In vitro gas methods for evaluation of feeds containing phytochemicals

Animal Feed Science and Technology 123–124 (2005) 291–302 In vitro gas methods for evaluation of feeds containing phytochemicals Harinder P.S. Makkar Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, PO Box 100, Wagramerstrasse 5, A-1400 Vienna, Austria Abstract Measurement of gas, a reflection of short chain fatty acids (SCFA), by in vitro gas methods provides information on effects of phytochemicals on rumen fermentation. However, to obtain complete information, it is necessary to measure other end products of fermentation, in particular microbial mass, especially for studies involving phytochemicals or bioactive moieties. Examples using tannins, saponins and alkaloids are discussed, highlighting the limited and often misleading information that can be obtained on potential effects of these compounds by measuring only gas production. In these studies, microbial protein was determined using purines or diaminopimelic acid as a marker, or by 15 N incorporation into microbes. In addition, the suitability of using the difference between apparently and truly degraded residues, as the estimate of microbial mass, for evaluation of tannin-free fibrous feeds, low-starch feeds, or when the objective is to investigate potential effects of phytochemicals other than tannins, is discussed. However for tannin-containing feeds, the presence of tannin–protein complexes in these residues, and/or solubilization of tannins from the substrate that do not contribute gas or microbial mass, produce artifacts in apparently and truly degraded values, rendering this approach invalid. For quantification of truly degraded substrate in tannin-rich samples, an indirect method based on level, and molar proportion, of SCFA and microbial mass is suggested. An approach for microbial mass determination, especially useful for tannin-rich samples, based on N balance is also discussed. An advantage of gas methods is that the fate of a phytochemical in the rumen can be investigated simultaneous with its effect on rumen fermentation. Such studies, using condensed tannins, alkaloids and saponins are presented. The in vitro gas method is a relatively simple and inexpensive tool to study potential effects, mechanisms of action and fates of phytochemicals in the rumen. The method Abbreviations: SCFA, short chain fatty acids; ATP, adenosine triphosphate; DAPA, diaminopimelic acid; PEG, polyethylene glycol; DM, dry matter; NDF, neutral detergent fibre; NDIN, neutral detergent insoluble N; IVDN, in vitro degradability of N; RNA, ribonucleic acid E-mail address: h.makkar@iaea.org. 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.06.003 292 H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 could also be used to screen for novel bioactive moieties such as those having antimethanogenic, antiproteolytic and antiprotozoal activity. © 2005 Elsevier B.V. All rights reserved. Keywords: Phytochemicals; Plant secondary metabolites; Tannins; Saponins; Bioactive compounds; Microbial protein; Gas method 1. Introduction In vitro methods to determine nutritional quality of feeds are important to nutritionists. These methods are less expensive, less time consuming and allow more control of experimental conditions than in vivo experiments. A number of gas measurement techniques and in vitro gas methods have been used by several groups to evaluate the nutritional value of feedstuffs (Getachew et al., 1998a). The in vitro gas method based on syringes (Menke et al., 1979) appears to be most suitable for use in developing countries where resources may be limited (Makkar, 2004), and it has been established in over 30 countries in the last 5 years through projects sponsored by FAO/IAEA. Increased interest in use of non-conventional feed resources has led to an increase in use of this technique, since gas production can provide useful data on digestion kinetics of both the soluble and insoluble fractions of feedstuffs. The ease of measuring fermentation end products makes this method more efficient than other in vitro methods for studies on phytochemicals, plant secondary metabolites and feed additives. For example, efficiency of microbial protein synthesis, and mechanisms of action of phytochemicals and other bioactive moieties including feed additives, can be studied with relatively inexpensive equipment, and in less time, since a large number of samples can be handled at one time. In addition, in vitro gas methods allow better monitoring of nutrient–phytochemical and phytochemical–phytochemical interactions. This paper reviews results of phytochemical–rumen microbe interactions, including principles and approaches that may be applied in studies of effects of other bioactive compounds, natural or synthetic, on rumen fermentation. 2. Importance of measuring microbial mass with gas measurement in studies on phytochemicals or other bioactive moieties The need to determine microbial mass while measuring gas production has been highlighted for evaluation of feed resources, particularly forages (Blümmel et al., 1997; Getachew et al., 1998a; Makkar, 2004). In this context, it is important to note that in vitro gas measurement reflects only short chain fatty acid (SCFA) production, and the relationship between SCFA and microbial mass production is not constant, probably because of variation of biomass production per unit ATP generated (Blümmel et al., 1997; Getachew et al., 1998a). In my laboratory, incubation of 0.6 mg/ml of various saponins in a gas method affected gas and microbial mass production to different extents (Table 1). For example, addition of Truly degraded substrate (mg) Gas (ml) Purines (␮mol) Efficiency of microbial protein synthesis ␮mol purine/ml gas ␮mol purine/mg truly degraded substrate Saponinsb Control Yucca saponins Quillaja saponins Acacia saponins 300.0 ± 10.3 a 320.7 ± 7.1 b 323.0 ± 1.0 b 297.6 ± 6 a 95.3 ± 0.9 a 91.2 ± 0.9 b 94.5 ± 1.9 a 83.5 ± 0.3 c 6.94 ± 0.36 a 9.11 ± 0.34 b 7.99 ± 0.34 c 8.38 ± 0.23 c 0.0728 0.0998 0.0845 0.1004 0.0231 0.0284 0.0247 0.0282 Alkaloidsc Control Lupanine Sparteine 264.7 ± 7.9 a 192.9 ± 9.2 b 232.9 ± 18.7 c 82.7 ± 1.2 a 59.1 ± 1.7 b 72.4 ± 1.1 c 6.28 ± 0.12 a 5.57 ± 0.08 b 6.0 ± 0.33 c 0.0759 0.0942 0.0828 0.0237 0.0289 0.0258 Means with different letters differ (P<0.05). a Data are from Makkar et al. (1998a,b), 500 mg hay (475 mg DM) was incubated in the syringes. b At 24 h of incubation. c At 18 h of incubation. H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 Table 1 Effects of saponins and alkaloids on gas production, purine content (index of microbial protein), truly degraded substrate and efficiency of microbial protein synthesisa 293 294 H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 Quillaja saponins did not affect gas production, but increased purine content and truly degraded substrate by about 7%. Since truly degraded substrate in an in vitro system can only lead to production of gas and microbial mass, this implies that all of the increase in truly degraded substrate by Quillaja saponins resulted in higher microbial protein mass (i.e., saponins increased efficiency of microbial protein synthesis). Had only gas production been measured, the conclusion could have been that Quillaja saponins had no effect on fermentation. In contrast, Acacia saponins decreased gas production, but increased microbial protein synthesis without affecting true digestibility. Thus saponins affected partitioning of degraded nutrients such that more microbial mass was produced at the cost of gas, and/or SCFA, production; again reflecting higher microbial efficiency. This higher microbial efficiency would not have been detected had only gas production been measured. The effect of Yucca saponins differed from those of Quillaja or Acacia saponins. Yucca saponins decreased gas, increased microbial protein synthesis and increased true digestibility (Makkar et al., 1998a), suggesting that measurement of gas only is not sufficient to describe the ‘true’ response of saponins. Alkaloids, namely lupanine and sparteine at a level of 5 mM in an in vitro gas method containing 500 mg of hay, decreased gas production and true degradability substantially, but the decrease in microbial mass was relatively lower (Makkar et al., 1998b). This resulted in higher efficiency of microbial protein synthesis, represented as ␮mol purines/ml gas or ␮mol purine/␮g truly degraded substrate (Table 1), which could be due to changes in microbial ecology by these alkaloids in a way that suppressed growth of those microbes having a high maintenance energy requirement. This is another example that demonstrates that a complete description cannot be obtained if only the gas produced in the in vitro gas method is measured. For studies on tannins, true degradability measured by digestion of syringe contents with neutral detergent (ND; Van Soest et al., 1991) does not represent true values. The presence of tannin–protein complexes in the fibre fraction obtained after digestion and/or solubilization of tannins from the substrate, and not contributing to the gas or microbial mass, distort these values (Makkar et al., 1997a; Getachew et al., 1998a; Makkar, 2004). Therefore, 15 N incorporation from buffer containing 15 N labeled ammonium bicarbonate, or measurement of purines or diaminopimelic acid (DAPA) has had to be used as an index of microbial protein production in studies where tannin-rich plants were incubated in an in vitro gas method. Studies in which effects of added purified quebracho tannins and tannic acid to hay, or a non-tannin-containing feed, have been investigated, and no artifact seems to be introduced in true degradability values determined using digestion with ND (Makkar et al., 1995a). In this study, an inverse relationship between 15 N incorporation and SCFA production per unit of truly degraded substrate occurred, again demonstrating that it is imperative to measure microbial mass. Polyethylene glycol, PEG (MW 4000 or 6000) is an inert compound having a high affinity for tannins, which it renders inactive (Makkar et al., 1995b). Hence it has been widely used in the in vitro gas method to study effects of tannins. Incubation of PEG with a tannin-rich Dichostachys cinerea leaf sample in an in vitro gas method enhanced gas production after 24 h incubation by 2.4, whereas increases in DAPA content, and 15 N incorporation, were only 1.3 and 1.2, respectively (Makkar et al., 1998b). This suggests higher availability of digestible nutrients, and of SCFA and microbial protein, on addition H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 295 of PEG, but lower efficiency of microbial protein synthesis. Similar conclusions have been drawn using a number of other tannin-containing feeds when incubated in low and high N media, and using purines as the marker for microbial protein (Getachew et al., 2000a). These observations also show that measurement of gas alone is not adequate to obtain a complete understanding of effects of tannins, or of a compound such as PEG, to alleviate effects of tannins, or any other phytochemical. 3. Determination of truly degraded substrate in tannin-rich diets As noted above, digestion of syringe contents with ND, used for conventional feeds, does not produce reliable results for tannin-rich feeds. An alternative approach could be determination of apparently fermented substrate (i.e., substrate required for production of gases, SCFA and water) followed by addition of the amount of microbial mass produced during fermentation. This approach is justified, since substrate truly degraded during fermentation in a closed in vitro gas system is converted into gases, SCFA, water and microbial mass. A good relationship (R2 = 0.94) existed between measured in vitro gas production and that calculated from SCFA for tannin-containing browses (Getachew et al., 2002). Addition of PEG to tannin-containing browses, when incubated in an in vitro gas method, increased gas production but did not affect the relationship between gas production and SCFA, which was: mmol SCFA = −0.00425 + 0.0222 (ml gas at 24 h) (1) Wolin’s stoichiometry (Wolin, 1960) is also valid for tannin-containing browses. Addition of PEG did not change molar proportions of SCFA, which were similar to those reported for high forage diets (acetate 0.72, propionate 0.23, butyrate 0.04). The browses examined (n = 39) had a wide range of crude protein (CP; 70–300 g/kg DM) and tannin as tannic acid equivalent (7–214 g/kg DM), suggesting that, for browses, SCFA production can be predicted from in vitro gas production (Getachew et al., 2002) using Eq. (1). The molar proportions of SCFA are influenced by diet, which is well documented for different classes of feeds. From the total amount of SCFA obtained from the gas value using Eq. (1), and molar proportions of each from the literature, it is possible to calculate (with reasonable accuracy) the total amount of substrate required for production of SCFA, gases and water (for calculations see Table 2 in Blümmel et al., 1997). Summing this value, and the value for microbial mass measured using a marker such as purines or DAPA is truly degraded substrate (mg) = total marker in syringe (mg) ×100/percent marker in rumen microbes 4. Determination of microbial mass using an N balance approach Using a gas method, two N balance approaches were used (Getachew et al., 2000a). 296 H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 4.1. First approach In a closed system, such as the in vitro gas method: Total N in syringe [before incubation] = microbial N + N bound to NDF (NDIN) +ammonia N in medium + N in amino acids and peptides [after incubation] (2) Since negligible amounts of amino acids and peptides are in the medium during fermentation (Krishnamoorthy et al., 1990), these can be ignored to give the equation: Microbial N in syringe (mg) = (total N at start of incubation, which is feed N + N in buffered medium) − NDIN + ammonia N in the medium, both at end of incubation (3) 4.2. Second approach Microbial N in syringe (mg) = N in apparently undegraded residue −NDIN, at end of incubation (4) The basis of this equation is that apparently undegraded residue after incubation is comprised of microbial, and undegraded feed, biomass. Then: Microbial mass (mg) = mg microbial N in syringe ×100/% N in the microbial fraction (5) The microbial fraction can be isolated, using differential centrifugation from incubations under similar conditions, freeze dried and subjected to Kjeldahl digestion for determination of N, which in a study was 77 g/kg DM (Makkar and Becker, 1999). To use these approaches, approximately 500 mg of feed is incubated in the in vitro gas method. After incubation, often to 24 h, when gas production is recorded, syringe contents are transferred to pre-weighed centrifuge tubes, and contents are centrifuged at 20,000 × g for 15–20 min at 4 ◦ C. The supernatant is pipetted and stored frozen until analyzed for ammonia N using a Kjeldahl method (Makkar and Becker, 1996). The pellet (i.e., apparently undegraded residue) is washed, lyophilized in the centrifuge tubes, and the tubes containing the pellet are weighed. The N in this residue is also determined. For determination of NDIN, syringe contents after incubation are digested with ND, filtered using crucibles, and the residue on the crucibles is dried and subjected to N analysis. The N in the syringe at the start of the incubation is added the amount of N in the buffered rumen liquor added to the syringe, and N in the buffered rumen liquor is determined similar to that for supernatant (Makkar, 2004). On incubation of six tannin-rich browses in the presence or absence of PEG, a strong correlation (R2 = 0.98) occurred between the two N balance methods. In addition, the pattern observed using these methods was similar to that observed with purines (Getachew et al., 2000a). H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 297 Microbial mass should be measured at a time when its rate of synthesis is maximum and actual degradation is minimal, as extensive microbial lyses could result in erroneous conclusions. The choice of methods for determination of microbial mass, and estimation of efficiency of microbial protein mass synthesis, and for investigation on the partitioning of nutrients to various products, depends on the facilities available and objective of the experiment. The in vitro gas method could be adapted to monogastrics to measure fibre degradability in the caecum or colon, but in this case selection of fibrous feeds should be for higher gas, or SCFA, and lower microbial protein production, since SCFA will be at least partially absorbed and provide energy, whereas microbial protein is not absorbed from the caecum or colon, and passes with faeces. In contrast, for ruminants in developing countries where forages tend to be deficient in CP, and CP-rich conventional sources (such as seed meals) are expensive, selection of feed components and diets should be driven by higher digestibility and a higher proportion of the digested feed being directed to microbial protein synthesis. Research on adaptation and use of the in vitro gas method to study fibre degradation by pig colon microbes is in progress in Colombia under an FAO/IAEA-sponsored project. 5. Tannin activity and in vitro gas methods The in vitro gas method, together with use of a tannin-inactivating agent such as PEG has been widely used to evaluate tannin activity in browses and tree leaves. The increase in gas production upon addition of PEG correlated (P<0.001, n = 37) with protein precipitation capacity of tannins (R2 = 0.76), total phenols and tannins (R2 = 0.76 for both total phenols and tannins), whereas the correlation (R2 = 0.41) with condensed tannins, as measured by the butanol–HCl method, was poor (Getachew et al., 2002). Based on relationships between these parameters, it was concluded that samples containing total phenols and tannin levels (g tannic acid equivalent/kg DM) up to 40 and 20, respectively, are not expected to precipitate protein or cause increases in gas production on addition of PEG to the in vitro gas production method and, therefore, are not likely to adversely affect ruminant productivity (Getachew et al., 2002). In in vivo studies conducted by five groups under a Joint FAO/IAEA Coordinated Research project, apparent digestibility coefficients of N correlated best with the proportional increase in gas on inactivation of tannins using PEG in the in vitro method for measuring gas production, total phenol and total tannins. However none of these values was as good predictor of feed intake, at least in the short term in vivo studies (Makkar, 2005). The in vitro gas method, coupled with use of PEG, provides useful information on the biological activity of tannins in the rumen and the whole gastrointestinal tract. In vitro gas methods have also been used to determine in vitro degradability of N (IVDN) from linear regression of ammonia N in the supernatant versus gas production observed on incubation of feeds for 24 h with graded levels of exogenous carbohydrate, such as starch or cellulose (Raab et al., 1983; Getachew et al., 1998b). This approach, with and without PEG, provides other useful information on tannin activity and protein degradability (i.e., the higher the proportional increase in IVDN on addition of PEG, the higher the tannin activity). In addition, the difference between IVDN values observed in the presence and absence of PEG indicates the amount of protein that can be potentially protected by tannins from 298 H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 degradation in the rumen. Whether this protein is fully available post-ruminally requires investigation (Getachew et al., 2000a). In the method of Raab et al. (1983) ammonia N disappearance with increasing energy (i.e., starch) availability was assumed to be due to its incorporation into microbial protein, and so the decline in ammonia N/ml of gas (i.e., slope being the rate of ammonia N uptake) was defined as the efficiency of microbial protein synthesis. This adaptation of the gas method was also used to investigate effects of PEG on tannin-containing feeds. The slope (i.e., rate of ammonia uptake) in the presence of PEG was lower than that in its absence (Makkar et al., 1998b), again suggesting that PEG decreases efficiency of microbial protein synthesis. The gas method has also been used to test a strategy to enhance this decrease in efficiency of microbial protein synthesis without sacrificing the PEG induced enhanced degradability of tannin-containing feeds. Getachew et al. (2001) tested a split application of PEG (i.e., an amount of PEG added in equal smaller portions to the incubation at different times during fermentation), which resulted in higher efficiency of microbial protein synthesis when compared to that observed with a one time addition of the same amount of PEG. This led to in vivo evaluation of a slow release form of PEG (i.e., PEG containing lick blocks) resulting in higher microbial protein supply post-ruminally, and higher performance in sheep when compared to feeding the same amount of PEG in one allocation (Ben Salem et al., 2004). 6. Fate of condensed tannins, saponins and alkaloids in the rumen In vitro gas methods with a phytochemical-containing feed, or a purified phytochemical, could also be used to study the fate of the phytochemicals. By collecting aliquots at different time intervals from the incubation medium of a gas method, the phytochemical (or added compound) could be monitored and investigations on its degradation by rumen microbes undertaken simultaneous to the study of their effects on rumen fermentation. Using this approach, no degradation of condensed tannins (Makkar et al., 1995a), or of lupanine and sparteine alkaloids (Aguiar et al., 1998), by rumen microbes occurred. In contrast, rumen microbes degraded Quillaja saponins to 16, 45 and 100% at 9, 12 and 24 h of incubation, respectively (Makkar and Becker, 1997). Rumen microbes also degraded Sapindus rarak saponins, and one degradation product identified at 12 h was hederagenin, which subsequently disappeared (Wina, 2005). Degradation pathway(s), as well as the metabolites produced and their physiological responses are not yet known. 7. Nutrient–phytochemical, phytochemical–phytochemical or nutrient–nutrient interactions in rumen Gas methods using 15 N-labeled rumen microbes have also been used to study influences of tannins and pectin on attachment of microbes to substrates. Although a positive linear relationship was found between gas production and microbial attachment at 24 h of incubation (R2 = 0.84, P<0.001), microbial attachment was not always well correlated with gas production when expressed as gas produced per unit of adherent 15 N-microbes. Furthermore, H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 299 tannins reduced attachment of rumen microbes to cellulose, and addition of PEG and pectin to the incubations improved attachment and gas production, but improvement with pectin was lower than that of PEG (Bento et al., 2005a). A study on interactions of tannins and pectin focusing on efficiency of microbial protein synthesis and gas production also demonstrated tannin-inactivating effects of pectin, although this effect was much lower than that with PEG (Bento et al., 2005b). These observations suggest that there could be considerable interaction between non-starch-polysaccharides and tannins, and that these interactions may influence functionality of microbes in the gastrointestinal tract and nutrient availability to animals. A gas method was used to evaluate phytochemical–phytochemical (tannin–saponin) interactions (Makkar et al., 1995a), and it was observed that simultaneous presence of these two phytochemicals did not counteract effects of either tannins or saponins. This method was also employed to evaluate the interaction between basal and supplementary diets, by incubating the basal diet and supplementary diet separately, as well as in combination, and monitoring gas production and other parameters (Getachew et al., 2000b; Blümmel et al., 1999, 2003). 8. Effects of bound tannins on rumen fermentation Incubation of NDF, or other fractions of a feed, after extraction of soluble tannins using aqueous organic solvents, with and without PEG, in a gas method allows evaluation of the nutritional importance of unextractable (i.e., bound) tannins. Addition of PEG during incubation of tannin-rich NDF led to increased gas production, suggesting that tannins released as a result of NDF degradation by rumen microbes are biologically active, and have the potential to influence rumen fermentation (Makkar et al., 1997b). Similar results were obtained on incubation of tannin-rich browses made free of extractable tannins by repeated use of acetone/distilled water (7:3, v/v) (Makkar et al., 1997b). 9. Saponins and rumen ecology In addition to investigating effects of S. rarak fruit saponins on end products of fermentation, including gas, SCFA and microbial mass, microbial ecosystem structure and activity have also been investigated. Using the conventional technique of counting protozoa, and the advanced one of using the membrane hybridization or breakdown of 14 C-leucine-labelled Prevotella byrantii, a considerable decrease in protozoal numbers by saponins has been recorded (Ningrat et al., 2002; Wina et al., 2005a,b). At a level of 1 mg of S. rarak saponin/ml of medium, or more, eukaryotic RNA (18S rRNA band) was not detected, suggesting almost complete removal of protozoa by these saponins (Wina et al., 2005a,b). Using various ribosomal RNA-targeted probes, S. rarak saponins at l mg/ml did not decrease methanogens, which did occur at 4 mg saponin/ml, suggesting a higher sensitivity of protozoa to saponins versus methanogens. Fibrolytic microbes responded differently, with no effect on Fibrobacter sp. up to a level of 4 mg saponin/ml, and a negative effect on Ruminococcus albus and R. flavefaciens at levels higher than 1 mg/ml. Similarly, these saponins decreased the fungi Chytriodiomycetes (Wina et al., 2005b). But, 300 H.P.S. Makkar / Animal Feed Science and Technology 123–124 (2005) 291–302 using a similar approach, no effect of Sesbania pachycarpa saponins was observed on fungi (Muetzel et al., 2003). The extent of changes in the microbial population observed in all these studies differed at different times of incubation. In vivo experiments using S. rarak fruit saponins at levels of 0.24, 0.48 and 0.72 g/kg body weight resulted in changes similar to those observed in vitro and increased body weight gain (Wina, 2005). Most of the studies on effects on fermentation, mechanism of action and fate of added compounds have been completed by addition of tannins to the in vitro gas method, by incubation of tannin-containing feeds with and without PEG, or by addition of saponins or alkaloids. Nevertheless, the approaches and principles could be applied for similar rumen fermentation studies on any bioactive moiety, be it natural or synthetic. For example, an in vitro gas method is being used in my laboratory to screen a large number of plants for antimethanogenic, antiproteolytic and antiprotozoal activities, and to study effects on rumen fermentation of the most promising. In addition, this method, along with the real time polymerase chain reaction, is being used in an FAO/IAEA Research Project to study effects of various phytochemicals and phytochemical-containing plants on methanogens, protozoa, fibre-degrading bacteria and fungi. 10. Conclusions In vitro gas methods are relatively simple and inexpensive tools to study potential effects, mechanisms of action, and fates of phytochemicals in the rumen. A potential application of the gas method is to investigate composition (e.g., protein, lipid, carbohydrate and mineral) of microbes as affected by bioactive moieties. In vitro gas methods, when used for evaluation of feeds free of, or containing, phytochemicals, requires validation against in vivo nutrient utilization and animal performance, which has largely been neglected to date. References Aguiar, R., Wink, M., Makkar, H.P.S., Blümmel, M., Becker, K., 1998. Effects of Lupinus alkaloids on in vitro rumen fermentation and their fate in the rumen. 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