Calibration and validation of a carbon oxidation system and determination of the bicarbonate retention factor and the dietary phenylalanine requirement, in the presence of excess tyrosine, of adult, female, mixed-breed dogs,.

Carbon oxidation methods have been used as rapid and sensitive methods to determine whole-body AA requirements in multiple species. The objectives of the current studies were to validate complete CO recovery, determine the bicarbonate retention factor, and estimate the Phe requirement, in the presence of excess Tyr, in adult dogs using the direct oxidation technique. In this series of studies, 2 oxidation chambers were constructed and calibrated to ensure accurate collection of breath CO. First, 104.6 ± 7.1% CO was recovered from chambers and suggests that the chambers were appropriately designed for complete and efficient CO recovery. Second, we determined bicarbonate retention in 5 dogs using repeated oral dosing of a bicarbonate tracer (NaHCO) with small meals. At isotopic and physiological steady state, 102.5 ± 2.6% of the delivered NaHCO was recovered in breath. Third, the Phe requirement, when Tyr was supplied in excess, was determined by the rate of appearance of CO in the breath (CO). Dogs ( = 5) were fed test diets with different concentrations of Phe ranging from deficient to excessive for 2 d prior to conducting the tracer studies. The mean Phe requirement (when Tyr was supplied in excess) was 0.535% of diet (upper 95% confidence interval = 0.645% diet) on an as-fed basis or 0.575% of diet (upper 95% confidence interval = 0.694% of diet) on a DM basis and was based on a calculated (modified Atwater calculation) dietary ME density of 3.73 Mcal/kg DM. These data support the use of carbon oxidation methods and oral dosing of isotope to measure whole-body requirements of indispensable AA in adult dogs and suggest the current recommendations may be low.

Tryptophan requirement of pregnant sows.

Amino acid requirements of sows may change from early to late gestation due to the accelerated growth of products of conception after d 70 of pregnancy. The objective of this study was to determine the Trp requirement, Phe kinetics, and energy expenditure in early (d 35 to 53) and late (d 92 to 111) pregnancy using the indicator amino acid oxidation method and indirect calorimetry. The same 6 second parity sows were fed 6 diets in a Latin square design in both early and late pregnancy. The diets based on corn, corn starch, and sugar provided 20 to 120% of the current recommended Trp intake (2.5 g/d) in early pregnancy and 60 to 180% in late pregnancy. Feed allowance was constant for each sow at 2.41 kg/d (SE 0.029). Expired air and blood were collected every 30 min for 5 1/2 h. After three 30-min periods to determine background (13)C enrichment in expired CO2 and plasma Phe, L[1-(13)C]Phe was given orally at a rate of 2 mg/(kg BW · h) with 8 1/2 hourly meals. Expired air and plasma were analyzed for (13)CO2 and (13)C-Phe enrichment, respectively. Requirements were determined as the breakpoint of 2-phase linear models. Sows grew from 167.7 kg (SE 3.93) at breeding to 211.9 kg (SE 5.18) post-farrowing and had litters of 14.5 piglets (SE 0.43) weighing 19.0 kg (SE 1.41) at birth. The Trp requirement was 1.7 g/d (SE 0.29, P = 0.001) in early pregnancy and 2.6 g/d (SE 0.37, P = 0.013) in late pregnancy, or 0.7 g/kg and 1.1 g/kg diet of total Trp, respectively, for a feed allowance of 2.4 kg/d. The Trp requirement in late pregnancy tended (P = 0.056) to be greater than in early pregnancy. Quantitative Phe kinetics were not affected by Trp intake except for a quadratic response of Phe oxidation and retention (P < 0.1) to Trp intake in early pregnancy. In late pregnancy, sows oxidized less Phe and retained more Phe (P = 0.001) than in early pregnancy, indicating that young, growing pregnant sows increase the efficiency of utilizing AA in late pregnancy to maintain protein synthesis in both maternal and fetal tissues. Oxidation and body protein breakdown contributed less to Phe flux in late than early pregnancy while protein synthesis contributed more (P < 0.01). Heat production and energy retention were not affected by Trp level or stage of gestation. To meet both energy and AA requirements in late gestation, a phase feeding program with 2 diets is recommended. The feed allowance in late pregnancy should be greater than in early pregnancy to account for the increased energy expenditure.

Isoleucine requirement of pregnant sows.

The objective of this study was to determine the Ile requirement in early (d 39 to 61) and late (d 89 to 109) pregnancy using the indicator AA oxidation method. The same 7 Large White × Landrace sows in their fourth parity were used in early and late pregnancy. Each sow received 6 diets based on corn, corn starch, and sugar in both early and late pregnancy at constant feed allowances (2.5 kg/d). Diets provided Ile at 20, 40, 60, 80, 100, and 120% of the Ile requirement (6.2 g/d based on the 1998 NRC) in early and 60, 80, 100, 140, 160, and 180% in late pregnancy. After determination of (13)C background in expired CO2 and plasma free Phe for 1.5 h when confined in respiration chambers, sows were fed the tracer, L[1-(13)C]Phe, a rate of 2.0 mg/(kg BW·h) over 4 h divided into eight 30-min meals. Expired CO2 and plasma free Phe were analyzed for (13)C enrichment above background. Requirements were determined as the breakpoint in 2-phase nonlinear models. Sow BW was 246.5 kg in early and 271.6 kg in late pregnancy. Daily gain of the 6 sows was similar in early (344 g/d) and late pregnancy (543 g/d). During pregnancy, sow maternal gain was 19.1 ± 4.4 kg and litters of 17.7 ± 0.8 piglets weighed 22.6 ± 0.9 kg at birth. The Ile requirement was 3.6 ± 1.2 g/d (P = 0.001) in early pregnancy with a Phe retention (-0.59 g/d) and energy retention (-0.31 MJ/d) that were not different from 0. This indicates that the fourth parity sows had requirements close to maintenance in early pregnancy. The Ile requirement in late pregnancy was 9.7 ± 1.9 g/d (P = 0.001) when sows retained 3.30 g/d of Phe and -1.45 MJ/d of energy. The greater Ile requirement in late pregnancy was probably caused by the increased conceptus growth after d 70 of pregnancy. Phenylalanine flux, oxidation, and nonoxidative disposal increased (P < 0.1) from early to late pregnancy, but body protein breakdown did not. Phenylalanine oxidation, nonoxidative disposal, and retention increased (P < 0.01) with increasing Ile intake in early pregnancy but were not affected by Ile intake in late pregnancy. Body protein breakdown did not respond to Ile intake in early or late pregnancy. Although energy retention was similar in early and late pregnancy, the respiratory quotient decreased (P = 0.047) from early (1.05) to late pregnancy (0.98), indicating lipid mobilization in late pregnancy when Ile was at or above the requirement. The results of this study show that the Ile requirement of sows increases from early to late pregnancy.

Protein intake but not feed intake affects dietary energy for finishing pigs.

The effects of dietary protein and feeding levels on dietary metabolizable (ME) and net energy (NE) content were determined in 24 pigs, each offered two diets at 2.0 times the energetic maintenance requirement or for ad libitum intake between 55 and 95 kg body weight. Within feeding levels, pigs received, in random order, low-protein (LP; 11.2% CP, 0.61% lysine) or high-protein (HP; 20.2% CP, 0.61% lysine) diets of similar digestible energy content. Dietary NE was calculated from heat production based on 24-h indirect calorimetry following a 7-day N-balance period. Feed intake was greater for LP than HP when fed for ad libitum intake (p = 0.001). Protein level did not affect daily gain (p > 0.1) but HP improved gain: feed (p = 0.003). Dietary ME and NE were not significantly affected by feeding level but were decreased by high protein intake (p < 0.07). Reducing dietary protein reduced urinary energy losses and increased energy retention but did not affect heat production. The effect of dietary protein restriction was already evident on the ME level and carried over to a similar degree to the NE level because the utilization of ME was not affected by protein level. Dietary ME and NE decreased by 0.012 MJ/kg (p = 0.014) and 0.018 MJ/kg (p = 0.062), respectively, for each gram per day N intake. The results suggest that although there was an effect of protein level on NE, the greatest effect occurred at the level of ME. However, the prediction of both ME and NE may be improved by adopting energy values for dietary protein that changes with dietary protein content.

Dietary lysine requirement of sows increases in late gestation.

Current AA recommendations for sows are to provide a fixed amount of AA intake throughout gestation; however, the demand for nutrients changes from maternal lean tissue in early gestation (EG) to fetal and mammary growth in late gestation (LG). The objective of this study was to determine the Lys requirement in EG (d 24 to 45) and LG (d 86 to 110) using the indicator AA oxidation method with simultaneous determination of heat production. Each of 7 Large White × Landrace sows received 6 diets in random order in both EG and LG. Three semisynthetic diets (14.0 MJ ME/kg) based on corn were formulated and mixed to produce a basal diet (60% of 1998 NRC Lys requirement) and high diets for EG and LG (150% and 185% of 1998 NRC Lys requirements, respectively). The 6 test diets provided Lys intakes of 7.5 to 19.3 g/d in EG and 8.1 to 23.7 g/d in LG. Sows were placed in respiration chambers, and expired air and blood were collected every 30 min for 5.5 h. The tracer AA, l-[1-(13)C]Phe, was given orally at a rate of 2 mg/(kg BW ⋅ h) over the last 4 h, divided into 8, 0.5-h meals. Expired air was measured for (13)CO(2) enrichment, and plasma was measured for l-[1-(13)C]Phe enrichment and free Lys concentration. Background (13)CO(2) was subtracted from plateau (13)CO(2) enrichment. Requirements were determined using a 2-phase nonlinear model. Mean maternal BW gain in gestation (43.7 kg; pooled SE, 1.2 kg), litter size (14.6 total born piglets; pooled SE, 0.8), and litter weight (19.4 kg; pooled SE, 0.9 kg) did not differ between parities. Sow weight gain and BW was greater (P = 0.001) in LG than EG. Lysine requirement was 9.4 and 17.4 g/d in EG and LG, respectively. Phenylalanine retention in LG was maximized at a Lys intake of 17.7 g/d. Heat production was more (P = 0.069) and energy retention less (P = 0.019) in LG than EG. Energy retention in LG was not different from 0. Quantitative Phe kinetics in EG were not affected by Lys intake. In LG, Phe retention increased with Lys intake (P = 0.004), whereas Phe oxidation decreased (P = 0.005). The Lys requirement was determined to be less than current recommendations in EG and more than current recommendations in LG. To meet the change in requirements, diets with increased lysine content are needed in LG. Increasing the feed allowance in LG is necessary to maintain a positive energy balance throughout gestation.

The threonine requirement of sows increases in late gestation.

Current AA recommendations for sows are to provide a fixed amount of AA intake throughout gestation based on the assumption that there is a constant demand for AA; however, the demand for nutrients changes from maternal lean tissue in early gestation to fetal and mammary growth in late gestation. The objective of this study was to determine the Thr requirement in early (d 35 to 53 and 25 to 55 for Exp. 1 and 2, respectively) and late (d 92 to 110 and 81 to 111 for Exp. 1 and 2, respectively) gestation using the indicator AA oxidation (IAAO) method with l-[1-(13)C]Phe as the tracer AA. A total of 14 multiparous sows were used: 6 in Exp. 1 and 8 in Exp. 2. Each sow received each of 6 diets in random order in both early and late gestation. A basal diet was formulated to contain Thr at 60% of the 1998 NRC recommendation in Exp. 1 and 20 and 60% of the 1998 NRC in Exp. 2 for early and late gestation, respectively. Crystalline l-Thr was added to create additional diets with approximately 10% incremental increases in Thr. Sows were placed in respiration chambers, and expired air and blood were collected every 30 min for 5.5 h. Tracer Phe [mg/(kg of BW·h)] was given orally over the last 4 h divided into eight 0.5-h meals. Expired air and plasma were measured for (13)CO(2) enrichment and free Thr concentration, respectively. Background (13)CO(2) was subtracted from plateau (13)CO(2) enrichment. Data were analyzed using a 2-phase nonlinear Mixed model. The overall litter size and litter weight were 13.5 ± 3.1 and 20.5 ± 3.9 kg, respectively. Based on IAAO, the Thr requirement in early gestation was 6.1 g/d (R(2) = 0.59, Exp. 1) and 5.0 g/d (R(2) = 0.71, Exp. 2). In late gestation, the Thr requirement based on IAAO was 13.6 g/d (R(2) = 0.60, Exp. 1) and 12.3 g/d (R(2) = 0.58, Exp. 2). Based on plasma Thr, the Thr requirement in early gestation was 7.0 g/d (R(2) = 0.90, Exp. 1) and 3.9 g/d (R(2) = 0.90, Exp. 2). In late gestation, the Thr requirement based on plasma Thr was 10.5 g/d (R(2) = 0.67, Exp. 2). There was a linear response to increasing Thr intake in late gestation in Exp. 1. Feeding a single amount of AA throughout gestation results in overfeeding AA in early gestation and underfeeding AA in late gestation. The 2-fold increase in Thr requirement in the last third of gestation suggests that phase feeding sows in gestation will more closely meet the demands for nutrients and that the requirement for essential AA in gestating sows should be re-evaluated in early and late gestation separately.

Methionine-hydroxy analogue was found to be significantly less bioavailable compared to dl-methionine for protein deposition in growing pigs.

When methionine (Met) is limiting in swine diets, it is commonly supplemented by using anhydrous dl-methionine (DLM, 99% purity) or liquid dl-methionine-hydroxy analogue free acid (MHA-FA, 88% purity). The objective of this experiment was to test the null hypothesis that the bioavailability of DLM and MHA-FA were not different for growing pigs, using the indicator amino acid (AA) (phenylalanine, Phe) oxidation (IAAO) method in a slope-ratio assay. Six barrows (mean BW during study: 21.1 kg) received seven dietary treatments with all pigs receiving all diets in random order at an intake of 95 g/kg BW0.75. The basal diet (BD) contained analyzed content of 15.1% CP, 0.20% Met, 0.73% Phe and all other AA in excess of requirement. The BD was supplemented with three graded levels of DLM or MHA-FA on an equimolar basis. Dietary treatments only varied in Met content and included: (i) BD, (ii) BD + 0.034% DLM, (iii) BD + 0.054% DLM, (iv) BD + 0.086% DLM, (v) BD + 0.029% MHA-FA, (vi) BD + 0.078% MHA-FA and (vii) BD + 0.107% MHA-FA, as analyzed. Indicator AA oxidation was determined during 4 h studies, where pigs were fed half-hourly meals each equal to 1/32 of their daily feed allowance. Each meal was mixed with 258.7 kBq (s.e. 2.6) of l-[1-14C]Phe with a prime of 3.5 times the half-hourly dose added to the first meal. The slope of the decrease in IAAO calculated by linear regression analysis was greater (P = 0.012) for DLM supplementation (9.87 ± 1.450 per g, 1.488 ± 0.215% per mmol) than for MHA-FA (6.48 ± 0.89 per g, 1.107 ± 0.152% per mmol). The ratio of slopes indicated a bioavailability of MHA-FA on a product basis, relative to DLM, of 65.7%. Bioavailability on an equimolar Met basis, calculated from the ratio of the slopes was 74.4% for MHA-FA, relative to DLM. In conclusion, these results indicate that the metabolic bioavailability of MHA-FA for growing pigs is appreciably lower than that of DLM on both an equimolar and a product basis.

An estimate of the methionine requirement and its variability in growing pigs using the indicator amino acid oxidation technique.

Although AA requirements for the mean of a population of growing pigs have been established using traditional methods, there are no estimates of the variability within the population and whether this variation differs among AA. With the increased use of supplemental Lys in pig diets, there will be an increased need to supplement Met, commonly the second or third limiting AA in corn-soybean diets. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs at a similar physiological stage. The objective of this study was to determine the mean Met requirement in individual gilts and to estimate the related variability. Six individually housed female pigs (initial BW = 8.8 kg, SD 1.5) each received diets providing 6 levels of dl-Met. The isonitrogenous and isoenergetic diets contained 0.187, 0.250, 0.290, 0.320, 0.350, and 0.377% Met (analyzed, as-fed basis). Cysteine (0.48%) and Lys (1.44%) concentrations were similar for all diets. Pigs were adapted for 6 d to the basal corn-soybean meal diet (0.187% Met), which was offered at 95 g/kg(0.75) of BW to ensure complete consumption of the test diets. During 4-h oxidation studies, 313.4 kBq, (SD 35.6) of L-[1-(14)C]Phe was mixed with each of 8 half-hourly meals, and expired CO(2) was collected. The breakpoint in Phe oxidation, representing the Met requirement, and its variability, was determined using 2-phase linear regression. Phenylalanine oxidation decreased as the Met content increased from 0.187 to 0.29%. Phenylalanine oxidation was not different (P > 0.2) for diets ranging from 0.320 to 0.377% Met. The dietary Met requirement varied from 0.320 to 0.373% for individual pigs. The mean Met requirement for individual pigs was determined to be 0.340% of diet (SD = 0.024%, CV= 7.1%), with 0.340, 0.364, and 0.388% covering the requirement of 50, 66, and 95% of the population, respectively. The present mean population estimate was similar to the recommended dietary Met concentration of 0.325% for pigs of this BW and feed intake. To maximize profitability, Met levels in starter pig diets should be determined, depending on the cost of crystalline Met and the fraction of the population whose requirement is to be met.

Effect of common antinutritive factors and fibrous feedstuffs in pig diets on amino acid digestibilities with special emphasis on threonine.

Most feedstuffs contain antinutritive factors (ANF) such as insoluble fibers, lignins, tannins, and lectins. Intake of these ANF has the ability to reduce nutrient digestibility and to increase endogenous protein losses, such as through increased intestinal mucus secretion. The objective of this experiment was to determine the apparent ileal digestibilities (AID) of AA of 6 ANF-enriched diets to estimate endogenous protein loss associated with these ingredients in diets for young pigs. Forty-two 10-kg BW pigs fitted with a simple T-cannula at the distal ileum were randomly assigned to 1 of 7 casein-based diets with: no supplement (control), 100 g/kg of canola meal (CM), 100 g/kg of wheat bran (WB), 150 g/kg of barley (BR), 22.5 g/kg of lignin (LG), 15 g/kg of kidney beans [as a lectin (LE) source], and 15 g/kg of tannins (TN). All diets were formulated to be similar in N, indispensable AA, and caloric contents. After a 7-d adaptation to the test diets, N balance was conducted for 5 d, followed by 24 h of collection of digesta for analyses of AA. Pigs fed BR had 17% lower ADG and 15% lower feed conversion ratio (P < 0.05) compared with control and CM pigs. Pigs fed diets containing WB and BR had lower N retention as a percentage of absorbed N compared with all other groups (P = 0.03). The AID for CP was lower in BR, WB, and LE pigs compared with control. Of the AA, AID of Thr was notably lowest in BR, WB, and TN pigs (P < 0.05). The standardized ileal digestibility was lower in WB and BR pigs for most indispensable AA. Altogether, these data suggest that hemicellulose fiber, at concentrations typical in commercial swine diets, reduces AID of AA by increasing endogenous losses. Understanding the differential effects of ANF on endogenous losses of individual dietary AA will improve the accuracy of diet formulation.

Relationship between bicarbonate retention and bone characteristics in broiler chickens.

Determination of the bicarbonate retention factor (BRF) is an important step during development of the indicator amino acid oxidation technique for use in a new model. A series of 4-h oxidation experiments were performed to determine the BRF of broilers aged 7, 14, 21, 28, 35, and 42 d using 4 birds per age group. A priming dose of 1.2 microCi of NaH(14)CO(3), followed by eight half-hourly doses of 1 microCi of NaH(14)CO(3) were given orally to each of 4 birds per age. The percentage of (14)C dose expired by the bird at a steady state was measured. These birds, as well as 12 additional birds matched for age and BW, were killed, and femur bone mineral density was measured by quantitative computed tomography to determine the relationship between bone development and bicarbonate retention at each age. There was a correlation (r = 0.50; P < 0.05) between total cross-sectional femur bone mineral density and bicarbonate retention at each age. A prediction equation (Y = 6.95 x 10(-2)X - 3.51 x 10(-5)X(2) + 27.58; P < 0.0001, R(2) = 0.79) where Y = bicarbonate retention and X = BW was generated to predict Y as a function of X. Bicarbonate retention values peaked at 28 d, during the stage of the most rapid bone deposition and the highest growth rate. A constant BRF was found from 1,900 to 2,700 g of BW of 35.15 +/- 1.095% (mean +/- SEM). This retention factor will allow the accurate correction of oxidation of (14)C-labeled substrates in broilers of different ages and BW in future indicator amino acid oxidation studies.

Estimate of the variability of the lysine requirement of growing pigs using the indicator amino acid oxidation technique.

Although AA requirements for the mean in a population of growing pigs are well established, there are no direct estimates of their variability within the population. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs. The objective was to determine the Lys requirement in individual pigs to derive a first estimate of the population mean requirement and its variability. Nine individually housed barrows (15 to 18 kg) were surgically implanted with venous catheters for isotope infusion. Pigs were offered, in random order, isonitrogenous and isoenergetic diets with one of seven Lys concentrations (4.8 to 15.5 g of Lys/kg diet, as-fed basis). The pigs were fed twice daily, except for study days when they received one-half of the daily allowance in eight equal hourly meals. After a validated minimum adaptation period, indicator (Phe) oxidation was determined for each dietary Lys level during a 4-h primed, constant infusion of L-[1-(14C)]Phe at a rate of 464 kBq/h. The Lys requirement was calculated using a two-phase linear regression crossover analysis within individual pigs. For each pig, Phe oxidation decreased linearly (P < 0.02) as the dietary Lys concentration increased until the requirement was reached; thereafter, Phe oxidation was not different. The true ileal digestible Lys requirement ranged from 7.5 to 10.6 g/kg of diet (as-fed basis) for the nine animals. The mean requirement for all pigs was 9.1 g/d (CV, 11.6%) or 93.9% (CV, 9.8%) of the predicted (NRC, 1998) requirement based on each pig's mean BW and energy intake. The measured and predicted requirements did not differ. The indicator AA oxidation method gave values for Lys requirement similar to conventional methods. The short (< 3 wk) experimental period allows, for the first time, the estimate of population variability, which provides for more accurate calculation of the effect of altering Lys intake on herd performance and production economics. This method is suitable to use with all dietary indispensable AA.

Feeding frequency and type of isotope tracer do not affect direct estimates of lysine oxidation in growing pigs.

Oxidation contributes to the inefficiency of lysine utilization for protein deposition. The influences of feeding frequency and type of isotope tracer on estimated lysine oxidation were studied in growing pigs fed lysine-limiting diets. Yorkshire gilts (n = 11) weighing 40-45 kg were fitted with venous catheters. They were fed, in 3 or 8 equal meals daily, a purified diet based on casein and cornstarch. Lysine intake limited the pigs' protein deposition to 70% of their potential. After a 5-d N-balance period, lysine oxidation was estimated by a primed, constant 26-h infusion of [1-14C]L-lysine and [6-3H]L-lysine. Feeding frequency and type of tracer did not affect lysine oxidation (P > 0.1). Increasing feeding frequency from 3 to 8 times daily reduced the variance and fluctuation of lysine oxidation by 46 and 30%, respectively. The mean lysine oxidation, as a fraction of the true ileal digestible lysine intake, was 9.2% based on the free lysine specific radioactivity (SRA) in plasma, 20.1% based on free lysine SRA in liver and 21.8% calculated from N-balance data. On the basis of liver free lysine SRA, tracer dilution methods and N-balance data give similar quantitative estimates of lysine oxidation (P > 0.10). Isotope tracer studies that cover one or more complete feeding cycles, i.e., feeding-to-feeding periods, can be used to obtain valid daily lysine oxidation values.

Development of the indicator amino acid oxidation technique in chickens: L-[1-(14)C]phenylalanine infusion dose and phenylalanine oxidation.

Amino acid requirements of broiler breeder chickens are not well known. The indicator amino acid oxidation (IAAO) technique was adapted for use in broiler breeders as a rapid and sensitive method to determine amino acid requirements. During IAAO, phenylalanine oxidation decreases, inversely to the changes in protein synthesis, as the intake of the limiting test amino acid increases from deficient to adequate. Above the adequate level, phenylalanine oxidation remains constant. Before IAAO can be employed, the optimum priming and constant infusion doses of phenylalanine must be determined. Prelaying catheterized birds aged 20 to 24 wk were placed in closed oxidation chambers attached to a breath collection apparatus. A constant L-[1-(14)C]phenylalanine dose of 3.5 microCi/kg BW/h and priming doses of 4.5, 5.5, and 7.0 microCi/kg BW were used to determine optimal prime:constant dose ratios, minimum time taken for breath 14CO2 excretion to become constant (plateau), and adequate percentage of phenylalanine oxidized. At this constant infusion rate, the optimal priming dose of L-[1-(14)C]phenylalanine was 5.5 microCi/kg BW, resulting in a prime:constant dose ratio of 1.6:1. By using this ratio, the average time taken for breath 14CO2 to reach plateau was 60 min. Average phenylalanine oxidation at plateau, corrected for bicarbonate retention, was 5.5 +/- 1.4% (mean +/- SD), which is adequate for IAAO studies using deficient-to-excess levels of test amino acids. To the authors' knowledge, this study is the first in chickens to establish a primed, constant infusion technique using L-[1-(14)C]phenylalanine. The IAAO technique will be used in future studies to determine amino acid requirements in chickens.

Development of the indicator amino acid oxidation technique in chickens: calibration of oxidation system and determination of bicarbonate retention factor.

The indicator amino acid oxidation technique has been widely used as a rapid and sensitive method to determine amino acid requirement. In this technique, the indicator 14C-amino acid is continuously infused and breath 14CO2 is collected to determine oxidation rate, which is used to predict requirement. However, a portion of the 14CO2 produced from amino acid oxidation is retained in the body and must be determined to correct oxidation rates based on breath 14CO2 collection. To develop the indicator amino acid oxidation technique for use in poultry, it is important to accurately measure expired and retained CO2; these measurements were accomplished using intravenous infusion of NaH14CO3. Oxidation chambers were constructed and calibrated to ensure complete collection of breath 14CO2. Hydrochloric acid was infused over 1 h into a beaker containing NaH14CO3 in four closed oxidation chambers, and the 14CO2 released was collected over 2 h. Of the initial radioactivity dose, 99.3 +/- 1.9% was recovered in collected 14CO2. We concluded that the oxidation chamber apparatus is adequate for complete recovery of expired 14CO2. In Experiment 2, we determined bicarbonate retention in five broiler breeder roosters from a primed, constant infusion of NaH14CO3 into the jugular vein for 4 h. At steady state, 86.0 +/- 1.4% of NaH14CO3 infusion was recovered in breath. Because 14% of infused 14C is retained within the body, the present results demonstrate the need to determine bicarbonate retention in chickens during oxidation studies and use it as a correction factor in subsequent experiments.

The effect of lean growth rate on puberty attainment in gilts.

Two hundred sixteen prepubertal Genex Manor hybrid F1 gilts were used to determine the impact of lean growth rate on sexual development of gilts. This study was composed of two experiments (Exp. 1 and Exp. 2). In Exp. 1, at approximately 96 d of age and 54 kg weight, gilts were allocated with respect to growth rate and litter origin to one of two dietary treatments: 1) a diet formulated to maximize lean growth potential (LP; n = 84) or 2) a diet formulated to produce a lower lean growth rate (LL; n = 84). In Exp. 2, at approximately 88 d of age and 50 kg weight, gilts were allocated with respect to growth rate and litter origin to one of two dietary treatments: 1) a diet formulated to maximize lean growth potential (LP; n = 24) or 2) a diet formulated to restrict lean growth further than was achieved in LL in Exp. 1 (RL; n = 24). All gilts were fed treatment diets for ad libitum consumption and housed in groups of six. Weight, backfat depth and loin depth, and feed intake were measured weekly. Starting at 135 d of age, gilts received 20 min of direct daily exposure to a boar as a pen group for pubertal stimulation. Puberty attainment was determined as the day gilts first exhibited the standing reflex in response to contact with a boar. At pubertal estrus, body weight, backfat depth, and loin depths were recorded. Diet affected (P < or = 0.05) estimated fat-free lean gain (LP, 424 vs LL, 347 g/d, Exp. 1; LP, 397 vs RL, 376 g/d, Exp. 2) during the growth period (start to stimulation). However, age at puberty was not affected by diet (LP, 157.3 vs LL, 157.6, Exp. 1; LP, 166.7 vs RL, 167.3, Exp. 2) or overall lean growth at stimulation (P > or = 0.05 in both experiments), confirming that innate variability in sexual development of commercial genotypes, rather than growth performance, determines onset of sexual maturity. A negative correlation between age at puberty and growth rate from 50 kg until puberty (P < or = 0.05) (LP, r = -0.40, LL, r = -0.36, Exp. 1; LP, r = -0.64, RL, r = -0.48, Exp. 2) was a consequence of reduced lean tissue growth during the stimulation period in later-maturing gilts.

Total sulfur amino acid requirement in young men as determined by indicator amino acid oxidation with L-[1-13C]phenylalanine.

BACKGROUND: Determining the sulfur amino acid (SAA) requirements of humans has remained elusive because of the complex nature of SAA metabolism. Current recommendations are based on nitrogen balance studies. OBJECTIVE: The goal of the present study was to determine the methionine requirement of men fed a diet devoid of cysteine (total SAA requirement). DESIGN: Six men were randomly assigned to receive 6 graded intakes of methionine: 0, 6.5, 13.0, 19.5, 26.0, and 32.0 mg x kg(-1) x d(-1). The total SAA requirement was determined by measuring the oxidation of L-[1-13C]phenylalanine to 13CO2 (F(13)CO2)). The mean total SAA requirement was estimated with use of a linear regression crossover analysis, which identified a breakpoint of the F(13)CO2 response to methionine intake. RESULTS: On the basis of the mean measures of F(13)CO2, the mean requirement and population-safe intake (upper limit of the 95% CI) of total SAAs were found to be 12.6 and 21 mg x kg(-1) x d(-1), respectively. CONCLUSION: Although the mean SAA requirement is consistent with current guidelines for the total SAA intake, the population-safe intake is substantially higher than the currently recommended total SAA intake.

Dietary cysteine reduces the methionine requirement in men.

BACKGROUND: Despite early evidence suggesting that dietary cysteine has a sparing effect on methionine requirements, some recent reports question the existence of a measurable sparing capacity. OBJECTIVE: The goal of the present study was to determine whether dietary cysteine could reduce the requirement for methionine in men consuming diets with and without cysteine. DESIGN: Six men were randomly assigned to receive graded intakes of methionine while fed a diet containing either no exogenous cysteine or an excess of cysteine (21 mg x kg(-1) x d(-1)). The methionine requirement was determined by measuring the oxidation of L-[1-13C]phenylalanine to 13CO2 and estimated by using a linear regression crossover analysis. RESULTS: The mean and population-safe (upper limit of the 95% CI) methionine requirements in the absence of exogenous cysteine were found to be 12.6 and 21 mg x kg(-1) x d(-1), respectively. The mean and population-safe methionine requirements in the presence of excess dietary cysteine were found to be 4.5 and 10.1 mg x kg(-1) x d(-1), respectively, representing a cysteine sparing effect of 64% in a comparison of mean methionine requirements and of 52% in a comparison of population-safe methionine intakes. Furthermore, the difference between population-safe intakes with and without dietary cysteine establishes a safe cysteine intake of 10.9 mg x kg(-1) x d(-1) in the presence of adequate methionine intakes. CONCLUSION: Our data suggest that dietary cysteine can reduce the exogenous requirement for methionine in men. These results strongly support the existence of a cysteine sparing effect in humans.

Tyrosine requirement of healthy men receiving a fixed phenylalanine intake determined by using indicator amino acid oxidation.

BACKGROUND: The currently accepted total aromatic amino acid requirement for adults is based on nitrogen balance measurements in individuals who received their intake of aromatic amino acids solely as phenylalanine. OBJECTIVE: The objective of this study was to determine the requirement for the amino acid tyrosine in healthy men receiving an adequate, but not excessive, intake of phenylalanine (9 mg x kg(-1) x d(-1)). DESIGN: The effect of a graded intake of tyrosine was determined in 6 healthy men consuming energy-sufficient diets containing 1 g protein x kg(-1) x d(-1). The tyrosine requirement was determined by using indicator amino acid oxidation methodology with L-[1-13C]lysine as the indicator. Subjects were studied at each of 7 tyrosine intakes. RESULTS: A graded intake of tyrosine had no effect on lysine flux. The mean tyrosine requirement was determined from the response of the oxidation of L-[1-13C]lysine to breath 13CO2. A 2-phase linear regression crossover analysis of breath 13CO2 identified the breakpoint and upper 95% confidence limit, which represents the mean and safe intakes, to be 6.0 and 7.0 mg x kg(-1) x d(-1), respectively. CONCLUSIONS: The safe intake of total aromatic amino acids calculated from the present results for tyrosine and our previous estimate for phenylalanine is estimated to be 21 mg x kg(-1) x d(-1). This intake is 1.5 times the currently recommended total aromatic amino acid intake of the FAO/WHO/UNU (1985), 14 mg x kg(-1) x d(-1). Furthermore, the absolute aromatic amino acid requirement may be dependent on the proportional balance of these amino acids in the diet.

The effect of graded intake of glycyl-L-tyrosine on phenylalanine and tyrosine metabolism in parenterally fed neonates with an estimation of tyrosine requirement.

Although tyrosine is considered indispensable during the neonatal period, its poor solubility has limited its inclusion in parenteral amino acid solutions to less than 1% of total amino acids. Dipeptides of tyrosine are highly soluble, have been shown to be well used and safe in animal models and humans, and, therefore, may be used as an effective means of providing tyrosine in the parenterally fed neonate. The goal of the present study was to determine the tyrosine requirement of the parenterally fed neonate receiving graded intakes of glycyl-L-tyrosine as a source of tyrosine. Thirteen infants receiving adequate energy (340 +/- 38 kJ. kg(-1).d(-1)) and protein (2.4 +/- 0.4 g.kg(-1).d(-1)) were randomized to receive parenteral nutrition with one of five graded levels of glycyl-L-tyrosine. The mean requirement and safe level of intake were estimated using a 1-(13)C-phenylalanine tracer and linear regression cross-over analysis that identified a break point in the response of label appearance in breath CO(2) (F(13)CO(2)) and phenylalanine oxidation to graded tyrosine intake. Based on the mean estimates of whole-body phenylalanine oxidation, the tyrosine mean requirement and safe level of intake were found to be 74 mg.kg(-1). d(-1) and 94 mg.kg(-1).d(-1), respectively. This represents 3.1 and 3.9% of total amino acids, respectively, considerably higher than levels found in present commercially available pediatric amino acid solutions. These data raise concern regarding the adequacy of aromatic amino acid intake in the parenterally fed neonate.

Steady state is not achieved for most plasma amino acids during 12 hours of fasting in the neonatal piglet.

Kinetics studies in neonates are important to establish the requirement for amino acids and to understand the mechanisms of normal and altered metabolism. During kinetics experiments, plasma amino acid concentrations should be in steady state. Our objective was to determine whether 12 h of fasting, after parenteral or enteral feeding, resulted in a steady state in concentrations of amino acids. Two-day-old piglets were implanted with catheters (d 0), and randomly assigned to either intragastric (i.g., n = 6) or i.v. (n = 6) feeding. On d 5, piglets were fasted for 12 h. During the first 2 h, plasma concentrations of almost all amino acids declined except asparagine (i.g. and i.v.), tyrosine (i.v.), and glycine (i.v.), which increased. Only i.g. glycine did not change. Between 2 and 12 h, the only indispensable amino acids that did not change were phenylalanine (i.v.) and histidine (i.g. and i.v.). The branched-chain amino acids increased during this period (i.v. and i.g.). The greatest change was tyrosine, increasing 13% (i.v.) and 32% (i.g.) per hour. After 12 h of refeeding, glycine, serine, threonine, and asparagine concentrations were lower than baseline (p<0.05) in the i.v. group. In i.g. fed piglets, only threonine remained below baseline (p<0.05), and arginine was greater than baseline (p<0.05). Differences between i.v. and i.g. may be the result of impaired small intestinal metabolism secondary to parenteral feeding. In neonatal pigs, most plasma amino acids were unstable during 12 h of fasting. Thus, kinetics studies that require a steady state must be conducted in the fed state.