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ANIMAL NUTRITION |
* Istituto di Zootecnica, Università Cattolica del Sacro Cuore, I-29100 Piacenza, Italy; and
European Union Regulatory Affairs Department, Alltech France, 92300 Levallois-Perret, France
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: glutathione peroxidase • horse • selenium • selenocysteine • selenomethionine
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, the Se concentration in grain and forages is highly variable; consequently, Se supplements have become an essential part of mineral premixes for horses, particularly in areas of Se deficiency (Lofstedt, 1997). Selenium nutrition is of importance due to its effects on immune function (Baalsrud and Overnes, 1986; Knight and Tyznik, 1990), exercise tolerance (Brady et al., 1978; Avellini et al., 1999), and thyroid hormones (Surai, 2006). Because the results of such experiments can be inconclusive or contradictory, there is a need for a more comprehensive understanding of Se metabolism, especially for equine species.
Selenium supplements can be in 2 forms, inorganic (usually Na selenite or selenate) or organic forms (Se yeast or high-Se grain). Selenium yeast is produced by growing specific strains of yeast in a Se-enriched media, and, although the distribution of Se forms in yeast varies among sources, SeMet is usually the predominant form (Rayman, 2004). In addition, Se yeast has recently been authorized for use within the European Union as a feed additive.
Inorganic and organic forms of dietary Se supplements are metabolized differently (Surai, 2006). Selenomethionine is actively transported through intestinal membranes during the absorption process and non-specifically incorporated into tissue proteins in place of Met during protein synthesis (Schrauzer, 2003). In contrast, inorganic Se is absorbed via passive diffusion and little is retained in tissue reserves. Consequently, a large proportion of inorganic Se is excreted in the feces and urine (Wolffram, 1999).
The aim of this study was to determine the effects of dietary Se sources, selenized yeast (Se yeast) or Na selenite (Na2SeO3), or Se dose (Se yeast only) on Se status, glutathione peroxidase (GPX) activity, and thyroid hormone status of mature horses.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Animals and Management Practices
Horses used in this study were raised within a private herd located in Piacenza province (Italy). A total of 25 mature Italian Saddle horses were selected on the basis of good health, age (mean age 13.6 ± 4.8 yr), and activity (slightly exercised). All horses were housed in a single barn with each horse occupying a single box stall (3 x 3 m), with sawdust as bedding material. Each stall was cleaned daily after the morning feed. Each stall was equipped with 2 troughs, one for the distribution of concentrate feed and the other for hay distribution. Fresh potable water was freely available from a water trough in each box stall. All horses were inspected daily, and records of all health-related problems and incidents that occurred throughout the trial and any treatment administered were kept.
All horses were individually fed and received the same basal diet that was formulated to meet the nutritional requirements of the type and size of horses used in this study (NRC, 1989). Feed was offered once in the morning (0800 h) and again in the afternoon (1800 h). At each feeding, horses were initially offered hay and then the concentrate mixture, which contained the mineral and vitamin supplement, 1 h later. On average, the basal diet consisted of 6 kg of grass hay plus 3 kg of concentrate (700 g of flaked barley, 200 g of oats, and 100 g of flaked corn/kg of concentrate). Representative samples of hay and concentrate feed were taken weekly during the study and pooled on a monthly basis for the analyses. Each horse was individually fed and refusals recorded daily.
Experimental Design
The study was conducted as a randomized complete block design comprising a continuous period of 112 d (from November 30, 2005 to March 23, 2006), in which all horses received the same basal diet that differed in only Se source (Se yeast vs. Na selenite) or dose of Se yeast [Sel-Plex Se yeast (Saccharomyces cerevisiae CNCM I-3060) containing 63% SeMet (Alltech, Nicholasville, KY)]. During the 2 mo preceding the beginning of the study, all horses received the same basal diet that had not been augmented with additional Se. At the end of this period, horses were blocked by BW, sex, age, and activity and were randomly allocated to 1 of 5 dietary treatments (5 horses per treatment): negative control (CTRL, background Se only), 3 different concentrations of Se yeast supplementation [SY02, SY03, and SY04; 0.77, 1.62, and 2.47 mg of Se/(head·d), respectively, to achieve 0.2, 0.3, and 0.4 mg of total Se/kg of DM, respectively], and 1 positive control supplemented with Na selenite [SS03: 1.62 mg of Se/(head·d) to achieve 0.3 mg of total Se/kg of DM]. The mean age of horses was 13.0 ± 5.1, 14.0 ± 3.3, 13.4 ± 5.8, 13.8 ± 5.9, and 13.8 ± 4.7 yr for CTRL, SY02, SY03, SY04, and SS03, respectively. The mean BW of horses was 594 ± 46, 591 ± 58, 585 ± 46, 583 ± 45, and 588 ± 58 kg for CTRL, SY02, SY03, SY04, and SS03, respectively. Each treatment included an equal number of females (n = 3), gelding males (n = 1), and males (n = 1). All Se supplements were offered daily to each individual horse by top-dressing the concentrate fed each morning using Ca carbonate as a carrier.
Measurements and Sampling
Climatic Conditions. Temperature and relative humidity of the inside and outside barn were recorded daily during the study period using 2 electronic probes (Gemini Data Logger, West Sussex, UK) located inside and 2 probes located outside of the barn. All electronic probes were connected to a data logger programmed to record every 10 min. Mean daily temperature and humidity and daily minimum and maximum temperature and humidity were calculated from temperature and relative humidity data recorded throughout the trial.
Blood Sampling.
Blood samples were obtained before the morning feed (0800 h) at d 0 (T0), 28 (T28), 56 (T56), 84 (T84), and 112 (T112). At each sampling point, 3 blood samples were collected by venipuncture from a jugular vein: two 7.5-mL Li-heparin-treated tubes (Monovet tube Se-free, containing 15 IU of Li-heparin/mL, Sarstedt, Princeton, NJ) and one 3-mL K3EDTA-treated tube (Venoject, containing 2.1 g of K3EDTA/ml, Terumo, Leuven, Belgium). Blood samples were placed immediately in an ice bath before processing. Within 2 min of sampling, 25 µL of whole blood was mixed with 1 mL of diluting reagent, according to the method of Paglia and Valentine (1967), using a commercial kit (Ransel kit, Randox, Crumlin, UK) and stored in an ice bath until analysis of red blood cell GPX (GPX-1) activity.
Laboratory Analyses
Feedstuffs.
Feedstuffs were analyzed for DM, CP, ether extract, crude fiber, ash, NDF, ADF, starch content, and water-soluble carbohydrate (Martillotti et al., 1987). Selenium content of the specific premixes and feedstuffs were determined using inductively coupled plasma mass spectrometry (ICP-MS; Elan 6100, Perkin Elmer, Norwood, MA). In addition, the content of Ca, P, Mg, Na, K, Cu, Fe, Mn, and Zn in each feedstuff was determined using inductively coupled plasma optical emission spectrometry (Optima 2100 DV, Perkin Elmer). The DE content of feedstuffs was estimated according to NRC (1989). The content of Met and Cys in pooled samples of hay and concentrate were determined (AFNOR, 1993).
Whole Blood.
Red blood cell GPX activity in whole blood samples diluted with the specific reagent was assessed by kinetic analysis according to the method of Paglia and Valentine (1967) using a commercial kit (Ransel kit, Randox). Samples were analyzed at 37°C by clinical analyzer (ILAB 600, Instrumentation Laboratory, Lexington, MA).
The first Li-heparin-treated tube of whole blood was stored at –20°C until determination of total Se. Briefly, total Se was determined by mineralizing 1 g of sample in a closed-vessel heating block system in the presence of 4 mL of HNO3 (16 mol/L) and 2 mL of H2O2 (9.8 mol/L). The solution was further diluted with water, and Se was determined by ICP-MS using the method of standard addition. The remaining blood from each sample was then pooled by treatment and sampling date (T0, T56, and T112) and freeze-dried before the determination of total Se, and the proportion of total Se as either SeMet or selenocysteine (SeCys). A proteolytic digestion was used to digest protein and to liberate selenized AA. The proteolytic digests were analyzed by reversed-phase HPLC ICP-MS using a collision cell after the purification of AA fractions by size-exclusion HPLC. Samples were spiked with 77SeMet before digestion to compensate for the recovery of SeMet. Quantification of SeMet was done using 77SeMet as an internal standard. Briefly, a 0.05-g portion of sample was incubated with urea 6 M, DL-dithiothreitol, and iodoacetamide (at 25°C for 5 h in the dark, pH = 7.5) to reduce and alkylate SeCys. Samples were spiked with 77SeMe and then extracted with a mixture of protease and lipase at pH 7.5 (20 h at 37°C). The supernatant was separated by centrifugation (9,000 x g for 10 min at room temperature) and purified by size exclusion liquid chromatography. The AA fraction was collected and preconcentrated by lyophilization. The residue was dissolved in water, and an aliquot was injected into the reversed-phase HPLC ICP-MS. An ICP-MS equipped with a collision cell was used as a detector (Bierla et al., 2008). The proportion of total Se comprised as either SeMet or SeCys was expressed in nanograms per gram of dry sample and in percentage of total Se. Whole blood Met and Cys concentrations were also measured (AFNOR, 1993).
Packed cell volume (PCV) was determined on an aliquot of the second Li-heparin-treated tube of whole blood after high-speed centrifugation (15,000 x g for 10 min at room temperature) of blood in a capillary tube. Blood treated with K3EDTA was used to determine the hemoglobin (Hb) content (Cell-Dyn 3700 hematology analyzer, Abbott Diagnostici, Roma, Italy).
Plasma. The remaining aliquot of the second Li-heparin-treated tube was centrifuged (3,500 x g for 15 min at 10°C), and the plasma fraction was decanted and divided into 3 subsamples that were subsequently stored at –20°C until analysis for plasma GPX (GPX-3) activity, plasma Se, and thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Plasma GPX activity was determined using the method described previously using clinical analyzer (ILAB 600, Instrumentation Laboratory) at a temperature of 37°C.
Total plasma Se was determined using the method described previously. The remaining plasma of each sample was pooled by treatment and sampling date (T0, T56, and T112) and freeze-dried before analysis for total Se, SeMet, and SeCys using the methods described previously. The proportion of total Se as either SeMet or SeCys was expressed in nanograms per gram of dry sample and in percentage of total Se. Thyroid hormones were determined by ELISA technique with a microplaque using a monoclonal antibody [T3, host mouse catalog 10-T35; T4, host mouse catalog 10-T30; enzyme-conjugated T3-HRP (horseradish peroxidase), catalog 65-IT35; enzyme-conjugated T4-HRP, catalog 65-IT50; Fitzgerald Industries International Inc., Concord, MA]. The intra- and interassay CV were 3.1 and 4.9%, respectively, for T4, and 2.9 and 4.0%, respectively, for T3.
Statistical Analysis
Results, with the exception of data pertaining to SeMet and SeCys, were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC) according to Littell et al. (1998). Sources of variation included treatment, time, and the treatment x time interaction. The random variable was horse within treatment. Preexperimental data were used for covariate adjustment where appropriate. The PCV values were used as a covariate in the analysis of total Se in whole blood. Each variable analyzed was subjected to 3 covariance structures: autoregressive order, compound symmetry, and spatial power (Littell et al., 1998). Using the largest Akaike information criterion and Schwarz Bayesian criterion, the spatial power was the covariance structure that fitted the model best. Results are presented in tables as least squares means and SED. A comparison was made between treatments SY03 (Se yeast) and SS03 (selenite) using the PDIFF option of SAS, because they had similar concentrations but different sources of Se. The dose response to concentration of Se yeast in the diet was considered for treatments SY02, SY03, and SY04. If a significant interaction was detected (P < 0.05), pairwise comparisons of treatment means at each sampling date and between sampling date within treatment were made using the PDIFF option of SAS.
Linear and quadratic regressions were developed using total Se in blood or plasma as the dependent variable and time as the independent variable. Linear regressions were also developed using GPX-1 activity as the dependent variable and total Se in blood as the independent variable. Correlation coefficients between total Se in blood and plasma and GPX activity in whole blood (GPX-1) and plasma (GPX-3) were also calculated.
Linear regressions were developed using the proportion of Se comprised as SeMet or SeCys in blood or plasma (ng/g of dry sample) as the dependent variable and time (d) as independent variable. The slope of the curves was compared when P < 0.10 for a 1-tail t-test, because greater values of the proportion of Se comprised as SeMet in blood and plasma were expected in Se yeast compared with the other treatments.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mean daily temperature inside the barn was 3.5 ± 1.4, 2.1 ± 1.2, 4.5 ± 1.6, and 8.9 ± 3.0°C for December, January, February, and March, respectively, with the minimum daily temperature only occasionally dropping below 0°C. The daily minimum temperature outside the barn during December, January, and the beginning of February was often below 0°C, with the lowest minimum temperature recorded as –5.8°C. In December and January, the maximum daily relative humidity was very often close to 100%, with minimum values often between 60 to 90%, particularly inside the barn.
Diet Characteristics and Se Content
The concentration of total Se in the negative control diet was 0.085 mg of Se/kg of DM and was similar to estimated values before the beginning of the study (Table 1). The total dietary Se concentrations of Se-supplemented treatments were also very close to values estimated before the beginning of the study. During the trial, the Se content of the diets was relatively constant with respect to treatment, with the high variability in the negative control. The chemical and nutritive characteristics of the basal diet indicated that the nutritional requirements of the horses involved in the study were met, except Cu and Zn (Table 2).
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Interactions between treatment and time for T4 (P < 0.05) and T3:T4 ratio (P < 0.01) were observed (Table 3), mainly because of the variability within the negative control. However, no effect of Se source or dose was observed on plasma T3 or T4 concentrations or T3:T4 ratio.
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All treatments supplemented with Se had greater total Se concentrations in whole blood and plasma when compared with the negative control (CTRL; P < 0.001; Table 3
). Linear dose effect and source effect were observed for total Se in blood, and Se values were greater in treatments supplemented with greater doses of Se (SY03 vs. SY02, P < 0.05 and SY04 vs. SY02, P < 0.05; Table 3) and in those horses supplemented with Se yeast (SY03) at T84 and T112 when compared with those receiving a comparable dose of selenite (SS03; Table 4). Total Se in blood in all treatments supplemented with Se (SY02, SY03, SY04, and SS03) was greater (P < 0.001) than the CTRL from T28 to the end of the study at T112. Nevertheless, with the exception of SY02 horses, the 16-wk experimental period was not sufficient for all treatments to achieve asymptotic, steady-state Se concentrations in whole blood. The response of total Se in blood differed between Se sources, whereby blood total Se increased more rapidly over time in those horses receiving the Se yeast supplement when compared with a comparable dose of selenite (SY03 vs. SS03; P < 0.05; Table 5).
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). Selenium source did not affect the Se content of plasma. Total Se in plasma increased over time in all supplemented treatments and was greater (P < 0.001) in all supplemented treatments when compared with the CTRL from T28 to the end of the study at T112 (Table 4). Furthermore, asymptotic steady state was achieved within 75 to 90 d of the beginning of the study in all supplemented treatments, with estimated values of 158, 185, 204, and 171 ng/g of for treatments SY02, SY03, SY04, and SS03, respectively (Table 5). Based on the equations in Table 5, total Se in plasma seemed to increase by 50 to 60% at T28, 85 to 93% at T56, and almost 100% at T84 in all supplemented treatments. Correlations were observed between total Se in blood and total Se in plasma (r = 0.83; P < 0.001), total Se and PCV in blood (r = 0.48; P < 0.001), and total Se and Hb in blood (r = 0.49; P < 0.001). GPX-1 and GPX-3
All treatments supplemented with Se had greater values of GPX-1 activity (P < 0.01) when compared with the negative control (CTRL; Table 3). Linear and quadratic dose effects were observed on GPX-1 activity, the dose effect being statistically significant (P < 0.05) between the low and intermediate doses of Se yeast (SY02 vs. SY03) or between the least and greatest dose of Se yeast (SY02 vs. SY04). Selenium source did not affect GPX-1 activity. In all treatments supplemented with Se (SY02, SY03, SY04, and SS03), there were increases in GPX-1 activity during the trial, and all supplemented treatments were greater (P < 0.05) than the negative control from T56 to study completion (T112; Table 4). Nevertheless, asymptotic GPX-1 activity did not seem to have been achieved in any of the Se-supplemented treatments after completion of the 16-wk experimental period.
Good correlations were observed between GPX-1 activity and total Se in blood (r = 0.86; P < 0.001) and total Se in plasma (r = 0.75; P < 0.001). The relationship between GPX-1 activity and total Se in blood was affected by Se source but seemed unaffected by Se dose. The coefficients of the linear regressions between GPX-1 activity and total blood Se are shown in Table 5. The regression coefficients differed (P < 0.05) between all Se yeast treatments and Na selenite (Table 5).
Plasma GPX activity (Table 3) were greater in Se yeast (SY03; P < 0.001) and Na selenite (SS03; P < 0.05) when compared with the negative control, but GPX-3 activity seemed to be unaffected by greater Se doses or Se source. Positive correlations were observed between GPX-3 activity and total Se in blood (r = 0.58; P < 0.001), between GPX-3 activity and total Se in plasma (r = 0.45; P < 0.001), and between GPX-3 activity and GPX-1 activity (r = 0.60; P < 0.001).
SeMet and Selenocysteine
The proportion of total Se as SeMet increased both in whole blood and plasma over time in Se yeast treatments (Figures 1a and 1b), with those increases being proportional to the amount of Se supplementation. The values of the SeMet:Met ratio in blood were related to the estimated SeMet:Met ratio in the diets (Table 6). Furthermore, the rate of increase in the proportion of total Se as SeMet over time was greater in whole blood (P < 0.05) and plasma (P = 0.10) of those horses offered Se yeast (SY03) when compared with those receiving a comparable dose of selenite (SS03; Figures 1a and 1b).
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), whereas SeMet, on average, only accounted for 15.2 and 10.0%, respectively. The proportion of total Se as SeCys in blood and plasma increased in all treatments supplemented with Se, and the rate of increase over time was greater than CTRL (P < 0.05).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, but above the concentrations that are considered as deficient (8 to 53 ng/mL in plasma) for horses (Puls, 1994). The values of total Se in whole blood observed at T0 were similar to the minimum of the reference range of 182 to 240 ng/mL for adult horses reported by Stowe (1998).
The values of total Se in plasma and blood observed in the current study with the CTRL horses averaged 88 and 175 ng/g in plasma and blood, respectively, which were obtained with a Se content in the basal diet of 0.085 mg/kg of DM. The results for plasma Se agree with those of Shellow et al. (1985), who reported a total Se in plasma of 65 ng/mL with a Se content in the basal diet of 0.06 mg/kg of DM. Greater values of total Se in plasma (106 ng/mL) were observed by Richardson et al. (2006) in horses fed greater Se concentrations in the basal diet (0.15 mg/kg of DM), approximately 1.5 times the requirements estimated by NRC (1989). The results of whole blood Se in this study were greater than those observed by Shellow et al. (1985). The variability observed during the trial in total blood Se in negative control horses could be related to the variability observed in the concentration of Se in the basal diet. Nevertheless, considering that plasma Se reflects short-term Se status and blood Se reflects longer-term Se status, the low variability of total Se in plasma of CTRL horses during the trial seems to indicate that other factors may be involved. The variability of PCV and Hb, considering the positive correlations between total Se and PCV in blood and also between total Se and Hb in blood, could also explain the variability in total blood Se observed in CTRL during the trial.
Increases in plasma Se concentration by the increases in dietary Se supplementation reported in this study were expected, because plasma Se concentrations are sensitive to changes in dietary Se intake (Shellow et al., 1985; Stowe and Herdt, 1992). Horses used in the current study responded to Se supplementation over a relatively short period of time, having notably greater plasma Se values in all supplemented horses compared with unsupplemented horses (CTRL) after 28-d exposure to experimental diets. This finding is similar to those of Shellow et al. (1985) and Richardson et al. (2006), the latter reporting that plasma Se concentrations of supplemented horses were greater than non-supplemented horses within 1 wk of supplementation. In the current study, plasma Se concentrations of supplemented horses reached a plateau between 75 and 90 d after the beginning of Se supplementation. These findings are similar to those of Shellow et al. (1985), who observed an increase in plasma Se up to 8 to 12 wk after the beginning of Se supplementation. These results indicate an increase in the distribution of Se from the liver to peripheral tissues, possibly through the actions of selenoprotein P 10 (SeP; Surai, 2006), and are implying the increase in Se storage within the body during the trial.
The plateau observed in this study for horses supplemented with Na selenite (171 ng/g) was greater than the value of approximately 150 ng/mL reported in other studies (Stowe, 1967; Maylin et al., 1980; Shellow et al., 1985; Richardson et al., 2006) in horses fed diets containing 0.30 to 0.40 mg of Se/kg of DM, whereas a similar plateau (158 ng/g) was obtained, in this study, in SY02 horses offered diets containing Se yeast (0.20 mg of Se/kg of DM). In addition, plasma Se contents of horses in this study, which received Se yeast supplement at similar concentrations to those reported by Stowe (1967), Maylin et al. (1980), Shellow et al. (1985), and Richardson et al. (2006), reached a plateau of 185 and 204 ng/g with 0.3 and 0.4 mg of Se/kg of DM, respectively. This tendency for greater values of total Se in plasma observed in this study with Se yeast compared with selenite agrees with the report by Richardson et al. (2006), in which greater Se in plasma was observed in horses supplemented with Zn-L-SeMet compared with those supplemented with Na selenite. In other trials and species, greater values of Se in plasma were observed with Se yeast compared with Na selenite (Surai, 2006).
The results observed in this study for total Se in plasma are related to increases of the proportion of Se comprised as SeMet. Increase of Se as SeMet observed in plasma was greater with Se yeast than with Na selenite. These increases in SeMet were expected, because SeMet is absorbed nonspecifically within the intestinal tract, and SeMet remains intact and is available for protein synthesis in place of Met (Weiss., 2003). Burk et al. (2001) demonstrated that Se from SeMet, but not from selenate or SeCys, can be incorporated into albumin, presumably as SeMet within the Met pool. Conversely, in the horses supplemented with Na selenite, the SeMet in plasma did not increase, because higher animals have no efficient mechanism for Met synthesis and are also unable to synthesize SeMet (Schrauzer, 2003). Furthermore, Se from inorganic supplementation is almost exclusively used for the production of selenoenzymes, whereas organic Se can be used to produce both selenoenzyme and also result in general labeling of all proteins containing Met, because absorbed SeMet can be used in place of Met in protein synthesis (Weiss, 2005).
Selenocysteine is the main form of Se in plasma. In plasma, Se is mainly transported by SeP, a glycoprotein containing up to 10 SeCys residues (Allan et al., 1999) and representing between 33 to 80% of total plasma Se in humans and rodents (Surai, 2006). Selenoprotein P 10 is involved in Se transportation, delivering Se at a cellular level. Indeed, SeP participates in the distribution of the Se from the liver to peripheral tissues (Surai, 2006). A greater content of Se in the liver of pigs (Mahan et al., 1999; Mahan and Peters, 2004) and newborn lambs (Rock et al., 2003) has been observed. Daniels (1996) postulated that there are 2 distinct metabolic pools of Se in the body. The main exchangeable metabolic pool includes all forms of Se derived from inorganic selenite-selenide, including endogenously synthesized selenoproteins (e.g., GPX, SeP, etc.), excretory Se metabolites (trimethylselenium ion), and various other intermediary products of selenite metabolism. This is an active pool providing Se for the synthesis of the primary functionally important selenocompounds (Daniels, 1996). The second Se pool consists of SeMet-containing proteins and can potentially contribute to the first pool via participation in selenoprotein synthesis. These findings support the view that SeMet is a non-specific form of Se that is metabolized as a constituent of the Met pool where it is randomly distributed and generally unaffected by specific Se metabolic processes and, therefore, can be considered as a storage form of Se in animals.
Increases in blood Se concentrations in response to dietary Se supplementation reported in this study were expected. Although, after a 112-d exposure to the experimental diets, a plateau was not reached in whole blood; it was achieved in plasma. Indeed, plasma or serum Se reflects short-term Se status, whereas erythrocyte Se reflects longer-term status and hair or nail reflect long-term Se status (Thomson, 2004). Changes in Se in erythrocytes are related to the rate of turnover of red blood cells, and the lifespan of equine erythrocytes is approximately 140 to 150 d (Mahan et al., 1999). The rate of turnover of equine red blood cells may explain why a plateau in GPX-1 activity was not achieved during the 112-d study. Because the majority of the GPX-1 is incorporated into red blood cells (RBC) at the time of erythropoiesis, a complete response of GPX-1 activity and Se in whole blood to Se supplementation will, therefore, require a time span equal to the average lifespan of the RBC (Stowe and Herdt, 1992). Previous studies have reported that the GPX-1 activity in the whole blood of horses required a period of at least 3 wk to respond to Se supplementation (Roneus and Lindholm, 1983) and 5 to 7 wk to increase substantially (Maylin et al., 1980; Knight and Tyznik, 1990) irrespective of the route of supplementation. In this study, GPX-1 activity increased in all supplemented horses at T28, and the increase became statistically significant at T56. This delayed response is generally attributed to the period of time necessary for newly formed RBC to form a substantial proportion of the mature RBC population (Maylin et al., 1980; Roneus and Lindholm, 1983; Knight and Tyznik, 1990).
The greater values of total Se in blood observed in horses supplemented with Se yeast compared with selenite is in line with those results obtained from trials with other species (Surai, 2006). Selenium in erythrocytes is distributed between GPX-1 and Hb, and, despite greater values of total Se in blood of horses supplemented with Se yeast, GPX-1 activity did not differ between organic and inorganic Se supplementation. These results agree with those of Richardson et al. (2006), who observed no difference in GPX-1 activity in horses supplemented with Zn-L-SeMet when compared with Na selenite. However, this is not consistent with previous reports with other species. Research with growing pigs indicated that Se from inorganic sources was more rapidly incorporated into GPX-1 protein in deficient or marginally replete animals than organic Se sources (Mahan et al., 1999). Little or no difference in GPX-1 activity in humans (Clausen and Nielsen, 1988; Brown et al., 2000) and sheep (Van Ryssen et al., 1989) was observed when comparing organic and inorganic Se sources. Similarly, Deagen et al. (1987) showed no difference in GPX-1 values when Na selenite or SeMet was fed to rats, but SeCys resulted in greater GPX-1 activity than the other 2 sources. In vitro studies (Ilian and Whanger, 1989) showed that 75Se selenite was incorporated into GPX, whereas 75Se from SeMet was mostly incorporated into Hb. In vivo studies confirmed the greater incorporation of Se into GPX when Se was supplied with selenite (Beilstein and Whanger, 1986) and selenate (Butler et al., 1991). These researchers observed that the majority of erythrocyte Se was present in GPX-1, and it was deposited to a greater extent in Hb than GPX when Se was supplied as SeMet or seleno yeast. These findings could explain the different relationships observed between total Se in blood and GPX-1 activity when comparing organic and inorganic Se supplements.
These findings are also supported by values of SeMet observed in whole blood. Indeed, the increase in SeMet was greater with Se yeast than with Na selenite and related to the amount of SeMet supplied in the diet. Most of the research in Se biochemistry and metabolism has been using inorganic Se, namely selenite or selenate. Selenite is taken up by red blood cells within several minutes, reduced to selenide by glutathione, and transferred to the liver. When SeMet is consumed, the Se moiety can be removed, reduced to selenide (H2Se), and used for synthesis of bioactive SeCys, which can be used in the active site of selenoenzymes. Alternatively, SeMet can remain intact and be used in protein synthesis (Weiss, 2003). The chemical species-specific metabolic pathway for Se was explained by the metabolic regulation through selenide as the assumed common intermediate for the inorganic and organic Se sources and as the intermediate metabolite between utilization for selenoprotein synthesis and methylation for the excretion of Se (Suzuki and Ogra, 2002).
The GPX-3 activity was unaffected by Se source, and the linear dose effect of dietary Se was not statistically significant. Previous studies showed no difference in GPX-3 activity of Se-deficient humans or rats after consuming different sources of supplemental Se (Levander et al., 1983; Deagen et al., 1987). This lack of effect on GPX-3 after Se supplementation was also reported by Shellow et al. (1985), in which the GPX-3 activity of horses failed to respond after 12 wk of supplementation, even when plasma Se concentrations showed an increase. In this study, an effect of dietary Se supplementation was noted with the exception of greater concentrations of Se supplementation in which the GPX-3 activity did not differ from the control group. Nevertheless, a correlation between GPX-3 activity and total Se in plasma was observed in this study, and GPX-3 can be responsible for only 6 to 17% of Se in plasma (Surai, 2006). Richardson et al. (2006) hypothesized that GPX-3 activity may not be a sensitive marker of Se status when horses are in an Se-adequate state. In addition, the fact that the values of GPX-3 activity at the greatest concentrations of Se supplementation did not differ from the control group indicates that some other factors may be involved in the fluctuation of GPX-3 activity. The results of the current study also indicated that GPX-3 activity was unaffected by Se source, agreeing with the results of Richardson et al. (2006), who did not observe an effect in horses supplemented with Zn-L-SeMet compared with Na selenite.
In the current study, concentrations of plasma T3 and T4 and the T3:T4 ratio were unaffected by Se dose or source. It is known that Se is required for the conversion of T4 to the more active T3 through the action of a selenoenzyme (enzyme type 1 deiodinase). Conversion of T4 to T3 can be impaired in experimentally induced Se deficiency, and a role for Se in thyroid hormone metabolism has been identified (Surai, 2006). In the current study, the Se status of the control group was marginal, and it is recognized that deiodinase activity is relatively protected in conditions of marginal Se availability (Sunde, 1997). In general, iodothyronine deiodinase is ranked greater in priority for available Se supply than cytosolic GPX and similar in ranking to that of phospholipid hydroperoxide GPX and selenoprotein P (Kohrle, 2000).
In conclusion, the results obtained in this study showed that Se supplementation with Se yeast compared with Na selenite did not modify GPX-1 activity but increased the content of total Se in blood, also as consequence of increased proportion of Se comprised as selenomethione. These results seem to support the view that SeMet is a nonspecific form of Se that is metabolized as a constituent of the Met pool where it is randomly distributed and unaffected by specific Se metabolic processes. Therefore, SeMet can be considered as a storage form of Se in animals and can contribute to endogenously synthesized selenoproteins, particularly during periods of decreased Se supply, providing Se for the synthesis of functionally important selenocompounds.
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2 We thank R. Lobinski (Laboratory UT2A, Pau, France) for selenium analyses, G. Brigati (Veterinary Office Cristella, Cremona, Italy) for veterinary assistance, and G. M. D’Aragona and E. D’Aragona (Agricultural Company Croara Vecchia, Piacenza, Italy) for their contribution to this research.
3 Corresponding author: luigi.calamari{at}unicatt.it
Received for publication November 22, 2007. Accepted for publication September 3, 2008.
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), or Se yeast (Sel-Plex Se yeast; Alltech, Nicholasville, KY) provided 0.18, 0.29, and 0.39 mg of total Se/kg of DM (SY02: 
; SY03: •;; and SY04: 
, respectively), or Na selenite provided 0.29 mg of total Se/kg of DM (SS03: 
). Rate increase over time in blood: CTRL vs. SY03 (P < 0.05) and SY04 (P < 0.01). Rate increase over time in plasma: CTRL vs. SY02 and SY04 (P = 0.10) and SY03 (P < 0.05). Rate increase over time: Se yeast (SY03) vs. selenite (SS03) in blood (P < 0.05) and plasma (P = 0.10).