METHIONINE REQUIREMENT OF THE FINISHING PIG ABSTRACT

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1 METHIONINE REQUIREMENT OF THE FINISHING PIG T. K. Chung~ O. A. Izquierdo, 1, K. Hashimoto" and D. H. Baker 3 University of Illinois 4, Urbana ABSTRACT Two pig experiments were conducted using a methionine (Met)-deficient feather mealcorn-soybean meal basal diet (13% CP; 3,400 kcal ME/kg diet,.126% Met,.456% cystine) supplemented with an amino acid mixture (lysine, tryptophan, histidine, threonine and phenylalanine) to determine the Met requirement of finishing pigs between 50 and 80 kg live weight. Using young chicks in a Met bioavailability growth assay and cecectomized adult cockerels in a Met digestibility assay, the Met-deficient basal diet was found to contain.115% bioavailable and. 110% digestible Met. These results gave a bioavailability estimate (relative to DL-Met set at 100%) of % and a true digestibility estimate of 87, % for Met in the basal pig diet. In Exp. t, 2I crossbred pigs averaging 61 kg initially were individually fed diets containing. 115,. 165 or.215 % bioavailable Met for 21 d. Average daily gain and gain:feed ratio increased quadratically (P <.05) as level of Met increased. In Exp. 2, 30 crossbred pigs averaging 53 kg were individually fed diets containing.115,.135,.155,.175 or.195% bioavailable Met for 27 d. Daily gain and gain: feed ratio responded linearly (P <.01) as Met level increased. Based on the results of Exp. 2, the bioavailable Met requirement of finishing pigs in the weight range 50 to 80 kg was estimated to be.182% of the diet. Assuming an 88% bioavailability of Met in commercial diets based on corn and soybean meal, the total Met level needed in practice would be.207%. If 55% of the finishing pig's sulfur amino acid need can be furnished by cystine, the total sulfur amino acid requirement would be.45% of the diet. (Key Words: Pigs, Methionine, Requirements.) J. Anim. Sci : Introduction The literature on sulfur amino acid (SAA) needs of finishing swine is confusing and provides little definitive information on which to set a minimum requirement (NRC, 1988). The study of Brown et al. (1974) indicated a SAA requirement no greater than.17% of the 1present address: Purina Italia, Milano, Italy. 2present address: Ajinomoto Co., Inc. 5-8, Kyobashi, 1 Chorne, Chuo-Ku, Tokyo 104 Japan. 3To whom reprint requests should be addressed. 4Dept. of Anim. Sci., supported in part by the Illinois Agric. Exp. Sta. (Project No ), by Moorman Mfg. Co., Quincy, IL., and by the Commodity Credit Corp., Washington, 1)12. Appreciation is expressed also to Degussa Corp., Allendale, NJ and Biokyowa Corp., St. Louis, MO for their contribution of amino acids. Received October 26, Accepled February 24, diet for maximum N retention; work by Allee and Trotter (1974) suggested that finishing pigs need less than.24% SAA for maximal growth and N retention. More recently, however, Roth and Kirchgessner (1987) completed a trial in which pigs in the 60- to 90-kg weight range responded to graded increments of methionine (Met) supplementation. Their results suggested that both weight gain and efficiency of feed utilization reached a plateau at.39% SAA. Because crystalline DL-Met is priced reasonably and is available readily, it is important to establish whether and under what conditions supplemental Met might elicit a response in finishing swine. To answer this question, however, more knowledge of the Met and SAA requirement and bioavailability is necessary. The primary objective of this investigation was to gain such knowledge. 2677

2 2678 CHUNG ET AL. TABLE 1. COMPOSITION OF THE METHIONINE-DEFICIENT PIG DIET a Ingredient % Cornstarch to Sucrose Feather meal Corn Soybean meal (dehulled) 7.40 L-lysine-HCl.41 L-histidine-HC1.H20.18 L-tryptophan.02 L-threonine.06 L-phenylalanine.13 Corn oil 2.00 Dicalcium phosphate (feed grade) 2.40 Ground limestone (feed grade).31 Trace mineral mix b.35 Vitamin mix c.10 Ethoxyquin (50 mg/kg diet) + nc.ontained 13% CP,.126% Met,.456% cystine and 3,400 kcal MF_/kg. b Trace mineral mix provided per kg of diet: Se,. 10 mg; I,.35 rag; Cu, 8.0 rag; Mn, 20.0 rag; Fe, 90.0 rag; Zn, mg; Co,.75 mg; NaCL, 2.73 g. c Vitamin mix provided per kg of diet: vitamin A, 6,600 IU; vitamin I23,660 IU; vitamin E, 44 IU; vitamin K, 4.0 mg; riboflavin, 2.2 rag; d-pantothenic acid, 13.1 rag; niacin, 33.0 rag; choline cldodde, 330 rag; vitamin B12, 35.1 ~tg. Experimental Procedure Diets. The basal diet (Table 1) was formulated to meet all NRC (1988) nutrient requirements of finishing pigs with the exception of Met. The diet contained 3,400 kcal ME/kg (NRC, 1988), 13% protein (determined by macro-kjeldahl),.126% Met and.456% cysfine. The Met and cystine levels of the intact protein sources (feather meat, corn, soybean meal) were determined individually by ionexchange chromatography of acid hydrolysates of performic acid oxidized subsamples. A slight modification of procedures described by Moore (1963) was used in the performic acid oxidation step. Excess performic acid was removed by lyophilizafion without the use of hydrogen bromide. After the Met and cystine were oxidized to Met sulfone and cysteic acid, respectively, and following removal of excess performic acid, samples were subjected to 6 N 5Beckman Model 119CL Amino Acid Autoanalyzer, Beckman Instrumems Co., Palo Alto, CA. 6Feather meal was analyzed for lanthionine in samples that had not been preoxidized with performic acid and found to contain 1.13%. HCI acid hydrolysis for 24 h and then Met sulfone and cysteic acid were quantified by chromatographic analysis 5. During oxidation, any lanthionine present (e.g., feather meal is known to contain lanthionine) was converted to lanthionine sulfone, a compound that coelutes with cysteic acid under our chromatographic conditions. Thus, cystine analytical values were corrected for lanthionine 6 and therefore represent cystine per se. Lanthionine has a theoretical cystine yield (after metabolic conversion of lanthionine to cysteine, pyruvate and ammonia) of 58% (Robbins et al., 1980; Baker et al., 1981). Feather meal was found to contain.59% Met and 3.83% cysfine; corn,.18% Met and.21% cystine; and soybean meal,.66% Met and.70% cystine. The basal pig diet (Table 1) was supplemented with lysine, threonine, histidine, tryptophan and phenylalanine to ensure that these amino acids would be adequate (NRC, 1988) such that bioavailable Met would be the single rate-limiting factor in the diet. All other dietary nutrients were calculated to either meet or exceed the nutrient needs of the finishing pig (NRC, 1988). Bioavailable Met and true digestible Met in the basal pig diet were estimated using chick growth and cecectomized cockerel digestibility assays, respectively (Parsons, 1985). Chick Bioavailability Growth Assay. Sixty crossbred male chicks with an average initial weight of 70 g were used in a 9-d chick bioavailability growth assay to determine bioavailable Met in the feather meal-cornsoybean meal protein blend contained in the Met-deficient basal diet (Table 1) used in the pig trials. Procedures for conducting the growth assay have been outlined previously (Sasse and Baker, 1973; Baker et al., 1981; Izquierdo et al., 1988). Triplicate groups of five chicks were fed each of the experimental diets from d 8 to d 17 posthatching. The standard curve procedure (Sasse and Baker, 1973; Izquierdo et al., 1988) was used for bioavailability assessment wherein a Metdeficient, cystine-adequate crystalline amino acid basal diet (Baker et al., 1979) was supplemented with 0,.05 and.10% DL-Met, or 15% of the feather meal-corn-soybean meal protein blend (36.5% feather meal, 36.5% corn, 27% soybean meal). Standard curve analysis (Table 2), based on the assumption of 100% bioavailability of DL-Met, indicated that

3 METHIONINE REQUIREMENT 2679 TABLE 2. BIOAVAILABILITY OF METHIONINE IN THE FEATHER MEAL-CORN-SOYBEAN MEAL PROTEIN BLEND DETERMINED BY A CHICK GROWTH ASSAY a Gala/feed, Suppl. Met Diet Wt, gc g/kg intake, nag 1. Basal diet b As % DL-Met As % DL-Met As % protein blend d r Pooled SEM 3 23 NA f adata represent means of triplicate groups of five chicks during the period 8 to 17 d posthatching; average initial weight was 70 g. bthe basal crystalline amino acid diet (Baker et al., 1979) contained.10% DL-Met and.35% L-cy~ine. ~ of chick gain (g) on supplemental Met intake (rag) was Y = X (r 2 =.98), where Y represents total chick gain and X represents supplemental Met intake. d Contained 36.5% feather meal, 36.5% corn and 27% soybean meal. ecalculated bioavailable Met intake (calculated from the standard curve, diets 1 to 3). This value divided by intake oftbe protein blend (i.e., 11.7 g) yielded a bioavallable Met level of.420% for the protein blend. fnot available due to heterogeneous variance. the protein blend contained.420% bioavailable Met. When translated into a bioavailable Met concentration in the basal pig diet (27.4% of.420), a value of.115% is obtained. Cecectomized Cockerel Digestibility Assay. Five adult, cecectomized Single Comb White Leghorn cockerels with an average initial weight of 2,350 g were used to determine digestible Met in the feather meal-cornsoybean meal protein blend. Following a 24-h fast, three cockerels were force-fed (via crop intubation in a single dose) 30 g of the protein blend (36.5% feather meal, 36.5% corn, 27% soybean meal), and two fasted cockerels served as negative controls for the calculation of endogenous Met excretion. Birds were kept in individual cages with raised wire floors on a 16-h illumination period, and water was available ad libitum during the excreta collection period. Details of cecectomy and procedures used in the 48-h excreta collection assay have been described elsewhere (Parsons, 1985; Hirakawa and Baker, 1986). Excreta voided during the subsequent 48-h were collected and processed according to the procedures described previously (Sibbald, 1979; Hirakawa and Baker, 1986). True Met digestibility of the protein blend was found to be 87.0% (Table 3). Thus, the pig basal diet was estimated to contain.110% digestible Met (.126 x 87.0%). Experimental Protocol for Pig Trials. A preliminary trial indicated that the Met-deficient feather meal-corn-soybean meal basal TABLE 3. METFIIONINE DIGESTIBILITY IN THE FEATHER MEAL-CORN-SOYBEAN MEAL PROTEIN BLEND DETERMINED BY THE CECECTOMIZED COCKEREL DIGESTIBILITY ASSAY a Met Met Met true intake, excreted, digestibility, Treatment mg mg d % Fasted 0 11 Protein blend c e adata represent means of two Single Comb White Leghorn cockerels for the fasted treamaent and three Single Comb White Leghorn cockerels for the protein blend treatment. Average initial weight of the birds was 2,350 g. b([met intake - (Met excreted - Met endogenous)]/met intake) 100%. CContained 36.5% feather meal, 36.5% corn, and 27% soybean meal; 30 g of the mixture was force-fed via crop intubation. dmet excreted during a 48-h collection period. ese of the true digestibility estimate was 2.2.

4 2680 CHUNG ET AL. TABLE 4. PERFORMANCE OF PIGS FED THE METHIONINE- DF.,FICIENT FEATHER MEAL-CORN-SOYBEAN MEAL DIET SUPPLEMENTED WITH GRADED LEVELS OF DL-METHIONINE (EXP. 1) a Bioavailable Daily Gain/feed, Diet Met, % gain, gb g/kgb 1. Met-deficient diet As % DL-Met As % DL-Met Pooled SEM adata represent mean values of seven individually fed barrows for a period of 21 d; average initial weight was 61 kg. bquadratic effect (P <.05). diet was capable of supporting excellent growth in finishing pigs when fortified with Met. Moreover, without Met fortification, growth rate was reduced markedly, indicating that a broad Met response range was possible. Two experiments were conducted with 51 crossbred (Duroc x Hampshire x Landrace) barrows averaging 61 kg initial weight in Exp. 1 and 53 kg initial weight in Exp. 2. From blocks based on ancestry and weight, individual pigs were assigned randomly to experimental treatments and pens. Each experimental diet was fed individually to seven pigs for 21 d in Exp 1 and to six pigs for 27 d in Exp. 2. Pigs were kept on a 24-h constant light schedule in an environmentally controlled finishing building. In both experiments, pigs were housed individually in 2.1-m x 1.4-m slatted-floor pens containing a nipple waterer and a selffeeder. In Exp. 1, the Met-deficient feather meal-corn-soybean meal basal diet (.115% bioavailable Met) was supplemented with 0,.05 and.10% DL-Met, resulting in concentrations of.115,.165 and.215% bioavailable Met. In Exp. 2, narrower increments of bioavailable Met (i.e.,. 115,. 135,. 155,. 175 and.195%) were employed for the purpose of further refining the requirement estimate. Pigs were allowed to consume feed and water ad libitum throughout the experimental period. Prior to initiating and terminating each experiment, pigs were fasted overnight and weighed the following morning. Weight gain and feed intake were monitored weekly. Statistical Analysis. Data from the pig trials were subjected to analysis of variance procedures appropriate for randomized completeblock designs. Orthogonal single degree-offreedom comparisons were made to test for Met linear and quadratic effects (Steel and Torrie, 1980). Data from Exp. 2 were subjected to broken-line analysis and also to curvilinear exponential analysis, in which the abscissa of the point whose ordinate was 90% of the upper asymptotic value represented the requirement estimate (Robbins et al., 1979). The chick bioavailability growth assay was analyzed using linear regression analysis and standard-curve methodology. The cecectomized cockerel digestibility assay was subjected to analysis of variance procedures appropriate for a completely randomized design. Results Experiment 1. The results of Exp. 1 are shown in Table 4. Supplementing the Metdeficient diet with 0,.05 and.10% DL-Met resulted in quadratic (P <.05) responses in daily gain and gain:feed ratio. The results of this trial suggested that maximal rate and efficiency of weight gain would likely occur at a bioavailable Met level between.165 and.215% of the diet. Experiment 2. Daily gain and gain:feed ratio increased linearly (P <.01) with each increase in Met supplementation (Table 5). The increase in weight gain between.175 and.195% bioavailable Met, however, was lower (P <.01) than that observed for increments below.175%. Broken-line analysis of the gain data estimated a breakpoint plateau at.182% bioavailable Met. The asymptotic fit predicted that 90% of maximal weight gain would occur at.188% bioavailable Met. Discussion Critical features of experiments designed to establish dietary requirements for amino acids have been discussed previously (Baker, 1986; Izquierdo et al., 1988). Baker et al. (1981) indicated that feather meal protein was frankly deficient in Met, which was found to be the first-limiting amino acid, Lysine, histidine and

5 METHIONINE REQUIREMENT 2681 TABLE 5. PERFORMANCE OF PIGS FED THE METHIONINE-DEFICIENT FEATHER MEAL-CORN-SOYBEAN MEAL DIET SUPPLEMENTED WITH GRADED LEVELS OF DL-METHIONINE (EXP. 2) a Bioavaitable Daily Gain/feed, Diet Met, % gain, gb g/kgb 1. Met -deficient diet As % DL-Met As % DL-Met As % DL-Met As % DL-Met Pooled SEM adata represent mean values of six individually fed barrows for a period of 27 d. Average initial weight was 53 kg. blinear effect (P <.01). tryptophan, however, also were found to be limiting. Hence, feather meal was a key ingredient in the Met-deficient basal diet. This diet proved to be responsive to Met supplementation, and it also allowed excellent weight gains of between 860 and 990 g when fortified adequately with Met. Curve fitting procedures predicted bioavailable Met requirements of. 182% (fitted brokenline) and.188% (curvilinear fit, 90% of upper asymptote). Careful examination of data in Table 5 suggests that maximal gain and gain: feed probably would occur at a Met level above.175% but below.195%. Thus, the magnitude of response between.155% and.175% was substantially greater than that between.175% and.195% Met (Table 5), which suggests that maximum gain and gain: feed would probably occur between the latter levels. For this reason, the broken-line estimate of.182% Met would seem defendable as a tentative requirement estimate, although admittedly the data in Table 5 do not lend themselves to precise selection of maximal response. The bioavailable Met requirement estimate of.182% translates into a bioavailable SAA requirement estimate of.40% if one assumes that 55% of the SAA requirement can come from cystine (Baker et al., 1969; Roth and Kirchgessner, 1987) 7. In the latter study, Met bioavailability apparently was not considered, nor was performic acid oxidation of the basal diet indicated as having been performed prior to SAA chromatographic analysis. Their basal 7This assumes that.22% of the (plethora of) cystine in the basal diet was utilized along with.18% Met for maintenance and tissue growth. diet (based on wheat, barley, manioc, meat and bone meal, fish meal, field beans and gelatin) was listed as containing.16% Met and.16% cystine. The diet responded (equally) to a small addition (.035%) of either DL-Met or L- cystine. Thus, assuming that analytical values of their basal diet were correct, and ignoring SAA bioavailability considerations, one arrives at a cystine replacement value of 55%. That the cystine replacement value may be higher for 60- to 90-kg pigs than the 50% value assumed for younger pigs (NRC, 1988) seems logical in that older pigs would be expected to have a higher SAA maintenance requirement than young pigs, and the total SAA requirement would be dominated more by cystine than by Met (Baker et al., 1966). Maximal growth and feed efficiency in the Roth and Kirchgessner (1987) study was achieved at.39% SAA (.23% Met +.16% cystine). Their estimate agrees well with our estimate of.40%, although our estimate is for bioavailable SAA, whereas theirs is for total dietary SAA. It should also be noted that the weight ranges covered in the two studies were similar. If we were to assume that 88% of the SAA in a corn-soybean meal diet is bioavailable to the pig (NRC, 1988), our.40% bioavailable SAA requirement estimate extrapolates to.45% on a corn-soybean meal basis. This estimates is considerably higher than the recent NRC (1988) SAA requirement estimate of.34%, although in their defense, they, like the British ARC (1981) committee, admitted that data were unsatisfactory for estimating SAA requirements of finishing swine. With a SAA requirement of.45% and a lysine requirement of.60% (NRC, 1988), the ratio of the SAA needed to the lysine needed is 75% for finishing pigs fed a corn-soybean meal diet. This ratio of SAA to lysine

6 2682 CHUNG ET AL. requirements for finishing swine is virtually identical to the ratio for young broiler chicks. With a purified amino acid diet, the bioavailable lysine requirement of chicks is.80% of the diet (Baker, 1978), whereas the bioavailable SAA requirement is.60% (Halpin and Baker, 1984). With a corn-soybean meal starter chick diet, NRC (1984) has estimated the lysine requirement at 1.20% of the diet and the SAA requirement at.93%. These requirement ratio calculations suggest that both pigs and poultry require SAA in the same proportion to lysine. At least three factors may have contributed to the low SAA requirement estimates of Brown et al. (1974) and Allee and Trotter (1974). Neither of those investigations considered SAA bioavailability in their dietary sources of SAA. Moreover, those studies did not use performic acid oxidation for SAA analysis in their basal diets. It is well established that Met and cystine are partially destroyed during acid hydrolysis, resulting in underestimation of many tabular values of Met and cystine in feed ingredients 8. Still another factor involves the performance level of the pigs employed in SAA requirement studies. Although not well documented, finishing pigs gaining weight at 800 to 900 g/d may require higher levels of SAA than those gaining at slower rates. Thus, pigs in both the recent German study (Roth and Kirchgessner, 1987) and those used herein gained in the 900 g/d category. Moreover, leaner pigs are used today than was the case 15 yr ago, and this, too, would have the effect of increasing SAA and all other amino acid requirements. Two methods were used to estimate Met efficacy in the basal diet used here: growth assay bioavailability in young chicks and digestibility in cecectomized adult cockerels. We had hoped that because cecectomy removes most of the lower gut fermentative activity in birds (Parsons, 1985), digestibility estimates would agree closely with growth assay bioavailability estimates. As shown in Tables 2 and 3, Met bioavailability was estimated at 91.3% + 2.5% and Met true 8Our studies have indicated that SAA analyses not employing preoxidation procedures underestimate Met by 15 to 30% (depending on which ingredient or diet is analyzed) and cystine by 20 to 40%. digestibility was estimated at %. Determining which of the two Met efficacy estimates is better is problematic. It would be surprising if bioavailability exceeded true digestibility! Nonetheless, our bioavailable Met requirement estimate of.182% extrapolates to.207% total Met when the 88% SAA bioavailability factor (NRC, 1988) in cornsoybean meal diets is considered. Implications The Met as well as the total SAA requirement of finishing swine is higher than that previously assumed. A realistic SAA requirement is important to proper formulation of experimental diets for pigs. Moreover, whether conceptual or real, studies designed to evaluate the ideal protein concept for use in pigs require that amino acid requirement estimates be as accurate as possible. With a SAA requirement of.45%, swine fed f'misher diets based on corn and soybean meal would not be expected to respond to SAA supplementation. Finishing swine fed diets containing barley or wheat in place of corn, however, could respond to Met addition. Literature Cited Allee, G. L. and R. M. Trotter Sulfur amino acid requirement of the finishing pig. J. Anim. Sci. 39:974 (Abstr.). ARC Nutrient Requirement of Pigs. Commonwealth Agricultural Bureaux-Agricultural Research Council, Slough, UK. Baker, D. H Nutrient bioavallability in feedstuffs: methodology for determining amino acid and B- vitamin availability in cereal grains and soybean meal. Proc. Georgia Nutr. Conf. pp Baker, D. H Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr. 116:2339. Baker, D. H., D. E. Becker, H. W. Norton, A. H. Jensen and B. G. Harmon Quantitative evaluation of the tryptophan, methionine and lysine needs of adult swine for maintenance. J. Nutr. 89:441. Baker, D. H., R. C. Blitenthal, K. P. Boebel, G. L. Czamecki, L. L. Southem and G. M. Willis Protein-amino acid evaluation of steam-processed feather meal. Poult. Sci. 60:1865. Baker, D. H., W. W. Clausing, B. G. Harmon, A. H. Jensen and D. E. Becker Replacement value of cystine for methionine for the young pig. J. Anita. Sci. 29:581. Baker, D. H., K. R. Robbins and J. S. Buck Modification of the level of histidine and sodium bicarbonate in the Illinois crystalline amino acid diet. Poult. Sci. 58:749. Brown, H. W., B. G. Harmon and A. H. Jensen Total sulfur-containing amino acids, isoleucine and tryptophan requirements of the finishing pig for maximum

7 METHIONINE nitrogen retention. J. Anita, Sci. 38:59. Halpin, K. M. and D. H. Baker Selenium deficiency and n.anssulfuration in the chick. J. Nun.. 114:606. Hirakawa, D. A. and D. H. Baker Assessment of lysine bioavailability in an intact protein mixture: comparison of chick growth and precision-fed rooster assays. Nun'. Res. 6:815. lzquierdo, O. A., K. J. Wedekind and D. H. Baker Histidine requirement of the young pig. J. Anim. Sci. 66:2886. Moore, S On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235. NRC Nutrient Requirements of Poultry. (Sth Rev. Ed.). National Academy Press, Washington, DC. NRC Nutrient Requirements of Swine. (9th Rev. Ed.). National Academy Press, Washington, DC. Parsons, C. M Amino acid availability in feedstuffs for poultry and swine. In: Baker, D. H., Parsons, C. M. (Ed.) Recent Advances in Amino Acid Nutrition. Ajinomoto Publ. Co., Tokyo, Japan. REQUIREMENT 2683 Robbins, K. R., J. R. Finley and D. H. Baker Studies on the utilization of lysinoalanine and lanthionine. J. Nutr. 110:907. Rohhins, K. R., H. W. Norton and D. H. Baker Estimation of nutrient requirements from growth data. L Nun.. 109:1710. Roth, F. X. and M. Kirchgesaner Biological efficiency of dietary methionine or cystine supplementation with growing pigs: a contribution to the requirement for S-containing amino acids. J. Anita. Physiol. Anita. Nun.. 58:267. Sasse, C. E. and D. H. Baker Availability of sulfur amino acids in corn and corn gluten meal for growing chicks. J. Anita. Sci. 37:1351. Sibhaid, I. R A bioassay for available amino acids and true metabolizable energy in feedsmffs. PouR. Sci. 58: 668. Steel, R.G.D. and J. H. Torrie Principles and Procedures of Statistics: A Biomen.ical Approach (2rid Ed.). McGraw-Hill Book Co., New York.