Review: Ruminal microbiome and microbial metabolome: effects of diet and ruminant host.

The rumen contains a great diversity of prokaryotic and eukaryotic microorganisms that allow the ruminant to utilize ligno-cellulose material and to convert non-protein nitrogen into microbial protein to obtain energy and amino acids. However, rumen fermentation also has potential deleterious consequences associated with the emissions of greenhouse gases, excessive nitrogen excreted in manure and may also adversely influence the nutritional value of ruminant products. While several strategies for optimizing the energy and nitrogen use by ruminants have been suggested, a better understanding of the key microorganisms involved and their activities is essential to manipulate rumen processes successfully. Diet is the most obvious factor influencing the rumen microbiome and fermentation. Among dietary interventions, the ban of antimicrobial growth promoters in animal production systems has led to an increasing interest in the use of plant extracts to manipulate the rumen. Plant extracts (e.g. saponins, polyphenol compounds, essential oils) have shown potential to decrease methane emissions and improve the efficiency of nitrogen utilization; however, there are limitations such as inconsistency, transient and adverse effects for their use as feed additives for ruminants. It has been proved that the host animal may also influence the rumen microbial population both as a heritable trait and through the effect of early-life nutrition on microbial population structure and function in adult ruminants. Recent developments have allowed phylogenetic information to be upscaled to metabolic information; however, research effort on cultivation of microorganisms for an in-depth study and characterization is needed. The introduction and integration of metagenomic, transcriptomic, proteomic and metabolomic techniques is offering the greatest potential of reaching a truly systems-level understanding of the rumen; studies have been focused on the prokaryotic population and a broader approach needs to be considered.

Not all saponins have a greater antiprotozoal activity than their related sapogenins.

The antiprotozoal effect of saponins varies according to both the structure of the sapogenin and the composition and linkage of the sugar moieties to the sapogenin. The effect of saponins on protozoa has been considered to be transient as it was thought that when saponins were deglycosilated to sapogenins in the rumen they became inactive; however, no studies have yet evaluated the antiprotozoal effect of sapogenins compared to their related saponins. The aims of this study were to evaluate the antiprotozoal effect of eighteen commercially available triterpenoid and steroid saponins and sapogenins in vitro, to investigate the effect of variations in the sugar moiety of related saponins and to compare different sapogenins bearing identical sugar moieties. Our results show that antiprotozoal activity is not an inherent feature of all saponins and that small variations in the structure of a compound can have a significant influence on their biological activity. Some sapogenins (20(S)-protopanaxatriol, asiatic acid and madecassic acid) inhibited protozoa activity to a greater extent than their corresponding saponins (Re and Rh1 and asiaticoside and madecassoside), thus the original hypothesis that the transient nature of the antiprotozoal action of saponins is due to the deglycosilation of saponins needs to be revisited.

The effect of an isoflavonid-rich liquorice extract on fermentation, methanogenesis and the microbiome in the rumen simulation technique.

Due to the antimicrobial activity of flavonoids, it has been suggested that they may provide a possible alternative to antibiotics to stimulate productivity and reduce the environmental load of ruminant agriculture. We hypothesised that an extract of liquorice, rich in prenylated isoflavonoids and particularly glabridin, might potentially improve the efficiency of nitrogen utilisation and reduce methane production in the rumen. When added to a long-term rumen simulating fermentor (RUSITEC), liquorice extract at 1 g L-1 decreased ammonia production (-51%; P < 0.001) without affecting the overall fermentation process. When added at 2 g L-1, decreases in not only ammonia production (-77%; P < 0.001), but also methane (-27%; P = 0.039) and total VFA production (-15%; P = 0.003) were observed. These effects in fermentation were probably related to a decrease in protozoa numbers, a less diverse bacteria population as well as changes in the structure of both the bacterial and archaeal communities. The inclusion of an isoflavonoid-rich extract from liquorice in the diet may potentially improve the efficiency of the feed utilisation by ruminants.

Microbial and chemical composition of liquid-associated bacteria in goats' rumen and fermenters.

This study was undertaken to investigate the relationship between chemical composition and microbial profile of rumen liquid-associated bacteria (LAB) in vivo (Murciano-Granadina goats) and in a rumen simulation system (single-flow continuous-culture fermenters). To achieve this aim, analyses of purine bases along with some molecular techniques (quantitative PCR to assess abundance and DGGE to identify biodiversity and bacterial profile) were carried out. A control diet (AHC) based on alfalfa hay (AH) and concentrate (C) in a 1:1 ratio and two experimental diets (AHCBI and AHCBII), in which concentrate was partially replaced with multinutrient blocks, were used. Diets AHCBI and AHCBII included multinutrient blocks differing in the relative amount of two-stage olive cake and the source of protein (sunflower meal vs. fava beans). We aimed to investigate the effect of these blocks on rumen microbiota to evaluate their potential as safe substitutes of cereal-based concentrates. Similar patterns of response to diet were found for chemical composition, microbial abundances and diversity in LAB isolated from goat's rumen and fermenters. Whereas bacterial density (log10 gene copies/g FM: 11.6 and 9.4 for bacteria and methanogens, respectively, in rumen) and diversity indexes (Shannon index: 3.6) were not affected by diet, DGGE analyses showed that bacterial community profile was affected. The cluster analysis suggested differences in bacterial profile between LAB pellets isolated from the rumen of goat and fermenters. A relationship between chemical composition and bacterial community composition in LAB pellets seems to exist. Changes in the former were reflected in the bacterial community profile. Further research is needed to clarify the relationship between chemical and microbial composition of ruminal bacterial pellets with diets of different quality.

Oral administration of cobalt acetate alters milk fatty acid composition, consistent with an inhibition of stearoyl-coenzyme A desaturase in lactating ewes.

Previous investigations have shown that cobalt (Co) modifies milk fat composition in cattle, consistent with an inhibition of stearoyl-coenzyme A desaturase (SCD) activity, but it remains unclear whether other ruminant species are also affected. The present study examined the effects of oral administration of Co acetate on intake, rumen function, and milk production and fatty acid (FA) composition in sheep. Twenty lactating Assaf ewes were allocated into 1 of 4 groups and used in a continuous randomized block design that involved a 15-d adaptation, a 6-d treatment, and a 10-d posttreatment period. During the treatment period, animals received an oral drench supplying 0 (control), 3 (Co3), 6 (Co6), and 9 (Co9) mg of Co/kg of BW per day, administered in 3 equal doses at 8-h intervals. Cobalt acetate had no influence on intake or milk fat and protein concentrations, whereas treatments Co6 and Co9 tended to lower milk yield. Results on rumen parameters showed no effects on rumen fermentation, FA composition, or bacterial community structure. Administration of Co acetate decreased milk concentrations of FA containing a cis-9 double bond and SCD product:substrate ratios, consistent with an inhibition of SCD activity in the ovine mammary gland. Temporal changes in milk fat composition indicated that the effects of treatments were evident within 3d of dosing, with further changes being apparent after 6d and reverting to pretreatment values by d 6 after administration. Effect on milk FA composition did not differ substantially in response to incremental doses of Co acetate. On average, Co decreased milk cis-9 10:1/10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, and cis-9,trans-11 18:2/trans-11 18:1 concentration ratios by 30, 32, 38, 33, 21, 24, and 25%, respectively. Changes in milk fat cis-9 10:1, cis-9 12:1, and cis-9 14:1 concentrations to Co treatment indicated that 51% of cis-9 18:1 and cis-9,trans-11 18:2 secreted in milk originated from Δ(9)-desaturation. In conclusion, results demonstrated the potential of oral Co administration for the estimation of endogenous synthesis of FA containing a cis-9 double bond in the mammary gland of lactating ruminants. Indirect comparisons suggest that the effects of Co differ between sheep and cattle.

In vitro-in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats.

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

Nutrient utilization, ruminal fermentation, microbial abundances, and milk yield and composition in dairy goats fed diets including tomato and cucumber waste fruits.

The effects of replacing 35% of cereals-based concentrate with feed blocks (FB) containing waste fruits of tomato, cucumber, or barley grain in diets for lactating goats on nutrient utilization, ruminal fermentation, microbial N flow to the duodenum, milk yield and quality, methane emissions, and abundances of total bacteria and methanogens were studied. Eight Murciano-Granadina goats (39.4 ± 5.39 kg of body weight, mean ± SD) in the middle of the third lactation were used and 4 diets were studied in a replicated 4×4 Latin square experimental design. Diets consisted of alfalfa hay (A) plus concentrate (C) in a 1:1 ratio (diet AC) or diets in which 35% of the concentrate was replaced with FB including wastes of tomato fruit, cucumber, or barley. In each period, 2 goats were randomly assigned to 1 of the dietary treatments. Intakes of FB including tomato, cucumber, and barley were 208 ± 65, 222 ± 52, and 209 ± 83 g of dry matter per animal and day, respectively. The replacement of 35% of concentrate with FB did not compromise nutrient apparent digestibility, total purine derivative urinary excretion, milk yield and composition, and total bacteria and methanogen abundances. Digestible energy and that in methane and urine were higher for AC than for FB-containing diets, whereas the metabolizable energy value was not affected by diet. The inclusion of tomato and cucumber fruits in FB decreased N in urine and CH(4) emissions compared with AC, which is environmentally relevant. However, tomato-based FB decreased microbial N flow in the rumen, whereas goats fed cucumber-based FB had the highest values for this measurement. Moreover, FB containing barley or tomato and cucumber led to lower rumen volatile fatty acid and NH(3)-N concentrations, respectively. Milk from goats fed diets including tomato and cucumber-based FB had higher linoleic, linolenic, and total polyunsaturated fatty acid concentrations than that from goats fed AC. Overall, our study suggests that tomato and cucumber FB could replace 35% of the concentrate in the dairy goat diet, reducing animal feeding cost and methane production, leading to higher polyunsaturated fatty acid proportions in milk, and without compromising nutrient utilization or milk yield.

Ricinoleic acid inhibits methanogenesis and fatty acid biohydrogenation in ruminal digesta from sheep and in bacterial cultures.

Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for CLA synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from 4 sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9,cis-12-18:2) or with a combination of LA and RA or LA and castor oil (LA, RA, and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA rumenic acid (cis-9,trans-11-18:2) accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. Butyrivibrio proteoclasticus but not B. fibrisolvens metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were decreased (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo studies are now required to confirm the potential of these additives.

Nitrogen and energy utilization in lactating dairy goats fed diets with different legume seeds.

The N and energy utilization of diets with different legume seeds was analysed in lactating goats. Four isonitrogenous and isoenergetic diets were used and in each case 30% of the protein was supplied by one of four different legume seeds: lupins (L), faba beans (FB), bitter vetch (BV) and vetch (V). A group of eight Granadina goats, allocated to a replicated 4 × 4 Latin Square, were used. The N and energy utilization of the different diets may be considered adequate; milk N/intake N was approximately 0.20, milk N/N balance was approximately 0.50 and milk energy/metabolizable energy was approximately 0.30. Under similar N and energy intakes, faecal and urinary excretion of N and the quantities in milk did not vary significantly. However, the partition of the N balance between milk production and deposit within the body varied depending on the diet. Although this N available to the metabolism was the highest for the FB diet, the quantity of this directed to milk was the lowest. Finally, the faecal and urinary excretion with respect to the quantity directed to milk production did not vary significantly among the different diets.

The role of microbes in rumen lipolysis and biohydrogenation and their manipulation.

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products - meat, milk and dairy - contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.

Nutritive evaluation of legume seeds for ruminant feeding.

Chemical composition, rumen degradability and the effect of particle losses, and intestinal digestibility of protein by using in situ-in vitro and in vitro techniques were stated for beans (Vicia faba), lupin (Lupinus albus), vetch (Vicia sativa) and bitter vetch (Vicia ervilia) and four diets including those legume seeds. In addition, the apparent digestibility of experimental diets was determined in goats. The legume seeds showed high protein content (206-319 g/kg dry matter). Effective degradability of protein for legumes and diets varied from 0.80 to 0.87 and 0.76 to 0.82, respectively, decreasing to 0.53-0.76 and 0.61-0.67, respectively, when particle loss was taken into account. Different intestinal digestibility values were obtained with both methodologies without significant relationship between them (y = 1.058-0.463x; R(2)=0.068; RSD = 0.140; p = 0.53). There were no differences in the apparent nutrients and energy digestibility among diets (p > 0.05). These legumes can supply rapidly degradable protein for microbial protein synthesis and contribute to the pool of amino acids available for the synthesis of milk protein and for retention in the body.

Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle.

The objective was to determine the concentration of total Se and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in postmortem tissues of beef cattle offered diets containing graded additions of selenized enriched yeast (SY; Saccharomyces cerevisiae CNCM I-3060) or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of edible muscle tissue were assessed 10 d postmortem. Thirty-two beef cattle were offered, for a period of 112 d, a total mixed ration that had been supplemented with SY (0, 0.15, or 0.35 mg of Se/kg of DM) or SS (0.15 mg of Se/kg of DM). At enrollment (0 d) and at 28, 56, 84, and 112 d following enrollment, blood samples were taken for Se and Se species determination, as well as whole blood GSH-Px activity. At the end of the study beef cattle were killed and samples of heart, liver, kidney, and skeletal muscle (LM and psoas major) were retained for Se and Se species determination. Tissue GSH-Px activity and thiobarbituric acid reactive substances were determined in skeletal muscle tissue (LM only). The incorporation into the diet of ascending concentrations of Se as SY increased whole blood total Se and the proportion of total Se comprised as SeMet, as well as GSH-Px activity. There was also a dose-dependent response to the graded addition of SY on total Se and proportion of total Se as SeMet in all tissues and GSH-Px activity in skeletal muscle tissue. Furthermore, total Se concentration of whole blood and tissues was greater in those animals offered SY when compared with those receiving a comparable dose of SS, indicating an improvement in Se availability and tissue Se retention. Likewise, GSH-Px activity in whole blood and LM was greater in those animals offered SY when compared with those receiving a comparable dose of SS. However, these increases in tissue total Se and GSH-Px activity appeared to have little or no effect in meat oxidative stability.

Selenium supplementation of lactating dairy cows: effects on milk production and total selenium content and speciation in blood, milk and cheese.

Forty multiparous Holstein cows were used in a 16-week continuous design study to determine the effects of either selenium (Se) source, selenised yeast (SY) (derived from a specific strain of Saccharomyces cerevisiae CNCM I-3060) or sodium selenite (SS), or Se inclusion rate in the form of SY in the diets of lactating dairy cows on the Se concentration and speciation in blood, milk and cheese. Cows received ad libitum a total mixed ration (TMR) with a 1 : 1 forage : concentrate ratio on a dry matter (DM) basis. There were four diets (T1 to T4), which differed only in either source or dose of Se additive. Estimated total dietary Se for T1 (no supplement), T2 (SS), T3 (SY) and T4 (SY) was 0.16, 0.30, 0.30 and 0.45 mg/kg DM, respectively. Blood and milk samples were taken at 28-day intervals and at each time point there were positive linear effects of Se in the form of SY on the Se concentration in blood and milk. At day 112, blood and milk Se values for T1 to T4 were 177, 208, 248 and 279 ± 6.6 and 24, 38, 57 and 72 ± 3.7 ng/g fresh material, respectively, and indicate improved uptake and incorporation of Se from SY. In whole blood, selenocysteine (SeCys) was the main selenised amino acid and the concentration of selenomethionine (SeMet) increased with the increasing inclusion rate of SY. In milk, there were no marked treatment effects on the SeCys content, but Se source had a marked effect on the concentration of SeMet. At day 112, replacing SS (T2) with SY (T3) increased the SeMet concentration of milk from 36 to 111 ng Se/g and its concentration increased further to 157 ng Se/g dried sample as the inclusion rate of SY increased further (T4) to provide 0.45 mg Se/kg TMR. Neither Se source nor inclusion rate affected the keeping quality of milk. At day 112, milk from T1, T2 and T3 was made into a hard cheese and Se source had a marked effect on total Se and the concentration of total Se comprised as either SeMet or SeCys. Replacing SS (T2) with SY (T3) increased total Se, SeMet and SeCys content in cheese from 180 to 340 ng Se/g, 57 to 153 ng Se/g and 52 to 92 ng Se/g dried sample, respectively. The use of SY to produce food products with enhanced Se content as a means of meeting the Se requirements is discussed.

Selenium persistency and speciation in the tissues of lambs following the withdrawal of dietary high-dose selenium-enriched yeast.

The objective was to determine the concentration of total selenium (Se) and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in post mortem tissues of lambs in the 6 weeks period following the withdrawal of a diet containing high-dose selenised yeast (HSY), derived from a specific strain of Saccharomyces cerevisae CNCM (Collection Nationale de Culture de Micro-organism) I-3060. Thirty Texel × Suffolk lambs used in this study had previously received diets (91 days) containing either HSY (6.30 mg Se per kg dry matter (DM)) or an unsupplemented control (C; 0.13 mg Se per kg DM). Following the period of supplementation, all lambs were then offered a complete pelleted diet, without additional Se (0.15 mg Se per kg DM), for 42 days. At enrolment and 21 and 42 days later, five lambs from each treatment were blood sampled, euthanased and samples of heart, liver, kidney and skeletal muscle (longissimus dorsi and psoas major) tissue were retained. Total Se concentration in whole blood and tissues was significantly (P < 0.001) higher in HSY lambs at all time points that had previously received long-term exposure to high dietary concentrations of SY. The distribution of total Se and the proportions of total Se comprised as SeMet and SeCys differed between tissues, treatment and time points. Total Se was greatest in HSY liver and kidney (22.64 and 18.96 mg Se per kg DM, respectively) and SeCys comprised the greatest proportion of total Se. Conversely, cardiac and skeletal muscle (longissimus dorsi and psoas major) tissues had lower total Se concentration (10.80, 7.02 and 7.82 mg Se per kg DM, respectively) and SeMet was the predominant selenised amino acid. Rates of Se clearance in HSY liver (307 µg Se per day) and kidney (238 µg Se per day) were higher compared with HSY cardiac tissue (120 µg Se per day) and skeletal muscle (20 µg Se per day). In conclusion, differences in Se clearance rates were different between tissue types, reflecting the relative metabolic activity of each tissue, and appear to be dependent on the proportions of total Se comprised as either SeMet or SeCys.