Dietary guanidino acetic acid is an efficacious replacement for arginine for young chicks 1

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1 Dietary guanidino acetic acid is an efficacious replacement for arginine for young chicks 1 R. N. Dilger,* 2 K. Bryant-Angeloni,* 3 R. L. Payne, A. Lemme, and C. M. Parsons * * Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801; Evonik Degussa Corporation, 1701 Barrett Lakes Blvd., Suite 340, Kennesaw, GA 30144; and Evonik Industries AG, Feed Additives Division, PO Box 1345, Hanau, Germany ABSTRACT Guanidino acetic acid (GAA) is synthesized in the liver and kidney from Arg and Gly and subsequently methylated by S-adenosylmethionine to form creatine. Four bioassays were carried out to determine the capacity of GAA to serve as a dietary replacement for Arg for growing chicks. Broiler chicks were fed Arg-deficient dextrose-casein (0.88% Arg) or corn-corn coproduct-soybean meal (1.0% Arg) basal diets during 9-d battery feeding trials involving 5 pens of 4 chicks per treatment. The dextrose-casein diet was shown to be markedly deficient in Arg as both weight gain and G:F increased (P < 0.01) due to addition of Arg, GAA, or creatine. The optimal level of added GAA was 0.12% of the diet, but this level of GAA or 1.0% creatine-h 2 O did not improve growth performance when added to an Arg-adequate diet. A second assay confirmed this level of optimal Arg in a 2 2 factorial arrangement of l-arg and GAA supplementation. Using a practical-type diet based on corn, corn gluten meal, distillers dried grains INTRODUCTION Current feeding strategies for poultry include greater accuracy in nutrient provision to not only support optimal growth performance but also reduce input costs and the environmental impact associated with feeding excess nutrients. Replacing protein-rich ingredients with crystalline amino acids is now economically feasible in many instances (e.g., Lys, Met, Thr), but the search for other efficacious products continues. Guanidino acetic acid (GAA) is a compound synthesized from Gly and with solubles, and soybean meal, similar improvements (P < 0.05) in G:F resulted from addition of 0.25% Arg, 0.12% GAA, or 0.15% creatine H 2 O. These results demonstrate that 0.12% supplemental GAA, like creatine, produces consistent growth responses in young chicks fed Arg-deficient diets. To provide further evidence of the capacity for GAA to serve as a dietary Arg replacement, the dextrose-casein diet was supplemented with 7 graded doses of Arg in the absence or presence of 0.12% GAA (14 total diets). Quadratic (P < 0.01) responses in weight gain and G:F responses to supplemental Arg were observed. Similar supplemental Arg requirements were estimated in the absence and presence of 0.12% GAA, but GAA elicited a greater improvement (P < 0.05) in G:F when added to Arg-deficient, compared with Arg-adequate, diets. Collectively, these data indicate that GAA can be used as an efficacious replacement for dietary Arg for young chicks. Key words: arginine, chick, creatine, guanidinoacetate, metabolism 2013 Poultry Science 92 : Poultry Science Association Inc. Received April 23, Accepted September 30, Supported, in part, by Evonik Degussa Corporation, Kennesaw, GA Corresponding author: rdilger2@illinois.edu 3 Current address: Akey, 10 Nutrition Way, PO Box 69, Brookville, OH Arg by the enzyme l-arginine:glycine amidinotransferase (AGAT) in the avian kidney and liver. Subsequently, GAA is methylated by S-adenosylmethionine to creatine, and finally, adenosine triphosphate donates a phosphorus moiety to form the high-energy compound, phosphocreatine (Meister, 1965). Thus, GAA may be important for poultry nutrition not only as a replacement for dietary Arg, an essential nutrient, but also to support overall energy homeostasis of the bird. The ability of creatine to spare dietary Arg was previously studied (Fisher et al., 1956a,b; Waterhouse and Scott, 1961; Austic and Nesheim, 1972), but guanidino acetic acid was only mentioned in those publications when discussing the role of Arg in the biosynthesis of creatine. More recent evidence (Ringel et al., 2008) suggests that GAA may also have growth and feed efficiency-promoting properties when added to practical corn-soybean meal diets. As discussed above, GAA is a precursor of creatine, and creatine biosynthesis from GAA may explain at least part of the GAA response. 171

2 172 Dilger et al. Importantly, GAA is a more suitable feed additive compared with creatine and Arg because it is less expensive than either of these compounds and is more chemically stable than creatine. In addition, GAA may be beneficial in poultry diets because it may be able to spare Arg; this is an important point considering Arg is the fifth limiting AA in corn-soybean diets for poultry (Han et al., 1992; Fernandez et al., 1994; Waguespack et al., 2009). The objective of these assays was to evaluate the capacity for GAA to serve as an efficacious dietary replacement for Arg in young chicks. MATERIALS AND METHODS All animal procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee. Four bioassays were conducted using male chicks (New Hampshire Columbian) obtained from the University of Illinois Poultry Research Farm. Chicks were housed in thermostatically controlled starter batteries with raised-wire flooring in an environmentally controlled room with continuous lighting. Water and experimental diets were provided ad libitum throughout the feeding period. Chicks were fed a diet adequate in all nutrients (NRC, 1994) from hatch to d 7 posthatch. Following an overnight fast, chicks were weighed, wing-banded, and randomized to dietary treatments on d 8 such that average initial pen weights and weight distributions were similar across treatments. At the conclusion of each growth assay, chicks and feeders were weighed, and BW gain, feed intake, and feed efficiency (i.e., G:F ratio) were calculated for each replicate pen of chicks. Basal Diets Two separate basal diets (Table 1) were formulated to evaluate the Arg replacement value of GAA. The semi-purified basal diet, based on dextrose and casein, was analyzed to contain 24.9% CP and 0.88% Arg, whereas the practical basal diet contained calculated concentrations of 20.0% CP and 1.0% Arg. Both types of basal diets were formulated to be singly deficient in Arg, but were otherwise nutritionally complete for chicks of this age (NRC, 1994). Assay 1 The objective of this assay was to determine whether or not GAA could be used as a dietary replacement for Arg in young chicks as compared with creatine. Dietary treatments included an unsupplemented (Arg-deficient) basal diet, and the basal diet plus 0.06 or 0.12% GAA. A fourth diet containing 1.0% supplemental l- Arg served as a positive control treatment. The Argdeficient basal diet (without and with 1.0% l-arg) was further supplemented with 0.39% GAA, 0.78% GAA, or 1.0% creatine H 2 O. Here, 1.0% creatine H 2 O provided 0.88% pure creatine substrate, which is isomolar to 0.78% GAA. Five replicate pens of 4 chicks received each of the 10 experimental diets during a 9-d feeding period (d 8 to 17 posthatch). Assay 2 This assay was conducted to confirm the results obtained with 0.12% GAA in assay 1 and to compare the responses from 0.12% GAA to an equal concentration of l-arg. Dietary treatments were arranged in a 2 2 factorial design and included a common semi-purified basal diet, singly deficient in Arg, and the basal diet plus 0.12% Arg, the same concentration of GAA, and their combination. Five replicate pens of 4 chicks received each of the 4 experimental diets during a 9-d feeding period (d 8 to 17 posthatch). Assay 3 This assay sought to determine whether GAA could serve as a dietary Arg replacement when included in a practical-type diet (Table 1). Dietary treatments included a common basal diet, singly deficient in Arg, and the basal diet supplemented with 0.25% Arg, 0.12% GAA, 0.25% Arg plus 0.12% GAA, or 0.153% creatine (isomolar to 0.12% GAA). Additionally, a methioninefortified corn-soybean meal broiler starter diet (23% CP, 1.25% Arg) was included as a positive control diet. Five replicate pens of 4 chicks received each of the 6 experimental diets during a 9-d feeding period (d 8 to 17 posthatch). Assay 4 This assay was conducted to estimate the minimal quantity of Arg necessary to maximize growth performance (i.e., Arg requirement) in the absence and presence of 0.12% GAA. The first series of diets included 7 graded concentrations of l-arg supplemented in the semi-purified, dextrose-casein diet. Supplemental l-arg concentrations ranged from 0% to 0.72%, which resulted in a total dietary range of 0.88% to 1.60% Arg. The second series of treatments was identical to the first in terms of graded Arg concentrations, but additionally contained 0.12% GAA at the expense of cornstarch. Five replicate pens of 4 chicks received each of the 14 experimental diets during a 9-d feeding period (d 8 to 17 posthatch). Statistical Analysis All data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute, 2004). Data were analyzed using pen means with procedures appropriate for a completely randomized design. Data are presented as mean values with pooled SEM estimates, and statistical significance was set at α = Differences among treatment means were evaluated using the least significance procedure (Carmer and Walker, 1985).

3 GUANIDINOACETATE SPARES DIETARY ARGININE 173 Table 1. Composition (%) of arginine-deficient basal diets Ingredient Semi-purified diet 1 Practical diet 2 Cornstarch 0.37 Dextrose Casein (84.8% CP) Corn (8% CP) Soybean meal (47% CP) Distillers dried grains with solubles (25.5% CP) Corn gluten meal (61% CP) 4.00 Solka floc 3.00 Soybean oil Dicalcium phosphate 1.70 Limestone 1.40 NaCl 0.40 NaHCO Amino acid mixture Mineral mix Vitamin mix Choline chloride dl-α-tocopherol acetate, 20 mg/kg + Ethoxyquin, 125 mg/kg + Bacitracin premix Analyzed to contain 0.88% Arg and 24.9% CP. Calculated to contain 1.0% Arg and 20.0% CP. Contained 0.30% dl-met and 2.00% Gly. 4 Contained 0.62% l-lys HCl, 0.26% dl-met, 0.12% l-thr, 0.03% l-trp, 0.06% l-val, 0.09% l-ile, and 0.60% Gly. 5 Provided per kilogram of diet: CaCO 3, 3 g; Ca 3 (PO 4 ) 2, 28 g; K 2 HPO 4, 9 g; NaCl, 8.8 g; CuSO 4 5H 2 O, 20 mg; ZnCO 3, 100 mg; MgSO 4 7H 2 O, 3.5 g; Fe 6 C 6 O 7 H 2 O, 500 mg; MnSO 4 H 2 O, 650 mg; H 3 BO 3, 9 mg; NaMoO 4 2H 2 O, 9 mg; KI, 40 mg; CoSO 4 7H 2 O, 1 mg; and Na 2 SeO 3, 215 μg. 6 Provided per kilogram of diet: FeSO 4 H 2 O, 228 mg; ZnO, 93 mg; MnO, 97 mg; CuSO 4 5H 2 O, 20 mg; ethylenediamine dihydroiodide, 934 μg; and Na 2 SeO 3, 219 μg. 7 Provided per kilogram of diet: retinyl acetate, 1,789 μg; cholecalciferol, 15 μg; dl-α-tocopheryl acetate, 20 mg; menadione dimethylpyrimidinol bisulfite, 2 mg; ascorbic acid, 250 mg; thiamine HCl, 20 mg; niacin, 50 mg; riboflavin, 10 mg; d-calcium pantothenate, 30 mg; vitamin B 12, 40 μg; pyridoxine HCl, 6 mg; d-biotin, 600 μg; folic acid, 4 mg; and ethoxyquin, 125 mg. 8 Provided per kilogram of diet: retinyl acetate, 1,514 μg; cholecalciferol, 25 μg; dl-α-tocopheryl acetate, 11 mg; menadione sodium bisulfite complex, 2.33 mg; niacin, 22 mg; riboflavin, 4.4 mg; d-calcium pantothenate, 10 mg; and vitamin B 12, 11 μg. 9 Provided 25 mg/kg of bacitracin methylene disalicylate. Data from assay 4 were subjected to an exponential regression analysis to allow simultaneous and separate modeling of responses to graded Arg concentration and the effect of added GAA. Exponential response of weight gain and G:F to graded Arg concentrations were fitted using the NLIN procedures of SAS/STAT software (SAS Institute, 2004). The statistical model allowed parameters to vary with supplemental GAA level, and took the following form: Y = {a + b [1 exp(c X)]} G1 + {d + e [1 exp(f X)]} G2, where Y = growth response (weight gain or G:F), X = Arg added above basal level (% of diet), a and d = intercept (performance on the basal diet), b and e = asymptotic response to dietary concentration of Arg, c and f = curvature steepness, and G1 and G2 are indicator variables (0 or 1) representing 0 and 0.12% added GAA, respectively. A modified Gauss-Newton iterative algorithm was used for simultaneous estimation of the aforementioned parameters. Optimal concentrations of supplemental Arg in the absence and presence of GAA were defined as 95% of the asymptotic response. Assay 1 RESULTS Weight gain and feed efficiency, or G:F ratio, responded markedly to Arg supplementation of the Argdeficient basal diet (Table 2). There was also a significant (P < 0.05) growth and feed efficiency response when 0.12% GAA was added to the Arg-deficient basal diet. At higher levels of GAA, no further response in weight gain or feed efficiency was observed; thus, the responses were maximized at 0.12% GAA in this assay. Higher levels of GAA were also added to the Arg-adequate diet, with no significant responses. Both weight gain and feed efficiency increased (P < 0.05) as a result of adding 1% creatine to the Arg-deficient diet; these responses were greater in magnitude relative to the isomolar addition of 0.78% GAA. When 1.0% creatine was added to the Arg-adequate diet, no significant responses were observed. These results established that

4 174 Dilger et al. Table 2. Performance of chicks fed graded doses of guanidino acetic acid (GAA) when added to an arginine-deficient dextrose-casein diet (assay 1) 1 Item Weight gain, g Feed intake, g G:F, g/kg the 0.12% GAA level yielded the optimal performance when supplied in the semi-purified dextrose-casein diet. Assay 2 Addition of 0.12% l-arg and 0.12% GAA yielded similar increases (P < 0.05) in weight gain and feed efficiency (Table 3). When the combination of GAA and Arg were added, an additional increase (P < 0.05) in gain and feed efficiency were obtained. Thus, the main effects of both Arg and GAA were significant for both gain (P < 0.05) and G:F (P < 0.01), but no interaction was observed. Assay 3 Diet 1. Basal diet e 195 g 569 d 2. As % l-arg 212 a 275 a 773 a 3. As % GAA 108 e 195 g 553 d 4. As % GAA 145 d 233 de 622 c 5. As % GAA 139 d 220 de 631 c 6. As % GAA 134 d 209 fg 641 c 7. As % GAA 209 ab 265 abc 789 a 8. As % GAA 197 b 249 cd 792 a 9. As % creatine H 2 O c 254 bc 680 b 10. As % creatine H 2 O a 268 ab 794 a SEM a g Means in columns with no common superscript letters differ (P < 0.05). 1 Data are means of 5 pens of 4 chicks fed the experimental diets from 8 to 17 d posthatch; average initial weight was 94 g. 2 The basal Arg-deficient diet was analyzed to contain 0.88% Arg. 3 Provided 0.88% creatine, an amount isomolar to 0.78% GAA. Weight gain was not significantly affected by any of the dietary treatments in this assay (Table 4). However, similar G:F responses (P < 0.05) were observed when levels of 0.25% Arg, 0.12% GAA, or 0.153% creatine were added to the Arg-deficient practical-type diet. Weight gain of chicks fed the corn-soybean meal Table 3. Response to guanidino acetic acid (GAA) in chicks fed an arginine-deficient dextrose-casein diet (assay 2) 1 Item Weight gain, 2 g Feed intake, g G:F, 2 g/kg Diet 1. Basal diet As % l-arg As % GAA As SEM Data are means of 5 replicate pens of 4 male chicks during the period of 8 to 17 d posthatch; average initial weight was 77 g. 2 Main effects of both Arg and GAA supplementation for weight gain (P < 0.05) and G:F (P < 0.01). 3 The basal Arg-deficient diet was analyzed to contain 0.88% Arg. positive control diet was similar to that of the supplemented diets, whereas the corn-soybean meal positive control diet yielded feed efficiencies that were greater than that obtained from all other diets. Assay 4 Growth performance of chicks responded markedly as dietary Arg increased from deficient to super-adequate levels (Table 5). Moreover, addition of 0.12% GAA increased (P < 0.01) weight gain, feed intake, and G:F relative to diets containing no added GAA. Although the overall Arg GAA interaction was not significant, an interaction was evident (P < 0.05) when comparing GAA responses in Arg-deficient versus adequate diets. When GAA was added to Arg-deficient diets (defined as containing less than 0.40% supplemental l-arg), it improved G:F by 8.2% over those with no GAA. However, when GAA was added to Arg-adequate diets (defined as containing more than 0.40% supplemental l-arg), it improved G:F by 4.3% over those with no added GAA. For diets without and with 0.12% GAA, weight gain responded similarly to Arg supplementation, reaching plateau responses at 172 and 177 g, respectively (Figure 1). Feed efficiency responses were also similar in diets without and with 0.12% GAA, with asymptotic plateau responses at and g/kg, respectively. Overall, gain and G:F responses to Arg supplementation fit the exponential model with correlation coefficients greater than 0.83, and no additional information was derived when expressing the dependent variable as Arg intake (data not shown). Based on the fitted exponential responses for weight gain, the optimal supplemental Arg concentration (95% of asymptotic response) was determined to be and 0.562% of the diet in the absence and presence of GAA, respectively. For G:F, the optimal supplemental

5 GUANIDINOACETATE SPARES DIETARY ARGININE 175 Table 4. Performance of chicks fed guanidino acetic acid (GAA) in an arginine-deficient practicaltype diet (assay 3) 1 Item Weight gain, g Feed intake, g G:F, g/kg Diet 1. Basal diet a 692 c 2. As % l-arg ab 728 b 3. As % GAA bc 733 b 4. As bc 739 b 5. As % creatine H 2 O abc 730 b 6. Corn-soybean meal diet c 760 a SEM a c Means in columns with no common superscript letters differ (P < 0.05). 1 Data are means of 5 pens of 4 chicks fed the experimental diets from 8 to 17 d posthatch; average initial weight was 112 g. 2 The basal Arg-deficient practical-type diet contained 1.0% Arg. 3 Provided 0.134% creatine, an amount isomolar to 0.12% GAA. 4 Methionine-fortified corn-soybean meal positive control diet (calculated to contain 23% CP and 1.25% Arg), which was the same starter diet provided to all chicks pretest. Arg concentration was and 0.663% of the diet in the absence and presence of GAA, respectively. These effects of GAA were observed when included in a semipurified, dextrose-casein basal diet with total dietary Arg concentrations ranging from 0.88% (unsupplemented control) to 1.60% (0.72% supplemental Arg). Thus, optimal concentrations of total dietary Arg were estimated at 1.40 and 1.44% for weight gain in the absence and presence of 0.12% GAA, respectively, and at 1.56 and 1.54% for G:F in the absence and presence of 0.12% GAA, respectively. DISCUSSION Using chick growth as a sensitive indicator of nutritional deficiency, the assays described herein demonstrate the ability of GAA to serve as a dietary replacement for Arg. When GAA was added to the Arg-deficient, dextrose-casein diet, there were marked improvements in both weight gain and efficiency of feed utilization, even though growth performance of the broiler chicks used in our studies may not be equivalent to that of modern broiler chicks. These results clearly show that GAA improves growth performance for chicks fed Arg-deficient diets, which is in agreement with work by Savage and O Dell (1960). In the Argdeficient practical-type diet, there was no weight gain response to GAA supplementation, but there was an improvement in G:F when l-arg, GAA, and the combination were fed. These improvements in G:F were similar to the responses obtained by adding creatine to the diet, which agrees with the work of Snyder et al. (1956). As an implication of this work, the presence of creatine in practical feed ingredients may help to explain the lower dietary Arg requirement known to exist in practical rations compared with purified diets (Fisher et al., 1956b). When GAA and creatine were fed at isomolar levels, the gain and G:F responses to GAA were similar to those of creatine for both the semi-purified and practical-type diets. Thus, the observed responses in growth performance may have resulted from GAA serving as a precursor for creatine synthesis. Edwards et al. (1958) suggested that any sparing action of GAA should be at- Table 5. Performance of chicks fed increasing levels of arginine without or with guanidino acetic acid (GAA) in a dextrose-casein diet (assay 4) 1 Arg, % Weight gain, 2 g Feed intake, 2 g G:F, 2 g/kg Supplemental Total 3 0% GAA 0.12% GAA 0% GAA 0.12% GAA 0% GAA 0.12% GAA SEM Data are means of 5 pens of 4 chicks fed the experimental diets from 8 to 17 d posthatch; average initial weight was 77 g. 2 Main effect of GAA supplementation (P < 0.01). 3 The basal Arg-deficient diet was analyzed to contain 0.88% Arg.

6 176 Dilger et al. Figure 1. Effect of supplemental guanidino acetic acid (GAA) on weight gain (A) and G:F (B) of chicks fed graded levels of Arg in an Arg-deficient basal diet (assay 4). Square and triangular symbols represent data from chicks receiving 0 and 0.12% supplemental GAA, respectively. The simultaneously fitted exponential responses allowed parameters to vary by supplemental GAA level. Indicator variables G1 and G2, 0 or 1, represented 0 and 0.12% added GAA, respectively. The fitted response for weight gain (Y in g) vs. supplemental Arg concentration (X in % of diet) was Y = { [1 EXP( X)]} G1 + { [1 EXP( X)]} G2. The optimal supplemental Arg concentration (95% of asymptotic response) was and 0.562% of the diet in the absence and presence of GAA, respectively. The fitted response for gain feed (Y in g/kg) vs. supplemental Arg concentration (X in % of diet) was Y = { [1 EXP( X)]} G1 + { [1 EXP( X)]} G2. The optimal supplemental Arg concentration (95% of asymptotic response) was and 0.663% of the diet in the absence and presence of GAA, respectively. Optimal Arg concentrations shown in each panel represent the total Arg concentration, which accounts for the analyzed dietary Arg contained in the semi-purified basal diet (0.88% Arg). Data points are means ± SEM values of 5 pens of 4 chicks. tributed to creatine synthesis as more Arg would theoretically be available for other functions, including protein anabolism. This theory was certainly tested in our studies as inclusion of 0.78% GAA is more than 10-fold higher than current European feeding recommendations, A. Lemme (Evonik Degussa GmbH, Hanau, Germany, personal communication). In the context of metabolic regulation of de novo GAA synthesis, a negative relationship appears to exist between creatine status and enzymatic activity of l-arginine:glycine amidinotransferase (Wyss and Kaddurah-Daouk, 2000), which may help to explain variable responses observed in the Arg-sparing ability of GAA. Further studies focusing on the ability for GAA to alter biochemical profiles associated with creatine metabolism are certainly warranted. In agreement with previous work (Fisher et al., 1956a,b), inclusion of GAA in a semi-purified dextrosecasein diet did not reduce the dietary requirement for Arg. In our studies, improvements in weight gain and G:F due to addition of 0.12% GAA predominantly occurred at the most deficient Arg levels. However, as discussed earlier, GAA elicited significant responses in feed efficiency of chicks fed Arg-adequate diets (i.e., a higher plateau response observed in assay 4), which resulted in no significant reduction of the Arg requirement (although it was reduced numerically by 3%). Although GAA may be used as a dietary Arg replacement when supplied in Arg-deficient diets, alternative functions for GAA, including those related to creatine synthesis, also need to be considered. Recent work (Ringel et al., 2008) suggests GAA could increase growth performance of broiler chicks fed Arg-adequate corn-soybean meal diets. Delineating the importance of GAA as a replacement for dietary Arg and intermediate in creatine synthesis will require further investigation. The optimal dietary Arg concentration for maximizing G:F (1.54%) estimated by exponential regression analysis was numerically higher than that for maximal weight gain (estimated at 1.38%). Differences in requirement estimates based on the response variable included in the model were previously observed for Lys (Han and Baker, 1991). The reason for the higher requirement of amino acids for feed efficiency versus weight gain is unknown but may be associated with changes in feed intake and body composition (lean vs. fat) as dietary concentrations of the limiting amino acid are increased (Baker et al., 1996). Overall, we conclude from these studies that dietary GAA is an efficacious replacement for dietary Arg when fed to young chicks. Whereas differences in supplemental Arg requirements due to GAA inclusion were not evident, there did appear to be GAA-dependent effects in growth performance in both Arg-deficient and Arg-adequate diets. REFERENCES Austic, R. E., and M. C. Nesheim Arginine and creatine interrelationships in the chick. Poult. Sci. 51: Baker, D. H., S. R. Fernandez, C. M. Parsons, H. M. Edwards III, J. L. Emmert, and D. M. Webel Maintenance requirement for valine and efficiency of its use above maintenance for accretion of whole body valine and protein in young chicks. J. Nutr. 126: Carmer, S. G., and W. M. Walker Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Educ. 14:19 26.

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