Selenomethionine as a Safer Substitute for Barium Selenate in Long

Sep 7, 2017 - While the rule directly affects animal management on EU farms, it may also impact overseas producers who intend to export meat from trea...
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Selenomethionine as a Safer Substitute for Barium Selenate in LongActing Injectable Se Supplements for Food-Producing Animals Scott O. Knowles*,† and Neville D. Grace‡ †

Food & Bio-Based Products Group, AgResearch Limited, Palmerston North 4442, New Zealand 26 Williams Road, RD 4, Palmerston North 4474, New Zealand

‡

ABSTRACT: Nutritional supplementation with selenium (Se) can prevent Se deficiency in food-producing animals. Injection with slow-release formulations is a preferred method for free-range grazing sheep and cattle, and barium selenate (BaSeO4) provides optimal efficacy. This chemical can become a health risk to humans if the concentrated depot of an injection site is consumed, and consequently such use is recently banned in the EU. A possible replacement is selenomethionine (SeMet), a naturally occurring form of Se supplementation hitherto only administered orally. In four animal studies we found that injection with SeMet maintained nutritionally adequate concentrations of Se in blood and tissues of lambs for at least 191 days and in blood and milk of dairy cows for at least 95 days. Stereoisomer forms L- and DL-SeMet were functionally equivalent. This is the first demonstration that injectable SeMet can deliver efficacy similar to BaSeO4 but with less risk of undesirable residues in edible tissues. KEYWORDS: selenium, barium selenate, selenomethionine, long-acting injection, meat, contamination

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INTRODUCTION Selenium is an essential dietary nutrient and deficiency occurs in farmed livestock in New Zealand, Australia, the United Kingdom, and other countries.1−6 In sheep, the deficiency is characterized by poor lamb growth and lower ewe fertility. In cattle, reduced milk yield and poor growth rate in calves have been observed.7,8 Clinical signs include white muscle disease in lambs that manifests as degeneration of the skeletal and cardiac muscle.9 Methods of treatment and prevention include Se supplementation via feedstuffs and water supply, oral dosing, intraruminal boluses, and injection. For the latter, highly effective slow-release formulations have been developed based on BaSeO4. When injected subcutaneously or intramuscularly, it forms a depot that delivers adequate Se supplementation for 8−12 months.1,7,10 The long-acting advantage for animal health brings some risk for consumers of sheep and beef meat, who might ingest residues that remain at the site of injection for up to 148 days.11,12 Such intake of Se could exceed the established safe level for human diets. In response, food safety authorities in some jurisdictions restrict nonalimentary use of BaSeO4. For example, the opinion of the EU European Medicines Agency to prohibit administration by injection into food-producing species was recently implemented as Regulation [EU] 2015/ 446 of March 17, 2015.13 The regulation is binding and applicable in all Member States. While the rule directly affects animal management on EU farms, it may also impact overseas producers who intend to export meat from treated animals to Europe. In circumstances where an injectable Se supplement is desired but BaSeO4 is banned, other forms of Se must be considered. For instance sodium selenate (Na2SeO4) or similarly sodium selenite are currently used. These watersoluble compounds dissipate quickly and safely from the site of injection.11 However, efficacy for the animal is concomitantly © XXXX American Chemical Society

short-lived, sometimes less than 4 weeks when administered to livestock at recommended dose rates.14 Larger doses for longer efficacy are contraindicated because rapid mobilization of excess Na2SeO4 can lead to liver toxicity.15 Published data on the efficacy and toxicity of injected selenomethionine (SeMet) is sparse and no formulations are yet manufactured.16,17 It may nevertheless prove to be a useful alternative. Selenomethionine is widely available as an oral Se supplement, most commonly as a constituent of selenized yeast added to the feed rations of ruminants18−20 as well as pigs and poultry. For free-ranging livestock without ready access to Se-rich feed supplements, oral delivery of SeMet may also be accomplished using intraruminal boluses.21 As an analogue of the amino acid methionine, SeMet from all sources becomes incorporated nonspecifically into the proteins of tissues throughout the body22 where it serves as a labile reservoir of Se to provide the animal with ongoing benefit. Indeed the deliberate accumulation of SeMet in the tissues of food-producing animals (e.g., in meat and milk) has been promoted as nutritionally valuable for consumers.23−25 Supplementary SeMet is indistinguishable from that which occurs naturally in animals and plants. An injectable supplement based on SeMet could have regulatory advantage over other forms of Se because, if residues persist, they would not be chemically foreign. Some characteristics of BaSeO4, Na2SeO4, and SeMet for Se supplementation of livestock are compared in Table 1. The potential of SeMet for administration by injection hinges on its capacity to prevent Se deficiency in animals and associated losses of farm productivity, the duration and extent of Received: June 19, 2017 Revised: August 16, 2017 Accepted: August 27, 2017

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DOI: 10.1021/acs.jafc.7b02809 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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The baseline Se nutritional status of sheep and cattle in these studies reflected the environmental Se status of the particular farms, where pasture herbage Se content is typically 0.01−0.08 mg Se/kg dry matter. Although this is considered low dietary intake by some international authorities,26,27 0.03 mg Se/kg dry matter is sufficient to raise blood Se concentrations in sheep and cattle to at least 250 nmol/ L and has repeatedly been shown to be adequate for animal growth, production, and reproduction under New Zealand conditions.28,29 Test Compounds. Injectable supplements were developed by AgResearch Ltd. and manufactured in small quantities to our specifications by Stockguard Laboratories Ltd. (Hamilton, NZ), a GMP compliant commercial facility under certificate NA/083 V/2004. All used a carrier of arachis oil (CAS 8002-03-07) and a suspending agent of white beeswax (CAS 8006-40-4). The BaSeO4 (CAS 7787-419) and Na2SeO4 decahydrate (CAS 10102-23-5) were from Sigma Chemicals (now Sigma-Aldrich, Australia). The DL-SeMet (48:52 ratio, CAS 2578-28-1) and L-SeMet (CAS 3211-76-5) were supplied by PharmaSe Inc. (part of Eburon Organics USA, Lubbock, TX). The nine formulations prepared for the Dose Factorial study also contained vitamin B12 (hydroxocobalamin, CAS 58288-50-9) microencapsulated in medical-grade lactide-glycolide polymer, at 3 mg B12 per mL.30 That study was part of a larger project to develop a dual-active animal nutritional supplement containing Se and Co-vitamin B12 suitable for livestock grazing in New Zealand and elsewhere, where grass and forages may not provide adequate amounts of these micronutrients.31 Vitamin B12 supplementation would not be expected to affect animal Se responses. Experimental Details. Dose Factorial Study. We compared the effects of three forms of Se supplement administered to lambs at three dose rates on blood Se concentrations. The study was carried out on the AgResearch Flock House sheep farm in the Rangitikei district of the North Island, New Zealand, during 2000−2001. From a flock of 200 ewes, 100 mixed sex lambs, 3−4 weeks old, and having a mean body weight (BW) of 14.1 ± 3.4 kg were identified with unique ear tags and randomized into 10 equivalent groups based on their pretreatment blood Se concentration. The animals were initially Sedeficient, with a mean blood Se concentration of the control and treatment groups of 68 ± 19 nmol/L, which is below the 250 nmol/L reference range.28 They were not deficient in cobalt or vitamin B12, as mean serum vitamin B12 concentrations were greater than the 350 pmol/L benchmark of adequate status.32 Supplements were injected subcutaneously into the anterior neck. Doses were 6, 12, or 24 mg of Se as BaSeO4; 2, 4, or 6 mg of Se as Na2SeO4; and 5, 7.5, or 10 mg of Se as DL-SeMet. Dose sizes were selected to manage risks to animal

Table 1. Comparison of the Properties of Supplemental Se for Use in Livestock chemical form water-soluble use as an oral supplement use as an injectable supplement forms a depot at site of injection long-acting efficacy after injection a

BaSeO4

Na2SeO4

SeMet

inorganic no rarely restricted significant yes

inorganic yes yes yes minor no

organic yes yesa novel unknown unknown

Typically as selenized yeast.

responses to treatment, its distribution and metabolism in tissues, and its safety to treated animals and food consumers. In this article we describe a series of proof-of-concept experiments that test the efficacy of three forms of injectable Se administered at various dose rates to sheep and cattle. Our results support further development of SeMet into a long-acting injectable Se supplement for food-producing animals.

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MATERIALS AND METHODS

Experimental Overview. Institutional and national guidelines for the care and use of animals were followed and all experimental procedures were approved in accordance with the New Zealand Animal Welfare Act (Public Act 1999 No. 142) by either the Crown Research Institutes Animal Ethics Committee or the AgResearch Ltd. Grasslands Animal Ethics Committee, both of Palmerston North, New Zealand. Key details about the study designs are shown in Table 2. In some cases, fuller descriptions of methods can be found in the related publications. All sheep studies took place on commercially run farms where the animals were managed under a New Zealand pastoral regimen, which typically involves rye grass/white clover pastures for free-range grazing, no or minimal access to dietary supplements or feed concentrates, no housing, and infrequent mustering and handling. Birth of lambs usually occurs in August−September during the southern hemisphere spring. Tail removal and castration of ram lambs occurs at 3−5 weeks of age. Lambs remain with their ewes until weaning at 14−18 weeks of age. In the cattle study, the dairy cows grazed pastures for approximately 80% of their metabolizable energy, with the remainder contributed by ensiled preserved forages, palm kernel expeller, and grains. The cows were not housed and were milked twice per day.

Table 2. Summary of the Designs of Four Studies Described in This Article design detail animals length of study (days) group sizes (n)c Se compounds tested

dose sizes (mg Se/animal)

dose rates (mg Se/kg BW)

selenium measurements other measurements related publications

dose factorial lambs 100 to 246 10 (a) DL-SeMet (b) BaSeO4 (c) Na2SeO4 (a) 5, 7.5, 10 (b) 6, 12, 24 (c) 2, 4, 6 (a) 0.36, 0.54, 0.72 (b) 0.43, 0.86, 1.73 (c) 0.14. 0.29, 0.43 blood, serum growth, injection site reactions 31

stereoisomersa

tissue response

cattleb

lambs 138 20 (a) DL-SeMet (b) BaSeO4

lambs 139 5 or 13 (a) DL-SeMet (b) L-SeMet

dairy cows 95 10 (a) DL-SeMet

(a) 10 (b) 24

(a) 15 then 20 (b) 15 then 20

(a) 119 to 185 either 1× or 2×

(a) 0.77 ± 0.12 (b) 1.84 ± 0.31

(a) 0.85 ± 0.05 (b) 0.97 ± 0.12

(a) 0.35 ± 0.01

blood, plasma, kidney, liver, muscle blood GSH-Px, growth

blood, serum, kidney, liver, lung, heart, muscle growth

blood, serum, milk

n/a

n/a

17

a On Day 67 of this study, both treated groups received a second dose of 20 mg Se, as 0.77 ± 0.14 mg Se/kg BW. bOn Day 49 of this study, one of the treated groups received a second dose of 130 to 182 mg Se, as 0.35 mg Se/kg BW. cEach study also included a carrier oil (no Se) control group.

B

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Journal of Agricultural and Food Chemistry safety and reflected our estimates for delivering similar efficacy rather than strict molar equivalency. The control group was given the carrier oil containing no Se. All lambs grazed with their ewes for 100 days until weaning, then the lambs grazed together as a single flock. The duration of the study varied by chemical form. Animal sampling and measurements continued only for as long as treatment effects were apparent (i.e., blood Se concentrations substantially greater than controls) because it would have been wasteful to continue unnecessarily. This was 246 days for the BaSeO4 groups (which was the maximum possible duration for on-farm management), 191 days for SeMet groups, and 100 days for the Na2SeO4 groups. Samples of blood were collected on Days 0, 31, 64, 100, 128, 158, 191, 219, and 246. Groups were weighed at monthly intervals to assess growth response. Tissue reactions at the site of injection were assessed on Days 7 and 15 by an independent veterinarian. Tissue Response Study. We compared the effects of supplementation with either BaSeO4 or SeMet on Se concentrations in a range of tissues collected during a serial slaughter of lambs. The study was carried out on the AgResearch Flock House sheep farm during 2001. Three groups of 20 3−4 week old lambs having a mean BW of 13.1 ± 2.1 kg were randomized and injected subcutaneously into the anterior neck with 24 mg Se as BaSeO4, 10 mg of Se as DL-SeMet or with the carrier oil containing no Se (control). Lambs grazed with their ewes for 89 days until weaning then as a single flock until the end of the study. Samples of blood were collected on Days 0, 19, 40, 89, and 138. On Day 0 two lambs per group and on Days 40, 89, and 138 six lambs per group were humanly slaughtered so that samples of kidney, liver, and skeletal muscles (longissimus dorsi, semitendinosus, triceps brachii) could be collected for Se determination. Stereoisomers Study. We compared the effects of supplementation with either L- or DL- forms of SeMet on blood and tissue Se concentrations in lambs. The study was carried out on a commercial sheep farm in the Hawkes Bay district of the North Island, New Zealand, during 2009. Three groups of either 13 (for controls) or 5 (for treatments) 4−5 week old lambs having a mean BW of 16.6 ± 1.9 kg were randomized and injected subcutaneously into the anterior neck with L-SeMet or DL-SeMet to provide 15 mg of Se or with the carrier oil containing no Se (control). On Day 63, the animals were transported to a research farm in the central North Island. On Day 67 both groups of treated lambs having a mean BW of 26.6 ± 4.5 kg were injected a second time with L-SeMet or DL-SeMet to provide 20 mg of Se. All lambs and ewes grazed together for 139 days. Samples of blood were collected from all animals on Days 0, 67, and 104 and from eight animals on Day 137. On Day 139 three lambs per group were humanely slaughtered to collect samples of kidney, liver, lung, heart, as well as skeletal muscle (a homogenized blend of meat from the bonedout carcass) for Se determination. Cattle Study. We evaluated the effect of DL-SeMet administered to dairy cows on blood and milk Se concentrations. The study was carried out on the AgResearch Flock House dairy farm during 2006. Three groups of ten dairy cows in mid lactation, 6.5 ± 2.2 years old and having mean BW of 448 ± 44 kg, were injected subcutaneously in the anterior neck with DL-SeMet at a dose rate of 0.35 mg of Se/kg BW (140−184 mg of Se) or with the carrier oil containing no Se (control). On Day 49, one of the groups of treated cows, having a mean BW of 473 ± 51, was injected a second time with DL-SeMet at 0.35 mg of Se/kg BW. All cows grazed with a commercial milking herd for 95 days. Samples of blood and milk were collected on Days −9, −2, or 0 as well as Days 5, 12, 26, 49, 54, 61, 76, and 95. Skimmed milk was produced by centrifugation and decanting. Collection of Tissues and Chemical Analyses. On each occasion listed above, blood was drawn from the jugular vein of lambs or the coccygeal (tail) vein of cattle into 9 mL vacutainers containing K-EDTA or no anticoagulant. Whole blood and serum (harvested by centrifugation at 2000 × g for 15 min) was stored at 4 °C until analysis, which was within 1 week. Samples of organs and tissues were collected from humanely slaughtered animals then minced, frozen, and stored at −20 °C. In these experiments no tissue samples were taken directly from the site of injection, so accumulation, dispersal, and residue were not assessed.

Blood and serum Se concentrations were determined by Alpha Scientific/Gribbles Veterinary Pathology (Hamilton, NZ) using a semiautomated fluorometric method with a limit of quantitation of 40 nmol Se/L (or 70 nmol for the Dose Factorial study).33 Tissue Se concentrations were determined using a nitric/perchloric digestion followed by inductively coupled plasma atomic emission spectrometry or by hydride generation and absorption spectroscopy.34 Glutathione peroxidase (GSH-Px) activity was measured in whole blood using a spectrophotometric procedure (limit of detection 0.10 kU/L at 25 °C).35 Statistical Analysis. Within each study, differences between treatment group means for Se concentrations in tissues were determined by t tests and ANOVA in Genstat 18th Edition (VSN International Ltd., 2015). Summary statistics in tables and text are presented as mean ± SD unless otherwise indicated. Figures depict mean ± SE values, and in some cases small error bars are obscured by chart markers. Linear regression lines and equations are shown in Figure 4, but statistical parameters were not estimated for these repeated-measures data. Areas under the blood Se concentration curves (AUC) were calculated in order to estimate dose−response efficiencies. This was done for each animal in the Dose Factorial study and for group means in the Stereoisomers study. Micronutrients like Se are never completely absent from blood, and therefore the observed rates of elimination of supplemental Se are approximations of true clearance. They reflect measurements made for finite time to the defined end of the study. Units for AUC are scaled from nmol to μmol and are (days · μmol Se/L). When the areas are normalized by dose size, the units for AUCnorm are (days · μmol Se)/(L · mg Se).

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RESULTS Dose Factorial Study. The effects of increasing dose sizes of injectable BaSeO4, Na2SeO4, and DL-SeMet on the blood Se concentrations of Se-deficient lambs are shown in Figure 1. Treatment with BaSeO4 was most effective at increasing and maintaining blood Se concentrations, with peak concentrations of 977, 2100, and 3310 nmol Se/L observed at 64 days for Se depots of 6, 12, and 24 mg/lamb, respectively. Concentrations remained greater than controls for at least 246 days after treatment (lamb age approximately 274 days). For example, mean values for the 6 mg of Se and control groups at 246 days were 414 versus 213 nmol Se/L (P < 0.001). Treatment with Na2SeO4 was least effective, with peak concentrations of 209, 340, and 642 nmol Se/L observed at 64 days for Se depots of 2, 4, and 6 mg/lamb, respectively. The dotted lines in Figure 1c are an indication that the large but transient “spike” in blood Se concentration that typically occurs within 1 day of treatment with Na2SeO4 was not captured in this experiment.14 For the DL-SeMet groups, peak blood concentrations of 1410, 2430, and 2540 nmol Se/L were recorded after 31 days for Se depots of 5, 7.5, and 10 mg/lamb, respectively. Concentrations remained elevated for 191 days, and mean values for the 10 mg of Se and control groups at 191 days were 406 versus 151 nmol Se/L (P < 0.001). Dose−response efficiency to affect animal Se status was evaluated using AUCnorm of blood Se concentrations (as [days · μmol Se]/[L · mg Se]). For BaSeO4 treatment with 6, 12, or 24 mg of Se, the mean values were 23 ± 5.2, 24 ± 2.8, or 22 ± 6.2, respectively. For DL-SeMet treatment with 5, 7.5, or 10 mg Se, the mean values were 27 ± 4.7, 26 ± 10.9, or 23 ± 3.2. Blood Se concentrations did not return to baseline during the study, so the AUCnorm values would have been greater if experimental durations were longer. The growth rates of lambs supplemented with 24 mg of Se as BaSeO4 or 10 mg of Se as DL-SeMet were not different up until weaning on Day 100 (182 versus 183 g/d, P = 0.90). Combined C

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Figure 1. Dose factorial study. Compared to the control group (○), a single subcutaneous injection of low (●), medium (◆), or high (■) doses of (A) BaSeO4 (6, 12, or 24 mg of Se), (B) DL-SeMet (5, 7.5, or 10 mg of Se), or (C) Na2SeO4 (2, 4, or 6 mg of Se) administered to lambs when 3−4 weeks of age increased the mean ± SE concentration of Se in whole blood. n = 10 lambs per group. The dotted lines in (C) are an indication that the large but transient “spike” in blood Se concentration that typically occurs within 1 day of treatment with Na2SeO4 was not captured in this experiment.14

growth rate of those animals was greater than that of the unsupplemented controls (182 versus 156 g/d, P = 0.02). Reactions at the site of injection were minor, typically raised bumps, and resolved over time (data not shown). At Day 15 they were assessed as more prevalent among the high dose BaSeO4 group than the high dose DL-SeMet group (70 versus 30%) but not different in their average size (356 versus 347 mm2). The treated animals showed more reactions than the controls (carrier oil only, 12% and 92 mm2). Tissue Response Study. The effects of injectable BaSeO4 or DL-SeMet on blood and tissues of Se-marginal lambs are shown in Figure 2. Both forms increased the concentrations of Se and the activity of GSH-Px, and these responses were significantly greater than the control group for at least 137 days after treatment (lamb age approximately 165 days). Peak concentrations occurred at similar times in muscle (shown as the average of three muscles collected), liver, and blood, but at later times for the BaSeO4 group in kidney and GSH-Px. Concentrations were uniformly low among the controls, except in kidney. BaSeO4 had an overall greater impact than DLSeMet, except in muscle. The peak response of GSH-Px activity lagged behind the peak response of blood Se concentration,

Figure 2. Tissue response study. Compared to the control group (○), a single subcutaneous injection of 24 mg of Se as BaSeO4 (■) or 10 mg of Se as DL-SeMet (◆) administered to lambs when 3−4 weeks of age increased the mean ± SE concentrations of Se in (A) muscle, (B) kidney, (C) liver (n = 2 to 6 per group per time point), and (D) blood (n = 6 to 20 per group per time point) and increased the activity of (E) blood glutathione peroxidase.

which is probably a consequence of incorporation of Se into GSH-Px protein and the turnover time of red blood cells. The mean BW of control lambs and lambs supplemented with BaSeO4 or DL-SeMet were not different at weaning on Day 89, being 34.8 ± 4.3, 36.7 ± 5.1, and 37.3 ± 5.1 kg, respectively. By Day 138 the continuing decline in Se status of the untreated controls revealed ongoing benefits of both forms of Se supplementation, with mean BW of 31.2 ± 3.6, 38.5 ± 5.1, and 40.0 ± 6.8 kg (P = 0.03 by ANOVA). Stereoisomers Study. The effects of two injections of either L-SeMet or DL-SeMet on the blood Se concentrations of D

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Journal of Agricultural and Food Chemistry Se-adequate lambs are shown in Figure 3. Peak concentrations of 3910 and 4160 nmol Se/L were recorded after 67 days (the

L-SeMet and DL-SeMet showed similar effects on the ranges of Se concentrations in serum and whole blood (Figure 4).

Figure 3. Stereoisomers study. Compared to the control group (○, n = 13), two subcutaneous injections of either L-SeMet (▲, n = 5) or DLSeMet (◆, n = 5) administered to lambs (15 mg of Se on Day 0 when 4−5 weeks of age and 20 mg of Se on Day 67) increased the mean ± SE concentration of Se in blood.

Figure 4. Stereoisomers study. Compared to the control group (○), two subcutaneous injections of either L-SeMet (▲) or DL-SeMet (◆) administered to lambs increased the Se concentrations in paired samples of blood and serum. Regression equations across 137 days of repeated measures were Control, y = 2.3x + 565; L-SeMet, y = 4.3x + 279; DL-SeMet, y = 4.3x + 220.

first blood collection post-treatment and just prior to the second “booster” injection) for L-SeMet and DL-SeMet, respectively. Concentrations remained greater than controls for at least 137 days after treatment (lamb age approximately 172 days). There appeared to be little difference in animal response due to stereoisomeric form of the supplement: AUC was 426 days μmol · Se/L for L-SeMet and 437 days μmol · Se/ L for DL-SeMet. Note that statistical power to detect a difference was low for this metric, at 60% (n = approximately 7.5 per group, mean difference = 240, SD = 180). Concentrations of Se in tissues collected from lambs at the end of the study are shown in Table 3. The SeMet supplements

Markedly less Se was found in serum, which reflects the association of Se with the proteins of red blood cells proliferating in these growing lambs. Among the treated groups, Se concentrations in whole blood were 4.7 ± 1.4 (range 2.2−8.5)-fold greater than in serum. In the control group, the mean ratio was 4.8 ± 1.8 although this varied during the study, being lower during Days 0−67 compared to Days 104−137 (3.7 versus 6.7; P < 0.001). The phenomenon may have been a consequence of grazing conditions and dietary Se intakes at the animals’ initial and final farm locations, which had different soil types and fertilizer histories. Cattle Study. The effects of one or two injections of DLSeMet on blood and milk Se concentrations of Se-marginal dairy cows are shown in Figure 5. Concentrations in blood remained greater than controls for at least 95 days. For example, mean values for the single-injection and control groups at 95 days were 1260 versus 259 nmol of Se/L of blood (P < 0.001). Significant Se enrichment of milk from those groups lasted only 54 days, when whole milk contained 175 versus 92 nmol of Se/L (P < 0.001). A preload or saturation effect was observed in all tissues; responses to the second dose of DL-SeMet were greater. Selenium concentrations in whole blood were 2.1 ± 0.8 (range 0.8−4.4)-fold greater than serum of the treated cows and 1.9 ± 0.5-fold greater than serum of the controls. There appeared to be little Se associated with the fat fraction of milk because, in 198 paired comparisons, skimmed milk was found to contain 97% (range 38−158%) of the Se content of the whole milk (data not shown).

Table 3. In the Stereoisomers Study, The Effect of Consecutive Injections of a SeMet Stereoisomer Administered to Lambs (15 mg of Se on Day 0 of the Study When Lambs Were 4−5 Weeks of Age, Followed by 20 mg of Se on Day 67) a

heart kidney liver lung muscle blood serum

control

L-SeMet

DL-SeMet

Pb

513 ± 29 5600 ± 242 500 ± 115 743 ± 106 333 ± 42 520 ± 57 140 ± 57

2860 ± 505 12200 ± 2700 2860 ± 510 2100 ± 318 2010 ± 237 2960 ± 384 470 ± 147

2600 ± 550 16600 ± 3020 2760 ± 921 2570 ± 891 2190 ± 661 2650 ± 1040 427 ± 146

0.58 0.13 0.88 0.44 0.68 0.65 0.74

Shown are the mean ± SD concentrations of Se in five tissues (nmol of Se/kg of fresh weight) collected on Day 139 and in blood and serum (nmol Se/L) collected on Day 137. N = 3 per treatment group. b P values from t test of difference between the stereoisomer treatment groups. a

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DISCUSSION In a series of experiments, we showed that SeMet can be formulated into an injectable supplement for Se-deficient sheep and cattle. It was effective at increasing simple biomarkers of animal Se status for 3−6 months, and the stereoisomer forms L- and DL-SeMet were found to be functionally equivalent. Duration and magnitude of SeMet benefits were much greater than those due to treatment with Na2SeO4 but somewhat less

increased Se content from 2- to 7-fold over controls, without difference due to stereoisomeric form. Selenium was highly concentrated in kidney, even in the control group. The other tissues had similar Se concentrations within each treatment group, suggesting nonspecific association of supplemental Se with proteins. E

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and 66 μg of Se. The sum total of 140 μg of Se is less than half of the EFSA tolerable upper limit, albeit not accounting for possible concentrations at the injection site. Many properties and attributes of SeMet support its potential as an injectable supplement that could substitute for current formulations that contain BaSeO4. Low Toxicity to Animals. Selenomethionine has a wide therapeutic range of safe dose levels with low acute toxicity,39 and chronic toxicity is lower than inorganic chemical forms.40 Small Doses Are Required for Nutritional Benefit. Dose rates of less than 1 mg of Se/kg of BW were effective (Table 2) and are comparable to the recommended injectable dose rates for Na2SeO4 and BaSeO4 of 0.1 and 0.5−1 mg of Se/ kg of BW, respectively.5,41 Response to increasing dose sizes was additive (Figure 1), approximately linear and similar in efficiency to BaSeO4, according to AUCnorm calculations. Thus, formulations can be titrated to suit animal requirements and severity of deficiency. Reasonably Long Efficacy. Injection with SeMet maintained nutritionally adequate Se concentrations in the blood and tissues of lambs for at least 191 days and in cattle for at least 95 days (Figures 1, 2, 3, and 5). Efficacy in sheep was not as long as BaSeO4 (246 days) per mg of Se administered but substantially longer than Na2SeO4 (64 days). A single injection of SeMet administered to lambs at 3−4 weeks of age maintained their Se status well beyond the usual age of slaughter at 16−20 weeks. Although SeMet is a more expensive ingredient than Na2SeO4, sufficiently long efficacy can offset the upfront expense if the cost of labor necessary for repeated treatments is also considered. Efficient Distribution to Body Tissues. Doses of SeMet larger than an animal’s immediate metabolic requirement lead to accumulation in proteins as selenoamino acids. Its appearance in a variety of tissues (Table 3) and effects on GSH-Px activity suggest that SeMet is well-utilized. Transfer of Se from dam to offspring is likely to be excellent. Selenomethionine fed to lactating ewes is a better source of Se than selenite for suckling lambs.16 When administered to ewes premating, BaSeO4 improves Se status of the developing fetus and newborn lamb1 and a similar benefit might be expected from SeMet. The many-fold increase observed for milk Se concentration suggests that SeMet treatment of cows could deliver Se to suckling calves (Figure 5). Studies are needed to determine if the duration is sufficient to substantially impact calf Se status. Accumulation of SeMet in the milk and edible tissues of treated animals offers opportunity for creating Se-enriched foods.23 Safer for Meat Consumers. Supplementary SeMet is indistinguishable from that which occurs naturally in animals and plants, which means that residues are not explicitly detectible. Being water-soluble, injected SeMet is unlikely to form a persistent, concentrated depot at the site of injection that could exceed safe levels for Se dietary intake. Further studies are required to confirm this. Although SeMet is already permitted for oral supplementation when added to the feed rations of ruminants,20 novel formulations of injectable SeMet would require regulatory approval. Flexible to Manufacture. In these experiments, the L- and DL- stereoisomers of SeMet were equally effective (Figure 4). This has been shown for stereoisomers infused into the abomasum (fourth stomach) of sheep42 but not for parenteral injections. Our results imply that future product development

Figure 5. Cattle study. Compared to the control group (○, n = 10), subcutaneous injection of one dose (◆, n = 10) or two doses (■, n = 10) of DL-SeMet administered to lactating dairy cows at 0.35 ± 0.01 mg of Se/kg of BW increased the mean ± SE concentrations of Se in (A) blood, (B) serum, and (C) whole milk.

than was observed for BaSeO4, the most widely used chemical form of Se for injection. This investigation addresses concerns among food safety authorities that BaSeO4 is a hazard to consumers of meat from supplemented livestock, due to the persistence of undesirable residue at the site of injection. The recommended site is the animal’s anterior neck region and, given best practice for slaughter and preparation of the carcass for sale, any contamination remaining from this depot of BaSeO4 would not be available for human consumption. However, mistakes can occur, for example, on-farm by misplaced injection into edible muscle or at the abattoir if sites are not excised. Residues of BaSeO4 in meat could exceed the tolerable upper intake levels for consumers established by EFSA for Se at 300 μg/ person per day36 and by the EC for barium (Ba) at 200 μg/kg body weight per day.37 This risk has motivated prohibition of injectable BaSeO4 for food-producing animals in the EU.13 For SeMet to be considered as a substitute, the tissues of injected animals must be safe to eat. Consumer exposure to Se can be estimated using the “daily food basket” approach to food intake, which presumes a maximum daily intake of 300 g of muscle, 100 g of liver, and 50 g of kidney.38 When lambs from the Stereoisomers study were slaughtered, the mean tissue Se concentrations for the DL-SeMet group were 2190, 2760, and 16600 nmol of Se/kg, respectively (Table 3). Converting units to μg/kg and multiplying by the presumed intakes yields 52, 22, F

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(2) Hartley, W. J.; Dodd, D. C. Muscular dystrophy in NZ livestock. N. Z. Vet. J. 1957, 5, 61−66. (3) Jolly, R. D. A preliminary experiment on the effect of selenium on the growth rate of calves. N. Z. Vet. J. 1960, 8, 13. (4) Cawley, G. D.; McPhee, I. Trials with a long acting parenteral selenium preparation in ruminants: sheep. Vet. Rec. 1984, 114, 565− 566. (5) Archer, J. A.; Judson, G. J. Selenium concentrations in tissues of sheep given a subcutaneous injection of barium selenate or sodium selenate. Aust. J. Exp. Agric. 1994, 34, 581−588. (6) Anderson, P. H.; Berrett, S.; Patterson, D. S. The biological selenium status of livestock in Britain as indicated by sheep erythrocyte glutathione peroxidase activity. Vet. Rec. 1979, 104, 235−238. (7) Tasker, J. B.; Bewick, T. D.; Clark, R. G.; Fraser, A. J. Selenium response in dairy cattle. N. Z. Vet. J. 1987, 35, 139−140. (8) Fraser, A. J.; Wright, D. F. The Relationship between Blood Selenium Levels and Calf Growth Responses to Selenium Supplementation. In Trace Elements in the Eighties; Palmerston North, New Zealand, August 7−8, 1984; Baker, M. J., Ed. New Zealand Trace Element Group: Palmerston North, New Zealand, 1986; pp 89−91. (9) Oldfield, J. E.; Schubert, J. R.; Muth, O. H. Implications of selenium in large animal nutrition (conference paper). J. Agric. Food Chem. 1963, 11, 388−390. (10) Grace, N. D.; Ankenbauer-Perkins, K. L.; Alexander, A. M.; Marchant, R. M. Relationship between blood selenium concentration or glutathione peroxidase activity, and milk selenium concentrations in New Zealand dairy cows. N. Z. Vet. J. 2001, 49, 24−28. (11) Kuttler, K. L.; Marble, D. W.; Blincoe, C. Serum and tissue residues following selenium injections in sheep. Am. J. Vet. Res. 1961, 22, 422−428. (12) Mallinson, C. B.; Allen, W. M.; Sansom, B. F. Barium selenate injections in cattle: effects on selenium concentrations in plasma and liver and residues at site of injection. Vet. Rec. 1985, 117, 405−407. (13) European Medicines Agency. Regulation [EU] 2015/446, amending Regulation (EU) No 37/2010 as regards the substance ‘barium selenate’. Off. J. Eur. Union 2015, 58, 18−20. (14) Meads, W. J.; Osborn, J.; Grant, A. B. The effect of single dose of selenium salts on whole blood levels of selenium in ewes on a selenium-deficient diet. N. Z. Vet. J. 1980, 28, 20−22. (15) Hopper, S. A.; Greig, A.; McMurray, C. H. Selenium poisoning in lambs. Vet. Rec. 1985, 116, 569−571. (16) Jenkins, K. J.; Hidiroglou, M. Transmission of selenium as selenite and as selenomethionine from ewe to lamb via milk using selenium-75. Can. Vet. J. 1971, 51, 389−403. (17) Knowles, S. O.; Grace, N. D. Parenteral selenomethionine for production of selenium-rich foods - complete patent specification for SMARTShot SeMet. U.S. Patent 8,633,248, January 21, 2014. (18) Knowles, S. O.; Grace, N. D.; Wurms, K.; Lee, J. Significance of amount and form of dietary selenium on blood, milk, and casein selenium concentrations in grazing cows. J. Dairy Sci. 1999, 82, 429− 437. (19) Chauhan, S. S.; Celi, P.; Leury, B. J.; Dunshea, F. R. High dietary selenium and vitamin E supplementation ameliorates the impacts of heat load on oxidative status and acid-base balance in sheep. J. Anim. Sci. 2015, 93, 3342−3354. (20) Juniper, D. T.; Phipps, R. H.; Ramos-Morales, E.; Bertin, G. Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle. J. Anim. Sci. 2008, 86, 3100−3109. (21) Knowles, S. O.; Grace, N. D.; Munday, R. Selenium Administration - complete patent specification for Senrich Bolus. World Patent WO/2008/121006-A1, September 30, 2010. (22) Ehlig, C. F.; Hogue, D. E.; Allaway, W. H.; Hamm, D. J. Fate of selenium from selenite or selenomethionine, with or without vitamin E, in lambs. J. Nutr. 1967, 92, 121−126. (23) Knowles, S. O.; Grace, N. D.; Knight, T. W.; McNabb, W. C.; Lee, J. Reasons and means for manipulating the micronutrient

work and eventual manufacture of injectable formulations will not require enantiomerically pure SeMet. In summary, long-acting injections of nutritional supplements are a practical approach to treating and preventing nutritional deficiencies in livestock, particularly for grazing flocks and herds that are infrequently handled and have no or minimal access to dietary supplements and feed concentrates.43 Proof-of-concept experiments have demonstrated that injectable formulations containing SeMet can deliver efficacy similar to BaSeO4 in sheep and cattle but with less risk of undesirable residues in edible tissues. The results support and encourage the development of novel SeMet products for markets where the use of BaSeO4 is restricted. Follow-up investigations should assess effects on farm productivity and quantify the risks to food consumers. Beneficiaries will include animal producers in the EU and elsewhere trying to raise healthy livestock in the face of changing legislative guidelines, and food manufacturers who must source sheep and cattle meat that has low risk of agrochemical residues. Overseas farms and producers who intend to export meat from treated animals to Europe may also be affected.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone 64-6-3518066. ORCID

Scott O. Knowles: 0000-0003-3482-5513 Author Contributions

S.O.K. and N.D.G. designed and supervised the studies, administered treatments, interpreted results, and cowrote each draft and revision of the article. N.D.G. collected blood and tissue samples from animals. S.O.K. analyzed the data and prepared the figures. Funding

This work was supported by various governmental contestable funding pools (NZ Foundation for Research Science and Technology, NZ Ministry of Science and Innovation, and NZ PreSeed Accelerator Fund) and from internal development grants (AgResearch PreSeed Fund). Notes

The authors declare the following competing financial interest(s): The authors are inventors of injectable nutritional supplements containing BaSeO4, SeMet, and microencapsulated vitamin B12 and are named on related patents held by a New Zealand government research institute. They maintain informal links with Stockguard Laboratories (NZ) Ltd. and Stockguard Animal Health Ltd. (now Virbac NZ), the companies that manufactured the Se and vitamin B12 products. Those companies had no input into the design and execution of the studies.

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ACKNOWLEDGMENTS The authors are grateful to John Parr and Dave Wildermoth for farm management, to Chris Miller, Andrea Death, John Rounce, and Jason Peters for technical support on-farm and in the laboratory, and to Dr. Emma Bermingham for assistance in organizing the Stereoisomers study.

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REFERENCES

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