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ANIMAL NUTRITION |
* Animal Science Department, Colorado State University, Fort Collins 80523-1171;
and
Zinpro Corporation, Eden Prairie, MN 55344
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: horse • selenium • glutathione peroxidase
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Selenium supplementation has been a concern in areas having a high incidence of white muscle disease in foals (Lofstedt, 1997). Equine Se nutrition is also of interest due to its effects on immune function (Baalsrud and Overnes, 1986; Knight and Tyznik, 1990; Janicki et al., 2000, 2001) and exercise tolerance (Brady et al., 1978; Ono et al., 1990; Avellini et al., 1999). Results of such experiments are often inconclusive or contradictory, indicating a need for a more comprehensive understanding of equine Se metabolism.
Inorganic and organic dietary Se sources are metabolized differently (Deagen et al., 1987; van Ryssen et al., 1989; Mahan et al., 1999). When fed to nonruminants, inorganic Se sources, such as sodium selenite, seem to be more rapidly incorporated into GPx than organic sources such as selenomethionine (Se-Met; Levander et al., 1983; Mahan and Parrett 1996; Mahan et al., 1999). On the other hand, Se-Met, the predominant organic form of Se in plants and yeast, is nonspecifically incorporated into tissues in place of methionine (Schrauzer, 2000) and as such seems better incorporated into tissues as compared with inorganic Se (Deagen et al., 1987; Mahan et al., 1999). Metabolism of selenocysteine, another organic Se source, seems to be similar to that of selenite (Deagen et al., 1987).
The purpose of the current study was to determine the effect of organic and inorganic Se sources on the Se status of horses by comparing plasma and skeletal muscle Se concentrations and GPx activities in plasma (P GPx-3), erythrocytes (RBC GPx-1), and skeletal muscle (M GPx-1).
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals were housed in groups of 3 by treatment and sex in 8 x 20-m dry lots of which 8 x 4 m was covered on 3 sides. Each shelter was equipped with 3 smaller pens to allow for individual feeding. Treatments were stratified across pens so that adjacent pens did not receive the same treatment. One gelding from the INORG group was injured halfway through the trial and was maintained in a stall for the duration of the study. Horses did not receive any forced exercise.
Diets
The CTRL (basal) diet consisted of 7.1 ± 1.7 kg of early bloom alfalfa hay (DM basis) per horse daily, plus 1.2 kg of a concentrated feed (DM basis), formulated to provide minerals (other than Se) and vitamins that were limited in the forage, to meet or exceed the NRC (1989) requirements for 18-mo-old, 425-kg of BW horses. Nutrient concentrations of the basal diet and ingredients are shown in Table 1. Alfalfa hay was weighed, divided into 2 equal feedings per day, and group-fed to each pen of 3 horses. The concentrate was fed to each horse individually one time per day. Animals also had ad libitum access to water and salt.
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Sample Collection
Animals were weighed at the beginning of the 28-d acclimation period and on d 0, 28, and 56 of the experimental period. On sample collection days, animals received their concentrate and supplement but no hay before BW and blood samples were obtained. Blood was collected between 1 and 3 h postfeeding, and the time between feeding and blood collection was consistent for each horse on all collection days.
Blood was drawn via jugular venipuncture into heparinized, trace mineral-free Vacutainer tubes (Becton-Dickinson, Rutherford, NJ) on d 0, 28, and 56 of the experimental period. Blood was stored on ice in a cooler for no more than 4 h and subsequently centrifuged at 1,500 x g for 15 min at 4°C. Plasma was harvested and stored in acid-washed polypropylene vials (CryoPro cryogenic vials, VWR, West Chester, PA) at –80°C. The RBC fraction was washed in 10 volumes of cold saline and centrifuged as just described. Erythrocytes were lysed by adding 4 volumes of cold deionized water and again centrifuged. The supernatant lysate was transferred to acid-washed polypropylene vials (CryoPro cryogenic vials, VWR) and stored at –80°C.
Muscle biopsies (approximately 200 mg of wet weight) were taken from 2 sites of the right-middle gluteal muscle using a percutaneous needle biopsy (Bergstrom Biopsy Tool, Bignell Surgical Instruments Ltd., West Sussex, UK) on d 0 and 56, as described by Snow and Guy (1976). Muscle samples were immediately washed in sterile Se-free saline, transferred to acid-washed vials, snap-frozen in liquid N, and stored at –80°C.
All samples were maintained at –80°C for 5 to 6 mo until analysis for Se and GPx activity, except during a 3-wk period immediately before analysis, when equipment failure necessitated storage of the samples at –20°C.
Sample Analysis
Dietary ingredients and plasma and middle gluteal muscle Se concentrations were determined by the Oregon State University Forage Analysis Services (Corvallis, OR) using a semiautomated fluorometric technique (Brown and Watkinson, 1977) with the modifications of Beilstein and Whanger (1986). Activity of P GPx-3, RBC GPx-1, and M GPx-1 was determined by the method of Paglia and Valentine (1967), using a Bioxytech GPx-340 Assay Kit (OXIS Research, Portland, OR). The assay provides an indirect measure of GPx activity.
The sample to be assayed for GPx was added to a solution containing glutathione (GSH), glutathione reductase, and NAD phosphate (NADPH), to which tert-butyl hydroperoxide was added. Sample GPx catalyzed the reduction of tert-butyl hydroperoxide, using reducing equivalents from GSH yielding oxidized glutathione. Oxidized glutathione was recycled back to GSH by glutathione reductase, using reducing equivalents from NADPH. The consumption of NADPH was accompanied by a decrease in absorbance at 340 nm. The change in NADPH concentration upon initiation of the reaction just described was directly proportional to GPx activity (i.e., 1 mU GPx activity/mL is equivalent to a decrease of 1 nmol of NADPH·mL–1·min–1). The NADPH concentration was calculated using the extinction coefficient (6,220 m–1·cm–1) at 340 nm.
Plasma and RBC lysate were diluted 1:1 and 1:9 (vol/vol), respectively, in assay buffer provided by the manufacturer (OXIS Research) to obtain values within the manufacturer’s recommended range of 5.6 to 24 mU/mL (i.e., 0.035 to 0.15 A340/min). Before analysis, middle gluteal muscle samples were thawed, blotted free of excess moisture, homogenized in Tris-HCl buffer (pH 7.5, containing 5 mM EDTA and 1 mM 2-mercaptoethanol) at a volume:weight ratio of 3 mL:1 mg using a tissue grinder (Kontes Duall Tissue Grinder, Fisher Scientific, Pittsburgh, PA), and centrifuged at 5,000 x g for 10 min at 4°C. The GPx assay was performed on the resulting undiluted supernatant. All samples were analyzed in duplicate. Each sample type (plasma, RBC, muscle) was analyzed within a single run within a single day. The intraassay CV was less than 5% for duplicate samples. A GPx control (Bioxytech cellular glutathione peroixdase control, OXIS Research) was run in duplicate at the beginning, middle, and end of each run to monitor the integrity of the assay.
Glutathione peroxidase enzyme activity was expressed per unit of hemoglobin in RBC and per unit of protein in plasma and muscle. Hemoglobin concentration was determined in undiluted RBC lysate using a Total Hemoglobin Assay (Sigma Diagnostics, St. Louis, MO). Total protein concentration of plasma was determined using a Total Protein Assay (Sigma Diagnostics); total protein concentration of muscle homogenate was determined using the microassay procedure of the BioRad Protein Assay (BioRad Laboratories, Hercules, CA).
All spectrophotometric readings were obtained using an 8452A Diode Array Spectrophotometer (Hewlett-Packard Co., Palo Alto, CA). The protein microassay was conducted using an ELX-800 Microplate Reader (BioTek Instruments Inc., Winooski, VT).
Statistical Analysis
Data were analyzed as a repeated measures design using the PROC MIXED procedure of SAS (version 8, SAS Inst. Inc., Cary, NC), according to Littell et al. (1998), and using individual horse as the experimental unit. The model included horse within treatment, treatment, time, and the treatment x time interaction. The appropriate covariate structure for the model for each response variable was determined using model-fitting statistics generated in SAS (1999). Data are presented as least squares means with SEM. When a significant treatment x time interaction was observed, differences between treatment means at each time point were determined using a simple t-test. A probability level of P < 0.05 was considered significant.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Results for plasma and middle gluteal muscle Se concentration, P GPx-3, RBC GPx-1, and M GPx-1 are shown in Table 3. Based on analysis of the Se supplements and total DM intakes, INORG and ORG diets provided 0.41 and 0.46 mg of supplemental Se per kg of DM total diet, respectively, as compared with the target formulation value of 0.45 mg/kg of DM total diet.
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Glutathione Peroxidase Activity
Mean P GPx-3 activity increased (P = 0.011) over the experimental period for horses fed all diets and was not affected by Se supplementation or source. Mean RBC GPx-1 activity also tended (P < 0.1) to increase over the experimental period for horses fed all diets. There was a tendency (P = 0.073) for a treatment x time interaction on RBC GPx-1 due to a tendency (P < 0.1) for greater activity on d 28 for ORG as compared with CTRL and INORG, and a tendency (P < 0.1) for greater activity on d 56 in INORG as compared with ORG. Mean RBC GPx-1 activity of INORG and ORG supplemented horses were not different from CTRL on d 56. Mean M GPx-1 activity decreased (P < 0.01) over the experimental period for horses fed all diets.
Body Weight
The BW increased (P < 0.001) over the experimental period for horses fed all diets but was unaffected by treatment. Mean BW was 418, 424, and 423 ± 23 kg on d 0 and 437, 443, and 435 ± 23 kg on d 56 for CONT-, INORG-, and ORG-fed horses, respectively.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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but above concentrations considered deficient (0.008 to 0.053 µg/mL) for horses (Puls, 1994). Treatment means for d 0 plasma Se concentrations of horses fed CTRL and ORG diets reflect marginal Se status (0.053 to 0.120 µg/mL) according to Puls (1994). The increase in plasma Se concentration in response to dietary Se supplementation reported in our study was expected because plasma Se concentration is sensitive to changes in dietary Se intake (Shellow et al., 1985; Stowe and Herdt, 1992). Plasma Se concentrations of horses in the current study responded to supplementation over a relatively short time, having plateaued by d 28. This finding is similar to Shellow et al. (1985), who reported that plasma Se concentrations of supplemented horses were greater than nonsupplemented horses after 2 wk of supplementation. It is worth noting that even though the CTRL diet provided 1.5 times the estimated requirement (NRC, 1989), the plasma Se concentrations of these horses were approximately 0.1 µg/mL, compared with 0.14 µg/mL previously reported by Shellow et al. (1985) for horses receiving a similar amount of dietary Se. In the current study, plasma Se concentrations of horses fed diets containing supplemental Se providing approximately 0.45 mg of Se/kg of DM total diet were similar to previously reported plateaus of approximately 0.15 µg/mL (Stowe, 1967; Maylin et al., 1980; Shellow et al., 1985).
Plasma Se concentrations of horses in the current study responded similarly to 56 d of Se supplementation with either sodium selenite or selenomethionine; however, the tendency for greater plasma Se concentrations on d 28 in horses fed selenomethionine may suggest a greater rate of plasma Se increase in Zn-L-selenomethionine- vs. sodium selenite-fed horses. This finding differs from that reported in rats (Deagen et al., 1987) and humans (Levander et al., 1983), where plasma Se concentration was greater after supplementation with Se-DL-methionine and Se-enriched wheat or Se-enriched yeast, respectively, as compared with inorganic sources. However, Se concentrations of supplemental diets used in the aforementioned experiments were approximately 20 times the requirement for rats and near the upper safe limit for humans, which may be one factor accounting for the differing plasma Se concentration response compared with that in the current study. The dietary Se intake has been shown to influence the effect of Se source (Se-enriched yeast vs. sodium selenite) on serum Se concentration in pigs (Mahan et al., 1999). Serum Se concentration was greater in pigs fed sodium selenite compared with Se-enriched yeast at a low dietary Se concentration (i.e., 0.05 mg/kg), but at greater dietary Se concentrations (i.e., 0.1, 0.2 and 0.3 mg/kg) serum Se concentration was greater for pigs fed Se-enriched yeast. However, dietary Se intake is not likely the sole explanation for the lack of difference in overall plasma Se concentration response between ORG and INORG groups. The supplemental Se intake of horses in the present experiment was approximately 4 times the estimated requirement (NRC, 1989). Janicki et al. (2001) reported that serum Se concentrations of pregnant mares fed 3 mg of Se/d, approximately 3 times that required, from Se-enriched yeast for approximately 55 d before foaling and 56 d postfoaling were greater immediately postfoaling and at 28 and 56 d postfoaling than those of mares fed either 1 or 3 mg of Se/d from sodium selenite. Several factors including differing physiological status, source of organic Se, and preexperiment Se status may be responsible for the differences in plasma Se concentration response between the current study and that of Janicki et al. (2001). The current estimate of dietary Se requirements for horses is 0.1 mg/kg of DM (NRC, 1989) and does not differentiate between physiological states. Whether the Se requirement of horses for growth differs from that of gestation is unknown; however, it is of interest to note that the dietary Se requirement determined for gestating rats was less than that for growing rats (Sunde et al., 2005). The source of organic Se used by Janicki et al. (2001), Se enriched yeast, has been estimated to contain approximately 40% selenomethionine, 15% selenocyteine, and lesser percentages of seleno AA analogs (Kelly and Power, 1995). Unfortunately, to the authors’ knowledge, no information is available in the literature regarding the response of plasma/serum Se concentration to dietary selenomethione compared with Se-enriched yeast. Preexperimental Se status of horses used in the current study may have also influenced the results. Although no treatment differences in plasma Se concentration existed on d 0 in the current study, horses fed the ORG diet had a mean plasma Se concentration reflective of marginal status according to Puls (1994) and tended to have a greater rate of increase in plasma Se concentration between d 0 and 28 compared with those fed the INORG diet. Horses fed the INORG diet had d 0 plasma Se concentrations between the range for marginal and adequate (Puls, 1994). Preexperimental Se status has been shown to influence the effect of Se source on plasma Se response of pigs (Mahan and Parrett, 1996; Mahan et al., 1999). Preexperiment Se status of horses used in the study by Janicki et al. (2001) was not reported.
This is the first report of which the authors are aware that describes the effect of diet on the Se concentration in equine skeletal muscle. The lack of a significant treatment x time effect accompanied by the significant effect of time indicates some Se accumulation occurred in the middle gluteal muscle regardless of dietary Se concentration or source. These results suggest that the middle gluteal muscle Se content in long-yearlings (~18 mo of age) is not sensitive to dietary Se amount or source under the present experimental conditions. Studies in other nonruminant species have indicated that Se concentrations in skeletal muscle are greater when Se-Met or Se-enriched yeast is fed compared with feeding an inorganic Se source or purified Se-Cys (Deagen et al., 1987; Mahan and Parrett, 1996; Mahan et al., 1999). The magnitude of this effect was lessened as muscle accretion declined in pigs approaching mature BW (Mahan and Parrett, 1996). Horses used in the current study were approximately 80% of mature BW based on estimates of mature BW at various ages (NRC, 1989). As a result, muscle accretion may have been relatively low in horses used in the current study. Additionally, the relatively short experimental period of 56 d may have contributed to the lack of treatment effect on middle gluteal muscle Se concentration in the current study.
Plasma GPx-3 activity was not affected by dietary Se supplementation or Se source. This finding agrees with previous studies where no differences in the P GPx-3 activity were reported in Se-deficient humans or rats after consuming different sources of supplemental Se (Levander et al., 1983; Deagen et al., 1987). The lack of effect due to Se supplementation on P GPx-3 is similar to previous reports where P GPx-3 activity of horses did not respond to 12 wk of supplementation, even when plasma Se concentration showed an increase (Shellow et al., 1985). Fluctuating P GPx-3 activity, similar to that in the current study, was reported for horses fed Se-adequate diets by Podoll et al. (1992). Dietary Se concentration in the current study was approximately 1.5 and 4 times the estimated requirement (NRC, 1989) for horses fed the CTRL and Se-supplemented diets, respectively. Plasma GPx-3 activity in rats plateaued with increasing dietary Se concentration, and the dietary Se concentration at the plateau breakpoint de-fines the dietary Se requirement (Sunde et al., 2005). Therefore, it is possible that dietary Se concentration of horses fed all 3 diets was well above the P GPx-3 activity plateau breakpoint, which could explain the lack of difference between dietary treatments. Plasma GPx-3 activity may not be a sensitive marker of Se status when horses are in a Se-adequate state.
The response of RBC GPx-1 to Se supplementation and source reported in the current study is not easily explained. The rapid (<4 wk) increase in the RBC GPx-1 activity of horses fed the ORG diet may indicate greater incorporation into RBC GPx-1. However, this is inconsistent with previous reports in other species. Research comparing inorganic and organic Se sources for growing pigs indicates that Se from inorganic sources is more rapidly incorporated into RBC GPx-1 protein in deficient or marginally replete animals than organic Se sources (Mahan et al., 1999). Little or no difference in RBC GPx-1 activity in humans (Clausen and Nielsen, 1988; Brown et al., 2000) and sheep (van Ryssen et al., 1989) was detectable when comparing organic or inorganic Se sources. Similarly, Deagen et al. (1987) found no difference in RBC GPx-1 values when SeNaO3 or Se-Met were fed to rats long term, but Se-Cys resulted in greater RBC GPx-1 activity than the other 2 sources. It is difficult to reconcile the rapid increase in RBC GPx-1 activity from d 0 to 28 followed by a decline in activity to d 56 for horses fed the ORG diet considering the life span of equine RBC is approximately 155 d (Morris, 1998). It is important to note that the initial increase in mean RBC GPx-1 activity observed in ORG fed horses is partially attributable to one extreme value on d 28, which represented a 170% increase from d 0, as compared with a 26% increase in the remaining horses fed ORG Se. Although this value was not considered an outlier after statistical analysis, analyzing the data without this value results in no difference in the RBC GPx-1 activity of the supplemented groups on d 28. Also, deletion of this data removes the apparent decline in mean ORG RBC GPx-1 activity between d 28 and 56. This horse was healthy and grew appropriately throughout the trial.
The greater d 56 RBC GPx-1 activity of horses fed INORG compared with CTRL- and ORG-fed horses aligns more closely with the relatively long life span of the equine RBC and previous reports in swine (Mahan et al., 1999). Previous studies have reported that the GPx activity in the whole blood of horses required at least 3 wk to respond to Se supplementation (Roneus and Lindholm, 1983) and 5 to 7 wk to increase significantly (Maylin et al., 1980; Knight and Tyznik, 1990) whether supplementation was given orally or parenterally. This delayed response is generally attributed to the period of time necessary for newly formed RBC to become a significant proportion of the mature RBC population (Maylin et al., 1980; Roneus and Lindholm, 1983; Knight and Tyznik, 1990). The increase in RBC GPx-1 activity of INORG supplemented horses after d 56 of supplementation in this study is similar to the increase in whole blood GPx activity after 5 wk reported in other experiments when selenite was fed as the supplemental source (Maylin et al., 1980; Knight and Tyznik, 1990).
The observed decline in M GPx-1 activity despite a rise in Se concentrations of this tissue indicates that factors other than Se status may play a role in determining its activity. Deagen et al. (1987) reported no difference in M GPx-1 activity of rats supplemented with 2 ppm of Se as NaSeO3, Se-Met, or Se-Cys for 9 mo, although there was a greater correlation of M GPx-1 and Se when NaSeO3 was the dietary source.
Before beginning our experiment, horses were fed a hay diet containing approximately 0.4 mg of Se/kg. Earlier investigations examining Se nutrition of horses have been almost exclusively conducted in geographically low-Se regions, (Stowe, 1967; Maylin et al., 1980; Roneus and Lindholm, 1983; Shellow et al., 1985; Janicki et al., 2001), and the effect of an inherently high Se diet on Se metabolism of horses has not been described.
Different Se status markers varied in their response to organic and inorganic Se supplementation in the current study. Horses receiving the CTRL diet exhibited lower Se concentrations in plasma, but not muscle, relative to supplemented groups; P GPx-3 activity increased. In combination, these results indicate that alterations in one response variable do not necessarily reflect changes in another.
Ultimately, no single source exhibited a clear advantage over the other in the current study. Plasma Se results suggest that zinc-L-selenomethionine is more effective at increasing short-term (i.e., d 0 to 28) Se status than sodium selenite; however, by d 56 the plasma Se concentrations were similar in horses fed either supplemental Se source. Plasma GPx-3 activity, RBC GPx-1 activity, and muscle Se concentrations were not conclusive in differentiating Se sources. Length of supplementation and initial Se status probably influenced the response to Se supplementation. More research is needed to establish which Se status markers are most useful in equine health.
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Footnotes |
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2 Corresponding author: paul.siciliano{at}colostate.edu
Received for publication July 30, 2005. Accepted for publication January 29, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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