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METABOLISM AND NUTRITION |
* Department of Poultry Science, North Carolina State University, Raleigh 27695-7608; Grupo Grica, Faculty of Agriculture, University of Antioquia, AA 1226, Medellin, Colombia; Akey, PO Box 5002, Lewisburg, OH 45338-5002; and Department of Animal Science, North Carolina State University, Raleigh 27695-7621
2 Corresponding author: jbrake{at}ncsu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: broiler • protein • lysine • amino acid • metabolizable energy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and consisted of a 3 x 4 factorial arrangement with 3 levels of ME (3,000, 3,100, and 3,200 kcal/kg) at each of 4 levels of dietary CP (21.9, 23.5, 25.2, and 26.9%). Prior to formulating the diets, the digestible AA content of ingredients was calculated using digestibility coefficients provided by the CVB (2003). The digestible Lys (dLys) content of all diets was set at 4.8% of the respective CP content and was 1.05, 1.13, 1.21, and 1.29%. Key essential AA in diets were adjusted using an ideal profile relative to dLys. To prevent any extracaloric effects between treatments that may have arisen from differences in the proportion of dietary ME that was derived from fat, the proportion of dietary ME derived from soy oil was held constant at 18.2% across all levels of dietary ME and CP. To reduce physical differences between diets the moisture level was held constant by amendment of the diets with water. An inert vermiculite filler was included to allow the application of these fundamental formulation principles across a wide range of dietary ME and CP (Table 2). To reduce the variation among dietary treatments that could originate from the mixing of ingredients and to ensure uniform gradations of dietary CP within each ME level, a summit-dilution blending technique was applied during the mixing of the diets. This consisted of mixing, pelleting, and crumbling a single batch of the highest CP (summit diets 4, 8, 12) and lowest CP diets (dilution diets 1, 5, 9). The 6 intermediate CP treatments (diets 2, 3, 6, 7, 10, 11) were subsequently derived by blending the respective summit and dilution diets within each level of ME.
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) were designed to determine the response of broilers to graded increments of dLys to a basal diet that contained 22.0% CP (low CP series) or 27.0% CP (high CP series). A third diet series (balanced CP series) was created by increasing dietary dLys from 1.08 to 1.32% while also increasing the dietary CP and all indispensable AA as a fixed percentage of dLys. With the exception of Lys, indispensable AA levels in all experimental diets were formulated to meet or exceed recommended minimum requirements (NRC, 1994). Prior to formulation of the diets (Table 4), all primary ingredients were subjected to CP and total AA analyses (AOAC, 2006). The digestible AA content of ingredients was calculated using digestibility coefficients provided by Degussa (AMINODat 2.0., 2001). Gradations of dLys of 0.85, 1.08, 1.19, and 1.30% in the low CP and 1.12, 1.22, 1.32, and 1.45% dLys in the high CP diet series were achieved by the addition of L-Lys HCl, at the expense of an inert vermiculite filler. To ensure uniform gradations of dLys in diets, a summit-dilution blending technique was again used (Figure 1). This consisted of mixing and pelleting a single batch of the diet with the highest dLys level (summit diets 4 and 8) and lowest dLys level (dilution diets 1 and 5) within the low and high CP series, respectively, and subsequently blending these in the respective proportions indicated in Table 3 to create the 2 intermediate levels of dLys within each level of CP. To further ensure good pellet quality, approximately half of the soy oil in the formulation was added to diets during the blending process after pelleting. To prevent an excessive amount of fines, rather than crumbling the diets, the knives of the pellet mill were set to cut a very small (
2 mm) pellet that was offered to the birds from 1 d of age. The gradations of dLys in the low and high CP diet series were designed such that the dLys expressed as a percentage of the dietary CP was 4.8% in diets 2 and 7. These 2 dietary treatments, while being intermediate in the low and high CP treatment series, respectively, also represented the lowest and highest levels of dLys in the balanced CP diet series. The 2 intermediate treatments in the balanced CP series (diets 9 and 10) were then derived by blending diets 2 and 7 in the proportions indicated in Table 3. Following the mixing and blending procedure, the CP of all diets was analyzed by LECO combustion analysis (AOAC Official Method 990.03, AOAC, 2006), whereas the AA concentration of summit and dilution diets was determined at the experiment Station Chemical Laboratories at the University of Missouri according to the AOAC (2006) methods 982.30 a and b.
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All data were analyzed using the Mixed Models procedure (SAS Institute, 2004) with a completely randomized design in experiment 1 and a randomized complete block design in experiment 2. Orthogonal polynomial contrasts were used to test the linear or quadratic nature of the response to incremental concentrations of dLys. As specific dietary treatments in experiment 2 were common to the high CP, low CP, or balanced CP diet series the broiler response to increments in dLys was analyzed separately within each diet series. Statements of statistical significance were based on P 
0.05 unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 5, respectively. The analyzed CP values of all diets in experiment 2 and the lowest and highest CP diets in experiment 1 were in general agreement with formulated values. However, the analyzed CP of the 2 intermediate diets in experiment 1 was 1.65 and 1.09% greater than the calculated values of 23.54 and 25.21%, respectively. As both intermediate diets had been mixed by blending the highest (26.88%) and lowest (21.88%) CP diets, for which the analyzed CP was close to formulated values, the deviation in the analyzed CP of the intermediate diets may be explained either by sampling error or analytical error. An examination of the broiler performance response suggested that the samples analyzed for the intermediate diets may not have been representative of the diets consumed, as the response in broiler BW gain from the lowest to the highest CP diets was strictly linear (P < 0.001) with no evidence of the curvature present in the analyzed CP values of the diets in experiment 1. The interim broiler performance to 7 and 14 d of age was reported elsewhere (Plumstead, 2005) and only performance to 21 d in experiment 1 or 20 d in experiment 2 was reported herein.
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. At the lowest concentration of dLys of 1.05% (21.9% CP) BW gain and AdjFCR at 21 d of age was 893 g and 1.35 g:g, respectively. However, in spite of the good performance obtained at the lowest concentration of dLys, a linear increase in 21 d BW gain of 61 g and a decrease in the AdjFCR to 1.26 g:g was obtained as dietary dLys was increased to 1.29% (26.9% CP; Table 6 and Figure 2). Broiler 21 d BW gain increased and AdjFCR decreased when dietary ME was increased from 3,000 to 3,100 kcal/kg with no further improvement to 3,200 kcal/kg of ME. Increasing dietary ME or dLys had no effect on feed intake to 21 d. A significant interaction of dLys and ME on feed intake was observed, which was attributed to an unusually high feed intake for birds receiving the 3,100 kcal/kg, 1.05 dLys (21.9% CP) diet. Due to the absence of consistent differences in feed intake between diets within a range of ME from 3,000 to 3,200 kcal/kg, an increase in dietary ME concentration resulted in a step-wise increase in the cumulative ME consumed by broilers to 21 d of age (Figure 3).
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). For broilers fed the low CP diets the dLys requirement at 95% of the asymptote in BW gain was estimated to be 1.19 ± 0.03%, which was equivalent to 1.30% total Lys. In contrast to the low CP diet series, the 20 d BW gain in both the high CP and balanced CP diet series increased in a linear manner with continued L-Lys supplementation up to the highest levels of 1.45% and 1.32% dLys, respectively. Whereas there was no significant response in the AdjFCR to increased dLys in the high CP diet series, a curvilinear response in AdjFCR was observed in the balanced CP diet series when dLys was increased to 1.32%. Feed intake between treatments in the low CP diet series was somewhat variable. Broilers that received the diet containing 0.85% dLys exhibited depressed BW gain and low feed intake, whereas feed intake on the 1.08 dLys treatment was higher than expected. The response in feed intake to dLys in the high CP diet series followed a linear trend that reflected the step-wise increase in BW gain, whereas dLys had no effect on feed intake in the balanced CP diet series.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lack of any consistent interaction between dietary ME and balanced CP concentration on BW gain and AdjFCR in experiment 1 suggested that the response in BW gain and adjFCR to increasing dietary concentrations of balanced CP from 21.9 to 26.9% CP was independent of the dietary ME level over the range from 3,000 to 3,200 kcal/kg. These results were in agreement with Gonzalez and Pesti (1993) who found no evidence of an optimum ME:CP ratio but that both ME and CP were important predictors of broiler performance. In contrast, the NRC (1984) and the CVB (2001) suggested that AA requirements should be expressed as a proportion of the dietary ME concentration. This was based on evidence that poultry adjusted their feed intake to differences in the dietary ME density to maintain a constant ME intake (NRC, 1984; Leeson et al., 1996). However, in the present study feed intake of broilers was not reduced when dietary ME was increased from 3,000 to 3,200 kcal/kg, which resulted in a step-wise increase in the cumulative ME intake to 21 d (Figure 3). The apparent lack of an effect of ME density on feed intake in experiment 1 was consistent with the revised observations of the NRC (1994) that modern broiler strains did not adjust their feed intake to changes in the dietary ME density. Differences in the effects of ME density on broiler feed intake observed by other authors may have been caused by differences in the range of dietary ME evaluated, the age of the birds, as well as by differences in the dietary formulation techniques used in the respective experiments. For example, Leeson et al. (1996) showed that broiler feed intake to 25 d was reduced by 200 g when the dietary ME was increased from 2,700 to 3,300 kcal/kg. However, in that study the range of ME of 600 kcal/kg was considerably wider than the range of 3,000 to 3,200 kcal/kg in the present study, which may explain differences in the response observed with increased energetic density of the diets. Furthermore, in the study by Leeson et al. (1996) changes in the dietary ME were achieved by increasing the dietary inclusion of an animal-vegetable fat blend from 1.15% to 8.65%. The linear correlation between dietary ME and added fat in that study makes it difficult to separate effects of ME and added fat on feed intake. To prevent extracaloric effects of dietary fat on pellet and crumble quality and bird performance, the diets utilized in experiment 1 of the present study were formulated in a manner that fixed the proportion of dietary ME derived from added fat at 18.4% and resulted in only small gradations of 0.42% in the added fat content of the diets over the range of 200 kcal/kg of ME. Thus the consistent feed intake obtained across the experimental diets in the present study suggested that when diets contained similar quantities of added fat and had a similar moisture content and physical texture, broilers grown to 21 d of age did not adjust their feed intake in response to increasing dietary ME concentration over the range from 3,000 to 3,200 kcal/ kg. The observations of the present study are supported by Richards (2003) who concluded that modern broilers selected for rapid growth do not regulate voluntary feed intake to achieve energy balance. This altered ability of broilers to adjust feed intake due to differences in ME density of the diet was postulated to result from continued selection for rapid juvenile growth rates, which may have altered hypothalamic mechanisms that regulate feed intake in broilers (Burkhart et al., 1983; Bokkers and Koene, 2003).
The AA requirement of growing broilers has been frequently estimated using an empirical method whereby graded levels of a single crystalline AA were added to a basal diet deficient in the AA in question and the response determined (D’Mello, 1982; Gous and Morris, 1985; Boorman and Burgess, 1986; Mack et al., 1999). An important aspect of this method was that the basal diet contained a fixed level of CP and adequate amounts of all other nutrients, whereas the degree of deficiency of the AA being examined should be severe enough to substantially impair performance (Mello, 1982). This method was applied in the low CP diet series in experiment 2 and shows a classic diminishing response curve with incremental dLys addition to a Lys deficient diet, allowing the dLys requirement of male broilers from 0 to 20 d of age of 1.19 ± 0.03% (1.30% total Lys) to be estimated (low CP diet series, Figure 4). Using similar dose-response methodology, Han and Baker (1991) reported a dLys requirement for 8 to 21 d of BW gain of Hubbard broilers of 0.96 to 1.01%, whereas Kidd and Fancher (2001) showed the dLys requirement of Ross 344 x Ross 508 broilers from 0 to 18 d of age to lie between 1.07 and 1.11%. The higher dLys requirement of 1.19% determined in the present study may have been caused by differences in broiler strain (Acar et al., 1991; Bilgili et al., 1992), environmental factors (Kidd and Fancher, 2001), the pellet or crumble quality of the diet (Kidd and Fancher, 2001; Greenwood et al., 2005), or the range of dLys evaluated in experimental diets (Abebe and Morris, 1990). Several workers also suggested that the Lys requirement of broilers increased when dietary CP of the diet was increased (Grau, 1948; Grau and Kamei, 1950, Velu et al., 1971, Morris et al., 1987, Abebe and Morris, 1990, Surisdiarto and Farrell, 1991). The high CP diet series in experiment 2 evaluated the response of broilers to graded L-Lys addition to a basal diet with 26.9% CP and high concentrations of all other indispensable AA. The absence of a quadratic response in BW gain of broilers fed the high CP diet series could most likely be attributed to the dietary dLys percentage of 1.12% in the basal diet with 27.0% CP not being low enough to greatly impair feed intake or performance. The inability to formulate diets with a high percentage CP and sufficiently low percentage dLys was indicated by Gous and Morris (1985) to be one of the limitations of the dose response methodology used to determine the requirement of broilers for a single AA. In contrast to the basal diet in the high CP diet series, the low CP basal diet with only 0.85% dLys severely impaired BW gain, which was most likely exacerbated by the depressed feed intake of broilers that was attributed to the severe imbalance of AA in the Lys deficient basal diet (Harper et al., 1970; Yanaka and Okumura, 1982). Furthermore, the continued linear response to L-Lys supplementation in the high CP diet series above the optimum dLys requirement of 1.19% determined in the low CP diet series can be explained by the observations by Abebe and Morris (1990) who suggested that as surplus protein depressed the utilization of the first limiting AA, higher concentrations of the limiting AA were required to maximize broiler BW gain.
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It can be concluded that our estimate of the dLys requirement of 1.19% obtained using traditional dose response methods was higher than previous estimates by other authors and was also shown to be dependent on the dietary CP percentage. Therefore, to prevent an imbalance of AA in the diets and obtain optimum broiler performance the dLys requirement should be expressed as a percentage of the dietary CP. When this principle was applied and a balance of all other AA was maintained, 21 d broiler BW and FCR responded positively to incremental dLys up to 1.32% (27.2% CP). The linear response in broiler performance was furthermore shown to be independent of the dietary ME concentrations in the range from 3,000 to 3200 kcal/kg. In practice, the nutrient density of diets will be limited by the associated exponential increase in diet cost as dietary concentrations of balanced CP will be increased. However, because broiler performance was shown to respond to increased cumulative intakes of balanced dietary CP and ME, similar improvements in CP and ME intake and early performance could also be expected by stimulating feed intake rather than increasing the CP or ME density of the diets.
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FOOTNOTES |
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Received for publication April 23, 2007. Accepted for publication August 27, 2007.
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REFERENCES |
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Acar, N., E. T. Moran Jr., and S. F. Bilgili. 1991. Live performance and carcass yield of male broilers from two commercial strain crosses receiving rations containing lysine below and above the established requirement between six and eight weeks of age. Poult. Sci. 70:2315–2321.[Web of Science]
AMINODat 2.0. 2001. Degussa Corporation Feed Additives. Hanau-Wolfgang, Germany.
AOAC. 2006. Official Methods of Analysis. 18th ed. Assoc. Off. Anal. Chem. Int., Washington, DC.
Baker, D. H., A. B. Batal, T. M. Parr, N. R. Augspurger, and C. M. Parsons. 2002. Ideal ratio (relative to lysine) of tryptophan, threonine, isoleucine, and valine for chicks during the second and third weeks post-hatch. Poult. Sci. 81:485–494.
Bilgili, S. F., E. T. Moran Jr., and N. Acar. 1992. Strain cross response of heavy male broilers to dietary lysine in the finisher feed: Live performance and further processing yields. Poult. Sci. 71:850–858.[Web of Science][Medline]
Bokkers, E. A. M., and P. Koene. 2003. Eating behavior, and preprandial and postprandial correlations in male broiler and layer chickens. Br. Poult. Sci. 44:538–544.[CrossRef][Web of Science][Medline]
Boorman, K. N., and A. D. Burgess. 1986. Responses to amino acids. Pages 99–123 in Nutrient Requirements of Poultry and Nutritional Research. C. Fisher and K. N. Boorman, ed. Butterworths, London, UK.
Burkhart, C. A., J. A. Cherry, H. P. Van Krey, and P. B. Siegel. 1983. Genetic selection for growth rate alters hypothalamic satiety mechanisms in chickens. Behav. Genet. 13:295–300.[CrossRef][Web of Science][Medline]
CVB. 2001. Tabellenboek Veevoeding 2001. Voedernormen landbouwhuisdieren en voederwaarde veevoeders. Centraal Vee-voederbureau, Postbus 2176:8203. AD, Lelystad, the Netherlands.
CVB. 2003. Veevoedertabel 2003. Gegewens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen. Centraal Veevoederbureau, Postbus 2176:8203. AD, Leylystad, the Netherlands.
D’Mello, J. P. F. 1982. A comparison of two empirical methods of determining amino acid requirements. World’s Poult. Sci. J. 38:114–119.[CrossRef][Web of Science]
Gonzalez, M. J., and G. M. Pesti. 1993. Evaluation of the protein to energy ratio concept in broiler and turkey nutrition. Poult. Sci. 72:2115–2123.[Web of Science][Medline]
Gous, R. M., and T. R. Morris. 1985. Evaluation of a diet dilution technique for measuring the response of broiler chickens to increasing concentrations of lysine. Br. Poult. Sci. 26:147–161.[CrossRef][Web of Science][Medline]
Grau, C. R. 1948. Effect of protein level on the lysine requirement of the chick. J. Nutr. 36:99–108.
Grau, C. R., and M. Kamei. 1950. Amino acid imbalance and the growth requirements for lysine and methionine. J. Nutr. 41:89–101.
Greenwood, M. W., K. R. Cramer, R. S. Beyer, P. M. Clark, and K. C. Behnke. 2005. Influence of feed form on estimated digestible lysine needs of male broilers from sixteen to thirty days of age. J. Appl. Poult. Res. 14:130–135.
Han, Y. M., and D. H. Baker. 1991. Lysine requirement of fast-growing and slow-growing broiler chicks. Poult. Sci. 70:2108–2114.[Web of Science][Medline]
Harper, A. E., N. J. Benevenga, and R. M. Wohlheuter. 1970. Effects of disproportionate amounts of amino acids. Physiol. Rev. 50:428–558.
Jorgensen, H., P. Sorensen, and B. O. Eggum. 1990. Protein and energy metabolism in broiler chickens selected for either body weight gain or feed efficiency. Br. Poult. Sci. 31:517–524.[CrossRef][Web of Science][Medline]
Kidd, M. T., and B. I. Fancher. 2001. Lysine needs of starting chicks and subsequent effects during the growth period. J. Appl. Poult. Res. 10:385–393.
Labadan, M. C., Jr., K. N. Hsu, and R. E. Austic. 2001. Lysine and arginine requirements of broiler chickens at two- to three-week intervals to eight weeks of age. Poult. Sci. 80:599–606.
Leeson, S., L. Caston, and J. D. Summers. 1996. Broiler response to diet energy. Poult. Sci. 75:529–535.[Web of Science][Medline]
Lemme, A. 2003. The "ideal" protein concept in broiler nutrition 2. Experimental data on varying dietary ideal protein levels. Pages 7–14 in Amino News volume 4 (2). Degussa AG, Hanau, Germany.
Locatelli, M. L., D. Hoehler, A. Lemme, P. J. A. Wijtten, and D. J. Langhout. 2003. Effects of varying dietary ideal amino acid levels in the starter diet on performance of male and female broilers. Poult. Sci. 82(Suppl. 1):28. (Abstr.)
Mack, S., D. Bercovici, G. De Groote, B. Leclerq, M. Lippens, M. Pack, J. B. Schutte, and S. van Cauwenberghe. 1999. Ideal amino acid profile and dietary lysine specifications for broiler chickens of 20 to 40 days of age. Br. Poult. Sci. 40:257–265.[CrossRef][Web of Science][Medline]
Mello, J. P. F. 1982. A comparison of two empirical methods of determining amino acid requirements. World’s Poult. Sci. J. 38:114–119.[CrossRef][Web of Science]
Morris, T. R., K. Alazzawi, R. M. Gous, and G. L. Simson. 1987. Effects of protein concentration on responses to dietary lysine by chicks. Br. Poult. Sci. 28:185–195.[CrossRef][Web of Science][Medline]
Morris, T. R., R. M. Gous, and C. Fisher. 1999. An analyses of the hypothesis that amino acid requirements for chicks should be stated as a proportion of dietary protein. World’s Poult. Sci. J. 55:7–22.[CrossRef][Web of Science]
National Research Council. 1984. Nutrient Requirements of Poultry. 8th rev. ed. Natl. Acad. Press, Washington, DC.
National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.
Plumstead, P. W. 2005. Response of young broilers to graded levels of dietary protein and amino acids. MS Thesis. North Carolina State Univ., Raleigh.
Quentin, M., I. Bouvarel, and M. Picard. 2005. Effects of the starter diet, light intensity, and essential amino acids level on growth and carcass composition of broilers. J. Appl. Poult. Res. 14:69–76.
Richards, M. P. 2003. Genetic regulation of feed intake and energy balance in poultry. Poult. Sci. 82:907–916.
SAS Institute. 2004. SAS/STAT User’s Guide. Release 9.1. SAS Inst. Inc., Cary, NC.
Surisdiarto, and D. J. Farrell. 1991. The relationship between dietary CP and dietary lysine requirement by broiler chicks on diets with and without the "ideal" amino acid balance. Poult. Sci. 70:830–836.[Web of Science][Medline]
van der Klis, J. D., and H. A. J. Versteegh. 1996. Phosphorus nutrition of poultry. Pages 71–83 in Recent Developments in Poultry Nutrition. P. C. Garnsworthy, J. Wiseman, and W. Haresign, ed. Nottingham Univ. Press, Nottingham, UK.
Velu, J. G., D. H. Baker, and H. M. Scott. 1971. Protein and energy utilization by chicks fed graded levels of a balanced mixture of crystalline amino acids. J. Nutr. 101:1249–1256.
Yanaka, M., and J. Okumura. 1982. Effects of excess tyrosine and level of feed intake on nitrogen and energy utilization in chicks. Poult. Sci. 61:2440–2446.[Web of Science][Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), high CP diet series (g), and the balanced CP diet series (
). In the low CP diet series dilution diet 1 and summit diet 4 were blended to create intermediate diets 2 and 3. For the high CP diet series dilution diet 5 and summit diet 8 were blended to create intermediate diets 2 and 3. The intermediate diets 2 and 7 formed the dilution and summit diets for the balanced CP diet series, respectively. These were blended to create intermediate diets 9 and 10.
) in mixed sex broiler chickens grown to 21 d of age expressed as a function of increments in formulated dietary digestible Lys in experiment 1. The main effect means ± pooled standard errors of 6 g for BW gain and 0.01 g:g AdjFCR are shown. Means represent the average response of 18 pens of 16 broilers each within each CP level. Both sexes were equally represented within each pen at placement. The response curve for BW gain was Y = 892.6 + 224.7 (X – 1.05) + 104.7 (X – 1.05)2 (R2 = 0.99; P < 0.001) and for AdjFCR: Y = 1.35 – 0.594 (X – 1.05) + 0.836 (X – 1.05)2 (R2 = 0.98; P < 0.001).
