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Journal of Animal Science - Article

 

 

This article in JAS

  1. Vol. 91 No. 5, p. 2158-2168
     
    Received: Aug 29, 2012
    Published: January 28, 2015


    3 Corresponding author(s): mbrummer@alltech.com
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doi:10.2527/jas.2012-5794

Measures of antioxidant status of the horse in response to selenium depletion and repletion1

  1. M. Brummer 32,
  2. S. Hayes*,
  3. K. A. Dawson and
  4. L. M. Lawrence*
  1. Department of Animal and Food Sciences, University of Kentucky, Lexington 40546
    Alltech Inc. Center for Animal Nutrigenomics and Applied Animal Nutrition, Nicholasville, KY 40356

Abstract

Selenium plays a role in the antioxidant mechanism via the selenoenzyme glutathione peroxidase (GSH-Px). Change in Se status because of Se depletion or supplementation is associated with a change in GSH-Px activity and could potentially affect antioxidant status. This study evaluated the impact of change in Se status on measures of antioxidant status and oxidative stress in adult horses. Twenty-eight horses were blocked by age and gender and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), high organic Se (SP), and high inorganic Se (SS). For 196 d, LS, SP, and SS received a low-Se diet (0.06 mg Se/kg DM) to allow for depletion of Se stores, whereas AS received an adequate Se diet (0.12 mg Se/kg DM). Then, for the next 189 d, LS and AS were maintained on the same diets, whereas SP was supplemented with Se-yeast and SS with sodium selenite to allow for a total dietary Se intake of 0.3 mg Se/kg DM. Blood samples were collected throughout the study. Variables of interest included whole blood Se and GSH-Px activity, serum vitamin E concentration, total antioxidant capacity (TAC), serum malondialdehyde (MDA), and triiodothyronine and thyroxine concentrations. Data were analyzed using ANOVA with repeated measures. Whole blood Se and GSH-Px activity decreased in LS, SP, and SS during the depletion phase and increased in SP and SS with supplementation (treatment × time, P < 0.001). At the conclusion of the supplementation period, GSH-Px activity was greater in SP and SS compared with AS and LS (P < 0.05). Vitamin E status remained adequate throughout the study, and no differences existed between treatments. Serum TAC did not change in response to Se depletion or repletion. Serum MDA was greater for AS than LS during depletion (P < 0.05) but similar across treatments after supplementation. Overall, change in Se status did not have a large impact on TAC or MDA, possibly because the horses maintained an adequate vitamin E status. However, Se supplementation at 0.3 mg/kg DM increased GSH-Px activity above that of the horses fed an adequate diet based on the 2007 NRC recommendations, indicating a potential benefit to feeding greater Se diets to horses kept in low-Se areas.



INTRODUCTION

Glutathione peroxidase (GSH-Px) was one of the first identified selenoenzymes, and its activity is influenced by the Se status of the body (Brown and Arthur, 2001). The enzyme GSH-Px is a component of the antioxidant system as it regulates hydrogen peroxide concentrations in the cell (Arthur, 1997; Ferguson and Karunasinghe, 2011). Therefore, antioxidant status might decline when Se status declines. Serum malondialdehyde (MDA), an end product of lipid peroxidation that occurs when cell membranes are damaged by reactive oxygen species (Ducharme et al., 2009) such as hydrogen peroxide (Surai, 2006), has been used as a measure of oxidative stress in horses. Although some studies have evaluated the relationship between dietary Se intake and GSH-Px activity in horses (Stowe, 1967; Shellow et al., 1985; Richardson et al., 2006), little is known about the relationship between Se status, GSH-Px activity, and antioxidant status in horses.

The study objectives were to evaluate the impact of Se depletion and repletion on GSH-Px activity, antioxidant status, and oxidative stress in the horse. The current recommended Se intake (NRC, 2007) for horses (1 mg/d for a 500-kg horse, or 0.1 mg/kg DM) is based on short-term studies that reported no advantage in terms of GSH-Px activity when exceeding 0.1 mg Se/kg DM. Therefore, a secondary objective was to determine if 0.3 mg Se/kg DM, fed over a longer period of time (189 d), would affect whole blood GSH-Px activity. We also evaluated the activity of the selenoenzyme iodothyronine deiodinase by determining the ratio of triiodothyronine (T3) to thyroxine (T4; Brown and Arthur, 2001). Serum vitamin E concentration was also measured, as vitamin E has both antioxidant properties (Ronéus et al., 1986) and a synergistic relationship with Se (Finch and Turner, 1996). We hypothesized Se depletion would cause a decrease in total antioxidant capacity (TAC) and an increase in MDA, whereas Se supplementation would increase TAC and decrease MDA.


MATERIALS AND METHODS

This research project was approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Animals

Twenty-eight mature horses (5 to 23 yr) were used in this study. The 28 horses included 8 geldings and 20 nonpregnant mares. The geldings consisted of 6 Thoroughbreds, 1 American Quarter Horse, and 1 Standardbred, and the mares consisted of 19 Thoroughbreds and 1 American Quarter Horse. All of the horses were housed on the same farm for at least 60 d before beginning the study, where they received a diet consisting of pasture and a fortified concentrate (0.3 to 0.6 mg/kg Se). None of the horses were subjected to any form of forced exercise during this study or for at least 60 d before the start of this study.

Experimental Design, Diets, and Treatments: Depletion Phase and Repletion Phase

The study was conducted in 2 phases as depicted in Fig. 1. The first phase was a Se depletion phase (196 d), immediately followed by a Se repletion phase (189 d). Preceding the onset of the depletion phase, horses were blocked by age and gender and randomly assigned within block to 1 of 4 treatment groups: low Se (LS), adequate Se (AS), high organic Se (SP), and high inorganic Se (SS). Mean ages for each treatment group were LS = 13.5 ± 4.5 yr, AS = 13.1 ± 3.9 yr, SP = 13.3 ± 5.3 yr, and SS = 13.1 ± 4.7 yr. Throughout the study, horses were kept on pasture. The horses were allocated to pastures so that all treatment groups were represented within each pasture. Before the onset and throughout the study, pastures were sampled periodically when forage availability was sufficient. Although the Se content of the pastures fluctuated, it remained marginal in Se (range: 0.03 to 0.08 mg Se/kg DM) at all times. Means for pasture Se concentration were 0.07 ± 0.02 mg and 0.06 ± 0.01 mg Se/kg DM during the depletion and repletion periods, respectively. When pasture availability declined in winter, horses were fed hay produced on the same farm (Se < 0.05 mg/kg DM) ad libitum. Approximately 0.9 kg of cracked corn (Se < 0.16 mg/kg DM) was fed to each horse during December, January, and February of the depletion period to provide additional energy. Horses had ad libitum access to water and an iodized salt block. The horses were weighed on a monthly basis.

Figure 1.

Experimental design and treatment allocation during the depletion (196 d) and repletion phases (189 d). Se source: sodium selenite, and total calculated dietary Se intake: 0.12 mg/kg DM; total calculated dietary Se intake: 0.06 mg/kg DM; Sel-Plex (Alltech Inc., Nicholasville, KY), and total calculated dietary Se intake: 0.3 mg/kg DM; and sodium selenite, and total calculated dietary Se intake: 0.3 mg/kg DM. 1Se source sodium selenite, total dietary Se intake 0.12 mg/kg DM; 2No Se added, total dietary Se intake 0.06 mg/kg DM; 3Se source Se-yeast (Sel-Plex, Alltech Inc. Nicholasville, KY), total dietary Se intake 0.3 mg/kg DM; 4Se source sodium selenite, total dietary Se intake 0.3 mg/kg DM.

 

During the depletion phase, AS received an adequate Se diet and served as a control, whereas the remaining 3 groups (LS, SP, and SS) received a low-Se diet, with the goal of depleting Se stores. In this phase, the 3 depleting groups were grouped together so that only 2 treatment groups existed: AS (n = 7) and LS (n = 21). One Thoroughbred mare had to be removed from the depleting (LS) group during the depletion phase; therefore, this treatment group consisted of n = 20. Adequate- and low-Se balancer pellets were used to manipulate dietary Se intake. To ensure that nutrients other than Se were provided at recommended levels (NRC, 2007), a custom-formulated low-Se (0.48 mg/kg DM) protein–vitamin-mineral balancer pellet was fed to the Se depleting (LS) horses, whereas AS received a similar balancer pellet with 2.52 mg Se/kg DM (Table 1; McCauley Bros. Inc., Versailles, KY). To feed the respective balancer pellets to the horses on an individual BW basis, individual feeding pens were constructed. The calculated total Se intake for the horses on LS was 0.06 mg/kg DM, or approximately 60% of the NRC (2007) recommended amount of 1 mg Se per day for a 500-kg horse, or 0.1 mg Se/kg DM. Horses on AS received a calculated total dietary Se concentration of 0.12 mg/kg DM, or approximately 120% of the NRC (2007) recommended dietary Se intake. Horses were fed their respective diets for a period of 196 d to allow for Se depletion. The 196-d duration of the depletion period was selected on the basis of the Se status of the horses as determined by the monthly blood samples obtained for whole blood Se and GSH-Px activity evaluation. On the basis of these results, the Se status was determined to be sufficiently altered in the LS group [both compared with published reference range (Stowe, 1998) and compared with AS] to proceed with the repletion phase.


View Full Table | Close Full ViewTable 1.

Dry matter content and energy and nutrient composition of pasture and hay sampled throughout the experimental period and balancer pellet fed during the Se depletion phase (DM basis)1,2

 
tem Pasture Grass hay Alfalfa hay Adequate Se balancer pellet Low-Se balancer pellet3
DM, % 92.2 89.7 89.7 89.2 90.1
DE, Mcal/kg 2.11 2.10 2.22 3.35 3.38
CP, % 13.3 16.3 16.2 36.5 34.8
ADF, % 41.8 36.1 34.2 9.2 8.5
NDF, % 59.9 59.7 50.6 15.4 14.9
Starch, % 0.47 1.50 1.9 5.1 7.8
Ca, % 1.06 1.10 1.06 4.07 4.16
P, % 0.36 0.47 0.4 2.16 2.07
Mg, % 0.22 0.29 0.24 0.45 0.47
K, % 1.59 1.77 2.15 2.05 1.98
Na, % 0.01 0.02 0.03 0.85 0.88
Fe, mg/kg 728.00 570.00 376.00 806.00 802.00
Zn, mg/kg 34.7 33.0 32.0 448.00 427.00
Cu, mg/kg 8.00 9.00 7.00 160.00 154.00
Se, mg/kg 0.07 0.07 0.06 2.52 0.48
1Analyzed by a commercial laboratory (Equi-analytical Laboratories, Ithaca, NY).
2Balancer pellet (McCauley Bros. Inc., Versailles, KY). Composition: soybean meal, 60%; ground oats, 8.25%; wheat bran, 7.5%; defluorinated phosphate, 7.5%; molasses, 6.6%; dehydrated alfalfa meal, 5.0%; calcium carbonate, 2.5%; salt, 1.25%; mineral premix, 0.5%; vitamin premix, 0.5%; soybean oil, 0.3%; and mold inhibitor, 0.1% (buffered propionic and sorbic acid).
3Mineral premix did not contain Se.

During the repletion phase, the horses in the AS group remained on the same adequate diet. The 7 horses initially allocated to the LS group remained on the low-Se diet. The remaining 2 depleted groups were supplemented with either an organic (SP; Se-yeast, Sel-Plex; Alltech Inc., Nicholasville, KY) or inorganic (SS; sodium selenite) Se supplement (Fig. 1). One Thoroughbred mare was removed from the repletion period because of an eye injury, and consequently, her entire data set was removed from the repletion phase of the study. Therefore, the number of experimental units per dietary treatment for the repletion phase was 6 horses each for LS and SS, whereas AS and SP consisted of 7 horses each. Throughout the repletion phase, horses had access to pasture, and hay was provided when pasture availability declined. Similar to the depletion phase, custom-formulated adequate Se (2.52 mg/kg DM) or low-Se (0.53 mg/kg DM) protein–vitamin-mineral balancer pellets (Table 2; McCauley Bros. Inc.) were fed. The same individual feeding protocol from the depletion phase was followed during the repletion phase, except that SP and SS supplements were top dressed on the balancer pellet. To account for the yeast component in the Se-yeast supplement fed to SP and brewer’s yeast that was used as the carrier for the sodium selenite supplement fed to SS, the AS and LS horses also received brewer’s yeast. A small amount of water was added to the balancer pellet and top-dressed supplement before feeding to improve the palatability and texture. The horses were monitored to ensure that all of the allotted supplement and balancer pellet was consumed.


View Full Table | Close Full ViewTable 2.

Dry matter content and energy and nutrient composition of pasture and hay sampled throughout the experimental period and balancer pellet fed during the Se repletion phase (DM basis)1,2

 
Item Pasture Grass hay Alfalfa hay Adequate Se balancer pellet Low-Se balancer pellet3
DM, % 87.6 92.1 91.2 89.2 90.9
DE, Mcal/kg 2.33 2.24 2.22 3.35 3.37
CP, % 24.2 21.6 15.8 36.5 36.9
ADF, % 31.9 34.6 38.6 9.2 7.7
NDF, % 49.3 53.6 49.6 15.4 14.7
Starch, % 1.60 2.80 1.40 5.10 3.60
Ca, % 0.91 0.97 1.31 4.07 3.75
P, % 0.52 0.53 0.40 2.16 2.41
Mg, % 0.33 0.40 0.20 0.45 0.44
K, % 2.84 2.75 2.97 2.05 2.14
Na, % 0.14 0.26 0.05 0.85 0.84
Fe, mg/kg 856.00 673.00 217.00 806.00 808.00
Zn, mg/kg 33.0 35.0 23.0 448.00 403.00
Cu, mg/kg 9.00 9.00 8.00 160.00 144.00
Se, mg/kg 0.06 0.06 0.02 2.52 0.53
1Analyzed by a commercial laboratory (Equi-analytical Laboratories, Ithaca, NY).
2Balancer pellet (McCauley Bros. Inc., Versailles, KY). Composition: soybean meal, 60%; ground oats, 8.25%; wheat bran, 7.5%; defluorinated phosphate, 7.5%; molasses, 6.6%; dehydrated alfalfa meal, 5.0%; calcium carbonate, 2.5%; salt, 1.25%; mineral premix, 0.5%; vitamin premix, 0.5%; soybean oil, 0.3%; and mold inhibitor, 0.1% (buffered propionic and sorbic acid).
3Mineral premix did not contain Se.

During the repletion period, the total dietary Se concentrations were calculated to be as follows: LS, 0.06 mg; AS, 0.12 mg; and SP and SS, 0.3 mg Se/kg DM. The horses were kept on their respective diets for a period of 189 d. This supplementation period was selected to ensure that the turnover of red blood cells would be sufficient for the identification of change in indicators of Se status (whole blood Se concentration and GSH-Px activity).

Blood Sampling Procedures

For indicators of Se status baseline blood samples were taken at the start of each phase and then at d 84, 140, 168, and 196 during the depletion phase and d 28, 56, 154, and 189 of the repletion phase. Additional blood samples were collected on d 56 and 112 of the depletion phase for complete blood count (CBC) analysis. Blood was collected in 7-mL lithium heparin blood collection tubes (Becton Dickson, Franklin Lakes, NJ) for analysis of whole blood Se and whole blood GSH-Px activity. Blood was also collected in 10-mL untreated blood collection tubes for serum separation (Becton Dickson) and tubes containing 7 mL EDTA (Becton Dickson) for CBC analysis. Whole blood was transferred to storage vials and kept at –80°C until analysis. Serum separation was conducted by allowing blood samples to clot for approximately 2 h at room temperature. Samples were then centrifuged at 2500 × g for 20 min at 4°C. Serum was aspirated, transferred to storage vials, and stored at –80°C until analyzed.

Laboratory Procedures

Whole blood GSH-Px activity was determined using an assay kit (Bioxytech GPx-340 Assay Kit; OXIS Research, Portland, OR; Richardson et al., 2006). This assay is based on the method developed by Paglia and Valentine (1967). The GSH-Px activity of each sample was calculated from the change in absorbance and expressed as units of enzyme activity per milligram hemoglobin (mU/mg Hb). Hemoglobin values were obtained from the CBC analysis. The interassay CV for whole blood GSH-Px activity was 3.28%. Whole blood Se concentration was analyzed (Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI) by means of inductively coupled plasma–mass spectroscopy. The interassay CV for whole blood Se concentration was 0.5%.

The CBC analyses were performed by a local commercial equine hospital (Rood and Riddle Equine Hospital, Lexington, KY; Morresey and Waldridge, 2010). Lymphocyte and neutrophil numbers were then calculated from the CBC analysis. Lymphocyte and neutrophil numbers were monitored as lymphocyte numbers have been found to increase with Se supplementation (Calamari et al., 2010).

Serum TAC was determined using a method that compares the ability of the antioxidants in the serum sample to inhibit the oxidation of 2,2′-azino-di-[3-ethylbenzthiazoline sulphonate] with that of Trolox, a water-soluble tocopherol analog (antioxidant assay kit; Caymenchemical; Ann Arbor, MI). Samples were analyzed in triplicate according to the manufacturer’s protocol (Caymenchemical). The TAC was determined for the baseline and d 154 samples collected for each phase. The interassay CV was 3.82%.

Serum MDA concentration was measured using a thiobarbituric acid-reactive substances (TBARS) method (Ducharme et al., 2009). This method was based on the reaction of MDA with thiobarbituric acid under acidic, high-temperature conditions, using MDA as standard. Samples were analyzed in triplicate according to the directions supplied by the kit manufacturer (Caymenchemical). Serum MDA was determined on the baseline and end point samples collected for each phase. The interassay CV for MDA concentration was 3.68%.

Serum vitamin E concentration was determined by HPLC analysis with UV detection (Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA). The interassay CV for serum vitamin E concentration was 4.8%.

A solid-phase 125I RIA (Coat-a-count; Siemens, Los Angeles, CA) was used to measure T3 and T4. The T3/T4 ratio was then calculated. The T3/T4 ratio was determined for the baseline and end point samples collected for both phases. The interassay CV for T3 and T4 were 2.47% and 3.14%, respectively.

Statistical Analysis

Data were analyzed as repeated measures ANOVA using the Proc MIXED function (SAS Inst. Inc., Cary, NC) with the least squares means separation procedure using the pdiff option in SAS. Data from the depletion and repletion phases were analyzed separately, with the exception of the serum vitamin E concentration, which evaluated change in vitamin E status over the course of the entire experiment. Each horse served as an experimental unit. The model included time, treatment, block, and treatment × time as fixed effects, and horse was included as a random effect. Data were tested for normality and were log transformed when required for statistical analysis. Data were back transformed and are presented in tables and figures as least squares means ± SE. Correlations between whole blood Se and GSH-Px were evaluated using the Proc CORR procedure of SAS. P < 0.05 was considered significant, and P < 0.10 was considered a trend.


RESULTS

Depletion Phase

Whole blood Se concentration and GSH-Px activity for the depletion phase are presented in Table 3. Whole blood Se concentration was similar between AS and LS at the onset of the depletion phase. Whole blood Se concentration was affected by treatment (P < 0.001), time (P < 0.001), and a treatment × time interaction (P = 0.007) during the depletion phase. Whole blood Se concentration in LS decreased until d 140, then stabilized, and at that time it was less than AS (P < 0.001). Whole blood Se concentration at the end of the depletion period (d 196) was different between the 2 treatment groups (P < 0.05). The Se concentration of AS decreased within the first 84 d but then stabilized. Whole blood GSH-Px activity for the depletion phase was affected by treatment (P < 0.001) and time (P < 0.001). Similar to Se, whole blood GSH-Px activity in AS decreased during the first 84 d of depletion and then stabilized, whereas GSH-Px activity in LS decreased throughout depletion phase. Mean GSH-Px activity was less in LS compared with AS at d 196 (P < 0.05). A positive correlation existed between whole blood Se and GSH-Px activity (r = 0.63; P < 0.001).


View Full Table | Close Full ViewTable 3.

Whole blood Se concentration and glutathione peroxidase activity of mature horses during the depletion phase (least squares means)1,2

 
Treatment (Trt)
SEM
P-values
Item LS AS LS AS Trt Time Trt × time
Se, ng/mL 4.42 7.47 <0.001 <0.001 0.007
    d 0 251.7w 261.8w
    d 84 202.1x 228.2x
    d 140 173.5ay 205.2bxy
    d 168 160.7y 182.9y
    d 196 165.1ay 211.8bx
Glutathione peroxidase, mU/mg Hb 2.04 3.44 <0.001 <0.001 0.916
    d 0 64.5aw 75.9bw
    d 84 54.4ax 62.8bxy
    d 140 52.7ax 65.5bx
    d 168 46.7ay 57.4bxy
    d 196 43.1ay 55.5by
a,bMeans within row and variable lacking common superscripts differ (P < 0.05).
w–yMeans within column lacking common superscripts differ (P < 0.05).
1Dietary treatments (total dietary Se): LS = low Se (0.06 mg/kg DM; n = 20), and AS = adequate Se (0.12 mg/kg DM; n = 7).
2Hb = hemoglobin.

Serum TAC (Table 4) was not affected by Se status during the depletion phase. However, there was an effect of time (P < 0.001) during the depletion phase, and TAC decreased from d 0 to 196 in both groups. Serum MDA concentrations were greater in AS (treatment, P < 0.023), and MDA increased in both AS and LS from d 0 to 196 (time, P = 0.041) during the depletion phase.


View Full Table | Close Full ViewTable 4.

Change in total antioxidant capacity (TAC) and malondialdehyde (MDA) concentration of horses during Se depletion phase (least squares means)1

 
Treatment (Trt)
SEM
P-values
Item LS AS LS AS Trt Time Trt × time
TAC, mM 0.020 0.034 0.742 <0.001 0.074
    d 0 1.181 1.118
    d 196 0.703 0.729
MDA, μM 0.230 0.397 0.023 0.041 0.831
    d 0 3.480 4.520
    d 196 4.110 4.940
1Dietary treatments (total dietary Se): LS = low Se (0.06 mg/kg DM; n = 20), and AS = adequate Se (0.12 mg/kg DM; n = 7).

The CBC data collected during the depletion phase (Table 5) indicated a trend (P = 0.057) for greater lymphocyte numbers for LS compared with AS. The number of neutrophils and the ratio of lymphocytes to neutrophils were similar between LS and AS. The T3/T4 ratio decreased over time during the depletion period (P = 0.018), but this decrease was not affected by Se status (data not shown).


View Full Table | Close Full ViewTable 5.

Change in lymphocyte and neutrophil numbers in horses during the depletion phase1

 
Treatment (Trt)
SEM
P-values
Item LS AS LS AS Trt Time Trt × time
Lymphocyte, × 103 0.16 0.27 0.057 <0.001 0.753
    d 0 3.53 2.82
    d 56 2.97 2.83
    d 84 2.74 2.32
    d 112 3.00 2.52
    d 140 3.00 2.46
    d 168 3.02 2.41
    d 196 2.84 2.33
Neutrophil, × 103 0.23 0.39 0.292 <0.001 0.334
    d 0 4.94 4.13
    d 56 4.85 4.66
    d 84 4.51 4.34
    d 112 4.32 4.02
    d 140 3.93 3.81
    d 168 4.61 4.01
    d 196 5.32 5.63
Ratio 0.13 0.22 0.274 <0.001 0.838
    d 0 1.50 1.48
    d 56 1.72 1.73
    d 84 1.75 1.94
    d 112 1.55 1.65
    d 140 1.38 1.65
    d 168 1.59 1.70
    d 196 2.04 2.47
1Dietary treatments (total dietary Se): LS = low Se (0.06 mg/kg DM; n = 20), and AS = adequate Se (0.12 mg/kg DM; n = 7).

Repletion Phase

Whole blood Se concentration during the repletion phase (Fig. 2) was affected by treatment (P < 0.001), time (P < 0.001), and a treatment × time interaction (P < 0.001). At the start of the repletion phase, whole blood Se concentration was similar among LS, SP, and SS. Within 28 d of starting the repletion phase, whole blood Se was comparable between AS, SP, and SS but greater than LS (P < 0.05). On d 154, whole blood Se concentrations in SP and SS were greater than AS (P < 0.05). Whole blood Se did not increase from d 154 to 189 in either SS or SP. As expected, LS and AS maintained their respective low or adequate Se concentrations.

Figure 2.

Whole blood Se concentration (ng/mL) of horses on different dietary treatments measured throughout the repletion phase (least squares means). Dietary treatments (total dietary Se): LS = low Se (0.06 mg/kg DM; n = 6); AS = adequate Se (0.12 mg/kg DM; n = 7); SP = organic Se [0.3 mg/kg DM (Sel-Plex; Alltech Inc., Nicholasville, KY); n = 7]; SS = inorganic Se [0.3 mg/kg DM (sodium selenite); n = 6]. Before d 0, LS, SP, and SS received the low-Se diet. a,bMeans within time point lacking common superscripts differ (P < 0.05). w–yMeans within treatment lacking common superscripts differ (P < 0.05); *Trend to differ (P < 0.10).

 

Whole blood GSH-Px activity during the repletion phase (Fig. 3) also was affected by treatment (P < 0.001), time (P < 0.001), and treatment × time (P < 0.001). Whole blood GSH-Px activity followed a similar but delayed response to the Se concentration data. At the beginning of the repletion phase GSH-Px activity was greater for AS in comparison with LS, SP, and SS. At d 154, the GSH-Px activity of SS was comparable to SP but greater than AS. At the final time point (d 189), the GSH-Px activity of SP and SS were similar (P = 0.653) but greater when compared with AS and LS (P < 0.03). Between d 154 and 189, GSH-Px activity plateaued within SS but continued to increase in SP. A strong, positive correlation existed between whole blood Se and GSH-Px activity (r = 0.82; P < 0.001).

Figure 3.

Whole blood glutathione peroxidase (GSH-Px) activity [mU/mg hemoglobin (Hb)] throughout the repletion phase (least squares means). Dietary treatments (total dietary Se): LS = low Se (0.06 mg/kg DM; n = 6); AS = adequate Se (0.12 mg/kg DM; n = 7); SP = organic Se [0.3 mg/kg DM (Sel-Plex; Alltech Inc., Nicholasville, KY); n = 7]; SS = inorganic Se [0.3 mg/kg DM (sodium selenite); n = 6]. Before d 0, LS, SP, and SS received the low-Se diet. a,bMeans within time points lacking common superscripts differ (P < 0.05). w–yMeans within treatment lacking common superscripts differ (P < 0.05).

 

Serum TAC (overall, 0.554 ± 0.005 mM) was not affected by Se status and remained similar throughout the repletion phase (data not shown). Similarly, serum MDA concentration (8.107 ± 0.332 µM) was unaffected by Se status and time during the repletion phase (data not shown). Serum vitamin E concentration (Fig. 4) monitored across both phases fluctuated over time (P < 0.001), but there was no effect of treatment or treatment × time.

Figure 4.

Serum vitamin E concentration (µg/mL) throughout the depletion and repletion phases. Dietary treatments (total dietary Se) during the depletion phase: LS = low Se (0.06 mg/kg DM; n = 20); AS = adequate Se (0.12 mg/kg DM; n = 7). Dietary treatments (total dietary Se) during the repletion phase: LS = low Se (0.06 mg/kg DM; n = 6); AS = adequate Se (0.12 mg/kg DM; n = 7); SP = organic Se [0.3 mg/kg DM (Sel-Plex, Alltech Inc., Nicholasville, KY); n = 7]; SS = inorganic Se [0.3 mg/kg DM (sodium selenite); n = 6]. Treatment, P = 0.746; time, P = 0.004; and treatment × time, P = 0.885.

 

Lymphocyte (overall mean 2.65 ± 0.06 × 103) and neutrophil (4.172 ± 0.077 × 103) numbers were similar between treatment groups throughout the repletion phase. The lymphocyte to neutrophil ratio was also unaffected by treatment (data not shown).

Although the T3/T4 ratio increased over time during the repletion phase (P = 0.073), the increase was unaffected by Se status (data not shown), and there was no change in the T3/T4 ratio within the LS group from the beginning of the depletion phase until the end of the repletion phase (data not shown).


DISCUSSION

Studies showing an increase in the activity of GSH-Px in response to Se supplementation introduced the concept of improved functional Se status through dietary Se supplementation (Brown and Arthur, 2001). In the present study, the change in whole blood Se and GSH-Px activity throughout the depletion and repletion phases followed the expected response, indicating that the Se status of the horse can be manipulated by dietary Se intake if sufficient time is allowed for the variables to adjust.

The adequate reference range for whole blood Se in a mature horse is estimated at 180 to 240 ng/mL (Stowe, 1998). Based on this reference range, the horses included in this study were of high Se status at the start of the study. For at least 60 d prior to the start of this study, all horses had received a fortified concentrate in addition to pasture. As is typical for commercial concentrates formulated for horses, the Se concentration in the concentrate (0.3 to 0.6 mg/kg) resulted in dietary Se intakes above the NRC (2007) recommendation and above the level provided by the AS diet in this study. Consequently, the initial decrease in Se concentration observed for AS from high Se status to within the adequate range at d 84 was not unexpected. The decrease in whole blood Se concentration of LS was greater than that of AS. Within 140 d of depletion, the whole blood Se concentration of LS was below the adequate reference range and remained so for the duration of the study. Yet the diet fed to the LS horses was still providing 60% of their estimated requirement (NRC, 2007). The observed decrease in whole blood Se concentration to below the reference range within 140 d in LS and the relatively stable concentration in AS indicate that the current Se recommendation (0.1 mg Se/kg DM) must be close to the minimum Se requirement for mature idle horses.

Similar to whole blood Se, GSH-Px activity decreased in both AS and LS during the depletion phase. It has been stated that erythrocyte GSH-Px activity will plateau when a whole blood Se concentration of 160 ng/mL is reached (Blackmore et al., 1982). Whole blood Se concentration remained above this concentration during the depletion phase in AS. Therefore, whole blood GSH-Px activity of AS was expected to remain similar throughout the depletion period. However, it declined regardless of adequate whole blood Se concentration and a Se intake slightly above the recommended daily Se intake (NRC, 2007). This observation indicates that whole blood GSH-Px is responsive to dietary Se intakes above the recommended level, supporting our hypothesis that 0.1 mg Se/kg DM may not allow for maximum GSH-Px activity in the horse. The whole blood GSH-Px activity reference range is 40 to 160 enzyme units/g Hb (Stowe, 1998), but the sensitivity of GSH-Px activity to storage times and assay conditions make it difficult to compare absolute GSH-Px activity values across studies. In this study, GSH-Px activity of LS approached the lower end of this reference range at the end of the depletion phase and remained below this reference range throughout the repletion phase, whereas AS always remained within the reference range throughout both phases. Whole blood GSH-Px activity appeared to increase faster in SS than SP between d 56 and 154 on the basis of greater GSH-Px activity in SS compared with AS at d 154 and the lack of difference between SP and AS at that time point. Sodium selenite supplementation has been reported to be more effectively incorporated into GSH-Px than organic Se in finishing pigs and reproducing gilts, as observed by a faster increase in GSH-Px activity (Mahan et al., 1999; Fortier et al., 2012). However, Mahan et al. (1999) reported that the effect was more prominent at lower levels of Se inclusion (0.05 and 0.10 mg/kg DM).

Within 28 d of the start of the repletion period, the whole blood Se concentration of SP and SS increased above LS and became similar to AS. This rapid response to supplementation has been described before in whole blood (Calamari et al., 2009) and plasma (Richardson et al., 2006). Calamari et al. (2009) reported that although plasma Se plateaued between d 75 and 90, whole blood Se concentration did not plateau during a 112 d Se supplementation study conducted with lightly exercised horses. In our study, whole blood Se concentrations for SP and SS did reach a plateau between d 154 and 189, indicated by the lack of change in Se concentrations between those time points. The Se concentration results for SS and SP indicate that whole blood Se concentrations will range from 225 to 278 ng/mL when horses are fed diets containing 0.3 mg Se/kg DM.

In this study, whole blood GSH-Px activity required 56 d to respond to supplementation in comparison with the 28 d for whole blood Se. This delay is likely due to the time required for the incorporation of GSH-Px in recently formed red blood cells (Knight and Tyznik, 1990). In the current study, both depletion and repletion phases exceeded the period needed for the complete turnover of the red blood cell population (approximately 150 d), thus allowing enough time for the incorporation of GSH-Px into recently formed red blood cells. Consequently, it was demonstrated that a dietary Se intake of 0.3 mg/kg DM resulted in greater GSH-Px activity, regardless of Se source when compared with a dietary Se intake of 0.12 mg/kg DM. In addition, maximum GSH-Px activity was achieved for SS between d 154 and 189. These results are in agreement with Calamari et al. (2009), who also reported greater GSH-Px activity with greater Se intakes. The current Se requirement of the horse is estimated at 0.1 mg/kg DM, but on the basis of whole blood GSH-Px activity in this study, horses may benefit from greater dietary Se levels if increased GSH-Px activity is desirable (e.g., in situations that may increase reactive oxygen species production).

Stowe (1967) and Shellow et al. (1985) found no additional benefit to feeding greater amounts of Se based on plasma GSH-Px response, but their study periods were less than 150 d. More recently, it has been suggested that the lack of detectable change in GSH-Px activity in response to supplementation levels above 0.1 mg/kg DM could be due to the shorter experimental periods used in some studies compared with the length of time required for complete red blood cell turnover in the horse (Richardson et al., 2006; Calamari et al., 2009). For instance, Richardson et al. (2006) reported that a 56-d supplementation period, comparing Zn-L-selenomethionine (total Se intake 5.1 mg Se/d) and sodium selenite (total Se intake 4.7 mg/d) to a control (total Se intake 1.3 mg Se/d), did not affect plasma, red blood cell, or muscle GSH-Px activity. The authors reported a trend for the organic supplementation group to have a faster red blood cell GSH-Px response within the first 28 d, but this was attributed to the response of a single horse within that treatment. Similarly, Karren et al. (2010) reported no difference in plasma GSH-Px between horses provided a total Se intake of 0.19 mg/kg (pasture) and intakes of 0.35 mg/kg DM (pasture and grain), 0.49 mg/kg DM (pasture and Se-yeast), or 0.65 mg/kg DM (pasture, grain, and Se-yeast) for 110 d.

At d 189, GSH-Px activity was greater for both SP and SS compared with AS. These results are in agreement with those of Calamari et al. (2009), who reported whole blood GSH-Px activity of horses supplemented at 0.29 or 0.39 mg Se/kg DM to be greater when compared with horses receiving 0.085 or 0.182 mg Se/kg DM for 112 d. They also found that horses receiving 0.29 mg Se/kg DM as Se-yeast had greater GSH-Px activity than horses receiving 0.29 mg Se/kg DM as sodium selenite on d 112. However, they reported that a plateau was not reached for GSH-Px activity within the 112 d feeding period. In the current study, a plateau was reached in GSH-Px activity for the SS group between d 154 and 189 but not for the SP treatment, which had greater GSH-Px activity at d 189 compared with d 154. This indicates that maximum GSH-Px activity was reached for SS within 154 d of supplementation, whereas GSH-Px activity was still increasing at this time in SP, even though whole blood Se concentrations did plateau in both SP and SS at this time.

The antioxidant mechanism is complex, consisting of a range of different nonenzymatic (vitamin E, vitamin C, carotenoids, ubiquinols, flavonoids, glutathione, and uric acid) and enzymatic (superoxide dismutase, catalase, GSH-Px, and thioredoxin system) antioxidants. When working in unison, the various components of the antioxidant system are capable of preventing, as well as repairing, oxidative damage (Ji, 1999; Urso and Clarkson, 2003; Surai, 2006; Battin and Brumaghim, 2009). A variety of different measures of antioxidant capacity exist, including oxygen radical absorbance capacity, trolox equivalent antioxidant capacity assay, and ferric reducing ability assay (Cao and Prior, 1998). Serum or plasma antioxidant capacity is a variable that is frequently included in equine exercise studies (Avellini et al., 1999; de Moffarts et al., 2005; Ogonski et al., 2008). Although the horses were not exercised during this study, we were interested in determining if the overall antioxidant capacity of the horses would be affected by low- or high-Se status. We hypothesized that antioxidant status and oxidative stress would be altered because of change in GSH-Px activity. On the contrary, no response to change in Se status was observed for TAC. It has been suggested that GSH-Px may only play a small role in the total cellular antioxidant system (Ho et al., 1997), and on the basis of these results, the same may be true for the extracellular total antioxidant system. However, it may also indicate a lack of specificity of the TAC assay to account for the GSH-Px-associated antioxidant capacity. More in depth evaluation is needed to fully understand the contribution of GSH-Px to the antioxidant mechanism in the horse.

Malondialdehyde is an end product of lipid peroxidation in biological membranes (Urso and Clarkson, 2003; Ducharme et al., 2009). Therefore, serum MDA concentration is frequently used as an indicator of oxidative stress, most commonly measured using the TBARS assay but also via HPLC and spectrophotometry (Urso and Clarkson, 2003). The TBARS assay has been used to evaluate MDA as an indicator of oxidative stress in the horse (Ducharme et al., 2009). Oxidative stress is normally a variable of interest in exercise-related studies because exercise has been shown to increase free radical production (Ji, 1999). However, we were interested in evaluating oxidative stress in these idle horses with low- and high-Se status to determine if reduced concentrations of GSH-Px would impact oxidative stress in nonexercising horses. The results indicated a greater MDA concentration for the AS horses compared with the LS horses during the depletion phase, with an increase in MDA for both treatments over time. Serum MDA concentration was similar between treatments during the repletion period. This lack of difference in MDA in horses with low, adequate, or high Se status at the end of the repletion phase may indicate that the antioxidant mechanism adjusted to account for the change in GSH-Px activity.

The lack of effect of Se on antioxidant status indicates that other components of the antioxidant system were minimizing the effects of Se depletion. Vitamin E acts as a lipid soluble antioxidant, protecting cell membranes and preventing lipid peroxidation (Ronéus et al., 1986). In horses, a serum vitamin E concentration above 2 µg/mL is considered adequate, and 1.5 to 2 µg/mL is regarded as a marginal status (NRC, 2007). Across the depletion and repletion period, serum vitamin E ranged from 1.9 to 8.1 µg/mL for samples from individual horses. The 1.9 µg/mL was the only marginal value that was observed throughout the entire study. Across all other time points, the serum vitamin E concentration for this specific horse ranged from 2.6 to 5.8 µg/mL. Thus, horses maintained an adequate vitamin E status throughout the depletion and repletion period. Vitamin E supplementation has been reported to alleviate “selenium responsive disorders” such as myopathies in horses and cattle or exudative diathesis in poultry (Finch and Turner, 1996). This synergistic relationship may explain the lack of effect of Se status on TAC and MDA concentrations. In addition, the assay used to evaluate TAC uses the tocopherol analog, Trolox, as the standard curve. As all horses were of adequate vitamin E status, this could in part also explain the lack of difference detected among treatments in terms of TAC. Throughout the depletion phase there was an overall decrease in TAC across both treatment groups without a substantial change in vitamin E status. This may indicate that other components of the antioxidant mechanism could have been changing during that period of time, even though the balancer pellet provided ensured that the horses were adequate in other nutrients that are known to affect antioxidant capacity such as Zn and vitamin A. Overall, high-Se status did not improve MDA or TAC measurements over that of horses on the adequate Se diet.

A preliminary study conducted in our laboratory found horses of greater Se status to have a greater number of lymphocytes. Similarly, Calamari et al. (2010) reported a trend for a linear dose effect of Se on lymphocyte numbers. In the current study, there was a trend for greater lymphocyte numbers in the LS group during the depletion phase. However, this was not observed for the repletion period. It has been proposed that GSH-Px prevents damage to phagocytic cells by neutralizing increased concentrations of H2O2 (Knight and Tyznik, 1990); however, neutrophil numbers were not affected by Se status during either the depletion or repletion phase.

Iodothyronine deiodinase (ID) is a selenoenzyme responsible for the conversion of the pro-hormone T4 to its active form, T3 (Brown and Arthur, 2001; Calamari et al., 2009; Muirhead et al., 2010) and calculating the ratio between T3 and T4 is thought to be a functional indicator of ID (Brown and Arthur, 2001; Calamari et al., 2009). The current study found that the ratio of T3 to T4 was unaffected by Se status. On the basis of in vitro studies, ID seems to be more protected from Se deficiency than GSH-Px (Köhrle, 2000). Calamari et al. (2009) reported a lack of effect of Se source or Se dose on plasma T3, T4, and the ratio of T3 to T4. Muirhead et al. (2010) evaluated the Se concentration, T3 and T4 concentrations in a group of horses in Prince Edward Island (Canada) and reported a correlation between T4 and Se concentrations but not T3 and Se. In contrast, Dalir-Naghadeh and Rezaei (2008) reported a decreased T3 to T4 ratio in lambs diagnosed with Se deficiency myopathy compared with healthy lambs. Recently, Fortier et al. (2012) reported a trend for greater T3 and T4 concentrations in gilts fed a Se-yeast supplement compared with gilts fed a sodium selenite supplement at a similar dietary level of 0.3 mg Se/kg. These authors further stated that the efficiency of conversion of T4 to T3 did not appear to be affected by the source of Se supplementation. The lack of effect of Se status on this selenoenzyme in our study may also indicate a greater priority of ID for available Se in the horse than GSH-Px.

In conclusion, this study demonstrated that a greater Se intake (0.3 mg Se/kg DM) for 154 d or more increased whole blood GSH-Px activity above that of horses fed an NRC (2007) adequate Se diet. The current dietary Se recommendation of 0.1 mg Se/kg DM may, therefore, not support maximal GSH-Px activity, which could be important in horses subjected to conditions that can induce oxidative stress, especially for horses kept in geographically low Se areas. Although antioxidant status and serum MDA were unaffected by change in GSH-Px activity, adequate vitamin E status may have played a role in the lack of effect of low Se status on MDA and TAC variables.

 

References

Footnotes