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© 2007 American Society for Nutrition J. Nutr. 137:2234-2241, October 2007

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Body Fat Deposition Does Not Originate from Carbohydrates in Milk-Fed Calves1,2

Joost J. G. C. van den Borne3,*, Gerald E. Lobley4, Martin W. A. Verstegen3, Jane-Martine Muijlaert3, Sven J. J. Alferink3 and Walter J. J. Gerrits3

3 Animal Nutrition Group, Department of Animal Sciences, Wageningen University, 6700 AH Wageningen, The Netherlands; and 4 Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, United Kingdom

* To whom correspondence should be addressed. E-mail: joost.vandenborne{at}wur.nl.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Milk-fed heavy calves utilize dietary protein with a low efficiency and often develop hyperglycemia and insulin resistance. Distributing the daily nutrient intake over an increasing number of meals increases protein deposition and improves glucose homeostasis. Therefore, we examined effects of feeding frequency (FF) and feeding level (FL) on the diurnal pattern of substrate oxidation and on the fate of dietary carbohydrates in milk-fed heavy calves. Eighteen milk-fed calves weighing 136 ± 3 kg were assigned to FF (1, 2, or 4 meals daily) at each of 2 FL (1.5 or 2.5 times maintenance), except for calves at FF1 (only at a low FL). Urea, leucine, and glucose kinetics were assessed for each treatment by use of [13C]urea, [1-13C]leucine, [U-13C], and [2-13C]glucose, respectively. FF altered the diurnal pattern, but not the total, of urea production production. Although urea production correlated well with nitrogen retention, oxidation of oral L-[1-13C]leucine did not. Dietary glucose was almost completely oxidized (80% based on [13C]glucose and 94% from indirect calorimetry measurements) regardless of FL. Fatty acid synthesis from glucose appeared to be negligible based on similar recoveries of 13CO2 from orally supplied [U-13C]glucose and [2-13C]glucose. The increased fat deposition at the higher FL originated almost exclusively from greater transfer of fatty acids to body lipid stores. These findings contrast with both glucose and lipid metabolism in growing pigs and indicate that alternative adaptive mechanisms operate in heavy milk-fed calves.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
During the past decades, skimmed milk protein in calf milk replacer has largely been substituted by more rapidly hydrolyzable (i.e. nonclotting) protein sources such as vegetable proteins and whey. This change, coupled with a high feeding level (FL)5 and low feeding frequency (FF; twice daily is common practice) results in rapid absorption of nutrients, such as amino acids, glucose, and galactose, soon after ingestion (1). The calves need adaptive mechanisms to cope with these high loads of nutrients, with partition toward either nutrient deposition or oxidation. Importantly, milk-fed heavy calves often develop insulin resistance (2), reflected in hyperglycemia (2) and low efficiency of dietary protein utilization (3). The insulin resistance may impact also on the capability to process large amounts of other rapidly absorbed nutrients toward anabolic outcomes.

Under such conditions, more efficient nutrient utilization may occur if the smaller amounts of nutrient are absorbed over short time intervals and this can be achieved by increasing FF at the same FL. Indeed, increased FF improves insulin sensitivity in calves, as indicated by lower postprandial insulin to glucose ratios and lower glucose excretion in urine (4). Furthermore, fat deposition is enhanced (5) by increasing FF, but whether this is due to either increased channelling of glucose into fatty acid synthesis de novo, as occurs in nonruminants (6), and/or better utilization of dietary fatty acids is not known. In other species, including obese and insulin-resistant human subjects (7,8), improved insulin sensitivity increases conversion of glucose to fatty acids and alters metabolic fuel selection, affecting diurnal patterns of glucose and fatty acid oxidation (OXF). Nonetheless, milk-fed heavy calves are ruminants ontologically but are maintained in a preruminant state by a liquid diet that supplies large amounts of digestible glucose and galactose. Fat synthesis from glucose is not a major route in ruminants, because acetate released by rumen fermentation provides ample precursor and there is not the reliance on either citrate lyase or malate dehydrogenase as observed in nonruminants (9). In the milk-fed heavy calf, however, it is unclear whether nutrient source or developmental physiology has the greater impact on the precursor for fat deposition.

Despite the improvements in the insulin-glucose axis with increased FF in these calves (8), the effect on amino acid metabolism is unclear. For example, increasing the FF did not improve protein utilization when skimmed milk protein was used as the sole protein source for milk-fed calves (10) but did when nonclotting whey protein was provided at a similar daily digestible nutrient intake (5). This suggested that the within-day pattern of amino acid oxidation and, thus, urea production may be linked to the altered diurnal amino acid availability, with this most pronounced when a rapidly hydrolyzable protein source is used.

Therefore, the aims of this study were to: 1) identify the mechanisms of glucose and fatty acid metabolism that operate in milk-fed heavy calves; and 2) quantify the effects of FF at a low and high FL on diurnal variation in amino acid oxidation. We hypothesized that, at equal daily nutrient intakes, an increased FF would lower glucose oxidation (OXCHO) whereas fatty acid synthesis de novo would increase and that there would be decreased urea production and postprandial leucine oxidation. Whole body protein and fat deposition as well as hormone and metabolite responses in this study have been reported elsewhere (4,5).


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Experimental design. The experimental design has been described in detail by Van den Borne et al. (5). Briefly, 18 male Holstein Friesian calves weighing 136 ± 3 kg were used in 9 trials, each with 2 calves of similar age. The trials consisted of 2 consecutive experimental periods each of 6 wk, 4 of which were for adaptation to the treatments. Period 1 was at low FL and period 2 at high FL. Within each trial, 1 particular FF was applied, because the visual and (limited) auditory contact between the individual calves could affect physical activity and cephalic phase reflexes. Animals used in a 2 x 3 factorial arrangement with FL [1.5 x metabolizable energy requirements for maintenance (MEm) and 2.5 x MEm] and FF (1, 2, or 4 meals per day). Six calves were studied per treatment. Animals on the high FL could not consume such an intake within a sensible period if this was offered as 1 meal and so this treatment was not included. Calves at FF 1 served as controls for the effect of experimental period, because FL was confounded with period in this study.

    Diets and feeding. Calves were fed according to their metabolic body weight (kg0.75). Feed supply was adjusted daily for a projected mean daily gain of 0.5 kg at the low FL and 1.5 kg at the high FL. The MEm was assumed to be 460 kJ/(kg0.75 · d). Whey was used as the only protein source in the milk replacer; this provides a rapidly hydrolyzable protein source that can be included at high levels in diets for milk-fed calves. The ingredients and analyzed nutrient composition of the experimental milk replacer are presented in Table 1. Nutrient analyses of the milk replacer have been described previously (5). Milk replacer was reconstituted with water (140 g/L) and supplied by bucket at a temperature of ~40°C. Roughage was not supplied. Feeding times were 0000 (FF 2 and 4), 0600 (FF 4), 1200 (FF 1, 2, and 4), and 1800 (FF 4). In addition, calves at FF 1 were supplied with 3 L of warm water (40°C) at 0600 and 1800 to prevent dehydration. Calves were allowed 15 min to consume the meal. Mean energy and protein intakes were 753 ± 3.4 kJ/(kg0.75 · d) and 6.9 ± 0.04 g/(kg0.75 · d) for calves at the low FL and 1228 ± 6.4 kJ/(kg0.75 · d) and 11.4 ± 0.07 g/(kg0.75 · d) at the high FL.


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  		TABLE 1 Ingredient and analyzed nutrient composition of the experimental diet

 
    Measurements. The calves were housed for 10 d in a metabolic cage within respiration chambers followed by 4 d when the calves were maintained in a metabolic cage within a climate-controlled stable (Fig. 1).


Figure 1
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  		FIGURE 1  Timeline of the experimental protocol. I.v. administration of [13C]bicarbonate (d 3) and [13C]urea (d 10–12) and oral administration of [U-13C]glucose (d 4), L-[1-13C]leucine (d 6), and [2-13C]glucose (d 8) to heavy milk-fed calves are indicated.

 
Oxidation of leucine was quantified from 13CO2 excretion after an oral bolus dose of 25.2 µmol L-[1-13C]leucine (99.0 atom%; Cambridge Isotope Laboratories) per gram dietary protein was added to the milk replacer offered at 1200. Glucose oxidation was determined from the amount of 13CO2 exhaled after an oral bolus dose of 2.5 µmol [U-13C]glucose (99.0 atom%; Cambridge Isotope Laboratories) per gram dietary lactose was added to the milk replacer supplied at 1200. The [2-13C]glucose (99.0 atom%; Cambridge Isotope Laboratories) was supplied similarly but at 15 µmol/g dietary lactose. Differences in oxidation of [U-13C]glucose and [2-13C]glucose would reflect, indirectly, fatty acid synthesis from glucose, with a greater reduction in the amount of 13CO2 released from [2-13C]glucose if pyruvate is converted to acetate, by the action of pyruvate dehydrogenase, and this is used for fatty acid synthesis (11). The labeled C-atom from [2-13C]glucose is not released if acetate is formed and used for fatty acid synthesis. Bicarbonate sequestration was determined from expired 13CO2 after injection of a bolus dose of 4.7 mmol [13C]sodium bicarbonate (99.1 atom%; Mass Trace) into an ear vein. The infusate was prepared in 10 mL sterile 0.15 mol/L NaCl and was injected within 2 min at 1400, i.e. 2 h after feeding for each treatment. During the 10 d in respiration chambers, nitrogen (N) and energy balances were performed (5) and exchanges of oxygen, carbon dioxide, and methane were measured over 6-min intervals, as described by Verstegen et al. (12). In addition, during the tracer studies, 13CO2 production was measured during 6-min intervals by nondispersive infrared spectrometry (Advance Optima Uras 14, ABB) as described by Alferink et al. (13). Sequestration of labeled bicarbonate was measured on d 3, leucine oxidation on d 6, and oxidation of [U-13C]glucose and [2-13C]glucose on d 4 and 8, respectively.

After the 10-d period in a respiration chamber, the calves were prepared with a venous catheter (16 gauge/1.70 x 1.60 mm; Secalon T, Becton Dickinson) in each jugular vein. The catheters were attached to the skin using Vicryl suturing (Ethicon) and were extended with a 200-cm 3-layer Lectroflex extension tube (Vygon) and a 25-cm Lectro-spiral extension tube (Vygon). Tape (Fermoflex and Elastoplast, Instruvet) was used to cover and secure the catheter. From d 11 at 1500 onwards, a solution of [13C]urea (99.0 atom%; Cambridge Isotope Laboratories; 1 mg/g dissolved in sterile 0.15 mol/L NaCl) was continuously infused at 0.11 mmol/h into the right jugular vein for 52 h preceded by a priming dose to enrich the body urea pool to 0.60 or 0.30 atom% excess for the low and the high FL, respectively. On d 13, blood samples were taken from the right jugular vein between 0700 and 1900 at 30-min intervals. Blood was immediately transferred into lithium heparin tubes (Vacutainer, Becton Dickinson) and stored on ice until plasma was collected after centrifugation at 1500 x g; 10 min. Plasma samples were stored at –20°C pending analyses.

Urea concentration was measured spectrophotometrically by the linked urease and glutamate dehydrogenase procedure (Cobas Integra 800; Roche Diagnostics). The enrichment of 13C in urea was measured as CO2 released by urease. Briefly, plasma was deproteinized by adding 5 mL methanol, mixing, and storing the tubes at –20°C for 2 h. After centrifugation at 1500 x g for 10 min, the supernatant was transferred into a 10-mL Exetainer tube (Labco) and 50 µL of 12 mol/L HCl was added and mixed. After evaporation to dryness in a water bath at 40°C under a gentle stream of nitrogen, 1 mL of (boiled) water and 300 µL of 1 mol/L HCl were added, mixed, and completely evaporated again. Then, 1.5 mL of a cold (4°C) buffer solution (27 g KH2PO4 and 53.5 g Na2HPO4/L, pH 7.0) containing urease (50 kU/L; Sigma Chemical) was added on ice. The tubes were covered with rubber lids, flushed with argon for 10 s to replace the air in the headspace, and then incubated at 37°C with gentle shaking in a water bath for 45 min. The reaction was stopped by injection of 0.5 mL 7 mol/L phosphoric acid and the 13C enrichment in CO2 measured in the headspace using a Finnigan Delta C continuous-flow isotope ratio mass spectrometer (Finnigan MAT). The enrichment was expressed as atom% 13CO2.

    Calculations. The derivative of a generalized Michaelis-Menten equation, as proposed by López et al. (14) for growth curves, was used to express 13CO2 production in time. The model was fitted to the 30-min means of 13CO2 excretion in breath (corrected for background enrichment) after ingestion of L-[1-13C]leucine, [U-13C]glucose, and [2-13C]glucose and after infusion of [13C]sodium bicarbonate:

Formula 1(1)

where y = 13CO2 production (micromoles per minute) at time t (minutes); b0, b1, and c (all > 0) are parameters that define the curve.

The nonlinear least squares regression procedure (PROC NLIN, SAS Institute) was used for curve fitting. Oxidation of the tracer metabolites was calculated by integration of the area under the 13CO2 excretion curve over a period of 24 h after administration of the isotope, Formula 1 and expressed relative to the amount of ingested or infused isotope by dividing the area under the curve by the dose of isotope corrected for enrichment and chemical purity. The time to peak was calculated as Formula 1. The maximum 13CO2 excretion (mmol/h) was calculated as Formula 1 and was expressed as percentage of the dose. Recoveries and maxima for the 13CO2 excretion after the ingestion of leucine and glucose tracers were corrected for bicarbonate sequestration.

Goodness of fit for predicted against experimental data were assessed from the mean squared prediction error (MSPE) (15). The MSPE and means were calculated over time until 90% of the total recovery was achieved. Values beyond this point were close to baseline and contributed disproportionately to the mean. The root MSPE was scaled to the observed mean (mean prediction error) and decomposed into errors due to overall bias, errors due to deviation of the regression slope from unity, and errors due to random variation. The correlation between predicted and observed values was also calculated (15).

The urea production was calculated across 30-min intervals from the changes in urea enrichment and concentration:

Formula 2(2)

where t = time (h), e = [13C]urea enrichment (as a fraction of total urea C), I = rate of infusion of [13C]urea (mmol/h), P = rate of urea production (mmol/h), V = volume of distribution of urea (L), and C = concentration of urea in V (mmol/L). For calculating urea production rates, distribution volume was assumed to be the body water mass, estimated as 60% of body weight, and urea was assumed to have similar concentration in all body water pools (16,17), with plasma water taken as representative for both concentration and enrichment. Urea production was determined as hourly means from 0700 until 1900. For comparison with daily urinary N excretion, urea-N production rates were extrapolated to a complete 24-h period.

OXCHO and OXF rates were calculated hourly from gas exchange rates with correction for urea-N production rates (protein oxidation). The constants of Brouwer were used to calculate hourly rates of OXCHO and OXF (see 18):

Formula 2


Formula 2

The same formulas were used for calculation of the mean daily OXCHO and OXF, but daily urinary N excretion values, instead of urea-N production rates, and mean daily rates of O2 consumption and CO2 production were used. The daily oxidation rate of amino acids (OXP) was calculated as 6.25 times the urinary N excretion [g/(kg0.75 · d)]. Responses of urea production, OXCHO, and OXF to a meal were calculated from hourly values as areas under the curve over a 6-h period after feed intake (AUC12–18). As the basal values could be affected by FL and FF, the responses in excess of preprandial values (i.e. 2 h before feeding) were also calculated ({Delta}AUC12–18).

    Statistical analysis. All data were analyzed for the effect of FF, FL, the interaction between FL and FF, and period by ANOVA using PROC MIXED in SAS. The factorial design was analyzed by a mixed model for the fixed effects of FF, FL, and period and the random effect of each calf [Eq. (3)].

Formula 3(3)

where Yijkl = dependent variable; µ = mean intercept; FFi = effect of FF i (i = 1, 2, 4); FLj = effect of FL j (j = 1, 2); Pk = effect of period k (k = 1, 2), and {varepsilon}ijkl = error term, which represents the random effect of calf within FF (l = 1,..., 6). If main effects were significant, post hoc t tests were used to compare individual treatments. Recovery of 13C, the maximum 13CO2 excretion, and the time of the maximum 13CO2 excretion were compared between treatments for each isotope tested. In addition, the 13C recoveries of [U-13C]glucose and [2-13C]glucose were compared. Hourly means and 12-h rates of urea production, and hourly means, {Delta}AUC12–18, and daily means of OXP, OXCHO, and OXF were compared between treatments. Pearson correlation coefficients were calculated for relationships between urinary N excretion and urea-N excretion for FF 2 and 4, and between mean daily OXP and L-[1-13C]leucine oxidation for all treatments. The SAS software package version 9.1 (SAS Institute) was used for all statistical evaluations.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    General. Two calves at FF 1 were excluded from the experiment because of illness and feed refusals. Another calf, at FF 2, was excluded from 1 of the experimental periods (at a low FL). The results were not affected (P > 0.10) by the experimental period. Therefore, the effect of experimental period was not included in the results.

    Urea production. FF had limited effect on hourly urea production rates, except that a greater FF increased (P < 0.05) urea production rates at 1130 for both FL (Fig. 2A,B), with a decrease observed at 1630 for the low FL (Fig. 1A). The higher FL increased (P < 0.05) or tended to increase (P < 0.10) hourly urea production rates at various timepoints (compare Fig. 2A,B). The 12-h urea production rate was not affected by FF but was greater (P < 0.001) with the higher FL (Table 2). The AUC12–18 for urea production increased (P = 0.004) with higher FL and tended to decrease (P = 0.053) with greater FF (Table 2), whereas the {Delta}AUC12–18 decreased with FF (P = 0.016) but was not affected by FL.


Figure 2
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  		FIGURE 2  Influence of FF 1, 2, and 4 at a low [690 kJ metabolizable energy (ME)/(kg0.75 · d); A] and high [1150 kJ ME/(kg0.75 · d); B] FL on the diurnal pattern of urea production in heavy milk-fed calves. Values are means ± SEM, n = 8 (FF 1), n = 5 (FF 2, in A), n = 6 (FF 2, in B), or n = 6 (FF 4). All calves were fed at 1200 ({downarrow}). Contrast comparisons (P < 0.05) for FF 1 vs. FF 2 (a), FF 1 vs. FF 4 (b), and FF 2 vs. FF 4 (c) are indicated.

 

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  		TABLE 2 Influence of FF (1, 2 or 4 meals/d) and FL (high vs. low) on the 12-h urea production rate, the AUC12–18, and the AUC12–18 in excess of preprandial values ({Delta}AUC12–18)12

 
    OXCHO and OXF: indirect calorimetry. At higher FF, the hourly OXCHO values were greater (P < 0.001) until 1330 but lower (P < 0.001) thereafter (Fig. 3). All hourly OXCHO values (except at 1330) were greater (P < 0.001) at the higher FL. Hourly OXF values were greater (P < 0.01) at the lower FF until 1330, whereas OXF increased (P < 0.05) with greater FF after 1630. All hourly OXF values (except 0730 and 1230) were lower (P < 0.05) at the higher FL.


Figure 3
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  		FIGURE 3  Influence of FF at a low [FL low; 690 kJ ME/(kg0.75 · d); A] and a high [FL high; 1150 kJ ME/(kg0.75 · d); B] FL on the diurnal pattern of OXCHO and OXF in heavy milk-fed calves. Values are means ± SEM, n = 8 (FF 1), n = 5 (FF 2 at FL high), n = 6 (FF 2 at FL low), or n = 6 (FF 4). All calves were fed at 1200 ({downarrow}). Contrast comparisons (P < 0.05) for FF 1 vs. FF 2 (a), FF 1 vs. FF 4 (b), and FF 2 vs. FF 4 (c) are indicated.

 
Daily OXP, as a percentage of the protein intake, decreased (P = 0.038) with greater FF but was not affected by FL (Fig. 4). Daily OXCHO expressed as percentage of the lactose intake was high with 94.2 and 91.5% oxidation at the low and high FL, respectively, but was not affected by either FF or FL. Daily OXF, as a percentage of the fat intake, tended to be affected by FF (P = 0.058) and was greater for the lower FL (77.7 vs. 30.6%; P < 0.001).


Figure 4
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  		FIGURE 4  Influence of FF at a low 690 kJ ME/(kg0.75 · d) and a high (1150 kJ ME/(kg0.75 · d)] FL on the OXP, OXCHO, and OXF expressed as percentage of their intake in heavy milk-fed calves. Values are means ± SEM, n = 8 (FF 1), n = 5 (FF 2, low FL), n = 6 (FF 2, high FL), or n = 6 (FF 4). a,bValues with different letters indicate significant differences (P < 0.05) between FF at the same FL. x,yValues with different letters indicate significant differences (P < 0.05) between FL at the same FF.

 
    L-[1-13C]leucine oxidation. Curve fits for 13CO2 from L-[1-13C]leucine were good and the MSPE averaged 17.6% (r = 0.94), with the majority of the prediction error (>95%) attributable to random variation. For [13C]bicarbonate, curve fits were also good (MSPE 16.3%; r = 0.97), with 72% of the injected dose recovered as 13CO2 for both FL. The proportion of the L-[1-13C]leucine oxidized was greater with increasing FF at the low FL but decreased with increasing FF at the high FL (FF x FL; P < 0.001; Table 3). The maximum of 13CO2 excretion tended to increase (P = 0.068) with greater FF. The time until maximum 13CO2 excretion was reached decreased (P < 0.001) with greater FF.


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  		TABLE 3 Influence of FF (1, 2 or 4 meals/d) and FL (high vs. low) on oxidation of orally provided L-[1-13C]leucine, [U-13C]glucose, and [2-13C]glucose in heavy milk-fed calves12

 
    Glucose oxidation: [U-13C]glucose and [2-13C]glucose. Curve fits were good and gave an MSPE of 17.0% for [U-13C]glucose (r = 0.95) and 17.6% for [2-13C]glucose (r = 0.92), with the majority of the prediction error (> 95%) attributable to random variation. Recoveries of the [U-13C]glucose dose as 13CO2 were high (mean 79.5%) and were unaffected by either FF or FL (Table 3). The maximum of 13CO2 production was greater (P < 0.001) with increasing FF. The time to maximum decreased (P < 0.001) substantially with greater FF. Recoveries of the [2-13C]glucose dose as 13CO2 averaged 78.8%, similar (P > 0.10) to [U-13C]glucose, and were unaffected by FF or FL (Table 3). The maximum 13CO2 production was greater (P < 0.001) with increasing FF, whereas the time to maximum decreased (P < 0.001).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
This study showed that urea production increased after milk intake in calves, was less at the lower FL and tended to decrease with greater FF. Extrapolated daily rates of urea production did not significantly decrease with greater FF. This contrasts with an increased protein deposition from N balance measurements and may indicate that longer-term (i.e. 24 h or longer) measurements of urea production are needed to detect differences in diurnal kinetics of amino acid oxidation. Oral glucose was almost completely oxidized and this was unaffected by both FF and FL, indicating that fatty acid synthesis de novo from glucose was negligible in milk-fed calves. The increase in fat deposition with increased feed intake originates almost exclusively from a reduced oxidation of fatty acids.

    Urea production. Amino acid oxidation was assessed by 2 approaches: ureagenesis based on [13C]urea kinetics and catabolism of oral L-[113C]leucine, in addition to the previously reported N balance data (5). Urea production rates showed marked diurnal fluctuations but, in general, increased after meal ingestion at 1200. Postprandial urea production (AUC12–18) was greater at the higher FL and tended to be greater with decreasing FF (larger meal size). In rats, larger meals also tended to increase amino acid oxidation (and thus urea production) in the postprandial state (19). After the 1200 meal, the hourly urea production rate approximately doubled in calves fed 1 or 2 meals daily, similar to findings in pigs (20) and humans (21). Although we hypothesized that urea production rates over 12 h would decrease with greater FF, this was not observed and apparently contrasts with the improvement in N retention found in this study (5). This may be due to differences in time scale between measurements (12 h vs. 10 d), diurnal changes in urea pool size, or the higher variance associated with the isotope-related measurements. Nonetheless, the daily urea-N production showed a strong positive correlation (r = 0.98; P < 0.001) with the 10-d urinary N excretion across FF.

As expected, urea production increased with increasing FL, as commonly observed across species (21–23). The urea-N production exceeded the urinary N excretion by 39%. Urea-N production is usually higher than the urinary N excretion due to entry and hydrolysis of urea in the gut lumen. In ruminants, 40–80% of the produced urea is returned to the gastrointestinal tract (24), but this recycling is usually lower in nonruminants. In exclusively milk-fed calves, hydrolysis of urea might be low due to the lack of fermentable substrates in the gastrointestinal tract, but even assuming that 75% of the urinary N originated from urea and a urea entry into the gastrointestinal lumen of 20% in milk-fed calves (W.J.J. Gerrits, unpublished data), hepatic urea production is still ~30% higher than urinary urea excretion in these heavy calves. A similar phenomenon was seen in human neonates (also milk-fed) in that only 20% of produced urea was excreted in urine (25).

    Dietary amino acid oxidation. The metabolic fate of dietary amino acids has often been studied by adding 13C- or 14C-labeled free amino acids to a meal (26–29). From breath 13CO2 or 14CO2 excretion, the proportion of the dose oxidized can be calculated, with this corrected to allow for bicarbonate sequestration (30). The recovery of [13C]bicarbonate averaged 72%, values similar to recoveries in human (31,32) and animal studies (33,34) after a bolus dose, and was independent of FL.

In response to greater FF, recovery of the L-[1-13C]leucine dose as 13CO2 increased at the low FL but decreased at the high FL (FF x FL; P < 0.001). The increased fractional oxidation at the low FL does not correspond with improved N balance (5) or urea production data and apparently contradicts our hypothesis that less L-[1-13C]leucine would be oxidized with increasing FF. Indeed, the correlation between recovery as exhaled 13CO2 from L-[1-13C]leucine and OXP calculated from N balance was considerably better at the high FL (r = 0.77; P = 0.003) than at the low FL (r = –0.34; P = 0.149). Several explanations may account for the apparent discrepancy between leucine oxidation and urea production. First, to obtain a quantitative value for leucine oxidation requires measurement of the flux through the free leucine pool, but this was not determined in the current study as it would not extend over a meal interval (12 h) with the dietary bolus dose method employed. Changes in leucine flux can influence markedly the net oxidation. For instance, Motil et al. (35) reported fractional leucine oxidation rates in response to protein intake in meal-fed humans of 16.3, 18.2, and 29.3% of the dose, whereas absolute oxidation rates were 11.8, 21.6, and 46.3 µmol/(kg · h), respectively. Second, ingestion of labeled leucine creates an isotopic gradient with the higher leucine enrichment in the gastrointestinal tract and this may overemphasize the contribution of enterocytes to whole body amino acid oxidation. Leucine can be extensively oxidized by enterocytes (36,37), but the proportion oxidized decreases with increasing FL (38). This would make a larger fraction of the dose available for peripheral tissue metabolism and may explain the better correlation with N balance at the higher FL. Third, a time lag between the absorption of free amino acids, such as the added L-[1-13C]leucine, and those originating from protein digestion may have resulted in a temporal amino acid imbalance and catabolism of L-[1-13C]leucine. Differences in oxidation patterns between free amino acids and intrinsically labeled proteins have been reported in rats (39). In summary, although the 13C-leucine breath test may be useful for clinical diagnosis (40) or studying the fate of dietary free amino acids (39), it is not a suitable method to measure postprandial amino acid oxidation in milk-fed calves.

    Glucose and fatty acid oxidation. Calculation of substrate oxidation from indirect calorimetry in fast-growing animals is often complicated by a high protein-free respiratory quotient (RQ), indicating de novo fatty acid synthesis from glucose (41,42). In our study, however, this RQ did not exceed unity. On the other hand, gluconeogenesis from noncarbohydrate precursors (e.g. alanine) results in a low RQ (42) and would lead to an overestimation of OXF and underestimation of OXCHO. Therefore, the estimates of OXCHO in this study should be considered as minimal values.

A milk-fed calf of 150 kg usually ingests >500 g of lactose in a single meal and portal glucose and galactose appearance is virtually complete within a few hours after feeding (1). Temporary storage of glucose (as additional glycogen) in well-fed animals is very small and therefore glucose is either oxidized or used as a precursor for de novo fatty acid synthesis. From the high 13C recovery of [U-13C]glucose as 13CO2 (mean 80%) and the high OXCHO as a percentage of the lactose intake (mean 94%), it appears that ingested carbohydrates are predominantly oxidized independent of either FF or FL. The lower glucose oxidation derived from the tracer compared with the gas exchange method may be a consequence of the label sequestration through glucose-glycogen interchange, thus reducing the number of 13C glucose molecules that enter the glycolytic pathway. The gas exchange method does not discriminate between oxidation of exogenous and endogenous glucose, so this more closely represents net catabolism of glucose. The net result of an increase in FL was 53–59% more OXCHO. At the same time, OXF decreased markedly at the higher FL. In energetic terms, this could be advantageous, because the efficiency of triglyceride synthesis from fatty acids is considerably greater than from glucose [90 vs. 74%, respectively; (43)]. Nonetheless, this is not simply a direct substitution, because regression analysis revealed that every 1 kJ increase in heat production from glucose resulted in only 0.5 kJ decrease in heat production from fatty acids (r = –0.79; P < 0.001). Partly, this is due to the additional energy utilized by the animal at the higher FL, possibly related to additional demands to support absorption or to drive elevated protein synthesis and urea production (44,45).

    Fatty acid synthesis from glucose. Other species generally tend to have increased rates of de novo fatty acid synthesis from glucose in the immediate postprandial period with decreasing FF, especially when carbohydrate-rich diets are offered (6). This is accompanied by increased enzyme activities associated with glucose and lipid metabolism and improved sensitivity of adipose tissue to insulin [reviewed in (6)]. This scenario is not true for milk-fed heavy calves, however, because OXCHO is virtually complete, regardless of the FF. Indeed, fatty acid synthesis de novo from glucose can be calculated from the difference between OXCHO plus urinary glucose excretion and digestible lactose intake plus gluconeogenesis from amino acids [as described in (41)]. This produced low values, 5.6 kJ/(kg0.75 · d), independent of both FF and FL and was <2% of total fat deposition at a FL of 2.5 x MEm (from 5), contrary to our hypothesis that more glucose is used for de novo fatty acid synthesis with increasing FF in milk-fed calves. This finding does not correspond with the higher activities of ATP citrate lyase observed in liver and adipose tissue from milk-fed calves compared with ruminant calves (46) and is also contrary to findings in pigs, where increasing the FL resulted in almost 700 g of the ingested starch retained as fat (41). Even at close to energy maintenance, pigs use substantial amounts (~150 g) of carbohydrates for fatty acid synthesis while also oxidizing body fat (41).

We speculate that the low level of de novo fatty acid synthesis from glucose in milk-fed calves relates to: 1) the (onto)genetic background of the calves belonging to a herbivorous species and may lack enough of the necessary enzyme systems for converting dietary glucose to fat; or 2) the high fat intake and the impact this exerts on the metabolic fate of dietary glucose. Potential mechanisms for the high OXCHO and low utilization of glucose for fatty acid synthesis remain to be studied in milk-fed calves.


   FOOTNOTES
 
1 Supported by the Netherlands Organisation for Scientific Research (NWO), the Product Board Animal Feed, and Orffa Additives BV. Back

2 Author disclosures: J. J. G. C. van den Borne, G. E. Lobley, M. W. A. Verstegen, J.-M. Muijlaert, S. J. J. Alferink, and W. J. J. Gerrits, no conflicts of interest. Back

5 Abbreviations used: AUC12–18, areas under the curve over a 6-h period after feed intake; FF, feeding frequency; FL, feeding level; ME, metabolizable energy; MEm, metabolizable energy requirements for maintenance; MSPE, mean square prediction error; OXCHO, glucose oxidation; OXF, fatty acid oxidation; OXP, amino acid oxidation; RQ, respiratory quotient. Back

Manuscript received 4 May 2007. Initial review completed 8 June 2007. Revision accepted 29 July 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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