Biol Trace Elem Res (2014) 161:272–287 DOI 10.1007/s12011-014-0107-4
Effect of Supranutritional Organic Selenium Supplementation on Postpartum Blood Micronutrients, Antioxidants, Metabolites, and Inflammation Biomarkers in Selenium-Replete Dairy Cows Jean A. Hall & Gerd Bobe & William R. Vorachek & Katherine Kasper & Maret G. Traber & Wayne D. Mosher & Gene J. Pirelli & Mike Gamroth
Received: 25 July 2014 / Accepted: 13 August 2014 / Published online: 21 August 2014 # Springer Science+Business Media New York 2014
Abstract Dairy cows have increased nutritional requirements for antioxidants postpartum. Supranutritional organic Se supplementation may be beneficial because selenoproteins are involved in regulating oxidative stress and inflammation. Our objective was to determine whether feeding Se-yeast above requirements to Se-replete dairy cows during late gestation affects blood micronutrients, antioxidants, metabolites, and inflammation biomarkers postpartum. During the last 8weeks before calving, dairy cows at a commercial farm were fed either 0 (control) or 105 mg Se-yeast once weekly (supranutritional Se-yeast), in addition to Na selenite at 0.3 mg Se/kg dry matter in their rations. Concentrations of whole-blood (WB) Se and serum Se, erythrocyte glutathione (GSH), and serum albumin, cholesterol, α-tocopherol, haptoglobin, serum amyloid A (SAA), calcium, magnesium, phosphorus, non-esterified fatty acids, and β-hydroxybutyrate were measured directly after calving, at 48 h, and 14 days of lactation in 10 cows of each group. Supranutritional Se-yeast supplementation affected indicators of antioxidant status and inflammation. Cows fed a supranutritional Se-yeast supplement during J. A. Hall (*) : W. R. Vorachek Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4802, USA e-mail: [email protected] G. Bobe : W. D. Mosher : G. J. Pirelli : M. Gamroth Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331-4802, USA G. Bobe : M. G. Traber Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-4802, USA K. Kasper : M. G. Traber School of Biological and Population Health Sciences, Oregon State University, Corvallis, OR 97331-4802, USA
the last 8-weeks of gestation had higher Se concentrations in WB (overall 52 % higher) and serum (overall 36 % higher) at all-time points, had higher SAA concentrations at 48 h (98 % higher), had higher erythrocyte GSH (38 % higher) and serum albumin concentrations (6.6 % higher) at 14 days, and had lower serum cholesterol concentrations and higher α-tocopherol/cholesterol ratios at calving and at 48 h compared with control cows. In conclusion, feeding Se-replete cows during late gestation a supranutritional Se-yeast supplement improves antioxidant status and immune responses after calving without negatively impacting other micronutrients and energy status. Keywords Acute phase response . Antioxidants . Dairy Cows . Metabolic biomarkers . Postpartum . Selenium-yeast supplementation
Abbreviations AIC Akaike information criterion BHBA β-hydroxybutyrate DM Dry matter FDA Food and Drug Administration GSH Glutathione ICP-MS Inductively coupled argon plasma emission spectroscopy NEFA Non-esterified fatty acids NRC National Research Council SAA Serum amyloid A Se Selenium SeCys Selenocysteine SeMet Selenomethionine TBARS Thiobarbituric acid reactive substances TMR Total mixed ration WB Whole blood
Supranutritional Selenium Supplementation for Dairy Cows
Introduction Selenium (Se) is an essential micronutrient for ruminant animals [1, 2]. In cattle, severe Se deficiency results in nutritional myopathy or “white-muscle disease” and male infertility, whereas subclinical Se deficiency causes immunosuppression and decreased performance [2–4]. To prevent Se deficiency, the U.S. Food and Drug Administration (FDA) permits Se supplementation of ruminant diets. To avoid Se toxicity, current FDA regulations limit the amount of dietary Se supplementation in ruminant animals to 0.3 mg/kg (as fed) of inorganic Na-selenite or Na-selenate, or organic Se-yeast, which is equivalent to 3 mg per beef cow per day [5]. We are interested in supranutritional Se supplementation (dosages greater than that required for optimal expression of selenoproteins, but less than maximal tolerable levels) to improve health and performance in livestock. Little is known how Se supplementation above FDA regulations affects health and performance of ruminants with adequate concentrations of Se (Se-replete animals). In sheep, supplementing Se-replete ewes with 24.5 mg Se/week as Seyeast improves body condition scores of ewes and growth rates of their progeny [6]. This is 5× the FDA upper limit for supplementing ruminant diets, but less than 5 mg/kg diet, which is considered the maximal tolerable level for ruminants [7]. None of the ewes receiving supranutritional Se yeast supplements showed adverse clinical signs at any time throughout two lambing seasons [6]. Benefits of supranutritional Se-yeast supplementation in ewes include improvements in immune function (increased Escherichia coli killing by neutrophils, increased antibody titers to the novel antigen keyhole limpet hemocyanin, and increased neutrophil expression of genes involved in bacterial pathogen recognition and response) [8, 9], and increased immunoglobulin G transfer to their progeny [10]. Others have shown in cannulated sheep that Se-yeast or nano-Se supplementation of 4 mg Se/kg of dry matter (DM) increases ruminal fermentation and nutrient digestibility [11], and that Na-selenite or Seyeast supplementation of 0.15 mg Se/kg of diet increases antibody titers against Pasteurella multocida P52 vaccination in Se-replete lambs [12, 13]. In Se-replete beef cattle, feeding Se-fertilized alfalfa hay improved their performance and immune function (increased antibody titers after vaccination with J-5 Escherichia coli bacterin) [14] and, likewise, we showed benefits in recently weaned beef calves fed Sefertilized alfalfa hay (increased antibody titers after vaccination with J-5 Escherichia coli bacterin, and increased growth rates and survival) [15, 16]. Others have shown that Se-yeast supplementation of 3 mg Se/day increases mitochondrial gene expression in liver of Se-replete beef heifers, and decreases expression of genes known to be upregulated during oxidative stress [17]. In dairy cows, we reported in a companion paper that supplementing Se-replete cows during the dry period with
273
105 mg Se/week as Se-yeast, equivalent to 5× the FDA allowed maximum supplementation rate for cows, improved immunoglobulin G transfer and Se status in their newborn calves [18]. Others have shown in dairy cows that supplementing Se-replete cows with 0.3 mg/kg Se-yeast compared with 0.3 mg/kg Na selenite during the last 60 days of pregnancy and up to 30 days lactation increases whole-blood (WB)-Se, serum-Se, and colostral-Se concentrations [19]. Supplementing Se-replete heifers with 3 mg Se/day as Seyeast tends to reduce the incidence of intramammary infections and clinical mastitis at calving [20]. Furthermore, Seyeast supplementation with 0.31 and 0.50 mg Se/kg of DM increases whole-blood (WB)-Se, serum-Se, and colostral-Se concentrations in lactating Se-replete dairy cows [21], and decreases plasma β-hydroxybutyrate (BHBA) and reactive oxygen metabolites in plasma during the hot weather season [22]. Dairy cows are more susceptible to various metabolic and infectious diseases during the first weeks after calving, which can be partly ascribed to decreased immune function, increased lipid mobilization, increased production of pro-oxidants, and decreased concentrations of several antioxidants shortly after calving [23–25]. Supranutritional organic Se supplementation may improve the antioxidant status and immune response because selenoproteins (proteins containing selenocysteine [SeCys]) are involved in regulating oxidative stress and inflammation [26]. We hypothesized that feeding Se-replete cows supranutritional Se-yeast during late gestation would improve their Se-status, their antioxidant status, and their immune response after calving. Thus, the objective of this study was to determine whether feeding Se-replete dairy cows a supranutritional Se-yeast supplement during late gestation affects blood indicators after calving. Biomarkers of antioxidant status (whole blood [WB]-Se, serum-Se, glutathione [GSH], and α-tocopherol), lipid transport (cholesterol), acute phase response (albumin, haptoglobin, and serum amyloid A [SAA]), macromineral status (calcium, magnesium, and phosphorus), and energy status (non-esterified fatty acids [NEFA] and β-hydroxybutyrate [BHBA]) were assessed.
Materials and Methods Animals and Study Design The experimental protocol was reviewed and approved by the Oregon State University Animal Care and Use Committee (ACUP Number: 4156). The study was conducted at a commercial dairy in Oregon (Columbia River Dairy, LLC, Boardman, OR). For 8 weeks before predicted calving, multiparous Jersey dairy cows (body condition score ranged from 2.50 to 4.25) were fed either 0 (control cows) or 105 mg Se-yeast once weekly (Se-yeast supplemented cows) on top of their
274
total mixed ration (TMR), which was formulated to meet National Research Council (NRC) recommendations for dry cows [7]. The TMR contained supplemental Na selenite at 0.3 mg Se/kg DM. Cows were head locked when they started to eat their TMR at a bunk feeder. The Se-yeast aliquot was placed directly in front of each cow for individual consumption. The organic Se source (Se-yeast, Prince Se Yeast 2000, Prince Agri Products Inc., Quincy, IL) had a guaranteed analysis of 2 g organically bound Se (78 % as selenomethionine, SeMet) per kg supplement [27]. We previously verified the Se concentration of the supplement [27]. The amount of Se-yeast fed to each cow was 52.5 g once per week. The dose of Seyeast supplement was calculated to provide 5× the maximum allowed FDA Se supplementation dose for ruminant diets, which is 3 mg/day. As an example of dose calculations, 3 mg Se/day is multiplied by 7 for the weekly amount of 21 mg and then by 5× to attain 105 mg Se/week as Se-yeast. After calving in the maternity area, cows were milked once and moved to a fresh pen, which was a freestall with headgates. Twice a day lactating cows were milked and fed a TMR that was formulated to meet NRC recommendations for fresh lactating dairy cows [7]. Again, the TMR contained supplemental Na selenite at 0.3 mg Se/kg DM. No supplemental Seyeast was provided postpartum. Sample Collection and Analysis Whole-blood Se and serum biomarkers of antioxidant status, lipid transport, inflammation, and energy status were measured in a subset of clinically healthy cows, 10 control and 10 Se-yeast supplemented cows. Selection criteria included cows with WB-Se concentrations measured after 6 weekly Se yeast treatments that were likely to be>300 ng/mL by 8 weeks of Se-yeast supplementation, indicating they were consuming the Se-yeast supplement, and that were available for blood collection at calving, and at 48 h and 14 days postpartum. Control cows were chosen based on their availability for blood collection at calving, and at 48 h and 14 days postpartum. In addition, control cows had WB-Se concentrations in the range of 120–300 ng/mL, which is the normal reference interval for WB-Se of adult cows at the Michigan State University Diagnostic Laboratory [14]. All Se-yeast supplemented cows received 7 (n=2) or 8 (n=8) weekly dosages of Se-yeast before calving. Cows received their eighth Se-yeast dosage within 1 (n=2), 3 (n=3), 4 (n=1), 5 (n=1), or 6 (n=3) days of calving. Blood samples were collected from the jugular vein of cows at the beginning of the study (8 weeks before expected calving date), after 6 weeks of Se-yeast supplementation, after parturition (at the time of first milking), at 48 h postpartum, and at 14 days postpartum. Blood samples were collected into evacuated EDTA tubes (2 mL; final EDTA concentration 2 g/L; Becton Dickinson, Franklin Lakes, NJ) for WB-Se analysis,
Hall et al.
into heparinized Vacutainer tubes (9 mL; Becton Dickinson) for erythrocyte GSH analysis, and into evacuated tubes without EDTA (10 mL; Becton Dickinson) for subsequent harvesting of serum. Samples were stored on ice until they were frozen at −20 °C. Colostrum was collected within 2 h of calving from control and Se-yeast supplemented cows and pooled, respectively, for feeding calves in a companion study [18]. Selenium concentrations in WB, serum, and pooled colostrum were determined using an inductively coupled argon plasma emission spectroscopy (ICP-MS) method with modifications as previously described [18]. In brief, Se concentrations were determined by a commercial laboratory (Center for Nutrition, Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing). Two hundred microliters of each sample was diluted 1:20 with a solution containing 0.5 % EDTA and Triton X-100, 1 % ammonia hydroxide, 2 % propanol, and 20 μg/kg of scandium, rhodium, indium, and bismuth as internal standards. All samples were analyzed on an Agilent 7500ce ionized coupled plasma mass spectrometer. Selenium, at mass 78, was analyzed in hydrogen mode to reduce spectral interference (Agilent Technologies, Santa Clara, CA). Serum concentrations of α-tocopherol were measured using reversed-phase HPLC with electrochemical detection, as previously described [28]. Plasma sample volumes of 100 μL per test were combined with alcoholic KOH and 1 % ascorbic acid, and then extracted with hexane and resuspended in 1:1 MeOH:EtOH. Injection volumes of 20 uL per sample were used for HPLC analysis. The peak areas of the tocopherol contents in the sample extracts were compared to those of authentic compounds; a standard curve was performed daily. Total GSH concentrations in metaphosphoric acid-treated, deproteinated, supernatant of erythrocyte lysates were measured in triplicates according to the OxiSelect™ Total Glutathione (GSSG/GSH) Assay Kit (Cell Biolabs, Inc.; San Diego, CA), as previously reported [29]. The assay is based on the method of Owen and Butterfield [30]: GSH reacts in the presence of NADPH, glutathione reductase, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase with Ellman’s reagent (5,5-dithiobis, 2-nitrobenzoic acid) to produce 2nitrobenzoic acid anion, a compound containing a chromophore that absorbs light at 412 nm. A GSH standard was included on each plate to determine total GSH concentration (μM) by the kinetic method. Commercially available kits were used to measure serum concentrations of albumin (Stanbio Albumin LiquiColor® Procedure No. 0285; Boerne, TX), cholesterol (Stanbio Cholesterol LiquiColor® Procedure No. 1010, Stanbio Laboratory), NEFA (ACS ACOD method, WAKO Diagnostics, Richmond, VA), BHBA (Stanbio BHBA LiquiColor® Proc. No. 2440, Stanbio Laboratory), haptoglobin (Catalog No. 2410–70, Life Diagnostics, Inc., West Chester, PA), SAA (Catalog No. KAA0021; Life
Supranutritional Selenium Supplementation for Dairy Cows
Technologies Corp., Grand Island, NY), calcium (Stanbio Total Calcium LiquiColor® Proc. No. 0150, Stanbio Laboratory), magnesium (Stanbio Magnesium LiquiColor® Proc. No. 0130, Stanbio Laboratory), and phosphorus (Stanbio Phosphorus Liqui-UV® Proc. No. 0830, Stanbio Laboratory) according to each manufacturer’s instructions, respectively. The sample volume per test was 2.5 μL for albumin, cholesterol, calcium, magnesium, and phosphorus, 5 μL for SAA and haptoglobin, and magnesium, 6 μL for BHBA, and 10 μL for NEFA. Samples were not diluted except for haptoglobin, which was diluted 2000-fold in three dilution steps, and for SAA, which was diluted 500-fold in two dilution steps, as recommended by the manufacturers. Absorbance was measured with a Bio Tek Synergy 2 Alpha microplate autoreader (BioTek; Winooski, VT). Samples were tested in duplicate. Statistical Analyses Statistical analyses were performed using SAS version 9.2 (SAS, Inc., Cary, NC, USA) software. Each blood parameter was tested for normal distribution using the Shapiro-Wilk test in PROC UNIVARIATE and normalized by natural logarithmic transformation if necessary. Concentrations of SAA, haptoglobin, BHBA, and NEFA were natural logarithm transformed. Data were measured over time and repeated-measures-in-time analysis was performed using PROC MIXED. The fixed effects were treatment (control and Se-yeast), day (0, 48 h, and 14 days postpartum), and their interactions. The lowest values of the Akaike information criterion (AIC) were used as the indicators for the most parsimonious variancecovariance matrix, which was the unstructured variancecovariance matrix for WB-Se concentrations and serum concentrations of α-tocopherol, α-tocopherol cholesterol ratio, SAA, haptoglobin, BHBA, and NEFA, and the compound symmetry variance-covariance matrix (equal variance and covariance) for serum concentrations of Se, GSH, albumin, cholesterol, calcium, magnesium, and phosphorus. Treatments were compared across sampling times and at each sampling time because significant interactions between treatment and sampling time were observed. All statistical tests were two-sided. Data are reported as least square means± SEM. After statistical analysis, least square means and SEM of natural logarithm transformed data were back-transformed for graphical representation. Statistical significance was declared at P≤0.05 and a tendency towards significance between 0.05
Effect of Supranutritional Organic Selenium Supplementation on Postpartum Blood Micronutrients, Antioxidants, Metabolites, and Inflammation Biomarkers in Selenium-Replete Dairy Cows Jean A. Hall & Gerd Bobe & William R. Vorachek & Katherine Kasper & Maret G. Traber & Wayne D. Mosher & Gene J. Pirelli & Mike Gamroth
Received: 25 July 2014 / Accepted: 13 August 2014 / Published online: 21 August 2014 # Springer Science+Business Media New York 2014
Abstract Dairy cows have increased nutritional requirements for antioxidants postpartum. Supranutritional organic Se supplementation may be beneficial because selenoproteins are involved in regulating oxidative stress and inflammation. Our objective was to determine whether feeding Se-yeast above requirements to Se-replete dairy cows during late gestation affects blood micronutrients, antioxidants, metabolites, and inflammation biomarkers postpartum. During the last 8weeks before calving, dairy cows at a commercial farm were fed either 0 (control) or 105 mg Se-yeast once weekly (supranutritional Se-yeast), in addition to Na selenite at 0.3 mg Se/kg dry matter in their rations. Concentrations of whole-blood (WB) Se and serum Se, erythrocyte glutathione (GSH), and serum albumin, cholesterol, α-tocopherol, haptoglobin, serum amyloid A (SAA), calcium, magnesium, phosphorus, non-esterified fatty acids, and β-hydroxybutyrate were measured directly after calving, at 48 h, and 14 days of lactation in 10 cows of each group. Supranutritional Se-yeast supplementation affected indicators of antioxidant status and inflammation. Cows fed a supranutritional Se-yeast supplement during J. A. Hall (*) : W. R. Vorachek Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4802, USA e-mail: [email protected] G. Bobe : W. D. Mosher : G. J. Pirelli : M. Gamroth Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331-4802, USA G. Bobe : M. G. Traber Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-4802, USA K. Kasper : M. G. Traber School of Biological and Population Health Sciences, Oregon State University, Corvallis, OR 97331-4802, USA
the last 8-weeks of gestation had higher Se concentrations in WB (overall 52 % higher) and serum (overall 36 % higher) at all-time points, had higher SAA concentrations at 48 h (98 % higher), had higher erythrocyte GSH (38 % higher) and serum albumin concentrations (6.6 % higher) at 14 days, and had lower serum cholesterol concentrations and higher α-tocopherol/cholesterol ratios at calving and at 48 h compared with control cows. In conclusion, feeding Se-replete cows during late gestation a supranutritional Se-yeast supplement improves antioxidant status and immune responses after calving without negatively impacting other micronutrients and energy status. Keywords Acute phase response . Antioxidants . Dairy Cows . Metabolic biomarkers . Postpartum . Selenium-yeast supplementation
Abbreviations AIC Akaike information criterion BHBA β-hydroxybutyrate DM Dry matter FDA Food and Drug Administration GSH Glutathione ICP-MS Inductively coupled argon plasma emission spectroscopy NEFA Non-esterified fatty acids NRC National Research Council SAA Serum amyloid A Se Selenium SeCys Selenocysteine SeMet Selenomethionine TBARS Thiobarbituric acid reactive substances TMR Total mixed ration WB Whole blood
Supranutritional Selenium Supplementation for Dairy Cows
Introduction Selenium (Se) is an essential micronutrient for ruminant animals [1, 2]. In cattle, severe Se deficiency results in nutritional myopathy or “white-muscle disease” and male infertility, whereas subclinical Se deficiency causes immunosuppression and decreased performance [2–4]. To prevent Se deficiency, the U.S. Food and Drug Administration (FDA) permits Se supplementation of ruminant diets. To avoid Se toxicity, current FDA regulations limit the amount of dietary Se supplementation in ruminant animals to 0.3 mg/kg (as fed) of inorganic Na-selenite or Na-selenate, or organic Se-yeast, which is equivalent to 3 mg per beef cow per day [5]. We are interested in supranutritional Se supplementation (dosages greater than that required for optimal expression of selenoproteins, but less than maximal tolerable levels) to improve health and performance in livestock. Little is known how Se supplementation above FDA regulations affects health and performance of ruminants with adequate concentrations of Se (Se-replete animals). In sheep, supplementing Se-replete ewes with 24.5 mg Se/week as Seyeast improves body condition scores of ewes and growth rates of their progeny [6]. This is 5× the FDA upper limit for supplementing ruminant diets, but less than 5 mg/kg diet, which is considered the maximal tolerable level for ruminants [7]. None of the ewes receiving supranutritional Se yeast supplements showed adverse clinical signs at any time throughout two lambing seasons [6]. Benefits of supranutritional Se-yeast supplementation in ewes include improvements in immune function (increased Escherichia coli killing by neutrophils, increased antibody titers to the novel antigen keyhole limpet hemocyanin, and increased neutrophil expression of genes involved in bacterial pathogen recognition and response) [8, 9], and increased immunoglobulin G transfer to their progeny [10]. Others have shown in cannulated sheep that Se-yeast or nano-Se supplementation of 4 mg Se/kg of dry matter (DM) increases ruminal fermentation and nutrient digestibility [11], and that Na-selenite or Seyeast supplementation of 0.15 mg Se/kg of diet increases antibody titers against Pasteurella multocida P52 vaccination in Se-replete lambs [12, 13]. In Se-replete beef cattle, feeding Se-fertilized alfalfa hay improved their performance and immune function (increased antibody titers after vaccination with J-5 Escherichia coli bacterin) [14] and, likewise, we showed benefits in recently weaned beef calves fed Sefertilized alfalfa hay (increased antibody titers after vaccination with J-5 Escherichia coli bacterin, and increased growth rates and survival) [15, 16]. Others have shown that Se-yeast supplementation of 3 mg Se/day increases mitochondrial gene expression in liver of Se-replete beef heifers, and decreases expression of genes known to be upregulated during oxidative stress [17]. In dairy cows, we reported in a companion paper that supplementing Se-replete cows during the dry period with
273
105 mg Se/week as Se-yeast, equivalent to 5× the FDA allowed maximum supplementation rate for cows, improved immunoglobulin G transfer and Se status in their newborn calves [18]. Others have shown in dairy cows that supplementing Se-replete cows with 0.3 mg/kg Se-yeast compared with 0.3 mg/kg Na selenite during the last 60 days of pregnancy and up to 30 days lactation increases whole-blood (WB)-Se, serum-Se, and colostral-Se concentrations [19]. Supplementing Se-replete heifers with 3 mg Se/day as Seyeast tends to reduce the incidence of intramammary infections and clinical mastitis at calving [20]. Furthermore, Seyeast supplementation with 0.31 and 0.50 mg Se/kg of DM increases whole-blood (WB)-Se, serum-Se, and colostral-Se concentrations in lactating Se-replete dairy cows [21], and decreases plasma β-hydroxybutyrate (BHBA) and reactive oxygen metabolites in plasma during the hot weather season [22]. Dairy cows are more susceptible to various metabolic and infectious diseases during the first weeks after calving, which can be partly ascribed to decreased immune function, increased lipid mobilization, increased production of pro-oxidants, and decreased concentrations of several antioxidants shortly after calving [23–25]. Supranutritional organic Se supplementation may improve the antioxidant status and immune response because selenoproteins (proteins containing selenocysteine [SeCys]) are involved in regulating oxidative stress and inflammation [26]. We hypothesized that feeding Se-replete cows supranutritional Se-yeast during late gestation would improve their Se-status, their antioxidant status, and their immune response after calving. Thus, the objective of this study was to determine whether feeding Se-replete dairy cows a supranutritional Se-yeast supplement during late gestation affects blood indicators after calving. Biomarkers of antioxidant status (whole blood [WB]-Se, serum-Se, glutathione [GSH], and α-tocopherol), lipid transport (cholesterol), acute phase response (albumin, haptoglobin, and serum amyloid A [SAA]), macromineral status (calcium, magnesium, and phosphorus), and energy status (non-esterified fatty acids [NEFA] and β-hydroxybutyrate [BHBA]) were assessed.
Materials and Methods Animals and Study Design The experimental protocol was reviewed and approved by the Oregon State University Animal Care and Use Committee (ACUP Number: 4156). The study was conducted at a commercial dairy in Oregon (Columbia River Dairy, LLC, Boardman, OR). For 8 weeks before predicted calving, multiparous Jersey dairy cows (body condition score ranged from 2.50 to 4.25) were fed either 0 (control cows) or 105 mg Se-yeast once weekly (Se-yeast supplemented cows) on top of their
274
total mixed ration (TMR), which was formulated to meet National Research Council (NRC) recommendations for dry cows [7]. The TMR contained supplemental Na selenite at 0.3 mg Se/kg DM. Cows were head locked when they started to eat their TMR at a bunk feeder. The Se-yeast aliquot was placed directly in front of each cow for individual consumption. The organic Se source (Se-yeast, Prince Se Yeast 2000, Prince Agri Products Inc., Quincy, IL) had a guaranteed analysis of 2 g organically bound Se (78 % as selenomethionine, SeMet) per kg supplement [27]. We previously verified the Se concentration of the supplement [27]. The amount of Se-yeast fed to each cow was 52.5 g once per week. The dose of Seyeast supplement was calculated to provide 5× the maximum allowed FDA Se supplementation dose for ruminant diets, which is 3 mg/day. As an example of dose calculations, 3 mg Se/day is multiplied by 7 for the weekly amount of 21 mg and then by 5× to attain 105 mg Se/week as Se-yeast. After calving in the maternity area, cows were milked once and moved to a fresh pen, which was a freestall with headgates. Twice a day lactating cows were milked and fed a TMR that was formulated to meet NRC recommendations for fresh lactating dairy cows [7]. Again, the TMR contained supplemental Na selenite at 0.3 mg Se/kg DM. No supplemental Seyeast was provided postpartum. Sample Collection and Analysis Whole-blood Se and serum biomarkers of antioxidant status, lipid transport, inflammation, and energy status were measured in a subset of clinically healthy cows, 10 control and 10 Se-yeast supplemented cows. Selection criteria included cows with WB-Se concentrations measured after 6 weekly Se yeast treatments that were likely to be>300 ng/mL by 8 weeks of Se-yeast supplementation, indicating they were consuming the Se-yeast supplement, and that were available for blood collection at calving, and at 48 h and 14 days postpartum. Control cows were chosen based on their availability for blood collection at calving, and at 48 h and 14 days postpartum. In addition, control cows had WB-Se concentrations in the range of 120–300 ng/mL, which is the normal reference interval for WB-Se of adult cows at the Michigan State University Diagnostic Laboratory [14]. All Se-yeast supplemented cows received 7 (n=2) or 8 (n=8) weekly dosages of Se-yeast before calving. Cows received their eighth Se-yeast dosage within 1 (n=2), 3 (n=3), 4 (n=1), 5 (n=1), or 6 (n=3) days of calving. Blood samples were collected from the jugular vein of cows at the beginning of the study (8 weeks before expected calving date), after 6 weeks of Se-yeast supplementation, after parturition (at the time of first milking), at 48 h postpartum, and at 14 days postpartum. Blood samples were collected into evacuated EDTA tubes (2 mL; final EDTA concentration 2 g/L; Becton Dickinson, Franklin Lakes, NJ) for WB-Se analysis,
Hall et al.
into heparinized Vacutainer tubes (9 mL; Becton Dickinson) for erythrocyte GSH analysis, and into evacuated tubes without EDTA (10 mL; Becton Dickinson) for subsequent harvesting of serum. Samples were stored on ice until they were frozen at −20 °C. Colostrum was collected within 2 h of calving from control and Se-yeast supplemented cows and pooled, respectively, for feeding calves in a companion study [18]. Selenium concentrations in WB, serum, and pooled colostrum were determined using an inductively coupled argon plasma emission spectroscopy (ICP-MS) method with modifications as previously described [18]. In brief, Se concentrations were determined by a commercial laboratory (Center for Nutrition, Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing). Two hundred microliters of each sample was diluted 1:20 with a solution containing 0.5 % EDTA and Triton X-100, 1 % ammonia hydroxide, 2 % propanol, and 20 μg/kg of scandium, rhodium, indium, and bismuth as internal standards. All samples were analyzed on an Agilent 7500ce ionized coupled plasma mass spectrometer. Selenium, at mass 78, was analyzed in hydrogen mode to reduce spectral interference (Agilent Technologies, Santa Clara, CA). Serum concentrations of α-tocopherol were measured using reversed-phase HPLC with electrochemical detection, as previously described [28]. Plasma sample volumes of 100 μL per test were combined with alcoholic KOH and 1 % ascorbic acid, and then extracted with hexane and resuspended in 1:1 MeOH:EtOH. Injection volumes of 20 uL per sample were used for HPLC analysis. The peak areas of the tocopherol contents in the sample extracts were compared to those of authentic compounds; a standard curve was performed daily. Total GSH concentrations in metaphosphoric acid-treated, deproteinated, supernatant of erythrocyte lysates were measured in triplicates according to the OxiSelect™ Total Glutathione (GSSG/GSH) Assay Kit (Cell Biolabs, Inc.; San Diego, CA), as previously reported [29]. The assay is based on the method of Owen and Butterfield [30]: GSH reacts in the presence of NADPH, glutathione reductase, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase with Ellman’s reagent (5,5-dithiobis, 2-nitrobenzoic acid) to produce 2nitrobenzoic acid anion, a compound containing a chromophore that absorbs light at 412 nm. A GSH standard was included on each plate to determine total GSH concentration (μM) by the kinetic method. Commercially available kits were used to measure serum concentrations of albumin (Stanbio Albumin LiquiColor® Procedure No. 0285; Boerne, TX), cholesterol (Stanbio Cholesterol LiquiColor® Procedure No. 1010, Stanbio Laboratory), NEFA (ACS ACOD method, WAKO Diagnostics, Richmond, VA), BHBA (Stanbio BHBA LiquiColor® Proc. No. 2440, Stanbio Laboratory), haptoglobin (Catalog No. 2410–70, Life Diagnostics, Inc., West Chester, PA), SAA (Catalog No. KAA0021; Life
Supranutritional Selenium Supplementation for Dairy Cows
Technologies Corp., Grand Island, NY), calcium (Stanbio Total Calcium LiquiColor® Proc. No. 0150, Stanbio Laboratory), magnesium (Stanbio Magnesium LiquiColor® Proc. No. 0130, Stanbio Laboratory), and phosphorus (Stanbio Phosphorus Liqui-UV® Proc. No. 0830, Stanbio Laboratory) according to each manufacturer’s instructions, respectively. The sample volume per test was 2.5 μL for albumin, cholesterol, calcium, magnesium, and phosphorus, 5 μL for SAA and haptoglobin, and magnesium, 6 μL for BHBA, and 10 μL for NEFA. Samples were not diluted except for haptoglobin, which was diluted 2000-fold in three dilution steps, and for SAA, which was diluted 500-fold in two dilution steps, as recommended by the manufacturers. Absorbance was measured with a Bio Tek Synergy 2 Alpha microplate autoreader (BioTek; Winooski, VT). Samples were tested in duplicate. Statistical Analyses Statistical analyses were performed using SAS version 9.2 (SAS, Inc., Cary, NC, USA) software. Each blood parameter was tested for normal distribution using the Shapiro-Wilk test in PROC UNIVARIATE and normalized by natural logarithmic transformation if necessary. Concentrations of SAA, haptoglobin, BHBA, and NEFA were natural logarithm transformed. Data were measured over time and repeated-measures-in-time analysis was performed using PROC MIXED. The fixed effects were treatment (control and Se-yeast), day (0, 48 h, and 14 days postpartum), and their interactions. The lowest values of the Akaike information criterion (AIC) were used as the indicators for the most parsimonious variancecovariance matrix, which was the unstructured variancecovariance matrix for WB-Se concentrations and serum concentrations of α-tocopherol, α-tocopherol cholesterol ratio, SAA, haptoglobin, BHBA, and NEFA, and the compound symmetry variance-covariance matrix (equal variance and covariance) for serum concentrations of Se, GSH, albumin, cholesterol, calcium, magnesium, and phosphorus. Treatments were compared across sampling times and at each sampling time because significant interactions between treatment and sampling time were observed. All statistical tests were two-sided. Data are reported as least square means± SEM. After statistical analysis, least square means and SEM of natural logarithm transformed data were back-transformed for graphical representation. Statistical significance was declared at P≤0.05 and a tendency towards significance between 0.05
