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Alternative selenium supplement for sheep
Animal Feed Science and Technology 261 (2020) 114390
Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Alternative selenium supplement for sheep K. Nedelkova,b, X.J. Chena,c, C.M.M.R. Martinsa,d, A. Melgara, M.T. Harpera, S. Räisänena, J. Oha, T.L. Felixa, E. Walle,1, A.N. Hristova,*
T
a
Department of Animal Science, The Pennsylvania State University, University Park, PA, 16802, USA Faculty of Veterinary Medicine, Trakia University, Stara Zagora, 6000, Bulgaria School of Computing, University of Ulster, Co. Antrim, Northern Ireland, BT37 0QB, United Kingdom d School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, 13635-900, Brazil e Pancosma, CH-1180, Rolle, Switzerland b c
A R T IC LE I N F O
ABS TRA CT
Keywords: Trace element GPx Sheep
This study investigated Se-dependent parameters in ewes after supplementation with an experimental Se product (P-Se) in comparison to sodium selenite. Six Dorset ewes averaging 93.0 (SE = 2.5) kg initial body weight were used in a replicated 3 × 3 Latin square design experiment balanced for residual effects. The study began with a 2-wk background, low-Se period and had 3 experimental periods of 3 weeks each, with 2-wk washout periods between them. The treatments studied were: (1) unsupplemented control (basal diet containing 0.065 mg Se/kg dry matter, DM); (2) basal diet supplemented with Na-selenite providing 0.25 mg Se/kg DM; and (3) basal diet supplemented with P-Se (containing 29 % Na-selenite and 71 % amorphous elemental Se) providing 0.27 mg Se/kg DM (P-Se). Blood samples (taken from the jugular vein) and spot urine and fecal samples were collected the day before the start of each experimental period, on the last day of each washout period, and once weekly during the experimental periods for analysis of Se concentration and glutathione peroxidase (GPx) activity (blood plasma only). Ewes supplemented with P-Se and Na-selenite had increased (P < 0.001) plasma Se concentration compared with ewes fed the control diet. Absolute concentration of Se in plasma was greater (P < 0.001) for ewes fed Na-selenite (80.9 μg/L) than for ewes fed P-Se (72.3 μg/L). Absolute plasma GPx activity was greater (P < 0.001) for ewes fed Na-selenite and P-Se compared with the control, but was not different between the Se treatments. Concentration of Se in fecal matter was greater (P < 0.001) for both Se sources than the control and fecal Se concentration was higher (P < 0.001) for P-Se compared with Na-selenite. Absolute Se concentration in urine was greater (P ≤ 0.003) for Na-selenite than the control and P-Se. Selenium supplementation of ewe diet increased Se concentration and GPx activity in blood plasma, regardless of Se source. Although P-Se had slightly inferior absorption, compared with Na-selenite, our data indicate that it can potentially be an effective source of dietary Se for mature ewes, but further studies are needed to confirm these results in other farm animal species and physiological stages.
Abbreviations: ADF, acid detergent fibre; NDF, neutral detergent fibre; BW, body weight; Na-selenite, sodium selenite; GPx, glutathione peroxidase; CP, crude protein; DM, dry matter ⁎ Corresponding author. E-mail address: [email protected] (A.N. Hristov). 1 Present affiliation : AVT NaturalProducts, Kerala, India. https://doi.org/10.1016/j.anifeedsci.2020.114390 Received 22 August 2019; Received in revised form 31 December 2019; Accepted 2 January 2020 0377-8401/ © 2020 Published by Elsevier B.V.
Animal Feed Science and Technology 261 (2020) 114390
K. Nedelkov, et al.
1. Introduction Selenium is an essential trace element for animal health, immune function, productivity, and reproductive performance in farm animals (Meyer et al., 2014). Animal diets are typically supplemented with Se to provide adequate amount of dietary Se and prevent Se-deficiency disorders. Due to potential toxicity of inorganic Se and high cost of organic Se sources, efforts have been directed towards discovering efficient, safe and cost-effective sources of Se (Pavlata et al., 2012a). In two recent studies, elemental Se was shown to have both increased bioavailability and decreased toxicity relative to sodium selenite in ewes (Sadeghian et al., 2012; Xun et al., 2012). These observations make elemental Se an interesting candidate for supplementing feed of ruminants to maximize health and performance with potentially lower inclusion rates, and at the same time limit environmental and toxicity risks. Importantly in both of those studies, nanoparticles were used, which can create a safety risk for handlers due to the risk of inhalation of ultra-fine particles less than 100 nm (Miller et al., 2010). Recently, a new Se product was developed (P-Se; Pancosma, Rolle, Switzerland) composed of an amino acid, sodium selenite and amorphous elemental Se (29 % of Se as Na-selenite and 71 % as elemental Se), spraydried into a red granular powder. We hypothesized that P-Se would have similar indicators of Se status compared with Se from Na-selenite in mature ewes. Thus, the objective of this study was to investigate Se-dependent parameters in ewes after supplementation with P-Se or with Na-selenite. 2. Materials and methods 2.1. Experimental design, animals and treatments All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University. Six Dorset cull ewes (initial BW 93.0 ± 2.5 kg) were used in this study. The ewes were housed in individual straw-bedded pens (3.75 × 1.45 m) at The Pennsylvania State University Beef and Sheep Research and Teaching Center. The study was a replicated 3 × 3 Latin square design with a 2-wk background, low-Se (no Se supplementation) period and 3 experimental periods of 3 wks each with 2-wk washout periods between them. The basal diet was formulated to meet or exceed the nutrient requirements for maintenance of a 90-kg BW ewe consuming 1540 g DM/d (based on NRC, 2007), except for Se. The current Se requirements for sheep estimated by using the factorial method are (as-fed basis) ∼ 0.3 mg/kg feed (NRC, 2007). The daily ration of each ewe consisted of 1.5 kg grass hay and 0.2 kg ground corn premix on an as is basis (Table 1). The 3 treatments were: 1) control, unsupplemented basal diet containing 0.065 mg Se/kg dietary DM; 2) basal diet supplemented with sodium selenite (Na-selenite) providing 0.25 mg Se/kg DM; and 3) basal diet supplemented with P-Se (Pancosma S.A., Rolle, Switzerland) providing 0.27 mg Se/kg DM. The investigated new Se product (P-Se) contained amorphous elemental Se that was spray-dried into a red granular powder (29 % of Se as Na-selenite and 71 % as elemental Se). The Se supplements were added to the diet through inclusion in ground corn premix. The premix was prepared in 10-kg batches using a commercial dough mixer (Hobart D-300 30 QT; Illinois Tool Works Inc., Glenview, IL) before the start of each experimental period. Each batch of Se premixes contained 3.3 mg /kg Na-selenite and 50 mg/kg P-Se, respectively. The ewes had free access to fresh potable water and were fed twice daily at 0830 h and 1630 h. 2.2. Feed analyses Grass hay and ground corn premix were sampled weekly throughout the experiment and composite samples (per experimental period) were ground through a 1-mm screen in a Wiley mill (Thomas Scientific, Swedesboro, NJ) and submitted to Cumberland Valley Analytical Services Inc. (Waynesboro, PA) to be analyzed by wet chemistry methods for amylase-treated NDF (Van Soest et al., 1991), ADF (method 973.18; AOAC International, 2000), CP (method 990.03; AOAC International, 2000), ash (method 942.05; AOAC International, 2000), and minerals (method 985.01; AOAC International, 2000). The data on net energy for maintenance (NEm) were reported by Cumberland Valley Analytical Services Inc. (Waynesboro, PA) where the energy estimates were calculated using the OARDC Summative Energy Equation of Weiss (1998). Ground, as described above, feed samples were sent to The University of Missouri Agricultural Experiment Station Chemical Laboratories (MU ESCL) for Se analysis by graphite furnace atomic absorption spectrometry (method 996.16; AOAC International, 2006). 2.3. Blood sampling Blood samples were collected via the jugular vein into 10-mL heparinized vacutainers containing Li-heparin (BD Biosciences, Franklin Lakes, NJ) the day before the start of each experimental period, on the last day of each washout period, and once weekly during each experimental period. Samples were collected 1 h before feeding and 1 h after feeding. Blood plasma was separated by centrifugation at 1,500 × g for 20 min at 4 °C and the plasma was apportioned into 1.0-mL aliquots and stored at −80 °C for further analyses. 2.4. Fecal and urine sampling Spot urine and fecal samples (approximately 20 ml and 100 g per sample, respectively) were collected the day before the start of each experimental period, on the last day of each washout period, and once weekly during each experimental period for analysis of Se 2
Animal Feed Science and Technology 261 (2020) 114390
K. Nedelkov, et al.
Table 1 Ingredient and chemical composition of the basal diet fed during the experiment. Item Ingredient, g/kg DM Grass hayac Corn grain, groundbc Trace mineral mixtured Composition,e g/kg DM (or as indicated) CP NDF ADF NEmf, Mcal/kg Ca P Se, mg/kg DM
881 116 3.0 96.5 586 367 1.28 3.7 2.6 0.065
a Grass hay was 927 g/kg DM and contained (DM basis): 93.0 g/kg CP, 652 g/kg NDF, 412 g/kg ADF, 1.17 Mcal of NEm g/kg, 4.10 g/kg Ca, 2.60 g/kg P. It also contained (as is basis; ± SE) 0.041 ± 0.005 mg Se/ kg. b Corn grain was 870 g/kg DM and contained (DM basis): 90.0 g/kg CP, 88.3 g/kg NDF, 29.4 g/kg ADF, 2.14 Mcal of NEm g/kg, 0.80 g/kg Ca, 3.00 g/kg P. Ground corn premix contained (as is basis; ± SE) 0.256 mg Se/kg and treatments resulted in the following Se concentrations: Naselenite, 1.906 ± 0.138 mg Se/kg and P-Se, 2.140 ± 0.092 mg Se/kg. c Selenium was analyzed in feed samples collected throughout the experiment (University of Missouri Agricultural Experiment Station Chemical Laboratories - MU ESCL). d Trace mineral mixture (Champion’s choice, Cargill Animal Nutrition, Cargill Inc., Roaring Spring, PA) contained (DM basis): NaCl, 940 g/kg; ZnO, 3.50 g/kg; MgO, 2.00 g/kg; FeCO3 2.00 g/kg; CuSO4, 0.30 g/kg; Ca (IO3)2, 0.30 g/kg; CoCO3, 0.07 g/kg. e Values calculated using the chemical analysis of the individual feed ingredients (Cumberland Valley Analytical Services Inc., Waynesboro, PA) and their inclusion rate in the diet. f Net energy for maintenance (NEm) concentration was calculated using the OARDC Summative Energy Equation of Weiss (1998).
concentration. Samples were collected approximately 2 h after feeding. Extra care was taken to separate fecal from bedding materials. Urine was collected by stimulating urination by holding off the animal's air and blocking the nostrils and mouth for a short period (approximately 30 s). After collection, spot urine samples were combined per experimental period and per animal resulting in a total of eighteen composite samples. The undiluted urine samples were stored frozen at −20 °C for subsequent Se analysis. Fecal samples were collected from the pen floor and were oven dried at 65 °C for 48 h, ground through a 1-mm sieve in a Wiley mill (Thomas Scientific) and saved in sealed Ziploc bags for Se analyses. 2.5. Selenium and glutathione peroxidase analyses Selenium concentrations in plasma, feces, and urine were analyzed at the University of Missouri Agricultural Experiment Station Chemical Laboratories (MU ESCL) in triplicate using the graphite furnace atomic absorption spectrometry as described by Elmer (1999). Glutathione peroxidase (GPx) activity was measured in blood plasma using a commercial kit (Item No. 703,102; Cayman Chemical Co., Ann Arbor, MI) with activity measured according to the decrease in absorbance at 340 nm (SPECTROstar Omega Microplate Reader; BMG Labtech, Inc., Cary, NC). Glutathione peroxidase activity was expressed as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP+ per minute per mL of sample at 25 °C. 2.6. Statistical analysis Data were analyzed using the MIXED procedure of SAS v 9.4 (SAS Institute Inc., Cary, NC) with treatment, sampling week, experimental period, and treatment × sampling week interaction included in the model and ewes and ewes within square as random effects. Plasma, urine and fecal Se data were analyzed as repeated measures with AR(1) covariance structure and were presented in absolute concentrations. Statistical significance was declared at P ≤ 0.05 and tendency was at 0.05 < P ≤ 0.10. Data are presented as least squares means unless indicated otherwise. 3
Animal Feed Science and Technology 261 (2020) 114390
K. Nedelkov, et al.
Table 2 Selenium intake (mg/kg as is basis or as indicated; ± SE) and effect of source of dietary Se on plasma Se, plasma GPx activity, and fecal and urine Se concentrations. Treatment1
P-value3
Item
Control
Na-selenite
P-Se
SEM2
Se supplementation rate (mg/kg ground corn premix) Analyzed Se, mg/kg DM Total dietary Se4, mg/kg DM Se fed, mg/head/day Plasma Se, μg/L Plasma GPx5, nmol/min/mL Fecal Se, μg/kg DM Urine Se, μg/L
0.00 0.22 0.065 ± 0.001 0.10 ± 0.001 57.4a 35.3a 113a 6.28a
1.50 1.66 ± 0.107 0.25 ± 0.024 0.39 ± 0.032 80.9c 57.1b 441b 24.4b
1.50 1.86 ± 0.082 0.27 ± 0.011 0.42 ± 0.019 72.3b 52.5b 526c 12.2a
2.21 1.63 26.5 2.63
< 0.001 < 0.001 < 0.001 0.003
a,b,c
Means with different letter superscripts differ at P < 0.05. Control = unsupplemented control; Na-selenite = sodium selenite, 0.39 mg Se/head/d; P-Se = experimental Se source, 0.42 mg Se/head/d. 2 Standard error of the mean (largest SEM shown in table), plasma Se: n = 108; plasma GPx: n = 106; fecal Se: n = 54; urine Se: n = 18 (n represents the number of observations used in the statistical analysis). 3 Main effect of treatment. Treatment × sampling week interaction for plasma Se concentration, plasma GPx activity and fecal Se concentration: P ≤ 0.001, 0.99, and 0.61, respectively. 4 Analyzed in feed samples collected throughout the experiment (University of Missouri Agricultural Experiment Station Chemical Laboratories MU ESCL). 5 GPx = glutathione peroxidase activity was expressed as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP + per minute per mL of sample at 25 °C. 1
3. Results and discussion 3.1. Plasma Se concentrations Effect of Se source on plasma Se absolute concentrations are shown in Table 2 and Fig. 1. Compared with the control, Se concentration in plasma was increased (P < 0.001) in ewes fed both Se treatments, and concentration was greater (P < 0.001) for Na-selenite compared with P-Se. There was a treatment × sampling week interaction (P ≤ 0.001) for plasma Se, and Fig. 1 shows the effectiveness of the washout periods between treatments. Plasma Se concentrations provide short-term data about Se status of the animal and can be used as an effective biomarker for assessing bioavailability of Se from various sources (Fairweather-Tait et al., 2010). Our results support the use of plasma Se as an index of Se availability with the data suggesting slightly inferior absorption efficiency of Se from P-Se.
Fig. 1. Weekly blood plasma Se data (mean ± SEM). Blood samples were collected 1 h before feeding and 1 h after feeding on D (day of background, low Se, washout and experimental periods) 14, 21, 28 and 35. Control = unsupplemented control; Na-selenite = sodium selenite, 0.39 Se /head/d; P-Se = experimental Se source, 0.42 mg Se/head/d. There was a treatment × sampling week interaction (P ≤ 0.001). Means within period labeled with different letters (a,b,c) differ at P < 0.05. 4
Animal Feed Science and Technology 261 (2020) 114390
K. Nedelkov, et al.
3.2. Plasma glutathione peroxidase activity Selenium status was also assessed by analyzing plasma GPx activity, which concentration was greater (P < 0.001) in the Sesupplemented ewes, compared with the control (Table 2). There was no effect of Se source on absolute plasma GPx activity. There were no treatment × sampling week interactions (P = 0.99) for the GPx activity data. These results are in agreement with previous reports in which Se supplementation led to rapid increase in plasma GPx activity in sheep (Chauhan et al., 2014). Gerloff (1992) stated that plasma GPx activity is considered to be an indicator of a short-term Se status. The form of Se can also play an important role in the rate or increase in GPx activity (Pavlata et al., 2012b). 3.3. Fecal and urine Se concentrations Compared with the control, the Se treatments resulted in greater Se concentration in feces, and absolute fecal Se concentration was higher (P < 0.001) for P-Se compared with Na-selenite. (Table 2). There was no interaction (P = 0.61) between treatment and sampling week for the fecal Se concentration data. The absolute Se concentrations in urine were greater (P ≤ 0.003) in ewes supplemented with Na-selenite than the control and P-Se-treated ewes (Table 2). Ruminants absorb Se far less efficiently than nonruminants and most of the ingested Se leaves the rumen with insoluble particulate matter (Suttle, 2010). Reportedly, in ruminants, the urinary excretion of Se is generally low and the primary route for excretion of orally administered Se is via feces (Tanner, 2008). In the study of Meyer et al. (2014), cows supplemented with inorganic or organic Se-sources over 3 wks at 2 inclusion rates had concentrations of Se in feces, ranging from 376 to 842 μg/kg, whereas concentrations in urine were 50–102 μg/L. Our data showed similar trends in fecal and urine Se concentration. The fecal Se concentration data suggest that P-Se, may have had slightly inferior absorption efficiency compared with Na-selenite. Considering that P-Se contains 29 % of its Se as Na-selenite, the observed difference in absorption efficiency is likely a result of lower absorption rate of elemental Se (71 % of Se in P-Se) in P-Se. Further studies are needed to investigate the post-absorption metabolism of this experimental Se product and its bioavailability in comparison with organic Se sources at possibly lower doses. 4. Conclusions Although Se from P-Se appeared to have slightly inferior absorption efficiency compared with Na-selenite, our data indicate that P-Se can be an effective source of dietary Se for mature ewes; however, further studies are needed to confirm these results in other farm animal species and physiological stages. CRediT authorship contribution statement K. Nedelkov: Investigation, Formal analysis, Data curation, Writing - original draft. X.J. Chen: Investigation, Data curation, Writing - review & editing. C.M.M.R. Martins: Investigation, Data curation, Formal analysis, Writing - review & editing. A. Melgar: Investigation, Data curation, Writing - review & editing. M.T. Harper: Investigation, Data curation, Writing - review & editing. S. Räisänen: Investigation, Data curation, Writing - review & editing. J. Oh: Methodology, Data curation, Writing - review & editing. T.L. Felix: Methodology, Writing - review & editing. E. Wall: Funding acquisition, Writing - review & editing. A.N. Hristov: Conceptualization, Formal analysis, Methodology, Supervision, Writing - review & editing. Declaration of Competing Interest The authors have no conflict of interest to declare. Acknowledgments This work was supported by the USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN04539 and Accession number 1000803. Partial financial support was also provided by Pancosma S.A. (Rolle, Switzerland). The authors thank Dr. Jacob Werner for assisting with euthanasia of the study animals and Wendall M. Landis and The Pennsylvania State University Beef Cattle and Sheep Center staff for their conscientious care of the experimental animals. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anifeedsci. 2020.114390. References AOAC International, 2000. Official Methods of Analysis, 17th ed. Association of Official Analytical Chemist International, Arlington, Arlington, VA. AOAC International, 2006. Official Methods of Analysis, 18th ed. Association of Official Analytical Chemist International, Arlington, VA. Chauhan, S.S., Celi, P., Leury, B.J., Clarke, I.J., Dunshea, F.R., 2014. Dietary antioxidants at supranutritional doses improve oxidative status and reduce the negative
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Animal Feed Science and Technology 261 (2020) 114390
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effects of heat stress in sheep. J. Anim. Sci. 92, 3364–3374. https://doi.org/10.2527/jas.2014-7714. Elmer, P., 1999. The THGA graphite furnace: techniques and recommended conditions. Recommended Conditions For Selenium. Perkin Elmer Corp, Shelton, CT, Perkin, pp. 7–51 Elmer Publication No. B3210. Fairweather-Tait, S.J., Collings, R., Hurst, R., 2010. Selenium bioavailability: current knowledge and future research requirements. Am. J. Clin. Nutr. 91, 1484–1491. https://doi.org/10.3945/ajcn.2010.28674J. Gerloff, B.J., 1992. Effect of selenium supplementation on dairy cattle. J. Anim. Sci. 70, 3934–3940. Meyer, U., Heerdegen, K., Schenkel, H., Dänicke, S., Flachowsky, G., 2014. Influence of various selenium sources on selenium concentration in the milk of dairy cows. J. Verbrauch. Lebensm. 9, 101–109. https://doi.org/10.1207/s15327914nc5402_7. Miller, A., Drake, P.L., Hintz, P., Habjan, M., 2010. Characterizing exposures to airborne metals and nanoparticle emissions in a refinery. Ann. Occup. Hyg. 54, 504–513. https://doi.org/10.1093/annhyg/meq032. NRC, 2007. Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids. The National Academies Press, Washington, DC. https://doi. org/10.17226/11654. Pavlata, L., Misurova, L., Pechova, A., Dvorak, R., 2012a. Comparison of organic and inorganic forms of selenium in the mother and kid relationship in goats. Czech J. Anim. Sci. 57, 361–369. https://doi.org/10.1016/j.jksus.2013.10.003. Pavlata, L., Misurova, L., Pechova, A., Husakova, T., Dvorak, R., 2012b. Direct and indirect assessment of selenium status in sheep – a comparison. Vet. Med. 57, 219–223. https://doi.org/10.17221/5951-vetmed. Sadeghian, S., Kojouri, G.A., Mohebbiand, A., 2012. Nanoparticles of selenium as species with stronger physiological effects in sheep in comparison with sodium selenite. Biol. Trace Elem. Res. 146, 302–308. https://doi.org/10.1007/s12011-011-9266-8. Suttle, N.F., 2010. Mineral Nutrition of Livestock, 4th edition. CABI publishing, Wallingford, UK. Tanner, G.E., 2008. The Influence of Calf Selenium Status on GPX-1 and 3 Activity and Liver GPX-1 Messenger Ribonucleic Acid. Master’s Thesis. Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College, LA. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2. Weiss, W.P., 1998. Estimating the available energy content of feeds for dairy cattle. J. Dairy Sci. 81, 830–839. https://doi.org/10.3168/jds.S0022-0302(98)75641-3. Xun, W., Shi, L., Yue, W., Zhang, C., Ren, Y., Liu, Q., 2012. Effect of high-dose nano-selenium and selenium–yeast on feed digestibility, rumen fermentation, and purine derivatives in sheep. Biol. Trace Elem. Res. 150, 130–136. https://doi.org/10.1007/s12011-012-9452-3.
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Contents lists available at ScienceDirect
Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Alternative selenium supplement for sheep K. Nedelkova,b, X.J. Chena,c, C.M.M.R. Martinsa,d, A. Melgara, M.T. Harpera, S. Räisänena, J. Oha, T.L. Felixa, E. Walle,1, A.N. Hristova,*
T
a
Department of Animal Science, The Pennsylvania State University, University Park, PA, 16802, USA Faculty of Veterinary Medicine, Trakia University, Stara Zagora, 6000, Bulgaria School of Computing, University of Ulster, Co. Antrim, Northern Ireland, BT37 0QB, United Kingdom d School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, 13635-900, Brazil e Pancosma, CH-1180, Rolle, Switzerland b c
A R T IC LE I N F O
ABS TRA CT
Keywords: Trace element GPx Sheep
This study investigated Se-dependent parameters in ewes after supplementation with an experimental Se product (P-Se) in comparison to sodium selenite. Six Dorset ewes averaging 93.0 (SE = 2.5) kg initial body weight were used in a replicated 3 × 3 Latin square design experiment balanced for residual effects. The study began with a 2-wk background, low-Se period and had 3 experimental periods of 3 weeks each, with 2-wk washout periods between them. The treatments studied were: (1) unsupplemented control (basal diet containing 0.065 mg Se/kg dry matter, DM); (2) basal diet supplemented with Na-selenite providing 0.25 mg Se/kg DM; and (3) basal diet supplemented with P-Se (containing 29 % Na-selenite and 71 % amorphous elemental Se) providing 0.27 mg Se/kg DM (P-Se). Blood samples (taken from the jugular vein) and spot urine and fecal samples were collected the day before the start of each experimental period, on the last day of each washout period, and once weekly during the experimental periods for analysis of Se concentration and glutathione peroxidase (GPx) activity (blood plasma only). Ewes supplemented with P-Se and Na-selenite had increased (P < 0.001) plasma Se concentration compared with ewes fed the control diet. Absolute concentration of Se in plasma was greater (P < 0.001) for ewes fed Na-selenite (80.9 μg/L) than for ewes fed P-Se (72.3 μg/L). Absolute plasma GPx activity was greater (P < 0.001) for ewes fed Na-selenite and P-Se compared with the control, but was not different between the Se treatments. Concentration of Se in fecal matter was greater (P < 0.001) for both Se sources than the control and fecal Se concentration was higher (P < 0.001) for P-Se compared with Na-selenite. Absolute Se concentration in urine was greater (P ≤ 0.003) for Na-selenite than the control and P-Se. Selenium supplementation of ewe diet increased Se concentration and GPx activity in blood plasma, regardless of Se source. Although P-Se had slightly inferior absorption, compared with Na-selenite, our data indicate that it can potentially be an effective source of dietary Se for mature ewes, but further studies are needed to confirm these results in other farm animal species and physiological stages.
Abbreviations: ADF, acid detergent fibre; NDF, neutral detergent fibre; BW, body weight; Na-selenite, sodium selenite; GPx, glutathione peroxidase; CP, crude protein; DM, dry matter ⁎ Corresponding author. E-mail address: [email protected] (A.N. Hristov). 1 Present affiliation : AVT NaturalProducts, Kerala, India. https://doi.org/10.1016/j.anifeedsci.2020.114390 Received 22 August 2019; Received in revised form 31 December 2019; Accepted 2 January 2020 0377-8401/ © 2020 Published by Elsevier B.V.
Animal Feed Science and Technology 261 (2020) 114390
K. Nedelkov, et al.
1. Introduction Selenium is an essential trace element for animal health, immune function, productivity, and reproductive performance in farm animals (Meyer et al., 2014). Animal diets are typically supplemented with Se to provide adequate amount of dietary Se and prevent Se-deficiency disorders. Due to potential toxicity of inorganic Se and high cost of organic Se sources, efforts have been directed towards discovering efficient, safe and cost-effective sources of Se (Pavlata et al., 2012a). In two recent studies, elemental Se was shown to have both increased bioavailability and decreased toxicity relative to sodium selenite in ewes (Sadeghian et al., 2012; Xun et al., 2012). These observations make elemental Se an interesting candidate for supplementing feed of ruminants to maximize health and performance with potentially lower inclusion rates, and at the same time limit environmental and toxicity risks. Importantly in both of those studies, nanoparticles were used, which can create a safety risk for handlers due to the risk of inhalation of ultra-fine particles less than 100 nm (Miller et al., 2010). Recently, a new Se product was developed (P-Se; Pancosma, Rolle, Switzerland) composed of an amino acid, sodium selenite and amorphous elemental Se (29 % of Se as Na-selenite and 71 % as elemental Se), spraydried into a red granular powder. We hypothesized that P-Se would have similar indicators of Se status compared with Se from Na-selenite in mature ewes. Thus, the objective of this study was to investigate Se-dependent parameters in ewes after supplementation with P-Se or with Na-selenite. 2. Materials and methods 2.1. Experimental design, animals and treatments All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University. Six Dorset cull ewes (initial BW 93.0 ± 2.5 kg) were used in this study. The ewes were housed in individual straw-bedded pens (3.75 × 1.45 m) at The Pennsylvania State University Beef and Sheep Research and Teaching Center. The study was a replicated 3 × 3 Latin square design with a 2-wk background, low-Se (no Se supplementation) period and 3 experimental periods of 3 wks each with 2-wk washout periods between them. The basal diet was formulated to meet or exceed the nutrient requirements for maintenance of a 90-kg BW ewe consuming 1540 g DM/d (based on NRC, 2007), except for Se. The current Se requirements for sheep estimated by using the factorial method are (as-fed basis) ∼ 0.3 mg/kg feed (NRC, 2007). The daily ration of each ewe consisted of 1.5 kg grass hay and 0.2 kg ground corn premix on an as is basis (Table 1). The 3 treatments were: 1) control, unsupplemented basal diet containing 0.065 mg Se/kg dietary DM; 2) basal diet supplemented with sodium selenite (Na-selenite) providing 0.25 mg Se/kg DM; and 3) basal diet supplemented with P-Se (Pancosma S.A., Rolle, Switzerland) providing 0.27 mg Se/kg DM. The investigated new Se product (P-Se) contained amorphous elemental Se that was spray-dried into a red granular powder (29 % of Se as Na-selenite and 71 % as elemental Se). The Se supplements were added to the diet through inclusion in ground corn premix. The premix was prepared in 10-kg batches using a commercial dough mixer (Hobart D-300 30 QT; Illinois Tool Works Inc., Glenview, IL) before the start of each experimental period. Each batch of Se premixes contained 3.3 mg /kg Na-selenite and 50 mg/kg P-Se, respectively. The ewes had free access to fresh potable water and were fed twice daily at 0830 h and 1630 h. 2.2. Feed analyses Grass hay and ground corn premix were sampled weekly throughout the experiment and composite samples (per experimental period) were ground through a 1-mm screen in a Wiley mill (Thomas Scientific, Swedesboro, NJ) and submitted to Cumberland Valley Analytical Services Inc. (Waynesboro, PA) to be analyzed by wet chemistry methods for amylase-treated NDF (Van Soest et al., 1991), ADF (method 973.18; AOAC International, 2000), CP (method 990.03; AOAC International, 2000), ash (method 942.05; AOAC International, 2000), and minerals (method 985.01; AOAC International, 2000). The data on net energy for maintenance (NEm) were reported by Cumberland Valley Analytical Services Inc. (Waynesboro, PA) where the energy estimates were calculated using the OARDC Summative Energy Equation of Weiss (1998). Ground, as described above, feed samples were sent to The University of Missouri Agricultural Experiment Station Chemical Laboratories (MU ESCL) for Se analysis by graphite furnace atomic absorption spectrometry (method 996.16; AOAC International, 2006). 2.3. Blood sampling Blood samples were collected via the jugular vein into 10-mL heparinized vacutainers containing Li-heparin (BD Biosciences, Franklin Lakes, NJ) the day before the start of each experimental period, on the last day of each washout period, and once weekly during each experimental period. Samples were collected 1 h before feeding and 1 h after feeding. Blood plasma was separated by centrifugation at 1,500 × g for 20 min at 4 °C and the plasma was apportioned into 1.0-mL aliquots and stored at −80 °C for further analyses. 2.4. Fecal and urine sampling Spot urine and fecal samples (approximately 20 ml and 100 g per sample, respectively) were collected the day before the start of each experimental period, on the last day of each washout period, and once weekly during each experimental period for analysis of Se 2
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Table 1 Ingredient and chemical composition of the basal diet fed during the experiment. Item Ingredient, g/kg DM Grass hayac Corn grain, groundbc Trace mineral mixtured Composition,e g/kg DM (or as indicated) CP NDF ADF NEmf, Mcal/kg Ca P Se, mg/kg DM
881 116 3.0 96.5 586 367 1.28 3.7 2.6 0.065
a Grass hay was 927 g/kg DM and contained (DM basis): 93.0 g/kg CP, 652 g/kg NDF, 412 g/kg ADF, 1.17 Mcal of NEm g/kg, 4.10 g/kg Ca, 2.60 g/kg P. It also contained (as is basis; ± SE) 0.041 ± 0.005 mg Se/ kg. b Corn grain was 870 g/kg DM and contained (DM basis): 90.0 g/kg CP, 88.3 g/kg NDF, 29.4 g/kg ADF, 2.14 Mcal of NEm g/kg, 0.80 g/kg Ca, 3.00 g/kg P. Ground corn premix contained (as is basis; ± SE) 0.256 mg Se/kg and treatments resulted in the following Se concentrations: Naselenite, 1.906 ± 0.138 mg Se/kg and P-Se, 2.140 ± 0.092 mg Se/kg. c Selenium was analyzed in feed samples collected throughout the experiment (University of Missouri Agricultural Experiment Station Chemical Laboratories - MU ESCL). d Trace mineral mixture (Champion’s choice, Cargill Animal Nutrition, Cargill Inc., Roaring Spring, PA) contained (DM basis): NaCl, 940 g/kg; ZnO, 3.50 g/kg; MgO, 2.00 g/kg; FeCO3 2.00 g/kg; CuSO4, 0.30 g/kg; Ca (IO3)2, 0.30 g/kg; CoCO3, 0.07 g/kg. e Values calculated using the chemical analysis of the individual feed ingredients (Cumberland Valley Analytical Services Inc., Waynesboro, PA) and their inclusion rate in the diet. f Net energy for maintenance (NEm) concentration was calculated using the OARDC Summative Energy Equation of Weiss (1998).
concentration. Samples were collected approximately 2 h after feeding. Extra care was taken to separate fecal from bedding materials. Urine was collected by stimulating urination by holding off the animal's air and blocking the nostrils and mouth for a short period (approximately 30 s). After collection, spot urine samples were combined per experimental period and per animal resulting in a total of eighteen composite samples. The undiluted urine samples were stored frozen at −20 °C for subsequent Se analysis. Fecal samples were collected from the pen floor and were oven dried at 65 °C for 48 h, ground through a 1-mm sieve in a Wiley mill (Thomas Scientific) and saved in sealed Ziploc bags for Se analyses. 2.5. Selenium and glutathione peroxidase analyses Selenium concentrations in plasma, feces, and urine were analyzed at the University of Missouri Agricultural Experiment Station Chemical Laboratories (MU ESCL) in triplicate using the graphite furnace atomic absorption spectrometry as described by Elmer (1999). Glutathione peroxidase (GPx) activity was measured in blood plasma using a commercial kit (Item No. 703,102; Cayman Chemical Co., Ann Arbor, MI) with activity measured according to the decrease in absorbance at 340 nm (SPECTROstar Omega Microplate Reader; BMG Labtech, Inc., Cary, NC). Glutathione peroxidase activity was expressed as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP+ per minute per mL of sample at 25 °C. 2.6. Statistical analysis Data were analyzed using the MIXED procedure of SAS v 9.4 (SAS Institute Inc., Cary, NC) with treatment, sampling week, experimental period, and treatment × sampling week interaction included in the model and ewes and ewes within square as random effects. Plasma, urine and fecal Se data were analyzed as repeated measures with AR(1) covariance structure and were presented in absolute concentrations. Statistical significance was declared at P ≤ 0.05 and tendency was at 0.05 < P ≤ 0.10. Data are presented as least squares means unless indicated otherwise. 3
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Table 2 Selenium intake (mg/kg as is basis or as indicated; ± SE) and effect of source of dietary Se on plasma Se, plasma GPx activity, and fecal and urine Se concentrations. Treatment1
P-value3
Item
Control
Na-selenite
P-Se
SEM2
Se supplementation rate (mg/kg ground corn premix) Analyzed Se, mg/kg DM Total dietary Se4, mg/kg DM Se fed, mg/head/day Plasma Se, μg/L Plasma GPx5, nmol/min/mL Fecal Se, μg/kg DM Urine Se, μg/L
0.00 0.22 0.065 ± 0.001 0.10 ± 0.001 57.4a 35.3a 113a 6.28a
1.50 1.66 ± 0.107 0.25 ± 0.024 0.39 ± 0.032 80.9c 57.1b 441b 24.4b
1.50 1.86 ± 0.082 0.27 ± 0.011 0.42 ± 0.019 72.3b 52.5b 526c 12.2a
2.21 1.63 26.5 2.63
< 0.001 < 0.001 < 0.001 0.003
a,b,c
Means with different letter superscripts differ at P < 0.05. Control = unsupplemented control; Na-selenite = sodium selenite, 0.39 mg Se/head/d; P-Se = experimental Se source, 0.42 mg Se/head/d. 2 Standard error of the mean (largest SEM shown in table), plasma Se: n = 108; plasma GPx: n = 106; fecal Se: n = 54; urine Se: n = 18 (n represents the number of observations used in the statistical analysis). 3 Main effect of treatment. Treatment × sampling week interaction for plasma Se concentration, plasma GPx activity and fecal Se concentration: P ≤ 0.001, 0.99, and 0.61, respectively. 4 Analyzed in feed samples collected throughout the experiment (University of Missouri Agricultural Experiment Station Chemical Laboratories MU ESCL). 5 GPx = glutathione peroxidase activity was expressed as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP + per minute per mL of sample at 25 °C. 1
3. Results and discussion 3.1. Plasma Se concentrations Effect of Se source on plasma Se absolute concentrations are shown in Table 2 and Fig. 1. Compared with the control, Se concentration in plasma was increased (P < 0.001) in ewes fed both Se treatments, and concentration was greater (P < 0.001) for Na-selenite compared with P-Se. There was a treatment × sampling week interaction (P ≤ 0.001) for plasma Se, and Fig. 1 shows the effectiveness of the washout periods between treatments. Plasma Se concentrations provide short-term data about Se status of the animal and can be used as an effective biomarker for assessing bioavailability of Se from various sources (Fairweather-Tait et al., 2010). Our results support the use of plasma Se as an index of Se availability with the data suggesting slightly inferior absorption efficiency of Se from P-Se.
Fig. 1. Weekly blood plasma Se data (mean ± SEM). Blood samples were collected 1 h before feeding and 1 h after feeding on D (day of background, low Se, washout and experimental periods) 14, 21, 28 and 35. Control = unsupplemented control; Na-selenite = sodium selenite, 0.39 Se /head/d; P-Se = experimental Se source, 0.42 mg Se/head/d. There was a treatment × sampling week interaction (P ≤ 0.001). Means within period labeled with different letters (a,b,c) differ at P < 0.05. 4
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3.2. Plasma glutathione peroxidase activity Selenium status was also assessed by analyzing plasma GPx activity, which concentration was greater (P < 0.001) in the Sesupplemented ewes, compared with the control (Table 2). There was no effect of Se source on absolute plasma GPx activity. There were no treatment × sampling week interactions (P = 0.99) for the GPx activity data. These results are in agreement with previous reports in which Se supplementation led to rapid increase in plasma GPx activity in sheep (Chauhan et al., 2014). Gerloff (1992) stated that plasma GPx activity is considered to be an indicator of a short-term Se status. The form of Se can also play an important role in the rate or increase in GPx activity (Pavlata et al., 2012b). 3.3. Fecal and urine Se concentrations Compared with the control, the Se treatments resulted in greater Se concentration in feces, and absolute fecal Se concentration was higher (P < 0.001) for P-Se compared with Na-selenite. (Table 2). There was no interaction (P = 0.61) between treatment and sampling week for the fecal Se concentration data. The absolute Se concentrations in urine were greater (P ≤ 0.003) in ewes supplemented with Na-selenite than the control and P-Se-treated ewes (Table 2). Ruminants absorb Se far less efficiently than nonruminants and most of the ingested Se leaves the rumen with insoluble particulate matter (Suttle, 2010). Reportedly, in ruminants, the urinary excretion of Se is generally low and the primary route for excretion of orally administered Se is via feces (Tanner, 2008). In the study of Meyer et al. (2014), cows supplemented with inorganic or organic Se-sources over 3 wks at 2 inclusion rates had concentrations of Se in feces, ranging from 376 to 842 μg/kg, whereas concentrations in urine were 50–102 μg/L. Our data showed similar trends in fecal and urine Se concentration. The fecal Se concentration data suggest that P-Se, may have had slightly inferior absorption efficiency compared with Na-selenite. Considering that P-Se contains 29 % of its Se as Na-selenite, the observed difference in absorption efficiency is likely a result of lower absorption rate of elemental Se (71 % of Se in P-Se) in P-Se. Further studies are needed to investigate the post-absorption metabolism of this experimental Se product and its bioavailability in comparison with organic Se sources at possibly lower doses. 4. Conclusions Although Se from P-Se appeared to have slightly inferior absorption efficiency compared with Na-selenite, our data indicate that P-Se can be an effective source of dietary Se for mature ewes; however, further studies are needed to confirm these results in other farm animal species and physiological stages. CRediT authorship contribution statement K. Nedelkov: Investigation, Formal analysis, Data curation, Writing - original draft. X.J. Chen: Investigation, Data curation, Writing - review & editing. C.M.M.R. Martins: Investigation, Data curation, Formal analysis, Writing - review & editing. A. Melgar: Investigation, Data curation, Writing - review & editing. M.T. Harper: Investigation, Data curation, Writing - review & editing. S. Räisänen: Investigation, Data curation, Writing - review & editing. J. Oh: Methodology, Data curation, Writing - review & editing. T.L. Felix: Methodology, Writing - review & editing. E. Wall: Funding acquisition, Writing - review & editing. A.N. Hristov: Conceptualization, Formal analysis, Methodology, Supervision, Writing - review & editing. Declaration of Competing Interest The authors have no conflict of interest to declare. Acknowledgments This work was supported by the USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN04539 and Accession number 1000803. Partial financial support was also provided by Pancosma S.A. (Rolle, Switzerland). The authors thank Dr. Jacob Werner for assisting with euthanasia of the study animals and Wendall M. Landis and The Pennsylvania State University Beef Cattle and Sheep Center staff for their conscientious care of the experimental animals. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anifeedsci. 2020.114390. References AOAC International, 2000. Official Methods of Analysis, 17th ed. Association of Official Analytical Chemist International, Arlington, Arlington, VA. AOAC International, 2006. Official Methods of Analysis, 18th ed. Association of Official Analytical Chemist International, Arlington, VA. Chauhan, S.S., Celi, P., Leury, B.J., Clarke, I.J., Dunshea, F.R., 2014. Dietary antioxidants at supranutritional doses improve oxidative status and reduce the negative
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