The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1774-1785

The Marginal Efficiency of Utilization of All Ileal Digestible Indispensable Amino Acids for Protein Gain Is Lower than 30% in Preruminant Calves between 80 and 240 kg Live Weight^1,2

Walter J. J. Gerrits^{*, , 3}, Johan W. Schrama^*, and Seerp Tamminga^*

^* Animal Nutrition Group, Wageningen Institute of Animal Sciences (WIAS), Wageningen Agricultural University, 6700 AH, Wageningen, The Netherlands and  TNO Nutrition and Food Research Institute, Department of Human and Animal Nutrition (ILOB), 6700 AA, Wageningen, The Netherlands

	ABSTRACT

Abstract Introduction Methods Results & Discussion References

A previous study showed that the marginal efficiency of utilization of digestible nitrogen for deposition in the body in preruminant calves is only ~30%. The study consisted of two similar experiments that were performed in two live weight ranges: 80-160 and 160-240 kg. In each experiment, 36 calves were allotted to one of twelve dietary treatments, consisting of six protein intake levels at each of two protein-free energy intake levels. This paper presents amino acid analyses of dietary and body proteins of these experiments with the following goals: 1) to identify possible limiting indispensable amino acids, and 2) to quantify the effect of protein and energy intake on the amino acid composition of deposited body proteins. The marginal efficiency of utilization of ileal digestible amino acids for deposition in the body did not exceed 30% for any of the indispensable amino acids. Increasing protein intake increased the ratio of indispensable to dispensable amino acids in deposited body proteins, likely caused by an increase in muscle-to-bone ratio in the carcass with a concomitant decrease in the proportion of collagen protein. It was concluded that the low marginal efficiency of utilization of digestible milk proteins for growth in preruminant calves of this weight range was not caused primarily by a severe limitation of a single indispensable amino acid in the diet.

	INTRODUCTION

Abstract Introduction Methods Results & Discussion References

In rapidly growing animals, the composition of the required digestible protein is largely determined by the amino acid (AA)⁴ composition of the deposited tissues. In pigs, the marginal efficiency of utilization of the ileal digested limiting AA can be as high as 74% (Bikker et al. 1994b) or 85% (Batterham et al. 1990). This implies that, for these animals, the AA composition of the empty body determines ~80% of the requirements for ileal digestible indispensable amino acids (IAA). The AA composition of the empty body, however, varies with body weight and nutritional inputs as a result of shifts in the contribution of individual tissues to the empty body (Bikker et al. 1994a). Furthermore, high rates of protein turnover may be associated with increased oxidative losses (Liu et al. 1995). Therefore, the contribution of tissues with a high turnover rate to total AA requirements may be higher than can be expected on the basis of their contribution to empty-body protein. Information on the AA composition of (preruminant) calves is scarce and is either restricted to selected tissues (e.g., Vervack et al. 1977) or obtained at a fixed body weight and feeding regimen (e.g., Williams 1978). In preruminant calves between 80 and 240 kg live weight, the efficiency of utilization of digestible milk proteins for protein deposition was shown to decrease from 60 to 35% with increasing protein intake. Marginal efficiency of protein utilization, i.e., the response of protein deposition rate to increased protein intake, was also low, i.e., ~30% of the extra ingested protein was deposited (Gerrits et al. 1996). This paper presents the results of the AA analysis of the feed and quantitatively collected body tissues of the calves used in these experiments (Gerrits et al. 1996) to address the following questions: 1) is the efficiency of utilization of ileal digestible milk proteins for deposition in the body of growing calves severely restricted by one limiting IAA? 2) Does the AA composition of deposited body proteins depend on the rate of protein and energy intake? If so, 3) are carcass, organs and hide + head + feet + tail (HHFT) fractions affected differently by these dietary treatments.

	MATERIALS AND METHODS

Abstract Introduction Methods Results & Discussion References

Two experiments were conducted to quantify the relationship between nutrient intake (protein, protein-free energy) and the rate of gain of live weight, protein and fat in Holstein-Friesian × Dutch-Friesian male calves. The experiments were similar in design, but were performed in two weight ranges: 80-160 and 160-240 kg live weight in Experiment 1 and 2, respectively. The experiments were conducted to provide essential data for the construction of a growth simulation model (Gerrits et al. 1997); the design was originally described by Gerrits et al. (1996).

Animals, housing and dietary treatments.  Male Holstein Friesian × Dutch Friesian calves (n = 90) were used in the two experiments. In Experiments 1 and 2, 8 and 10 calves were used as a reference group, respectively. These calves were slaughtered at the beginning of the experiment (80 and 160 kg live weight in Experiment 1 and 2, respectively) and analyzed for body composition, as described later for the calves slaughtered at the target weight. In each experiment, 36 calves were grouped in three blocks, based on live weight; calves were randomly assigned per block to one of the 12 dietary treatments, which consisted of six protein intake levels at each of two energy intake levels (Table 1). Proteins, carbohydrates and fat differ in their ATP yield per MJ. Therefore, the energy intake levels were kept constant on a protein-free basis. Moreover, the ratio of energy intake from carbohydrates to energy intake from fat was kept constant at ~1 in both experiments. For each experiment, two basal milk replacers that varied in protein and protein-free energy content were used. All experimental treatments could be realized by mixing these basal diets in different ratios and by varying the quantity fed. The ingredient and analyzed nutrient composition of the basal milk replacers is presented in Table 2. When formulating the basal diets for Experiment 2, it was decided to minimize the effect of possible mixing faults, leading to a reduction in the protein content of basal diet 2. The mixing ratios of the two basal diets were subsequently adapted.

The calves were weighed weekly, and the feed intake was adjusted twice weekly according to the measured live weight and projected rates of weight gain. Milk replacers were reconstituted with water and supplied at a temperature of ~39°C. Calves were fed individually twice daily, at 0800 and 1600 h. Calves were housed individually in wooden, slat-floored crates of 1.75 × 0.75 and 1.85 × 0.85 m in Experiment 1 and 2, respectively. At an average live weight of 120 and 200 kg in Experiment 1 and 2, respectively, plastic bags were harnessed to the calves to allow quantitative collection of feces over a 5-d period after a 5-d adaptation period. Fecal samples were pooled per calf over the collection period and stored at 20°C pending analysis. Both experiments were approved by the ethical committee of the TNO Nutrition and Food Research Institute, Zeist, the Netherlands.

Slaughter procedures and sample preparation.  The calves were slaughtered in the week in which their live weight was closest to the target weight: 160 and 240 kg in Experiment 1 and 2, respectively. Calves were killed by stunning and exsanguination. Body components were split into the following three fractions: 1) carcass; 2) hide, head, feet and tail (HHFT); and 3) organs. The organ fraction included blood, the emptied gastrointestinal tract and all other organs. For analysis of the carcass fraction, the composition of the right carcass half (split longitudinally) was considered representative. All fractions were weighed and stored in plastic bags at 20°C. The frozen carcass, organ and HHFT fractions were cut into small blocks and minced in a commercial butcher's mincer (45 L, 2 speeds, Rohwer). The three fractions were sampled separately and stored in sealed plastic bags at 20°C. Due to the heterogeneity of the HHFT fraction, large samples (~1.5 kg) were obtained from the mincer, autoclaved for 10 h at 124°C with a known amount of water and minced using a laboratory disperser before analysis. All samples were analyzed for dry matter, nitrogen, crude fat and crude ash, as described by Gerrits et al. (1996). Amino acid analyses were performed in pooled samples. Thawed samples of the fresh material of the individual fractions were pooled per treatment. Samples of the calves of the initial slaughter groups were pooled to one sample per fraction in each experiment. Samples of the carcass, organ and HHFT fractions were pooled, based on their nitrogen content, which had been analyzed previously (Gerrits et al. 1996). Samples of different batches of the basal diets were pooled into one sample per basal diet, based on the size of the batch in which they were produced.

Amino acid analyses.  After pooling, the samples obtained were dried (for modified procedures, see Gerrits et al. 1996) and extracted using petroleum ether (boiling range 40-60°C) (ISO 1985). The extracted tissue was ground over a 0.5-mm screen and analyzed for dry matter according to ISO 6496 (ISO 1983). The ground, air-dried extracted tissues were subsequently used for the AA analysis. According to our unpublished observations, the autoclaving procedure for the HHFT samples did not affect their dry matter, nitrogen and crude fat content. It was expected, however, to affect the AA composition of these samples. A test was performed to quantify the loss of individual AA during the autoclaving procedure. Large samples (~3 kg) of the HHFT fraction of four calves, chosen randomly from the experiments, were further ground in the butcher's mincer (45 L, two speeds, Rohwer) using sufficient liquid nitrogen to obtain a finely ground material. The material was split into two parts; one part was sampled directly and treated as the other fractions describe above, and the other part was pretreated by autoclaving as described above. After the fat extraction, the residue obtained of the nonautoclaved HHFT material was further ground using a mixer mill (Retsch MM2000, Haan, Germany; beaker volume 10 mL, 2 stainless steel balls of 9 mm, amplitude 100, time 2 min) to avoid an increase in temperature in the samples due to grinding.

The extracted, air-dried material was hydrolyzed to AA for 22 h using 6 mol/L HCl at 108°C under reflux and analyzed for AA composition. The amino acid analyses were performed with an amino acid analyzer (Biotronik LC 5001, Eppendorf-Biotronik, Maintal, Germany) using postcolumn derivitization with ninhydrin as the coloring reagent, according to the manufacturer's recommendations. For the determination of the sulfur AA, the samples were oxidized at 0°C with performic acid for 16 h before the acid hydrolysis (Moore 1963). Tryptophan was analyzed after alkaline hydrolysis (16 h, 120°C) using lithium hydroxide, followed by HPLC separation as described by Slump et al. (1991) and UV detection at 280 nm.

Calculations.  In each experiment, the AA composition of the deposited protein was calculated for each treatment as the sum of the amount of AA in the three fractions, subtracted by the amount of AA present at the start of the experiment. The latter was estimated from initial live weight and the AA composition of the reference group. Deposition of AA in the fractions was calculated analogously. Corresponding rates of AA deposition were calculated as deposition divided by the length of the experimental period.

The efficiency of utilization of an ileal digestible AA for protein deposition was calculated as the deposition of that AA divided by its ileal digestible intake. The latter was estimated on the basis of the measured ingested quantity and the measured apparent fecal N digestibility in these experiments (Gerrits et al. 1996). For the calculation of the amount of each AA apparently digested at the terminal ileum, two assumptions were made. First, the true ileal digestibility of milk proteins is 100%, as found in preruminant calves by Tolman and Beelen (1996) with the ¹⁵N dilution technique. Consequently, the AA profile of endogenous losses equals the AA profile of ileal digesta of calves fed milk proteins. This profile was calculated from profiles given by Tolman and Beelen (1996) and Lallès et al. (1990). Second, the ileal N digestibility is 2.5% lower than the fecal N digestibility in calves fed milk proteins (Hof 1980, Tolman and Beelen 1996), the latter determined in the present experiments.

Statistical analyses.  Due to illness, one (treatment 2) and three animals (1 and 2 in treatments 2 and 12, respectively) had to be excluded from Experiment 1 and 2, respectively. Samples of these calves were not used for preparation of the pooled samples. The statistical analyses were performed on data obtained from pooled samples. Therefore, each treatment group was considered as the experimental unit and all regression procedures described below were weighted for the number of animals, pooled per treatment. The effects of protein and protein-free energy intake on the composition of the deposited protein in the body fractions or on the efficiency of utilization of ileal digestible AA were analyzed using the following model:

(1)

in which Y is the dependent variable, µ is the average intercept, E_i is the fixed effect of protein-free energy intake level i, ₁ is the effect of protein intake, _2i is the interaction between protein intake and protein-free energy intake level i, X_j is the digestible protein intake of the calves of treatment j (g N/d), is the average experimental digestible protein intake (g N/d), _ij is the error, i = 1, 2 and j = 1 ... 6.

The marginal efficiency of utilization of all ileal digestible AA for deposition in the body was calculated as the slope of the regression line between the rate of intake of apparent ileal digestible AA and the rate of deposition of that AA. On the basis of the reported marginal efficiencies for nitrogen (Gerrits et al. 1996), diminishing returns were not hypothesized to be present for individual IAA. This hypothesis was tested in a prelimenary analysis, using model (1), with the intake of ileal digestible IAA_k (k = 1 ... 12) as X-variables, expanded with the quadratic term X² and the interaction between X² and E_i. This preliminary analysis revealed no interaction between X² and E_i for any of the IAA. With this interaction term omitted, the quadratic component was significant for tryptophan (Experiment 1), threonine, isoleucine and leucine (Experiment 2). It was decided to present the linear effects only, except for these AA, in which case the marginal efficiency was computed at the lowest as well as the highest realized level of intake. These computations were made by using the first derivative of the quadratic model, with E_i omitted from the model. These values are presented in the text.

Regression procedures were carried out using the General Linear Models procedure of SAS (SAS Institute, Cary, NC); the probability level at which differences were considered significant was P < 0.05.

	RESULTS AND DISCUSSION

Abstract Introduction Methods Results & Discussion References

Intake of ileal digestible amino acids.  The analyzed AA composition of the protein of the basal diets is presented in Table 2 and was in good agreement with that of milk proteins (Walstra and Jenness 1984). Table 3 shows the estimated intake of ileal digestible amino acids of the animals for all treatments, averaged over the experimental weight range. Table 3 reflects the experimental design, corrected for net endogenous amino acid losses at the terminal ileum. Although the effect of protein-free energy intake was significant for a number of AA, it was very small and thus of minor quantitative importance. It was a consequence of the difference in amino acid composition between skim milk and casein protein, which constitute the major part of the protein in basal diets one and two, respectively. Compared at the average nitrogen intake level, basal diet 1 provided 75 and 86, and 31 and 59% of the daily feed intake at the low and high protein-free energy intake level in Experiment 1 and 2, respectively. Therefore, compared at the average nitrogen intake level, the calves at the high protein-free energy intake level received a slightly smaller part of their dietary protein from casein. Casein contains less cystine than skim milk protein, which is reflected in the analysis of the basal diets (Table 2). Consequently, the relative increase in cystine intake with increasing protein intake was lower than that of other AA.

Effect of the autoclaving procedure on the recovery of amino acids in the HHFT fraction.  The recovery of each amino acid was calculated as the amino acid concentration in the autoclaved sample over the concentration in the non-autoclaved sample, expressed in grams amino acid per kilogram dry matter. The recovery of cystine, methionine, glutamic acid, alanine, lysine and arginine was (P < 0.05) <100%: 56 ± 1.3, 98 ± 0.4, 96 ± 1.0, 95 ± 1.5, 96 ± 0.6 and 97 ± 0.7%, respectively. For the other amino acids, the recovery did not differ from 100% (P > 0.05). Apart from the loss of cystine, the loss of most amino acids during autoclaving was within the limits of accuracy of the amino acid determination. The high recoveries may well have been due to the absence of carbohydrates in the HHFT samples. Cystine is known to be labile at high temperatures, being partly converted into lanthionine (Papadopoulos 1984). The results of the AA analysis of the HHFT fraction, presented in this paper, are corrected for an incomplete recovery if the recovery for an amino acid was <100% (P < 0.05).

The efficiency of deposition of ileal digested amino acids in the body.  The effects of protein and protein-free energy intake on the efficiency of utilization of apparent ileal digestible AA for protein deposition are presented in Table 4. The change in AA deposition rate per unit increase in the rate of intake of that AA (i.e., the marginal efficiency of utilization of apparent ileal digestible AA) is also presented in this table. The efficiency of utilization of AA decreased substantially with increasing protein intake for almost all AA in both experiments. Increasing protein-free energy intake increased this efficiency for almost all AA in both experiments. This is in agreement with the effects of protein and protein-free energy intake on the efficiency of nitrogen deposition found in these experiments (Gerrits et al. 1996). The marginal efficiency was not affected by protein-free energy intake in either experiment. The results presented are therefore the average values.

Dispensable amino acids (DAA).  The average experimental efficiency of utilization of alanine and glycine exceeded 100%. For every mole of glycine intake, ~3 mol were deposited. In addition to the needs for tissue growth, glycine is required for a number of physiologic processes such as the synthesis of nucleic acids, bile acids, hemoglobin and glutathione (Reeds and Hutchens 1994). Although the low glycine content in milk proteins also contributed to this high efficiency, it is clear that a large amount of glycine has to be synthesized every day. Glycine is released in degradative pathways of various AA; it therefore is difficult to say whether amino acids were preferentially degraded to meet the needs for glycine. The efficiency of utilization of other DAA was low, but this does not necessarily mean that the intake can be reduced without affecting growth rate.

Conditionally dispensable amino acids.  Of the conditionally dispensable AA, arginine showed the highest efficiency of utilization (~90%). The maximum rate of arginine synthesis has been shown to be insufficient for maximum growth in growing pigs (Fuller 1994) and ruminants (Davenport et al. 1990). It has been suggested that at least 40% of the arginine deposited has to be supplied through the diet (Fuller 1994). It is therefore unlikely that arginine supply was a limiting factor, i.e., the quantity of arginine ingested was in almost all cases higher than the quantity retained. The efficiency of utilization of cystine was also considerably higher than that of the IAA. Based on these data, the possibility that considerable amounts of methionine were used for cystine synthesis (leading to a low efficiency of methionine utilization) cannot be excluded. This may be supported by the absence of the effect of protein intake on the efficiency of utilization of cystine (Table 4). Alternatively, however, this may have been the consequence of a lower contrast in cystine intake compared with the other AA, as discussed previously.

Indispensable amino acids.  The average experimental efficiency of utilization of apparently digestible IAA for protein retention was highest for threonine and lysine (42 and 38% in Experiment 1, respectively). Even at low protein intake levels (e.g., 45 g digestible N/d), the efficiency of utilization did not exceed 50% for any of the IAA in either experiment. The marginal efficiency of threonine and lysine was highest among the IAA, i.e., 20-23% in both experiments. Within the measured range, there was no evidence of diminishing returns in the relationship between AA intake and AA retention for any of the IAA, except for tryptophan (Experiment 1) and threonine, isoleucine and leucine (Experiment 2). For these IAA, the marginal efficiencies were calculated to have decreased from 25 to 18, 29 to 21, 21 to 14 and 21 to 16% when their ileal digestible intake increased from 2 to 7, 6 to 27, 9 to 37 and 15 to 63 g/d, respectively. At intakes below the measured range, however, marginal efficiencies must have been higher. This is evidenced by positive intercepts at zero intake for all IAA in both experiments, which is physiologically impossible. Obviously, the relationships presented in this paper cannot be extrapolated beyond the measured range.

The calculation of ileal digestible AA was based on a number of assumptions, each of which adds some uncertainty to the estimation of the apparent ileal digestibility values, thus affecting the reported efficiencies. First, the true ileal digestibility of milk proteins was assumed to be 100%. This assumption, however, was relevant to only the AA composition of the net ileal endogenous losses, not to the quantity. Second, the gap between ileal and fecal nitrogen digestibility was assumed to be 2.5%, with fecal nitrogen digestibilities determined in the experiments. Obviously, underestimation of this gap leads to overestimation of the apparent ileal digestibility values and would thus contribute to the reported low efficiency of IAA utilization. Additional calculations, however, showed that increasing this gap to 5% would result in an absolute increase in the marginal efficiency of <1.5% for all IAA in both experiments. For cystine, the absolute increase was 18 and 10% in Experiment 1 and 2, respectively, emphasizing the quantitative importance of cystine in ileal net endogenous losses.

The experimental diets were not designed to be limiting in one particular IAA. This is difficult when dealing with milk proteins, and even impossible when trying to avoid large quantities of synthetic IAA. We chose to quantify relationships between protein and energy intake and protein and fat gain, as described by Gerrits et al. (1996). In these experiments we tried to detect separate phases in which protein deposition depended on either protein intake (hypothesized at low protein intakes) or on energy intake (hypothesized at high protein intakes). These were shown to be absent in preruminant calves in this weight range. From the results of the AA analyses, the efficiency of utilization of all AA could be calculated, although the intake of all AA was varied simultaneously. These data therefore do not allow direct calculation of requirement values for growth for individual IAA. However, if the extremely low marginal efficiency of nitrogen utilization, reported by Gerrits et al. (1996), had been caused by a severe dietary IAA imbalance, this would have resulted in a high marginal efficiency of utilization of at least one of the IAA. The marginal efficiencies found were very low and within the rather narrow range of 11-29% for all IAA. Theoretically, the marginal efficiency of the limiting IAA for protein gain can be very high (Reeds and Hutchens 1994). In young pigs, values as high as 74% (Bikker et al. 1994b) and 85% (Batterham et al. 1990) have been reported for lysine. The results presented in this paper clearly show that dietary IAA imbalance was not the main reason for the low marginal efficiency of protein utilization of calves in this weight range. It implies that in preruminant calves of this weight range, the AA profile of deposited body protein is not necessarily reflecting the composition of so-called ideal protein for growth. The reason for the low marginal efficiencies, however, remains unclear. Factors known to affect the marginal efficiency of protein utilization include the following.

First, the marginal efficiency may decrease with age. Although in most experiments, effects of age and intake are confounded, younger calves seem to have a higher marginal efficiency compared with older calves. In studies with younger calves fed milk proteins, the marginal efficiency of nitrogen utilization is higher than the 30% found in the current experiments (Gerrits et al. 1996); Donelly and Hutton (1976) found a marginal efficiency of 45% for calves weighing between 40 and 70 kg live weight, and from experiments, presented by Tolman and Beelen (1996), a value of 69% could be calculated for calves of 70 kg live weight. In calves between 40 and 70 kg live weight, IAA requirement values could be estimated by increasing the intake of a specific IAA and measuring the N-balance (summarized by Williams 1994). In older calves (>100 kg live weight), according to our unpublished observations, this is far more difficult to achieve.

Second, the inefficiency of protein utilization may to some extent be the consequence of the diurnal pattern of feed intake. Protein must be deposited in the fed state to match postabsorptive losses, which have been shown to vary with protein intake (Millward et al. 1990). If this is the case, however, one should expect an improvement of nitrogen utilization with increased meal frequency. This effect could not be shown in preruminant calves when the feeding frequency was increased from once to six times daily (Williams et al. 1986).

Third, metabolically very active tissues, for example portal-drained viscera, may account for a disproportionate part of whole-body IAA metabolism. Recent studies with piglets (Stoll et al. 1997 and 1998) and sheep (MacRae et al. 1997) have shown that a considerable amount of IAA absorbed from the intestinal lumen are metabolized during first pass of the gastrointestinal tissue. In piglets, on average, 60% of the intake of IAA was recovered in the portal blood (Stoll et al. 1998). In sheep, this percentage varied between 61 and 83% for the IAA, independent of the level of feed intake (MacRae et al. 1997). Moreover, portal-drained viscera were shown to metabolize IAA from the arterial circulation as well (MacRae et al. 1997, Stoll et al. 1997). It is noteworthy that in the experiment of Stoll et al. (1998), the portal balance of cystine did not significantly differ from zero. These experiments indicate the following: 1) metabolism of IAA by portal-drained viscera could possibly account for a substantial part of the low efficiencies of IAA utilization reported in this paper, and 2) metabolism of particular IAA in specific tissues may lead to an imbalanced IAA profile at the site of deposition.

The high proportion of dietary IAA metabolized in the gastrointestinal tissue of piglets, however, is remarkable. If this were to be the fate of incremental intakes of IAA as well (which the work of MacRae suggests for sheep), it implies that the marginal efficiency of utilization of the most limiting dietary IAA for protein gain would also likely be <60% in pigs. This is in contrast with the serial slaughter experiments of Bikker et al. (1994b) from which one can calculate much higher marginal efficiencies, i.e., 58 and 74% for ileal digestible protein and the limiting ileal digestible IAA (lysine), respectively. Ignoring the effect of feeding frequency and body weight range of the pigs, a quantitative comparison of the data obtained from these transorgan measurements with those found in the whole body by means of serial slaughter experiments could resolve this apparent anomaly.

Fourth, as discussed previously, IAA may selectively be utilized to meet the needs for conditionally DAA, for example, utilization of methionine for the production of cystine. With regard to requirements for DAA, possibilities are numerous. For example, potential ruminants may preferably utilize AA for the production of precursors for gluconeogenesis, even if dietary glucose supply is abundant. Furthermore, IAA may be degraded to provide precursors for the urea cycle. The latter has been hypothesized for ruminants and was recently discussed by Lobley and Milano (1996). Briefly, removal of NH₃ by the ruminant liver possibly incurs a penalty on AA metabolism. The extent to which this happens is a matter of debate. In preruminant calves, NH₃ would originate from AA metabolism of peripheral, specifically intestinal, tissues.

Normally, calves will switch from a milk- to a plant-based diet before reaching 80 kg live weight. Our experiments were conducted with calves weighing >80 kg receiving a milk-based diet. It is not clear to what extent the metabolism of ingested IAA changes with age, regardless of the type of diet. Such potential changes in metabolism may explain the effects of age discussed above by any of the mechanisms proposed.

In summary, it is difficult to explain the low efficiency of utilization of IAA. As can be seen in Table 4, increased intakes of protein-free energy (lactose and fat in a similar energy ratio) increased the efficiency of utilization of amino acids for growth. As shown previously, however, there are no protein- and energy-dependent phases in protein deposition in preruminant calves, i.e., increased energy intake led to increased protein deposition rates, even at low protein intakes. The results of these experiments demonstrate the absence of a single IAA limiting the rate of protein deposition in calves fed milk protein. Alternative hypotheses to explain the low efficiency of utilization of IAA for growth may include effects of age, specific preference of particular tissues, for example, the gastrointestinal tract, to utilize IAA, leading also to an IAA imbalance at the site of deposition, and catabolism of IAA to meet the needs for DAA or conditionally DAA.

The amino acid composition of the whole body and the individual tissues.  The results of the AA analyses are presented in Table 5 (whole body), Table 6 (carcass fraction), Table 7 (organ fraction) and Table 8 (HHFT fraction). The whole-body AA composition of the calves of the initial slaughter group (Table 5) compared well with the results of Williams (1978), obtained with Friesian calves of ~70 kg live weight, with the exception of proline (70 vs. 81 g/160 g N), valine (48 vs. 39 g/160 g N) and isoleucine (36 vs. 28 g/160 g N). According to Simon (1989), the whole-body AA composition is comparable across species. The AA profiles obtained in these experiments resemble those of pigs (e.g., Bikker et al. 1994a, Wünsche et al. 1983), but clearly differ from that of rats, whose protein contains more cystine and glycine and less tyrosine, phenylalanine, histidine and arginine (Rafecas et al. 1994).

The AA profiles of the individual fractions clearly differed (Tables 6-8). The ratio between indispensable (including cystine, tyrosine and arginine) and dispensable AA (IAA/DAA) was highest in organ protein (1.1), followed by carcass protein (1.0) and was lowest for the HHFT fraction (0.6). The HHFT fraction was particularly rich in glycine, proline and arginine, indicating a high collagen content of this fraction (Nguyen en Zarkadas 1989, Williams 1978). The AA profiles of carcass protein were in good agreement with the values found by MacRae et al. (1993) for growing lambs, with the notable exception of threonine, which appeared lower in the calf carcass (42 vs. 50 g/kg total amino acid).

The effect of body weight.  Although these experiments were not designed to compare the AA composition at different body weights, the ratio IAA/DAA seemed to decline with increasing body weight. This could be due to the higher contribution of HHFT-N to total body-N in Experiment 2 (see Table 9). This is in contrast to what may be expected given an increasing volume-to-surface ratio with increasing body size. The increased contribution of HHFT-N to total body N with increasing body weight was confirmed, however, in a recent slaughter experiment (P. L. van der Togt, Wageningen Institute of Animal Sciences, The Netherlands, personal communication). Also, within experiments, the ratio IAA/DAA was higher for the initial slaughter group than for the deposited protein. This ratio, however, was affected by the experimental treatments. It seems, nevertheless, to contrast with findings in pigs, in which this ratio increases between 20 and 45 kg live weight (Bikker et al., 1994a). In preruminant calves, little information is available on changes in AA profiles within specific tissues with increasing body weight; in somewhat older cattle, however, there are indications that the collagen content in muscle (Nguyen and Zarkadas 1989) and bone (Mello et al. 1975) increases with age.

Effects of protein and protein-free energy intake on the composition of the deposited protein.  The effects of protein and protein-free energy intake on the composition of the deposited protein in the whole body and the individual fractions are presented in Tables 5-8. In general, the measured effects in Experiment 2 were larger than those in Experiment 1, which may have been due to the somewhat larger contrasts in protein and protein-free energy intakes in Experiment 2.

Most treatment effects on the AA composition of deposited protein at the whole-body level can be explained by a shift in the proportion of different tissues to total body protein. These changes are reflected in part by a change in the contribution of the analyzed fractions to total body protein. Table 9 shows the distribution of whole-body protein over the analyzed fractions. Unlike protein-free energy intake, protein intake did not affect this distribution in either experiment. The effect of protein intake on the AA profile of the deposited protein is therefore not caused by differences in distribution between fractions. However, there is evidence for shifts in the proportional contribution of tissues within a fraction, i.e., increasing protein intake increased the ratio of IAA to DAA in the deposited protein in both experiments. Threonine, leucine, isoleucine and tryptophan, in particular, were increased at the expense of glycine, alanine and proline. This indicates an increase in the ratio between muscle and collagen protein with faster growing animals and is caused mainly by changes in the carcass fraction, as will be discussed later in this paper.

Increasing protein-free energy intake generally increased the proportion of protein deposited in the carcass fraction at the expense of the HHFT fraction (Table 9). This could explain the effects of protein-free energy intake on the content of aspartic and glutamic acid, glycine and isoleucine (Experiment 2) and proline (Experiment 1). Carcass and HHFT protein clearly differed in their contents of these AA. Protein-free energy intake also affected the AA composition of the HHFT and carcass fractions, which may help explain some of the effects on the deposition of whole-body protein. Although the significance of these effects was not always consistent between experiments, they were mainly in a similar direction. It is remarkable that increasing protein-free energy intake in Experiment 1 led to an increased proportion of body protein present in the organ fraction, whereas this effect was absent in Experiment 2 (Table 9). The effect measured in Experiment 1 agrees with the findings of Bikker et al. (1995) in growing pigs.

Protein deposition in the carcass fraction accounted for 60-65% of the deposited protein in the whole body (Table 9). Therefore, the major treatment effects on the AA profile of the deposited protein in the whole body followed the effects found on the deposited carcass protein (compare Tables 5 and 6). Effects of protein-free energy intake on the AA composition of the deposited carcass protein were absent in both experiments. Increasing protein intake increased the ratio of IAA/DAA (although not significantly in Experiment 1). In particular, the content of isoleucine and tryptophan (Experiments 1 and 2), threonine, valine, leucine, lysine (Experiment 2) and phenylalanine (Experiment 1) increased, and alanine, arginine (Experiment 1), glycine and proline (Experiment 2) decreased with increasing protein intake. These effects are likely caused by an increasing muscle-to-bone ratio in the carcass as protein intake increases, with a concomitant decrease in the proportion of collagen protein. Compared with muscle protein, collagen protein is particularly rich in proline and glycine, and poor in most IAA (Nguyen and Zarkadas 1989). Bone protein is rich in glycine, proline, phenylalanine, tyrosine and valine and poor in glutamic and aspartic acid, tryptophan and threonine (Mello et al. 1975). This effect corresponds with other observations made in these experiments, for example, a decreasing ash-protein ratio with increasing protein intake (Gerrits et al. 1997).

Protein intake did not affect the AA composition of deposited HHFT protein (Table 8), whereas protein-free energy intake had an effect. Although the significance of this effect was not equal between experiments, they were in a similar direction for most AA. In general, increasing protein-free energy intake increased the proportion of the branched-chain AA, tyrosine and tryptophan, and lowered the proportion of glycine and proline in the deposited HHFT protein. This suggests a lower proportion of collagen protein at the high protein-free energy intake level. The effect of protein-free energy intake on the serine content in Experiment 2 (P < 0.001) was absent in Experiment 1.

In conclusion, the results of these experiments show the following: 1) the marginal efficiency of utilization of all ileal digestible IAA for protein gain is <30% in preruminant calves between 80 and 240 kg live weight. We concluded that this was not caused primarily by a severe limitation of a single IAA in the diet. 2) The AA composition of deposited body proteins varies with the rate of protein intake, i.e., increasing protein intake increases the ratio of IAA/DAA, likely caused by an increasing muscle-to-bone ratio in the carcass with a concomitant decrease in the proportion of collagen protein. 3) Carcass, organs and hide + head + feet + tail fractions clearly differ in AA profile. In addition, effects of protein and protein-free energy intake at the whole-body level reflect changes mainly in carcass fraction. Dietary effects on the organ and hide + head + feet + tail fractions were different.

	FOOTNOTES

Manuscript received 6 January 1998. Initial reviews completed 13 February 1998. Revision accepted 8 June 1998.

	ACKNOWLEDGMENTS

The authors gratefully acknowledge the excellent, extensive laboratory work performed by G. Post, M. van 't End and L. J. G. M. Bongers.

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