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ANIMAL NUTRITION |
* Animal Science Research Group, School of Agriculture, Policy and Development University of Reading, Earley Gate, Reading RG6 6AR, UK; and
ALLTECH France, EU Regulatory Affairs Department, 14 Place Marie-Jeanne Bassot, 92300 Lavallois-Perret, France
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: biochemistry • ruminant • selenium • selenomethionine tolerance
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), exudative diathesis in chicks (Patterson et al., 1957), and muscular degeneration in calves and lambs (Schubert et al., 1961). In the early part of the 1970s a specific biological role for Se became apparent with the discovery of the selenoprotein glutathione peroxidase (GSH-Px; Rotruck et al., 1973). During the last 30 yr, a further 24 Se-dependent proteins have been identified, which include the thioreductases, deiodinases, and selenoproteins P and W (Underwood and Suttle, 2001).
Diets for ruminant animals are often of plant origin, and the Se concentration within plants can be extremely variable. Consequently, concentration of dietary Se can be deficient, and the addition to the diet of a Se supplement may be required. Selenium supplements are in 2 forms: inorganic mineral salts, typically sodium selenite (Na2SeO3) or selenate (Na2SeO4), or organic forms such as Se-enriched yeasts (SY). Although inorganic and organic forms of Se are approved in the United States, only recently has an organic Se source (Saccharomyces cerevisiae CNCM I-306) been approved for use within the European Union (EU). Recent studies by Juniper et al. (2006) have indicated improved bioavailability of Se when using selenized yeast (Saccharomyces cerevisiae CNCM I-306). There were positive linear effects of increasing Se dose rate on Se concentration in blood, milk, feces, and urine in diets containing up to the maximum permitted dose of 0.568 mg/kg of DM.
The NRC (1980) placed the maximum tolerable dose of Se for all species at 2.0 mg/kg of DM, as this dose produced no signs of toxicosis. However, a number of studies have examined the long-term administration of high-dose Se, in the form of sodium selenite, to sheep (Cristaldi et al., 2005; Davis et al., 2006), whereby the authors reported that the tolerance level in sheep was well in excess of that recommended by the NRC. Despite these studies, similar investigations were required to examine the administration of SY at EU-agreed tolerance levels of at least 10 times the maximum permitted inclusion rate.
The objective of this study was therefore to assess and evaluate any adverse effects, in terms of animal health, as determined by measuring key enzymes that are indicative of hepatic or renal dysfunction, animal performance, and voluntary feed intake following the inclusion of at least 10 times the maximum dietary inclusion rate of Se permitted within the EU (currently 0.568 mg of Se/kg of DM) or 20 times that permitted within the United States (0.3 mg/kg of DM), for a period of at least 60 d.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Between October 2003 and June 2005, a series of individual Se tolerance studies were conducted on a range of productive ruminant animal types, namely dairy cows (n = 28, study duration = 60 d, milk yield = 33.7 kg/d, days in milk = 106, BW = 645 kg, parity = 3.8), beef cattle (n = 56, study duration = 75 d, BW = 250 kg, age = 10 mo), calves (n = 56, study duration = 75 d, BW = 40 kg, age = 2 wk), and lambs (n = 32, study duration = 91 d, BW = 9 kg, age = 2 wk). Each study had 2 treatment groups, namely a control group (control) and a high-dose dietary Se yeast group (SY). Animals were paired by BW (beef cattle, calves, and lambs) or by parity and milk yield (dairy cows) and then randomly allocated to 1 of the 2 dietary treatments within each pair.
Diets
Dietary treatments within each individual study consisted of the same basal diet that differed only in the dietary concentration of Se. Dairy cows were offered ad libitum access to a total mixed ration (TMR) that contained 375, 125, 250, 135, 100, and 15 g/kg of DM of corn silage, grass silage, cracked wheat, soybean meal, rapeseed meal, and mineral supplement, respectively (for the nutritional values of the diets, see Table 1). Beef cattle were offered ad libitum access to a predominantly corn silage TMR that contained 550, 130, 150, 150, and 20 g/kg of DM of corn silage, grass silage, ground wheat, soybean meal, and mineral supplement, respectively. Calves were offered 4 L/d (2 equal meals in a.m. and p.m.) of a proprietary calf milk replacer (declared Se value of 0.3 mg/kg of DM; Volac, Herts, UK) and ad libitum access to a proprietary coarse calf weaning mix (Associated British Nutrition and Agriproducts, Peterborough, UK). Diets within each of these studies were supplemented with high-dose SY [Sel-Plex produced by a specific strain of Saccharomyces cerevisiae (CNCM I-3060), manufactured and supplied by Alltech (Nicholasville, KY)] or unsupplemented, depending on the treatment designation. Lambs were offered 2 L/d (2 equal meals a.m. and p.m.) of a proprietary ewe milk replacer (declared Se value of 0.3 mg/kg of DM; Volac, Herts, UK) and ad libitum access to a pelleted weaning ration (Dairy Direct, Bury St. Edmunds, UK) that contained 360, 340, 165, 90, 25, and 20 g/kg of DM of ground barley, wheat bran, ground wheat, soybean meal, molasses, and vitamin supplement, respectively. Both ewe milk replacer and pelleted feed were supplemented with high-dose SY (Sel-Plex) or unsupplemented, depending on treatment designation.
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Feed Analyses.
Representative samples of component feeds, TMR, or complete diets were taken weekly and later frozen (–20°C). At the end of the study, feed samples were bulked, subsampled, and then analyzed for a range of nutritional characteristics. Samples were analyzed for DM, CP, NDF, starch, and ash using wet chemistry methods (Ministry of Agriculture, Fisheries, and Food, 1993). Metabolizable energy was estimated from the neutral cellulase and gamminase digestibility of feed after the neutral detergent extraction of soluble cell contents (Ministry of Agriculture, Fisheries, and Food, 1993).
Total Se was determined by mineralizing 1 g of each sample in 4 mL of 16 M HNO3 and 2 mL of 9.8 M H2O2 within a closed-vessel heating block system. The solution was further diluted with water, and Se was subsequently determined using inductively coupled plasma mass spectrometry (ICPMS; Perkin Elmer Elan 6100 ICPMS, Cambridge, MA).
Feed Preparation. The TMR for beef cattle and dairy cows were mixed fresh daily using a self-propelled feeder wagon (Calan Data Ranger, American Calan Inc., Northwood, NH) fitted with a weighing device (Weigh-Tronix Model 1015, Fairmount, MN), which recorded the individual weights of the different feed ingredients added to the hopper. Selenium-enriched yeast was incorporated into the mineral supplements (Dairy Direct) of SY-designated treatments at the point of manufacture using an appropriate rate to achieve the minimum Se dose required (>5.68 mg of Se/kg of DM). The mineral mix was subsequently incorporated into SY-designated TMR at the time of mixing. Control mineral mixes were of the same basic formulation but contained no additional Se.
Selenium-enriched yeast was incorporated into the vitamin and mineral supplements of the SY lamb weaning pellet at the point of manufacture using an appropriate rate to achieve the minimum Se dose required (>5.68 mg/kg of DM). The control pelleted feed contained the same basic mineral formulation, which had not been supplemented with additional Se.
Selenium-enriched yeast was incorporated at an appropriate rate into the SY coarse calf mix at point of feeding following the premixing of the SY product into a 10 g/kg fraction of the intended mix. The premixture was then thoroughly mixed for a minimum of 5 min with the remaining fraction of the coarse calf mix, using a dual auger counter action feed mixer (CEDAR design, IAE, Staffs, UK).
Selenium-enriched yeast was incorporated into SY-designated milk replacer (calf and lamb) during reconstitution (at an appropriate rate to achieve the required dose) after the mixing and feeding of the control-designated milk replacer. Milk powders were reconstituted according to manufacturer’s recommendations and the milk mixer thoroughly cleaned between feeds.
All feeds were offered fresh daily to individual animals following the removal and recording of the previous day’s refusal. Refusals of solid feed (TMR and weaning rations) were maintained at least 110 g/kg of the previous day’s intake.
Animal Performance. Feed intake was recorded daily for all studies. A weighing device fitted to the hopper of the self-propelled feeder wagon weighed the amount of feed dispensed daily to dairy cows and beef cattle. Feed refusals from dairy cows and beef cattle were weighed daily using a set of calibrated digital scales (Sartorius CP12001S, Sartorius Ltd., Epsom, UK). Feeds offered and refused to calves and lambs were weighed daily using a calibrated set of digital scales (Sartorius CP12001S). Live weights for beef cattle, calves, and lambs were recorded on 2 consecutive days at enrollment onto the study, once weekly throughout the study, and on 2 consecutive days at the conclusion of the study using a Ritchie weighing device (Ritchie Industries Inc., Conrad, IA) fitted with an analog scale (Salter, Tonbridge, Kent, UK). Animals were weighed at the same time of day following feeding. Daily rates of gain and subsequent G:F were calculated from these data.
Dairy cows were milked twice daily at 0500 and 1500 through a Fullwood Herringbone Parlor with automatic cow identification, automatic cluster removal, and Full-flow In-Line Milk Meters (Fullwood Co., Ellesmere Port, UK) to record and sample milk. Individual milk yields were recorded automatically for all cows at each milking. Milk samples were taken manually at 2 consecutive milkings at d 0, 20, 40, and 60 of study and analyzed using infrared reflectance spectroscopy (Milkoscan Model 203B, Foss Products, York, UK) to determine milk fat, protein, and lactose concentrations.
Blood Biochemistry and Hematology. Tail vein blood samples were taken from dairy cows at enrollment and on d 20, 40, and 60 of the study. Blood samples were taken via jugular venipuncture from calves at enrollment and on d 20 and 75 of the study. Blood samples were taken from beef cattle via jugular venipuncture at enrollment and on d 25, 50 and 75 of the study and from lambs at enrollment and on d 28, 56, and 91 of the study. All samples were taken using the Vacutainer System (BD Diagnostics, Oxford, UK) using a 21-ga needle. Samples consisted of four 5-mL Vacutainers (1 lithium heparin, 1 K3EDTA, 1 oxalate/sodium fluoride, and 1 serum tube) filled per animal. Measurements of blood biochemistry included the activity of the enzymes alanine transaminase, alkaline phosphatase, creatine phosphokinase, gamma-glutamyl transpeptidase, lactate dehydrogenase, aspartate aminotransferase, and GSH-Px and the concentrations of albumin, globulin, total protein, glucose, urea, and inorganic phosphate. Hematological measures were made for hemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell hemoglobin, platelets, and the white blood cell differentials segmented neutrophils, banded neutrophils, lymphocytes, monocytes, eosinophils, and basophiles. Standard analytical techniques were used for these analyses (Compton Paddock Laboratories, Yattendon, Berkshire, UK).
Selenium.
An additional 2 lithium heparin tubes were taken to determine total Se and selenomethionine (SeMet) content. Total Se was determined as previously described in this current paper. Samples for SeMet were determined using the method described by Palacios et al. (2005). Briefly, samples were initially incubated for 5 h with DL-dithiothreitol and iodoacetamide to reduce them and alkylate the selenocysteine. Samples were then spiked with 77SeMet and subsequently incubated for 24 h at 37°C with a mixture of protease and lipase maintained at a pH 7.5. After incubation, the mixture was centrifuged and the supernatant was separated and purified by cell exclusion liquid chromatography. Aliquots of the supernatant were analyzed by reversed-phase HPLC-ICPMS using an ICPMS equipped with a collision cell (Perkin Elmer Elan 6100 ICPMS, Waltham, MA).
Statistical Analysis
Statistically significant differences between the 2 treatment groups for animal performance measurements within each study that were recorded on a weekly basis throughout each study were determined by AN-OVA using GLM (SAS Inst. Inc., Cary, NC). There were 28, 56, 56, and 32 individual animal observations for each performance parameter for dairy cows, beef cattle, calves, and lambs, respectively. Sources of variation within the model included treatment (1 df).
Statistical differences between treatment groups for each biochemistry and hematology value were determined by ANOVA using the MIXED procedure of SAS. Each individual animal formed the repeated subject, and sequential time point the repeated term. There were 28, 56, 56, and 32 individual animal observations at each time point for each measured biochemical and hematological parameter for dairy cows, beef cattle, calves, and lambs, respectively. Sources of variation within the model included treatment (1 df), time point (2 df for calves; 3 df for beef cattle, dairy cows, and lambs), and the treatment x time interaction. Results for physical performance, biochemistry, and hematology are presented as treatment least squares means with the SEM. Statistical significance was defined as P < 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The nutritional compositions of the diets used in this series of tolerance studies are shown in Table 1
. Within study there was very little difference between the 2 treatment groups with respect to DM, CP, ME, starch, NDF, and ash. However, as planned, dietary Se concentrations of the control and SY treatments varied. The control diets, irrespective of study, contained only background Se that was present within the component feeds. This is reflected in the generally low concentration of Se seen in the control diets with values ranging between 0.15 and 0.20 mg of Se/kg of DM. In contrast, the Se contents of SY rations were markedly higher, with values ranging between 5.86 and 6.63 mg of Se/kg of DM, which complied closely with EU regulations (Council Directive 87/153/EC amended by 2001/79/EC) regarding the minimum tolerance target dose for registration purposes (minimum of 5.68 mg of Se/kg of DM complete feeding stuffs).
Animal Performance
Feed and Selenium Intake.
There were no treatment effects on DMI (Table 2). However, intakes of Se, when expressed as a function of BW, both between treatment groups within each study and between studies receiving similar dietary Se concentrations, were notably different. Of those animals receiving SY diets, lambs had the highest Se intake at 0.25 mg of Se/kg of BW, whereas dairy cows, beef cattle, and calves had intakes of 0.165, 0.134, and 0.11 mg of Se/kg of BW, respectively. Selenium intakes were lowest in control group animals with estimates ranging between 0.005 and 0.006 mg of Se/kg of BW, irrespective of species.
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). Similar thyroid function impairment as a result of Se-deficient diets has also been reported in cats (Yu et al., 2002) and rats (Arthur et al., 1991). Conversely growth retardation has been reported in rats as a consequence of the administration of toxic concentrations of Se in the form of sodium selenite. It was found that there was a significant reduction in circulating concentrations of IGF-I and its associated regulatory binding proteins (Gronbaek et al., 1995). However, the doses administered within this series of studies were neither suboptimal nor toxic and the results were in line with other studies which have also reported no adverse effects on growth rates following the long term administration of Se at well above the maximum recommended within the EU and United States (Lawler et al., 2004; Taylor, 2005; Cristaldi et al., 2005; Davis et al., 2006).
Milk Yield and Composition.
There were no treatment effects on milk yield or milk fat, protein, and lactose contents. Milk Se content increased markedly in those animals receiving the SY-supplemented diets, increasing from a pretreatment value of 15.3 ± 1.41 µg/L to 456 ± 13.6 µg/L by d 20 of the study, a value that remained relatively constant throughout the remainder of the study. The quantity of total Se in milk as SeMet also increased markedly in those cows receiving SY-supplemented rations, increasing from a pretreatment value of 4.5 ± 0.3 ng of SeMet/g of DM, which accounted for 28% of total Se, to 571 ± 3.4 ng of SeMet/g of DM by d 20, which accounted for 96% of total Se. Between 97 and 99% of the total Se contained within the SY used in these studies is in organic form, of which 63% is SeMet (Commission Regulation 1750/2006). Seleno-methionine is readily absorbed across the gut wall via methionine transporter mechanisms (Weiss, 2003) and once absorbed can enter the methionine pool and be incorporated into general proteins, where it can also act as a biological pool for Se (Suzuki and Ogra, 2002). It is probable that the marked increase in milk SeMet is a consequence of the incorporation of SeMet rather than methionine into milk proteins.
Blood Biochemistry and Hematology
The results of the extensive analysis for blood biochemistry and hematology are shown in Table 3. The most notable and consistent difference between treatment groups with respect to dairy cattle, beef cattle, and calves was that GSH-Px activity was higher (P < 0.05) in SY-supplemented animals when compared with controls. Glutathione peroxidase was the first of the selenoproteins to be identified (Rotruck et al., 1973) and is often used as an indicator of whole-animal Se status. However, as whole-blood Se values increase, the GSH-Px enzyme becomes saturated, and further increases in whole-blood Se tend not to be reflected in commensurate increases in GSH-Px activity (Neve, 1995). However, Se uptake and incorporation into erythrocyte GSH-Px is dependent upon the rate of erythrocyte turnover, which ranges between 60 and 120 d depending upon the species (Underwood and Suttle, 2001). It is therefore possible that the duration of each individual study was not sufficient to allow maximal expression of erythrocyte GSH-Px activity.
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). This difference in white blood cell differentials is not evident in the calf or dairy studies, although it has been reported that subclinical Se toxicosis in pregnant beef cattle can affect the bovine immune system (Yaeger et al., 1998). There were very few other statistically significant differences between the 2 treatment groups, and those differences that achieved statistical significance were not biologically meaningful and were within expected ranges (Boyd, 1984).
Total whole blood Se and SeMet content of whole blood with respect to time for dairy cows, beef cattle, calves, and lambs are shown in Figures 1 to 4, respectively. In all cases whole-blood Se values increased markedly in SY-supplemented animals over the course of each study, with Se concentrations tending to plateau toward the latter part of the study. In addition, mean whole-blood Se values were higher (P < 0.001) in SY-supplemented animals regardless of animal type when compared with controls, although whole-blood Se concentrations did not achieve those that are often associated with the onset and manifestation of the symptoms of selenosis (2.1 mg of Se/mL; Underwood and Suttle, 2001).
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) and wether sheep (Cristaldi et al., 2005) that had received comparable doses of sodium selenite. This disparity between ovine and bovine species could be a consequence of greater feed transit times allowing for greater uptake of Se from the gastrointestinal tract. However, Se balance was not conducted in this series of studies, and therefore it is not possible to definitively say whether this is the case or not. The proportion of SeMet that comprised total Se was also markedly higher in SY-supplemented animals, accounting for between 40 and 75% of total Se, whereas SeMet accounted for approximately 10% of total whole blood Se in control animals. This marked increase in the SeMet content of whole blood could once again reflect the relatively high proportion of Se that comprises SeMet within the SY product (63%) and the efficiency with which SeMet is transported across the gastrointestinal tract via methionine transporter mechanisms. However, in the absence of a positive control of inorganic Se, it would be difficult to speculate as to whether this is definitively the case.
The incorporation into ruminant diets of high concentrations of Se in the form of SY (Saccharomyces cerevisiae CNCM I-3060) providing in excess of 10 times and nearly 20 times the maximum recommended Se doses within the EU and United States, respectively, although increasing markedly whole-blood and milk Se and SeMet concentrations, did not result in any adverse health effects or influence animal performance as a consequence of Se toxicosis. In light of these results it would be feasible to suggest that maximum permitted Se doses with regard to SY could be increased.
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Footnotes |
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2 Corresponding author: d.t.juniper{at}reading.ac.uk
Received for publication November 23, 2006. Accepted for publication September 5, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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