to generate a gradient of standard solutions. The standard solution gradient and diet and serum samples were analyzed by high-performance liquid chromatography (Waters-2695, USA) using a C18 column (250 mm × 4.6 mm; 5.0 μm, Waters-XSELECT, USA). The initial mobile phase was methanol:water (95:5) and a flow rate of 1.0 mL/min. The injection volume was 20 μL, and the detector was a photodiode array detector. The concentration of VE was calculated according to the China Standard Press method (GB17812-2008).

The concentrations of serum glucose (GLU), triglyceride (TG), cholesterol (CHO), low-density lipoprotein cholesterol (LDL), highly density lipoprotein cholesterol (HDL), creatinine (CRE), total protein (TP), and albumin (ALB) (kits supplied by Zhongsheng Beikong Biotechnology LLC, Beijing, People’s Republic of China) were analyzed by an automatic biochemistry analyzer (Hitachi 7020, Hitachi High Technologies, Inc., Ibaraki, Japan). The concentrations of blood urea nitrogen (BUN), maleic dialdehyde (MDA), and total glutathione (T-GSH) as well as the enzyme activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-ST), glutathione reductase (GR), thioredoxin reductase (TrxR), and thioredoxin peroxidase (TPX) were determined using respective diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, People’s Republic of China).

Statistical Analysis

The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F:G) were calculated for each animal using the following formulas: ADG = (final weight−initial weight)/days; ADFI = sum of daily feed intake/days; and F:G = ADFI/ADG. The crude protein (CP) concentration in the diets, feces, and urine was calculated as N × 6.25.

The apparent digestibility (AD) coefficient of nutrients was calculated as follows: AD = (a−b)/a × 100%, where a is the nutrient intake from feed and b is the nutrient excretion in feces. The analysis of N metabolism used the following equations: N deposition = N intake−fecal N− urinary N; net protein utilization (NPU) (%) = N deposition/N intake × 100%; and biological value (BV) (%) = N deposition/(N intake−fecal N) × 100%. Se metabolism-related parameters were calculated with the following equations: total Se excretion = fecal Se excretion + urinary Se excretion; Se deposition = Se intake−total Se excretion; Se digestibility = Se deposition/Se intake × 100%.

The data were subjected to ANOVA in a completely randomized design with 4 × 2 factorial arrangement of treatments using the GLM procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). For all statistical analyses, significance was declared at P < 0.05. The following model was considered for analysis:

$$ {Y}_{\mathrm{i}\mathrm{j}\mathrm{k}}=\upmu +{A}_{\mathrm{i}}+{B}_{\mathrm{j}}+{(AB)}_{\mathrm{i}\mathrm{j}}+{\upvarepsilon}_{\mathrm{i}\mathrm{j}\mathrm{k}} $$

where Yijk is the characteristic that was measured, μ is the overall means, Ai is the effect of dietary VE, Bj is the effect of dietary Se, (AB)ij is the effect of the interaction between VE and Se, and εijk is the random error term. When interactions were significant, the PDIFF option of LSMEANS in SAS was used to separate the means, and the effects of the main factors were not considered.

Results

Growth Performance and Nutrient Digestibility

The basal diet contained 0.096 ± 0.004 (mean ± variance) mg Se/kg, and VE was not detected, which is shown in Table 1. The trial diets were supplemented with VE 0, 100, 200, and 400 mg, which were contained 0, 50, 100, and 200 mg VE/kg, respectively, and added with 0 and 0.2 mg Se/kg, which included 0.096 and 0.268 mg Se/kg. Different levels of VE and Se in diets significantly affected the growth performance of blue foxes (Table 2). Dietary levels of Se markedly influenced BW of different groups of blue foxes at 60 days (P < 0.05), and an interaction of VE and Se was observed (P < 0.05). Moreover, BW of male blue foxes was significantly influenced by the concentrations of VE in diets (P < 0.05). ADG greatly increased with dietary Se levels in male blue foxes during the first 30 days (P < 0.05), but there was no significant effects on female blue foxes. From 30 to 60 days of the feed experiment, ADG of blue foxes was affected by the dietary amounts of VE and Se as well as the interaction of VE and Se levels (P < 0.05). Between days 30 and 60, ADFI was impacted by the interaction of VE and Se levels in female blue foxes (P < 0.05), and it increased with the amount of Se in male blue fox diets (P < 0.05). During the first 30 days, F:G was higher in male blue foxes fed diets supplemented with VE but with Se (P < 0.05). Regardless of gender Se supplementation and the interaction of dietary VE and Se influenced F:G in the period from day 30 until day 60 (P < 0.05).

Table 2 Effects of different vitamin E and selenium supplementation on growth performance of growing furring blue foxes
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There were significant effects of different VE and Se supplementation levels on AD and Se metabolism of growing furring blue foxes which are shown on Table 3. There were no significant effects of different VE and Se supplement levels in diets on female blue foxes AD (P > 0.05). Interestingly, adding Se to male blue fox diets enhanced the digestibility of DM, CP, and CF (P < 0.05). However, the concentrations of VE and the interaction of VE and Se in male blue fox diets did not influence AD in growing furring male blue foxes (P > 0.05). The Se excretions were impacted by the levels of Se in blue fox diets (P < 0.05). Fecal and total Se excretions from female blue foxes as well as the total Se excretion from male blue foxes were significantly influenced by the amounts of VE in the diets (P < 0.05). The total Se excretion in groups supplemented with 400 mg VE/kg was significantly higher than that in other groups (P < 0.05), and groups supplemented with 200 mg VE/kg excreted more Se than groups fed diets not supplemented with VE (P < 0.05). In male and female foxes, Se deposition was significantly affected by the VE levels in diets (P < 0.05). In addition, a significant effect of dietary Se levels on Se deposition was found from male blue foxes (P < 0.05).

Table 3 Effects of different vitamin E and selenium supplementation on nutrients apparent digestibility as well as on the metabolism of nitrogen and selenium of growing furring blue foxes
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Serum Antioxidant Parameters

There were significant effects of different dietary levels of VE and Se on serum antioxidant parameters of blue foxes (Table 4). T-AOC was significantly influenced by the levels of Se in diets as well as the interaction of VE and Se addition (P < 0.05) in male but not in female. MDA concentrations were higher in female foxes receiving VE supplemented diets, whereas dietary Se influenced the MDA concentrations in male blue foxes (P < 0.05). The activities of SOD were increased in female serum of the group with 200 mg VE/kg and 0 mg Se/kg supplementation (P < 0.05). Regardless of gender, the SOD activities were significantly influenced in serum from foxes fed diets enriched with VE and Se (P < 0.05). The activities of GSH-PX were much lower in the groups without Se added in both female and male foxes (P < 0.05). In female foxes, the GSH-ST activities were significantly higher in the serum of groups with 100 or 200 mg VE/kg and 0 mg Se/kg in diet, but the GSH-ST activities were much lower in the serum of groups with 200 or 400 mg VE/kg and 0.2 mg Se/kg in diet (P < 0.05). The activities of GSH-ST were decreased with Se levels in male diets (P < 0.05), and the GSH-ST activities were affected by an interaction between VE and Se (P < 0.05). The activities of GR in female serum were not significantly affected by the levels of VE and Se (P > 0.05). However, the VE levels and the interaction between VE and Se supplementation significantly affected the activities of GR in males (P < 0.05). The TrxR-activity was decreased in female blue foxes receiving Se supplemented diets (P < 0.05), but not influenced by diets in males (P > 0.05). The TPX activity was lower in serum from female foxes in the group without VE and Se addition as well as in the group with 400 mg VE/kg and 0.2 mg Se/kg addition (P < 0.05). The highest TPX activities in male blue fox serum were found in the group with 400 mg VE/kg and 0 mg Se/kg supplementation (P < 0.05). Interestingly, the concentrations of T-GSH were highest in female serum in the group with 100 mg VE/kg and 0 mg Se/kg addition but lowest in the group without VE and Se addition (P < 0.05). The T-GSH concentrations in male blue fox serum were influenced by the concentrations of VE and Se in diets (P < 0.05), but no interaction of VE and Se supplementation was observed (P > 0.05).

Table 4 Effects of different vitamin E and selenium supplementation on serum antioxidant parameters of growing furring blue foxes
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Metabolism Parameters

The effects of different VE and Se levels in diets on blood metabolism parameters of blue foxes are shown in Table 5. The concentrations of TP in female fox serum were markedly influenced by the concentrations of Se in diets (P < 0.05), and they were significantly impacted by the interaction of VE and Se in male fox serum (P < 0.05). The effects of supplementation with VE and Se in female diets on the ALB concentrations were not significant (P > 0.05), but the interaction of VE and Se influenced the concentrations of ALB in male fox serum (P < 0.05). The GLB concentration in female blue foxes serum was lower in female blue foxed fed diets supplemented with Se (P < 0.05), but not influenced by diet in males (P > 0.05). The concentrations of GLU and TG in serum were significantly influenced by VE and Se supplementation, and the interaction of VE and Se was observed on the concentrations of GLU and TG in serum (P < 0.05). The GLU concentrations were significantly higher in the groups with 0.2 mg Se/kg and 0 mg VE/kg compared to the other groups (P < 0.05). However, the blue foxes fed diets enriched with 200 or 400 mg VE/kg and 0 mg Se/kg diet had higher serum TG levels compared with the other feeding groups (P < 0.05). The levels of serum CHO were not changed with supplementation of VE and Se in female foxes (P > 0.05), but serum CHO levels were significantly influenced by the amount of VE addition in male diets (P < 0.05). Moreover, the HDL concentrations were greatly impacted by the VE levels in diets (P < 0.05), and the lowest serum HDL concentrations were found in the group without VE and Se added in diets. However, there were no significant effects of different dietary VE and Se supplementation levels on serum LDL concentrations (P > 0.05). The BUN concentrations in serum from blue foxes were significantly affected by the interaction between dietary VE and Se levels (P < 0.05). There were no significant effects of VE and Se addition to diets on the concentrations of Se in female serum (P > 0.05), but dietary VE significantly influenced the concentrations of Se in male serum (P < 0.05). Furthermore, supplementation with VE affected the serum VE concentrations, but the effect was more pronounced in foxes fed diets supplemented with VE and Se (P < 0.05).

Table 5 Effects of different vitamin E and selenium supplementation on serum metabolism parameters of growing furring blue foxes
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Discussion

The present study showed that the growth performance was better in foxes fed with suitable VE or Se addition compared with those receiving diets without VE and Se and those fed diets supplemented with high amounts of VE. Ebeid et al. [7] and Eiben et al. [30] demonstrated that the addition of VE or Se to diets results in higher BW and lower F:G in growing rabbits. Työppönen et al. [31] reported that the requirement of VE in growing mink is 60 to 80 mg/kg diets. When growing mink are fed high oxidizing sea fish, additional VE should be added to diets [14], because VE as a natural antioxidant reacts with reactive oxygen metabolites (ROM) to inactivate them, which destroys VE in the process. Foxes are carnivores, indicating that fat plays a more significant role in meeting their energy requirements. Unfortunately, higher concentrations of PUFA in fox diets may decrease their antioxidant capacity. Therefore, fox diets should be supplemented with sufficient VE levels. Chauhan et al. [32] found that dietary VE and Se supplementation increased ADFI and ADG of lambs during the 3-week finisher period. Se is widely distributed in the environment [33], and fur animals can obtain sufficient levels of Se from marine fish [11]. Due to inclusion of fishmeal, no clinical signs of Se deficiency were observed in the present study. Kiiskinen and Maekelae [34] reported that several Finnish mink diets contain 0.05–0.42 mg Se/kg in dry matter. The Se concentrations of basal diets were contained in the research of Kiiskinen and Maekelae [34], which is comparable to the Se concentrations in the basal diet used in our study.

Results in male blue foxes showed that Se concentrations in diets significantly enhanced the AD of DM, CP, and CF, but did not affect the nutrient AD in female blue foxes. Adkins and Ewan [35] and Nuijten et al. [36] found that supplementation with VE and Se in pig diets significantly increased AD of nutrients. In addition, dietary Se supplementation may partly increase the utilization of DM and CP in goats [37]. However, Tian et al. [38] observed that dietary levels of Se did not affect the nutrient AD in growing finishing pigs, which was similar to the results obtained for female foxes in the present study. This difference may be due to the different physiological digestive characteristics of male and female foxes, which should be studied in the future.

In the present study, the total excretion of Se of foxes increased with the level of Se supplementation in diets. Absorbed Se accumulates in the liver and metabolized and a large portion is then transferred into the serum [39]. Se that is not absorbed is expelled in feces as an insignificant source of environmental Se accumulation [40]. Dietary Se supplementation increased the urinary Se excretion in blue foxes, which is in line with other studies [41,42,43]. A relative increase in Se urinary excretion occurs with increasing dietary Se to decrease Se retention and maintain Se homeostasis [43]. Se supplementation in blue fox diets increased the urinary excretion of Se. In 1957, Schwartz and Foltz established Se as an essential trace element in nutrition for prevention of disease [44]. In addition, dietary levels of VE greatly influenced total Se excretion and deposition.

The present study demonstrated that supplementation of blue fox diets with VE and Se may affect the antioxidant capacity of the body and alter the concentration of antioxidant enzymes in serum. It is well known that supplementing VE and Se in feed causes multiple alterations in the antioxidant system [45, 46]. VE is a chain-breaking antioxidant that prevents the propagation of free radical reactions [45]. The nutritive effect of Se in animals is manifested in the composition of selenocysteine that is used as building block in selenoproteins [47]. Increasing dietary levels of antioxidants may decrease in vivo measure of oxidative damage [46].

In the present study, dietary VE and Se levels affected T-AOC and the level of MDA in the serum of foxes. Lan et al. [48] found that VE affects the activity of T-AOC and the concentrations of MDA in rat serum. Se supplementation to blue fox diets enhanced the serum GSH-PX activity in the present study which was similar to results obtained in poultry diets [49]. The GSH-Px enzyme contains selenocysteine residues as its active site [50]. Ebeid et al. [7] reported that VE and organic Se supplementation in rabbit diets increased serum GSH-PX concentrations. In the present study, the activity of SOD was influenced by diet. Interestingly, SOD activities were highest in groups fed with 200 mg VE/kg and approximately 0.1 mg Se/kg diet. The SOD enzyme also participates in the antioxidant defense system [51]. Akiyama et al. [52] suggested that SOD activation is a characteristic feature of ROM production. Interestingly, the concentrations of TG in serum were also higher in the abovementioned group. Kurahashi found that the concentrations of TG in wild-type mice serum were higher than in SOD1-knockout mice serum [53], which indicated that SOD could stimulate lipid secretion into the blood. Ziaie et al. [54] indicated that VE is a potent ROM scavenger that decreases the total SOD activity. In the present study, the activity of GSH-ST was significantly changed with the levels of VE and Se in diets. GSH-ST is part of a superfamily that is composed of antioxidant enzymes [55]. In female fox serum samples, the activity of TrxR was markedly affected by the Se levels in diets. TrxR is an enzyme that contains selenocysteine residues, which are essential for its catalytic activity [56]. Hence, the results indicated that the dietary Se levels may influence the synthesis of selenocysteine residues and further affect the activities of related enzymes. The current study found that the activity of TrxR in serum was not influenced by dietary levels of VE and Se in male blue fox diets. Dietary Se levels did not influence the expression of TrxR 3 in livers and kidneys tissue of mice [57] or of TrxR 1 in pig either [58]. The activity of TPX was significantly influenced by the dietary VE and Se contents in the present study. TPX is a major cellular protein disulfide reductase, which serves as electron donor for reducing enzymes [59]. Suitable concentrations of VE and Se in fox diets can improve the activities of TPX. The serum of T-GSH was also affected by the dietary VE and Se concentrations in this study. It is well known that VE together with glutathione and a membrane-bound enzyme can prevent against damaging effects of reactive oxygen species on PUFA in biomembranes (lipid peroxidation) [60]. Exposure to reactive oxygen and nitrogen species can increase the GSH concentrations by increasing the rate of GSH synthesis [60]. Hence, the serum of T-GSH was lower in the groups with the highest VE supplementation in diets.

In the present study, interactions between VE and Se affected serum TP and ALB in male blue foxes, but not in female. There were significant effects of dietary Se supplementation on the concentrations of GLB in females, but there was no effect on the GLB in male serum. Shinde et al. [61] revealed no significant difference in the concentrations of TP, ALB, and GLB in the serum of male buffalo calves fed different VE or Se supplemented diets. Some studies in animals indicated that high doses of Se might have gender-dependent effects on the immune system [33]. Marsh et al. [62] showed that high levels of Se impaired the antibody responses of male chickens but not of female chickens, and similar results have been found for male and female rats [63]. Adding VE to blue fox diets decreased the concentrations of serum GLU in the present study. The research in type-2 diabetes mellitus patients found that a significant decrease in fasting blood GLU was seen in groups supplemented with VE [64]. In the present study, the concentrations of TG in serum of foxes with Se supplementation were lower than in those fed diets without Se addition. Crespo et al. [65] indicated that Se is involved with TG in the metabolism. In the present study, there was no significant effect of VE and Se supplementation in blue fox diets on serum LDL, which agreed with the findings of Corino et al. [66] and Ebeid et al. [7] in growing rabbits. The concentrations of HDL were influenced by the concentrations of VE in diets, but they were not changed by the dietary Se addition. Shinde et al. [61] found that HDL concentrations in serum of calves were affected supplementation with VE and Se. In some studies, Se supplementation has been associated with modest reductions of CHO and increased HDL levels in serum of children [28, 67]. González-Estecha et al. [68] found significant correlations between serum Se and TG, CHO, and LDL in humans. Dietary treatments with VE significantly affected the CHO concentrations in male serum, which contradicted the findings of Shinde et al. [61] in male buffalo calves fed different dietary concentrations of VE and Se. Moreover, the effects of different levels of VE and selenomethionine supplementation on serum biochemistry in laying hens have suggested that a combination of VE and Se added to the diet decreases serum CHO, TG, and LDL concentrations but increases HDL concentrations [69]. In the present study, interaction effects of different VE and Se supplementation levels were found on BUN. Sugden et al. [70] found that Se supplementation in ewe diets significantly increased the levels of BUN. Similar effects of VE and Se supplementation on blood chemistry have been reported in male calves [62]. The different results may be due to differences in species of animals, and further studies are needed. Regardless of gender, serum VE concentrations were significantly affected by dietary supplementation of VE and Se. Jewell et al. [46] found that increasing levels of dietary VE in dog feed caused significant increases in serum VE levels. These results further confirmed a synergistic effect between VE and Se [33].

Conclusion

The present study demonstrated that appropriate dietary levels of VE and Se in growing furring foxes were beneficial for feed conversion ratio, the nutrient digestibility, and the antioxidant abilities. In addition, some metabolic parameters were influenced by diet. These findings suggested that the suitable dietary concentration of VE is 200 mg/kg in combination with approximately 0.1 mg Se/kg from raw material.