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Influence of specific management practices on blood selenium, vitamin E, and beta‐carotene concentrations in horses and risk of nutritional deficiency

Abstract Background Selenium or alpha‐tocopherol deficiency can cause neuromuscular disease. Beta‐carotene has limited documentation in horses. Objective To evaluate the effect of owner practices on plasma beta‐carotene concentration and risk of selenium and alpha‐tocopherol deficiencies. Animals Three‐hundred and forty‐nine adult (≥1 year), university and privately owned horses and mules. Methods Cross‐sectional study. Whole blood selenium, plasma alpha‐tocopherol, and plasma beta‐carotene concentrations were measured once. Estimates of daily selenium and vitamin E intake, pasture access, and exercise load were determined by owner questionnaire. Data were analyzed using t tests, Mann‐Whitney tests, parametric or nonparametric analysis of variance (ANOVA), Kruskal‐Wallis test, Spearman's correlation and contingency tables (P < .05). Results Nearly 88% of the horses received supplemental selenium; 71.3% received ≥1 mg/d. Low blood selenium concentration (<80 ng/mL) was identified in 3.3% of horses, and 13.6% had marginal concentrations (80‐159 ng/mL). Non‐supplemented horses were much more likely to have low blood selenium (odds ratio [OR], 20.2; 95% confidence interval [CI], 9.26‐42.7; P < .001). Supplemental vitamin E was provided to 87.3% of horses; 57.7% received ≥500 IU/d. Deficient (<1.5 μg/mL) and marginal (1.5‐2.0 μg/mL) plasma (alpha‐tocopherol) occurred in 15.4% and 19.9% of horses, respectively. Pasture access (>6 h/d) and daily provision of ≥500 IU of vitamin E was associated (P < .001) with higher plasma alpha‐tocopherol concentrations. Plasma beta‐carotene concentration was higher in horses with pasture access (0.26 ± 0.43 versus 0.12 ± 0.13 μg/mL, P = .003). Conclusions and Clinical Importance Suboptimal blood selenium and plasma alpha‐tocopherol concentrations occurred in 16.7% and 35.5% of horses, respectively, despite most owners providing supplementation. Inadequate pasture access was associated with alpha‐tocopherol deficiency, and reliance on selenium‐containing salt blocks was associated with selenium deficiency.


| INTRODUCTION
Deficiencies of the antioxidants selenium and alpha-tocopherol can result in severe and frequently irreversible neuromuscular disease in horses. Clinical signs can include weakness, atrophy, rhabdomyolysis, abnormal gait or limb position, and fasciculations. Selenium deficiency consistently has been associated with skeletal and cardiac muscle damage, and inadequate alpha-tocopherol status has been linked to degenerative myeloencephalopathy and neuroaxonal dystrophy, motor neuron disease, and vitamin E-deficient myopathy in horses. [1][2][3] In addition, supplementation of horses with these antioxidants has been correlated with attenuation of oxidative stress markers and positive effects on fertility and immune function. [4][5][6][7] Soils of volcanic origin and acidic pH contain minimal selenium, and the risk of selenium deficiency is increased in nonsupplemented animals subsisting on grass or hay grown in selenium-deficient soils. 8,9 Grasses and plants produce alpha-tocopherol via photosynthesis, but content degrades substantially after harvest and storage as hay. 10,11 Horses predominantly fed cured hay rations are therefore at risk of alpha-tocopherol deficiency. Additional factors including poor gastrointestinal absorption and genetics also have been hypothesized to contribute to alpha-tocopherol deficiency. 12,13 Beta-carotene is also predominantly found in fresh green forage and is subject to storage degradation. 14 Increased intake of beta-carotene, a vitamin A precursor, has been associated with improved fertility and immune function in several species. [15][16][17] The National Research Council (NRC) recommends 1 mg of selenium daily for 400 kg horses in heavy or very heavy work, and to increase blood selenium concentrations in 1 to 6-year-old horses, with evidence that up to 3 mg daily might improve immune function. 15 The recommended daily intake of vitamin E is 1 IU/kg body weight, with 2 IU/kg body weight recommended for foals and exercising horses. 15 Although studies have identified an impact of both formulation (natural versus synthetic) and dose on alpha-tocopherol status of horses, 18 current NRC guidelines do not specify a recommended formulation.
Many owners actively provide these nutrients in fortified feeds or directed supplements to prevent deficiency. However, the many available products have variable composition and efficacy, and current labeling guidelines do not require specification of vitamin E formulation. [19][20][21] Currently, no dietary recommendations are available for beta-carotene, and deliberate supplementation is uncommon.
Our main objective was to determine the impact of owner supplementation and management practices on whole blood selenium and plasma alpha-tocopherol concentrations in horses located in a region where clinical disease from both deficiencies occurs. Plasma betacarotene concentration also was evaluated because it is influenced by similar management practices that affect alpha-tocopherol, and currently has limited documentation in horses. It was hypothesized that owners often supplement horses inadequately, which, in combination with other specific management factors, could lead to a moderate prevalence of deficiency of these critical nutrients.

| Experimental animals and geographical region
For this cross-sectional study, subject recruitment was accomplished via internet postings, an informational brochure, and regional veterinarians.
Enrolled horses were located at 49 different properties in Oregon and were contained within an area of approximately 31 400 square miles.
Soil selenium concentration within this area ranged from 0.12 ± 0.03 to 0.27 ± 0.16 ppm (average, 0.17 ppm); soils containing <0.6 ppm are considered selenium deficient. 22,23 Terrain in this region varied from valley with a cool summer Mediterranean climate (Köppen climate classification Csb) to high desert with a semiarid climate (Köppen climate classification BSk). Yearly, average precipitation across the sampled region ranged from 15 to 70 inches. 24 A total of 349 horses and mules were enrolled, but data from 11 horses subsequently were excluded as a result of young age (<1 year old; n = 6) or small body mass (miniature horses and ponies; n = 5) so as to achieve greater homogeneity of body mass and estimated nutrient requirements for adult horses weighing approximately 400 to 500 kg. Two horses with recent history of injection with a vitamin E/selenium-containing product and 5 horses with incomplete dietary information also were excluded, leaving 331 horses.
Study horses were affiliated with Oregon State University (OSU, n = 16) or were privately owned (n = 315). Animal use protocols were approved by the Institutional Animal Care and Use Committee of OSU.

| Questionnaire
Owners, caretakers, or both completed a questionnaire regarding the management and feeding of each horse (Supplementary Figure S1).
The questionnaire recorded signalment, use, frequency and duration of exercise, ration composition (including access to green pasture and source, amount and type of hay), and supplement provision. Exercise load was partitioned into (1) no forced exercise, (2) low exercise (<180 minutes per week), and (3) high exercise (>180 minutes per week) based on NRC definition. 15 Horses receiving oil or feed supplements containing ≥10% of calories from fat were defined as receiving dietary fat supplementation. Adequate green pasture access was defined as access to fresh, green grazing for >6 hours per day, which was estimated to provide a minimum 500 IU of alpha-tocopherol based on the average alpha-tocopherol content of fresh grass and estimated hourly pasture intake of a 500 kg horse. 10,25 Follow-up telephone conversations with all owners to review completed questionnaires further clarified management history, including pasture quality, frequency, amount, and specific supplements and feeds provided over the preceding 6 months, including any changes in ration or exercise during that time period.
Estimates of the total amount and form of selenium (mg of selenomethionine versus sodium selenite) and vitamin E (IU of natural versus synthetic forms) in each horse's daily ration were collated from information provided in the questionnaire and independent evaluation of commercial feed and supplement labels by 2 of the authors (M.O. Pitel and E.C. McKenzie). Dietary selenium contributions were grouped into the selenomethionine category if specified on the label as "selenomethionine," "selenium yeast," or "organic selenium" and into the sodium selenite category if specified on the label as "sodium selenite," "inorganic selenium," or "selenium." Similarly, dietary vitamin E was grouped into the natural category if specified on the label as "natural vitamin E," "d-alpha-tocopherol," "d-alpha-tocopheryl acetate," "d-alpha-tocopherol acetate," or "d-alpha-tocopheryl succinate" and grouped into the synthetic category if specified on the label as "synthetic vitamin E," "dl-alpha-tocopherol," "dl-alpha-tocopheryl acetate," or "vitamin E supplement." Any provided hay was assumed to contribute minimal selenium and alpha-tocopherol to each ration, because all hay was Oregon sourced. [8][9][10][11] Access to a selenium-containing salt block was documented but no estimates were made in regard to salt's contribution to total selenium content of each ration, because consumption could not be reliably confirmed or quantified.

| Blood collection
A total of 15 mL of blood was collected into EDTA from the jugular vein of each horse, at a single time point. Sample acquisition from all horses was accomplished over 3 weeks in August 2017 to limit seasonal effects on studied variables. Plasma samples for alpha-tocopherol and beta-carotene analysis were protected from light after collection, centrifuged within 30 minutes, placed into cryovials, and frozen in a liquid nitrogen Dewar before being stored in a −80 C freezer until analysis at the Linus Pauling Institute of OSU using previously described methods. 26

| Statistical analysis
To analyze the effects of selenium supplementation, horses receiving adequate supplementation, defined as ≥1 mg of selenium daily, were divided into 3 groups based on the predominant selenium form provided: (1) >90% of selenium as selenomethionine (SeM); (2) >90% as sodium selenite (NaSe); and (3) a combination of both forms with each representing <90% of daily intake (SeM+NaSe). Horses classified as inadequately supplemented with selenium (InSe) were receiving some selenium, but a total amount <1 mg daily. Horses with no directed selenium supplementation were considered nonsupplemented (NonSe), including horses with access to a selenium-containing salt block.
To analyze the effects of vitamin E supplementation, horses were divided into those with adequate (P, >6 h/d grazing) and inadequate Analysis of the association between exercise load and whole blood selenium concentration and the association between selenium supplementation group and whole blood selenium were performed using ordinary 1-way analysis of variance (ANOVA) and Tukey's multiple comparison test. Comparison of the average total selenium intake in each of the selenium supplementation groups for horses receiving ≥1 mg of selenium daily was performed using a Kruskal-Wallis test with Dunn's multiple comparisons test. The association between exercise load and plasma alpha-tocopherol concentration was performed using a Kruskal-Wallis test with Dunn's multiple comparisons test. The association between fat supplementation and plasma alpha-tocopherol and between green pasture access of >6 h/d and plasma alpha-tocopherol were performed using a Mann-Whitney test. The association between vitamin E supplementation groups in horses grazing ≤6 h/d and plasma alpha-tocopherol was performed using Brown-Forsythe and Welch ANOVA testing and Dunnett's multiple comparison test. Comparison of the average total vitamin E intake in each of the vitamin E supplementation groups for horses receiving ≥500 IU/d was performed using a Kruskal-Wallis test with Dunn's multiple comparisons test. The association between green pasture access and plasma beta-carotene concentration was performed using a Mann-Whitney test.
Contingency tables were used to generate odds ratios (OR) for: (1) selenium supplementation categories versus whole blood selenium concentration categories and (2)

| Selenium
Whole blood selenium concentration was normally distributed in the study population. Average whole blood selenium concentration for all sampled horses was 210 ± 66 ng/mL (n = 331). Deficient whole blood selenium concentrations (<80 ng/mL) were identified in 11  Table 1).
Total daily selenium intake was not normally distributed. A total of 290/331 horses (87.6%) were receiving some additional selenium in their ration, of which 236 (71.3%) were considered adequately supplemented, receiving ≥1 mg/d (2.89 ± 1.46 mg/d) in various formulations, with no significant difference in daily selenium intake among T A B L E 1 Estimated daily selenium intake (mean ± SD, mg/d) of all horses (n = 331) separated into groups based on selenium form and amount, and contingencies (column totals) of selenium supplementation groups and whole blood selenium concentration groups  9.26-42.7; P < .001) more likely to have deficient blood selenium concentrations than horses receiving any amount of feed selenium supplementation ( Figure 1). A higher proportion of inadequately supplemented horses had marginal blood selenium concentrations compared to adequately supplemented horses (24.1 versus 6.4%, P < .001). Of adequately supplemented horses, blood selenium concentration was significantly higher in the NaSe+SeM compared to the NaSe group (P = .02, Figure 1), despite no significant difference in daily selenium intake between the 2 groups (P = 1). No other differences in blood selenium concentration were noted among adequately supplemented horses. Regardless of supplement type provided, the risk of deficiency was significantly lower in horses supplemented with ≥1 mg of selenium daily (P < .001, Table 1). Whole blood selenium concentration was significantly higher in horses in the low (219 ± 66 ng/mL, n = 116) and high (219 ± 57 ng/mL, n = 100) exercise groups compared to horses with no forced exercise (194 ± 70 ng/mL, n = 114; P = .01 and P = .02, respectively).

| Alpha-tocopherol
Plasma alpha-tocopherol concentration was not normally distributed in the study population. Average plasma alpha-tocopherol concentration from all sampled horses was 2.56 ± 1.13 μg/mL (n = 331;  (Figure 3). Total daily vitamin E intake in horses receiving ≥500 IU/d, regardless of pasture access, was significantly higher in the NatE+SynE-P, NatE+SynE-NP, and NatE-NP groups compared to the SynE-NP group (P = .02, F I G U R E 1 Box and whisker plot of whole blood selenium concentration versus supplementation groups; horses with no directed selenium supplementation, including 26 horses with access to a selenium-containing salt block (NonSe), horses receiving some selenium but <1 mg daily (InSe), and horses receiving ≥1 mg of selenium per day, as either >90% sodium selenite (NaSe); >90% selenomethionine (SeM); or a combination of both at <90% each (SeM+NaSe). The box describes first, second (median) and third quartiles, "x" signifies the mean, whisker length is 1.5*IQR and dots are outliers. Number of horses included in each supplementation group is provided below the graph. Superscripts *, # and^indicate significant difference in whole blood selenium concentration from NonSe (P < .001), InSe (P < .001), and NaSe (P = .02) groups, respectively, via ordinary 1-way ANOVA and Tukey's multiple comparison test T A B L E 2 Plasma alpha-tocopherol (mean ± SD, μg/mL) of all horses (n = 331) separated into groups based on supplementation form and amount ; or a combination of both at <90% each (NatE+SynE). The box describes first, second (median) and third quartiles, "x" signifies the mean, whisker length is 1.5*IQR and dots are outliers. Number of horses included in each supplementation group is provided below the graph. Superscript * indicates significant difference in plasma alpha-tocopherol concentration from the InE/NonE-NP group (P = .04, P = .03, and P = .004 for SynE-NP, NatE-NP, and SynE+NatE-NP, respectively) via Brown-Forsythe and Welch ANOVA testing and Dunnett's multiple comparison test P = .04 and P = .003, respectively; Table 3) and also was significantly higher in the NatE-NP compared to the SynE-P group (Table 3; P = .01).
A very weak, positive correlation was found between plasma alpha-tocopherol concentration and estimated daily vitamin E intake for horses with both adequate (n = 174) and inadequate (n = 157) pasture access (Spearman's R = 0.250, P = .004 and R = 0.314, P < .001, respectively; Figure 4). A weak, positive, correlation (Spearman's R = 0.316, P < .001) also was found between plasma alpha-tocopherol and whole blood selenium concentrations (n = 331, Figure 5). No significant association of plasma alpha-tocopherol concentration with exercise load or fat supplementation was identified.

| DISCUSSION
In the population of our study, subclinical deficiency of 2 critical nutrients was prevalent, with 16.7% and 35.5% of all studied horses exhibiting suboptimal selenium and alpha-tocopherol concentrations, respectively. These deficiencies occurred despite owners providing a very large range of feed and supplement products containing selenium and vitamin E to the majority of horses.
All horses in our study were considered at risk of selenium deficiency because they were located in a region with low soil selenium content and fed locally sourced hay. 8,9,23 Although previous work has identified improved absorption of selenomethionine compared to sodium selenite in horses, 29 in our study, form of delivered selenium F I G U R E 4 Correlation of plasma alpha-tocopherol concentration and total IU of daily oral vitamin E supplementation (regardless of supplement type) for horses with (P, >6 h/d, n = 174) and without (NP, ≤6 h/d, n = 157) adequate fresh, green pasture access. Spearman's correlation for P: R = 0.250, P = .004 and for NP: R = 0.314, P < .001 F I G U R E 5 Correlation of plasma alpha-tocopherol and whole blood selenium concentration for all horses (n = 331). Significant Spearman's correlation R = 0.316, P < .001 F I G U R E 6 Box and whisker plot of plasma beta-carotene concentration for horses with (P, >6 h/d) and without (NP, ≤6 h/d) adequate fresh, green pasture access. The box describes first, second (median) and third quartiles, "x" signifies the mean, whisker length is 1.5*IQR and dots are outliers. Superscript * indicates significant difference in plasma (beta-carotene) based on pasture access (P = .003) via Mann-Whitney test had minimal influence on deficiency and total amount of delivered selenium was more important. Nearly 90% of horses in our study were receiving some directed amount of selenium daily, and no horses receiving ≥1 mg daily selenium had deficient blood concentrations (<80 ng/mL), regardless of form provided. Suboptimal blood selenium concentrations were most frequent in non-or inadequately supplemented horses, including a substantial proportion of horses with access to a selenium-containing salt block. These findings corroborate previous reports that daily intake of at least 1 mg of selenium prevents deficiency in horses. 30,31 However, supplementation by selenium-containing salt block, although popular, was inadequate, and should be discouraged as a sole form of supplementation. Supplemented salt blocks range in selenium content from between 1 ppm to a maximum of 120 ppm. The voluntary daily salt intake of horses is highly variable, but an average intake of 1 to 2 oz per day has been reported. 15 A small but significant correlation was found between whole blood selenium and plasma alpha-tocopherol concentrations, likely reflecting the close relationship between these 2 antioxidants. When deficiency in 1 is suspected, testing for both is likely prudent, particularly for horses residing in areas of selenium-deficient soil. A seasonal effect on both whole blood selenium and plasma alpha-tocopherol concentrations has been reported previously, 28,41 and in our study samples were collected over a brief period to limit seasonal influences.
A strong, positive correlation also was identified between plasma alpha-tocopherol and beta-carotene concentrations. This correlation was likely the result of a comparable influence of dietary and management practices on these nutrients, particularly pasture access. Plasma beta-carotene concentrations were highly variable among horses, but generally were significantly higher with adequate pasture access, consistent with prior work. 42 Because beta-carotene is not typically supplemented, high plasma concentrations usually indicate that horses have access to fresh green forage.
Our study had some limitations. Clinical or subclinical evidence of disease or dysfunction associated with nutrient deficiencies was not evaluated and because we relied on owner-reported practices, results may have been affected by recall bias and incomplete reporting. To mitigate possible inaccuracies associated with owner reporting, all owners were contacted by telephone after submission of questionnaires to verify and complete survey responses, and daily intake of selenium and vitamin E was independently estimated. Analysis of selenium and vitamin E content was not performed on hay samples in our study to confirm the minimal contribution of forage to daily intake of these nutrients, instead minimal contribution was assumed based on existing literature relevant to the geographic region and the reported impact of processing on alpha-tocopherol content of hay. [8][9][10][11] In conclusion, suboptimal selenium and alpha-tocopherol status was identified in a large proportion of horses in our study, including horses with apparently adequate supplementation according to current NRC recommendations. Furthermore, common methods of supplementation, such as providing selenium in a salt block, clearly were inadequate for preventing deficiency. This finding indicates that supplementation plans must take into account a range of influencing factors, and that periodic testing should be utilized to confirm successful maintenance of selenium and alpha-tocopherol status, even when apparently adequate supplementation regimens are employed.
Because diseases of deficiency typically represent readily preventable disorders of management, our results indicate that improved management practices, guided by veterinarians, would benefit a large number of horses kept in circumstances similar to those of the study population.

ACKNOWLEDGMENTS
Scott Leonard and Maret Traber for assistance with vitamin E analysis.

CONFLICT OF INTEREST DECLARATION
Dr. Robert Stuart is the director and owner of Stuart Products, 112 Bedford Road, Bedford, Texas, which produces a vitamin E supplement and other nutritional products.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Approved by the Oregon State University IACUC.

HUMAN ETHICS APPROVAL DECLARATION
The project did not meet the definition of "human subject" under the Common Rule (45 CFR 46) and therefore did not require review by the ethics committee at OSU that oversees research on humans.