Central nesfatin-1 reduces the nocturnal food intake in mice by reducing meal size and increasing inter-meal intervals

Nesfatin-1 is well established to reduce food intake upon brain injection in rats, while in mice its anorexigenic action and brain expression are largely unexplored. We characterized the influence of intracerebroventricular (icv) and peripheral (intraperitoneal, ip, subcutaneous, sc) injection of nesfatin-1 on dark phase ingestive behavior using an automated feeding monitoring system and co-localized NUCB2/nesfatin-1 immunoreactivity in the associated brain areas. Nesfatin-1 (0.3, 1 or 3 μg/mouse, icv) caused a dose-related reduction of 4-h dark phase food intake by 13%, 27%, and 46% respectively. Nesfatin-1 (3 μg/mouse, icv) action had a 2-h delayed onset, 82% peak inhibition occurring at 3-4 h post-injection and was long lasting (30% reduction for 12 h period post-injection). Nesfatin-1 (3 μg/mouse, icv)-treated mice had a 46% lower meal frequency associated with 2-times longer inter-meal intervals and a 35% reduction in meal size compared to vehicle during the 1-4 h post-injection (p < 0.05). NUCB2/nesfatin-1-immunopositive neurons were found in hypothalamic (supraoptic, paraventricular, arcuate, dorsomedial, lateral) and brainstem (dorsal vagal complex) feeding regulatory nuclei. When injected peripherally, neither food intake nor feeding microstructure parameters were altered. These results demonstrate that NUCB2/nesfatin-1 is prominently expressed in mouse hypothalamus and medulla and acts in the brain to curtail the dark phase feeding by inducing satiation and satiety indicated by reduced meal size and prolonged inter-meal intervals respectively. The lack of nesfatin-1 effect when injected peripherally at a 23-times higher dose indicates a primarily central site of the anorexigenic action for nesfatin-1 in mice.     

Cholecystokinin and bombesin act independently to decrease food intake in the rat

The satiating effects of cholecystokinin-octapeptide (CCK-8) and bombesin (BBS) when injected alone and in combination were compared in intact rats. When injected alone, both CCK-8 and BBS elicited a dose-related decrease of 30-minute food intake. Injections of BBS were less potent than the equivalent doses of CCK-8 in producing satiety. BBS reached an asymptotic level of suppression of approximately 40 percent at a dose of 2 micrograms/kg, whereas injections of 4 micrograms/kg of CCK-8 resulted in a 72 percent suppression of food intake. When the two peptides were administered in a single injection, the resulting suppression of food intake was equivalent to that which would be predicted if their effects were completely additive. These results support the hypothesis that CCK-8 and BBS act via independent mechanisms to induce satiety. A preliminary model of peptidergic satiety, based on this hypothesis, is proposed.     

Cholecystokinin-33 inhibits meal size and prolongs the subsequent intermeal interval

There are various forms of the satiety gut-brain peptide cholecystokinin (CCK), a short, widely utilized form or CCK-8, and a long, putatively more effective form or CCK-33. The issue of which of these forms is a more effective satiety peptide is not resolved. Here, we compared the satiety responses, including the sizes of the first three meals (MS) and intermeal intervals (IMI) as well as their calculated satiety ratios (SR), evoked by both peptides. CCK-8 and 33 (1, 3 and 5 nmol/kg, i.p) reduced the size of the first meal similarly, only CCK-33 prolonged the first IMI and increased SR and both peptides failed to affect second and third MS and IMI. As such, CCK-33 is a more effective satiety peptide than CCK-8. The current results confirm previous findings which showed that both peptides reduce food intake by inhibiting meal size, whereas only CCK-33 reduces food intake by prolonging the intermeal interval.     

圈养赤斑羚夏季日食量及表观消化率的探讨

<正>野生动物营养学是野生动物生态学和管理学的重要组成部分,是了解野生和圈养野生动物种群生存和繁殖的核心内容之一(Robbins,1983)。动物采食是一个复杂的、动态的、生物和非生物因素相互作用和相互影响的过程(Van Soest,1994),同时受动物自身、环境和饲料等多种因素的影响,有时还存在互作(NRC,1996)。采食量决定营养物质的摄入量,决定着动物能够从环境中获得营养物     

The use of different models for the estimation of residual feed intake (RFI) as a measure of feed efficiency in meat sheep

The concept of residual feed intake (RFI), in determining differences among animals in converting feed into body tissue, was first raised in 1963. Feed efficiency is typically calculated as a function of liveweight gain (LWG) and feed intake (FI). Historically two versions of the same model were proposed, one where FI was adjusted for liveweight (LW) and LWG, and the other where LWG was adjusted for FI and LW. Variation in LWG or FI could then be partitioned into two parts; that which is expected and can be attributed to differences in FI or LWG; and that which is the residual portion, which is the deviation from the expected value based on regression, and therefore not accounted for by differences in FI or LWG. Based on this definition, it is the residual portion which is the measure of efficiency. Both within a livestock industry and between different livestock industries there is no set model for calculating RFI. This paper evaluated four models used to calculate RFI and one model used to calculate residual LWG (RLWG) at a standard level of nutrition. They were the main model currently in use in the Australian beef cattle industry (RFI_B), the original models proposed in 1963 (RFI₁₉₆₃; RLWG₁₉₆₃); a French model which included ultrasound measures of muscle and fat depth (RFI_F) and the use of the Australian feeding standards to calculate predicted intake and thus RFI (RFI_SCA). Using feed intake, liveweight and body composition data generated from the same group of sheep (n = 52) at two ages (6 mo, 13 mo), the relative merits of each model were evaluated and compared to the other models, to determine the most appropriate model to calculate RFI for sheep. For all the models except that used to calculate RLWG, over half of the variation in FI could be explained by the model. The amount of variation in FI accounted for depended on the parameters included and the dataset, with less variation in FI explained by the specific models in the older animals. The RFI_F model, which included measures of body composition, accounted for the greatest proportion of the variation in FI and as such suggests that the inclusion of body composition parameters is likely to more accurately reflect true biological efficiency.     

Determination of the daily selenium intake in slovakia

Three models were used to determine the daily dietary Selenium intake in Slovakia. The Selenium content of food produced and consumed in the Slovak Republic was used to estimate and calculate the daily Selenium intake based on food consumption data per capita and seven days, (24 h) eating protocol models. In a duplicate portion model, Selenium was analyzed in a whole day hospital diet during an eight-day period. According to these models the daily dietary Selenium intake was 38.2 μg; 43.3 ±6.5 μg for men and 32.6 ±6.6 μg for women; 27.1 ±7.8 μg for normal and 32.3 ±4.8 μg for nourishing hospital diets. The main contributors of Selenium to daily intake were the following: eggs, pork, and poultry. The obtained results indicate that the daily dietary intake of Selenium of the Slovak people is below the recommended values.     

Flock size and habitat-dependent food and energy intake of foraging Goldfinches

Summary During the breeding season Goldfinches (Carduelis carduelis L.) feed on milky ripe seeds of about 20 food plants. Individual Goldfinches joining a flock reduce the time spent vigilant with increasing flock size. Therefore birds feeding in flocks get an increased intake of kernels per time unit. This was measured for five different food plants (Dactylis glomerata (Gramineae), Knautia arvensis (Dipsacaceae), Senecio vulgaris, Taraxacum officinale, Tragopogon pratensis (Compositae)). In large-sized flocks, birds fed up to 2.3 times more kernels, than when feeding solitarily. In addition, visibility in the vegetation leads to a further increase of kernel intake. Thus feeding under good conditions as in recently mown areas, can raise kernel intake to the seven fold per time unit as compared to solitary feeding. The maximum ingestion rate of kernels was 98 per min which implies a head up-and-down movement every 0.6 s. The calculated energy intake of birds per time unit is lowest in Senecio and highest in Tragopogon. Thus the birds, when feeding on Tragopogon in larger flocks, can gain an energy intake 16 times higher than that reached when feeding on Senecio, despite of a higher kernel intake rate. The energy intake individual Goldfinches gain at the particular plant species is markedly increased with flock sizes up to eight birds, with larger flocks the intake increases only slightly.     

Intermittent nicotine administration modulates food intake in rats by acting on nicotine receptors localized to the brainstem

Previous studies have shown nicotine (NIC) administration leads to decreased food intake, while other investigations have reported that NIC stimulates c-Fos expression in the brainstem. Whether there is a causal relationship between NIC effects on ingestion and its effect on brainstem neurons is uncertain, however we hypothesized that blocking NIC action in the brainstem would prevent, to some extent, the hypophagic effects of NIC. In the present study, cannulas were placed in the fourth ventricle of rats. A dose of NIC or saline was injected i.p. in four equal injections during the dark phase for four days. At the start of the second day of injections the NIC receptor antagonist mecamylamine (MEC) or artificial cerebrospinal fluid (a-CSF) was infused intracerebroventricularly (i.c.v.). Thus, four experimental groups were examined: a-CSF + SAL; a-CSF + NIC; MEC + SAL; MEC + NIC. Meal patterns were recorded using a computerized system and water intake and body weight were measured daily. Peripheral NIC injections suppressed food intake by decreasing meal size, whereas infusion of the NIC receptor antagonist MEC (4 microg) into the fourth ventricle blocked the NIC suppression of food intake. Moreover, the MEC effect was due primarily to an increase in dark phase meal size, which suggests neurons localized to the brainstem transmit NIC signals that regulate feeding behavior by affecting meal size.     

Peripheral activation of corticotropin-releasing factor receptor 2 inhibits food intake and alters meal structures in mice

The orexigenic effect of urocortins (Ucns), namely Ucn 1, Ucn 2 and Ucn 3 through activation of corticotropin-releasing factor (CRF) receptors, has been well characterized after injection into the brain but not in the periphery. We examined the role of CRF receptor subtype 2 (CRF₂) in the regulation of food intake using intraperitoneal (ip) injection of Ucns and the selective CRF₂ antagonist, astressin₂-B, and CRF₂ knockout (−/−) mice. Meal structures were monitored using an automated episodic solid food intake monitoring system. Ucn 2 (3, 10 or 30 μg/kg, ip) induced a rapid in onset, long lasting and dose-dependent decrease (38%, 66% and 86%, respectively at 4 h) of cumulative food intake after an overnight fast in mice. Ucn 3 anorexic effect was 10-times less potent. Astressin₂-B (30 or 100 μg/kg) injected ip, but not intracerebroventricularly, blocked the inhibitory effect of ip Ucn 1 and Ucn 2 (10 μg/kg). Fasted CRF_2−/− mice did not respond to ip Ucn 1 (10 μg/kg). Meal microstructure analysis of the 4-h re-feeding response to an overnight fast showed that Ucn 2 (10 μg/kg, ip) decreased meal size and duration, but increased meal frequency. In mice fed ad libitum, Ucn 2 (30 μg/kg) injected ip before the dark phase decreased the 4-h nocturnal meal size and duration without influencing meal frequency while the 10 μg/kg dose had no effect. These data indicate that Ucns, through peripheral CRF₂ receptor-mediated induction of satiation, inhibit the eating response to a fast more potently than the physiological nocturnal feeding in mice.     

Cholecystokinin octapeptide decreases intake of solid food in man

Cholecystokinin octapeptide (CCK-OP) was reported to decrease the intake of liquid food in lean and in obese man. This study investigated the effect of CCK-OP on the consumption of real life food, i.e., of standardized sandwiches. Sixteen young non-obese females and males participated, after an overnight fast, each in two experiments. After a basal 30 min, saline or CCK-OP, 1.5 or 3.0 Ivy Dog Units/kg body weight/15 min, was infused in random double blind fashion, while sandwiches were placed in front of the subjects. For the next three 15-min periods, the subjects were instructed to eat as much as they liked. In the first 15 min after 3.0 as well as 1.5 U CCK-OP/kg/15 min significantly fewer sandwiches (50 and 17 percent) were eaten than after saline (p less than 0.01 and p less than 0.05) and less hunger was reported (p less than 0.02 and p less than 0.05). Self-reported activation decreased only with 3.0 U CCK-OP (p less than 0.005). Reports of well-bring , electroencephalogram, heart rate, and respiration were not altered. The results support the notion that CCK is involved in the regulation of food intake.     

Nesfatin-1(30-59) but not the N- and C-terminal fragments, nesfatin-1(1-29) and nesfatin-1(60-82) injected intracerebroventricularly decreases dark phase food intake by increasing inter-meal intervals in mice

Nesfatin-1 is an 82 amino acid N-terminal fragment of nucleobindin2 that was consistently shown to reduce dark phase food intake upon brain injection in rodents. We recently reported that nesfatin-1(1-82) injected intracerebroventricularly (icv) reduces dark phase feeding in mice. Moreover, intraperitoneal injection of mid-fragment nesfatin-1 (nesfatin-1(30-59)) mimics the food intake-reducing effects of nesfatin-1(1-82), whereas N-terminal (nesfatin-1(1-29)) and C-terminal fragments (nesfatin-1(60-82)) did not. We therefore characterized the structure-activity relationship of nesfatin-1 injected icv to influence the dark phase meal pattern in mice. Mouse nesfatin-1(1-29), nesfatin-1(30-59), nesfatin-1(60-82) or vehicle was injected icv in freely fed C57Bl/6 mice immediately before the dark phase and food intake was monitored using an automated episodic feeding monitoring system. Nesfatin-1(30-59) (0.1, 0.3, 0.9 nmol/mouse) induced a dose-related reduction of 4-h food intake by 28%, 49% and 49% respectively resulting in a 23% decreased cumulative 24-h food intake compared to vehicle at the 0.3 nmol/mouse dose (p<0.05). The peak reduction occurred during the 3rd (-96%) and 4th hour (-91%) post injection and was associated with a reduced meal frequency (0-4h: -47%) and prolonged inter-meal intervals (3.1-times) compared to vehicle (p<0.05), whereas meal size was not altered. In contrast, neither nesfatin-1(1-29) nor nesfatin-1(60-82) reduced dark phase food intake at equimolar doses although nesfatin-1(60-82) prolonged inter-meal intervals (1.7-times, p<0.05). Nesfatin-1(30-59) is the active core of nesfatin-1(1-82) to induce satiety indicated by a reduced meal number during the first 4h post injection. The delayed onset may be indicative of time required to modulate other hypothalamic and medullary networks regulating nocturnal feeding as established for nesfatin-1.     

Effect of group size on feed intake, growth and feed efficiency of juvenile perch

Feed intake, growth and feed efficiency of juvenile perch Perca fluviatilis were significantly affected by group size, with fish in groups of 12 having lower feed intake, higher growth and higher feed efficiency than solitary individuals. The same trend was seen for fish in groups of 12 compared to fish in groups of four, and for fish in groups of four compared to solitary individuals, although the differences were not always significant. The high growth and low feed efficiency for fish in groups may be caused by benefits obtained from shoaling such as reduced levels of stress.     

Influence of hay particle size at different concentrate and feeding levels on digestive processes and feed intake in ruminants. 2. passage,digestibility and feed intake

In order to study the main effects of particle size, three ruminally fistulated cows (550 to 580 kg BW) were fed a constant low concentrate level (3.56 kg DM/d, 20% of total DMI) and a fibre‐rich hay (approximately 60% NDF in DM) in long (28.7 mm), chopped (9.2 mm) and fine ground (2.9 mm) form in a 3 x 3 Latin square design. In another three factorial experiment with 8 wethers (4 animals were ruminally fistulated, mean BW = 68 kg) the main effects and interactions of the above mentioned hay particle sizes at two concentrate levels (10.4 to 13.3% and 29.5 to 40.1% of DMI, resp.) and two intake levels (restricted and ad libitum) were investigated. In comparison to long hay (28.7 mm), feeding of chopped hay (9.2 mm) at low concentrate levels, increased not only the hay intake (7% in dairy cows and 13% in sheep) but also the intake of digestible organic matter (12% in dairy cows and 32% in sheep), due to an increase in the apparent digestibility of OM by 3.8% in dairy cows and 8.2% in sheep. Ad libitum feeding of fine ground hay in combination with low concentrate amount in the ration increased the passage rate in the hindgut and consequently the hay intake, but not the intake of DOM, due to a significant depression of digestibility, especially of fibre fractions (4 to 7% in dairy cows and 4.5 to 14% in sheep), in comparison to 28.7 and 9.2 mm hay particle sizes. The digestibility decreased significantly with restricted feeding of fine ground hay in sheep only in comparison to 9.2 mm particle size. A threefold increase of concentrate amount levelled out all effects of the particle size reduction. The effect of particle size was more pronounced in sheep than in dairy cows.     

Acute activation of ER alpha decreases food intake, meal size, and body weight in ovariectomized rats

Estradiol exerts many of its actions by coupling with two nuclear estrogen receptor (ER) proteins, ER alpha, and ER beta. While the acute, anorexigenic effect of estradiol appears to involve such a mechanism, the relative contributions of ERalpha and ERbeta are equivocal. To address this problem, food intake was monitored in ovariectomized (OVX) rats following acute administration of a selective ER alpha agonist (4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, PPT; dose range = 0-200 microg), a selective ER beta agonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN; dose range = 0-600 microg), and a physiological (4 microg) dose of estradiol benzoate (EB). While PPT-treated rats displayed dose-dependent decreases in daily food intake and body weight, neither of these measures was influenced by any dose of DPN. In addition, DPN failed to modulate the anorexigenic effect of PPT when the two ER agonists were coadministered. Meal pattern analysis revealed that the anorexigenic effect of 75 microg PPT (a dose of PPT that produced a similar decrease in daily food intake as 4 microg EB) was mediated by a decrease in meal size, not meal number. Thus, PPT, like EB and endogenous estradiol, decreases food intake by selectively affecting the controls of meal size. The finding that acute administration of 75 microg PPT failed to induce a conditioned taste aversion suggests that the anorexigenic effect of this dose of PPT is not secondary to malaise. Taken together, our findings demonstrate that selective activation of ER alpha decreases food intake, body weight, and meal size in the ovariectomized rat.     

Sire and liveweight affect feed intake and methane emissions of sheep confined in respiration chambers

Daily methane production and feed intake were measured on 160 adult ewes, which were the progeny of 20 sires and 3 sire types (Merino, dual-purpose and terminal) from a genetically diverse flock. All animals were housed in individual pens and fed a 50/50 mix of chaffed lucerne and oaten hays at 20 g/kg liveweight (LW), with feed refusals measured for at least 10 days before the first of three 22-h measurements in respiration chambers (RC). Feed was withdrawn at 1600 h on the day before each RC test to encourage the ewes to eat the entire ration provided for them in the RC. After the first 1-day RC test, the sheep were returned to their pens for a day, then given a second 1-day RC test, followed by another day in their pens, then a third RC test. After all animals had been tested, they were ranked according to methane emissions adjusted for feed intake in the RC and on the previous day, enabling 10 low and 10 high methane animals to be chosen for repeat measurement. No variation between sires nor consistent effects of LW on feed eaten (%FE, expressed as per cent of feed offered) was evident in the 10 days before the first RC measurement. However, significant differences between sires (equivalent to an estimated heritability of 41%) were identified for %FE during the 2^nd and 3^rd days of RC testing (2 and 4 days after the initial RC test). The analysis of all data showed that methane emissions in the RC were related to feed intake on the day of testing and the two previous days (all P<0.0005). Before correcting for feed intake on previous days, there was some variation between sires in methane yield, equivalent to an estimated heritability of 9%. Correction for feed intake on the 2 previous days halved the residual variation, allowing other effects to be detected, including effects of LW, twins reared as singles, test batch, RC and test-day effects, but estimated sire variation fell to zero. In order to avoid potential biases, statistical models of methane emissions in the RC need to consider potential confounding factors, such as those identified as significant in this study.     

Replacing soybean meal with flax seed meal: effects on nutrient digestibility,rumen microbial protein synthesis and growth performance in sheep

Flax seed meal (FSM) is rich in various nutrients, especially CP and energy, and can be used as animal protein feed. In animal husbandry production, it is a long-term goal to replace soybean meal (SBM) in animal feed with other plant protein feed. However, studies on the effects of replacing SBM with FSM in fattening sheep are limited. The aim of this experiment was to study the effects of replacing a portion of SBM with FSM on nutrient digestibility, rumen microbial protein synthesis and growth performance in sheep. Thirty-six Dorper × Small Thin-Tailed crossbred rams (BW = 40.4 ± 1.73 kg, mean ± SD) were randomly assigned into four groups. The dietary treatments (forage/concentrate, 45 : 55) were isocaloric according to the nutrient requirements of rams. Soybean meal was replaced with FSM at different levels (DM basis): (1) 18% SBM (18SBM), (2) 12% SBM and 6% FSM (6FSM), (3) 6% SBM and 12% FSM (12FSM) and (4) 18% FSM (18FSM). The rams were fed in individual pens for 60 days, with the first 10 days for adaptation to diets, and then the digestibility of nutrients was determined. There was no significant difference in DM intake, but quadratic (P < 0.001) effects on the average daily gain and feed efficiency were detected, with the highest values in the 6FSM and 12FSM groups. For DM and NDF digestibility, quadratic effects were observed with the higher values in the 6FSM and 12FSM groups, but the digestibility of CP linearly decreased with the increase in FSM in the diet (P = 0.043). There was a quadratic (P < 0.001) effect of FSM inclusion rate on the estimated microbial CP yield. However, the values of intestinally absorbable dietary protein decreased linearly (P < 0.001). For the supply of metabolisable protein, both the linear (P = 0.001) and quadratic (P = 0.044) effects were observed with the lowest value in the 18FSM group. Overall, the results indicated that SBM can be effectively replaced by FSM in the diets of fattening sheep and the optimal proportion was 12.0% under the conditions of this experiment.     

Identification of polymorphisms influencing feed intake and efficiency in beef cattle

Feed efficiency is an economically important trait in beef cattle. Net feed efficiency, measured as residual feed intake (RFI), is the difference between actual feed intake and the predicted feed intake required for maintenance and gain of the animal. SNPs that show associations with RFI may be useful quantitative trait nucleotides for marker-assisted selection. This study identified associations between SNPs underlying five RFI QTL on five bovine chromosomes (BTA2, 5, 10, 20 and 29) with measures of dry matter intake (DMI), RFI and feed conversion ratio (FCR) in beef cattle. Six SNPs were found to have effects on RFI (P < 0.05). The largest single SNP allele substitution effect for RFI was -0.25 kg/day located on BTA2. The combined effects of the SNPs found significant in this experiment explained 6.9% of the phenotypic variation of RFI. Not all the RFI SNPs showed associations with DMI and FCR even though these traits are highly correlated with RFI (r = 0.77 and r = 0.62 respectively). This shows that these SNPs may be affecting the underlying biological mechanisms of feed efficiency beyond feed intake control and weight gain efficiency. These SNPs can be used in marker-assisted selection but first it will be important to verify these effects in independent populations of cattle.     

A meta-analysis of nutrient intake,feed efficiency and performance in cattle grazing on tropical grasslands

It is essential to quantify the potential of tropical grasslands to allow significant feed efficiency for grazing livestock in controlled conditions such as at pasture. We conducted a quantitative analysis of published studies reporting the experimental results of average daily gains (ADG) and diet characteristics obtained specifically under grazing conditions (17 publications and 41 experiments), which have been less studied compared with controlled conditions in stalls. The database was analyzed to determine the average and range of values obtained for ADG (g/kg BW), dry matter digestibility, intake (DMI) and digestible DMI (DDMI, g/kg BW) and feed conversion efficiencies (FCE), as well as to predict the response of these parameters to the main strategies investigated in the literature – that is, mainly the stocking rate (SR) and the concentrate intake (CI). The ADG reached 1.2 kg BW per day and was directly linked to DDMI (ADG=−1.63+0.42 DDMI −0.0084 DDMI², n=90, r.m.s.e=0.584, R²=0.93). The DDMI, which was representative of the nutrient input, was driven mainly by DMI rather than dry matter digestibility, whereas these two parameters did not correlate (r=0.068, P=0.56). The average global FCE (0.11 g ADG/g DDMI) showed a greater association with the metabolic FCE (0.17 g ADG/g DMI) than the digestive FCE (0.62). The CI (g DM/kg BW) increased ADG (ADG=2376+CI 56.1, n=16, r.m.s.e.=441, R²=0.95). The SR expressed as kg BW/ha decreased the individual ADG by 1.19 g/kg BW per additional ton of BW/ha, whereas the global ADG calculated per ha increased by 0.57 per additional ton BW/ha. When the SR was expressed as kg BW/ton DM and per ha rather than as kg BW/ha, the impact on the individual ADG decreased by 0.18 or 0.86 g per additional ton BW/ha, depending on the initial BW of the cattle. These results provide a better view of the potential performance and feeding of cattle in tropical grasslands. The results provide an improved quantification of the relationships between diet and performance, as well as the overall quantitative impact of SR and supplementation.