Postprandial responses and gut permeability in calves fed milk replacer with different macronutrient profiles or a whole milk powder

There is a significant difference in the macronutrient profile of current milk replacer (MR) formulations and bovine whole milk. This study aimed to investigate how the macronutrient profiles of 3 different MR formulations containing varying amounts of fat, lactose, and protein, and a whole milk powder (WP), affect postprandial responses and gut permeability in male Holstein calves. Sixty-four calves (45.4 ± 4.2 kg [mean ± SD)] and 1.8 ± 0.6 d of age) were blocked in order of arrival to the facility and randomly assigned to one of 4 treatments within each block of 4 calves. Treatments included a high-fat MR (25.0% DM fat; 22.5% protein, 38.6% lactose; HF; n = 14), a high-lactose MR (44.6% lactose, 22.5% protein, 18.0% fat; HL; n = 17), a high-protein MR (26.0% protein, 18.0% fat, 41.5% lactose; HP; n = 17), and a WM powder (26.0% fat; 24.5% protein, 38.0% lactose; WP; n = 16). Calves were fed 3.0 L (135 g/L) 3 times daily at 0600, 1200, and 1800 h with a teat bucket. Milk intake was recorded daily for the first 28 d after arrival, and blood sampling and BW measurements were performed at arrival and on d 7, 14, 21, and 27. Gut permeability was estimated from fractional urinary excretion of indigestible markers (Cr-EDTA, lactulose, and D-mannitol) administered as a single dose on d 21. Digestibility was determined simultaneously from a total collection of feces over 24 h. Postprandial responses were assessed on d 28 by sequential blood sampling over 7.5 h. Dry matter intakes of MR over 28 d were slightly greater in HL and HP than in WP. Recovery of Cr-EDTA and D-mannitol over a 24-h urine collection was greater in WP and HP than in HL fed calves. Apparent total-tract digestibility of crude ash, protein, and fat did not differ among treatments; however, DM digestibility was lower in WP than in other treatment groups. In addition, abomasal emptying, reflected by the AUC for acetaminophen, was slower in WP than in HF and HL. The area under the curve (AUC) for postprandial plasma glucose was lower in HL than in WP and HF and lower in HP than in WP. The AUC for postprandial serum insulin was greater with HP than in WP and HF, whereas HL was not different from the other treatments. Postprandial triglycerides were greater in WP, and postprandial adiponectin was higher in HL than in the other groups. The high content of lactose and protein in MR had a major impact on postprandial metabolism. This raises the possibility of optimizing MR formulations to maintain metabolic homeostasis and influence development.


INTRODUCTION
Compared with bovine whole milk (WM), most milk replacers (MR) for calves currently contain higher levels of lactose (~45 vs. 35% DM) and lower levels of fat (~16 vs. 30%; Echeverry-Munera et al., 2021;Wilms et al., 2022).Although protein content in MR and WM are rather similar, high-protein MR formulations (≥26%) are also available (Daniels et al., 2008;USDA, 2011).Low-fat formulations (<20%) for calves seem to result from the historical availability of whey and lean growth objectives for calves.This practice has been supported by studies showing increased body fat deposition in response to the higher fat content in MR (Tikovsky et al., 2001;Hill et al., 2008;Bartlett et al., 2006).Increased fat deposition in the mammary glands of postweaning heifers has been negatively associated with milk production (Sejrsen and Purup, 1997), but there is currently no evidence to suggest that fat deposition in the mammary glands of preweaning calves has adverse effects on subsequent milk production.In fact, a high plane of nutrition in the preweaning phase has been associated with an increase in mammary parenchyma mass which may partly explain differences in future milk yield (Geiger, 2019).
An imbalanced macronutrient profile in MR has implications for neonatal metabolism.Prolonged feeding of MR with high lactose content (up to 55% DM) resulted in impaired glucose homeostasis and insulin sensitivity in veal calves (Hugi et al., 1997).However, at shorter feeding durations, MR with high lactose (44 to 46% lactose) fed twice daily did not affect the insulin sensitivity of calves of 4 d (Welboren et al., 2021a) and 4 wk of age (Stahel et al., 2019).Likewise, a high protein intake from infant formula increases the blood concentrations of AA in neonates, which stimulates insulin secretion and may increase susceptibility to insulin resistance later in life (Michaelsen and Greer, 2014;Luque et al., 2015).In calves, Wilms et al. (2022) reported higher serum IGF-I in the blood of calves fed MR with high lactose and high protein compared with calves fed MR high in fat.These collective findings in infants and calves suggests that glucose-insulin homeostasis could be altered when feeding high protein and high lactose MR for a prolonged duration at high planes of nutrition.
Increasing the fat intakes from liquid feed reduced the number of therapeutic interventions (Berends et al., 2020), the number of days with abnormal fecal scores (Amado et al., 2019), and mortality in preweaned calves (Urie et al., 2018).In contrast, previous work reported an increase in gut permeability assessed by indigestible markers when feeding MR high in fat (23 to 25% fat) compared with high lactose MR (Amado et al., 2019;Welboren et al., 2021b).This was attributed to a lower mRNA expression of genes related to tight junction proteins or to an increase in chylomicron synthesis in response to an HF diet (Welboren et al., 2021b).However, there are no well-established permeability reference values for calves fed WM (Mellors et al., 2023), and thus, the biological relevance of these differences remains to be determined.
Breastfed infants are the gold standard for growth, health, and development in infant nutrition.Likewise, WM likely has an optimal nutrient composition; thus, formulating MR closer to WM could benefit calf health and metabolism.However, studying calves fed WM is challenging because the composition and quality of WM varies widely.Studies examining feeding of WM or MR showed improved growth in WM fed calves (Moallem et al., 2010;Zhang et al., 2019), a different metabolic profile (Lepczyński et al., 2015;Kesser et al., 2017;Wilms et al., 2022), and differences in diarrhea incidence (Bascom et al., 2007;Yoho et al., 2013).Al-though extensive data are available in MR fed calves, postprandial dynamics and gut permeability have not been studied in WM fed calves.
It was hypothesized that feeding MR with a high fat content would lead to similar postprandial dynamics and gut permeability as feeding WM, whereas feeding MR with a high lactose or protein content would lead to a different result.Therefore, this study aimed to investigate how the macronutrient profile in MR affects postprandial responses and gastrointestinal health of male dairy calves fed 3 times daily in the first 4 wk of life.

MATERIALS AND METHODS
This study was conducted at the Calf Research Facility of Trouw Nutrition Research & Development (Sint Anthonis, the Netherlands) between April and July 2019.All procedures described in this article complied with the Dutch Law on Experimental Animals, which complies with ETS123 (Council of Europe 1985 and the 86/609/EEC Directive) and were approved by the animal welfare authority (Centrale Commissie Dierproeven, CCD, the Netherlands), under project application code AVD2040020173425.

Animals and Experimental Design
This experiment was conducted in a randomized complete block design.A total of 68 male Holstein Friesian calves were purchased at birth from 7 neighboring dairy farms.A standardized protocol for colostrum management was used on the farm of origin, which included 3 feedings of colostrum in the first 24 h: 3.0 to 4.0 L within the first 3 h after birth, followed by 2 feedings of 2.0 L. Colostrum quality was monitored at the farm of origin and a Brix value of 22% or greater, indicating an IgG content of 50 mg/L or greater (NAHMS, 2007) was required.Thereafter, meals of 3.0 L MR containing 135 g/L (13.5% solids; Sprayfo Excellent, Trouw Nutrition, Deventer, the Netherlands) were offered twice daily until the day of collection.Calves were brought to the research facility between 1 and 3 d after birth, and blood IgG concentrations were measured within 48 to 72 h after birth using a portable Multi-Test analyzer (DVM Rapid Test II, Vetlab, Palmetto, FL) to monitor successful colostrum administration.The mean BW at arrival was 45.4 ± 4.2 kg (mean ± SD), and the age was 1.8 ± 0.6 d.On arrival, calves were assigned to one of 17 blocks based on the day of arrival and the day of birth to minimize age differences within a block.Within each block, calves were randomly assigned to one of 4 treatments (17 calves per treatment group) and were exposed to their respective diet up to 4 wk  2212, Build 16.0.15928.20278).Treatment allocation was performed by an individual who was not involved in treatment administration or sampling.Treatments were blinded to animal caretakers by randomly assigning a letter (A, B, C, or D) to each treatment.Health was monitored daily, and a standardized protocol was followed in case of disease.Administration of medical treatments and oral rehydration solution (Sprayfo OsmoFit, Deventer, Netherlands) was recorded.Four calves were removed from the study after admission because they had severe diarrhea requiring intravenous administration of bicarbonate and saline solutions (n = 3) and critically low milk intake (n = 1).One calf belonged to the WP treatment group, and 3 calves to the HF treatment group.Calves were removed during the second (n = 1) and third (n = 3) wk of the experiment, and incomplete data collected from these animals were not included in the statistical analyses.
Treatments included a high-fat MR (25.0%fat; 22.5% protein, 38.6% lactose; HF; n = 14), a high-lactose MR (44.6% lactose, 22.5% protein, 18.0% fat; HL; n = 17), a high-protein MR (26.0%protein, 18.0% fat, 41.5% lactose; HP; n = 17), and a WM powder (26.0%fat; 24.5% protein, 38.0% lactose; WP; n = 16).Ingredients and analyzed nutrient composition of MR and the WP treatment (whole milk powder [WMP] 26%, Arla Foods, Denmark) are presented in Table 1.Each MR treatment (HF, HL, and HP) included the same raw materials, but varying inclusion of fat, protein, and lactose.Skim milk powder accounted for 50% of each MR formula.The HF treatment was designed to be close to WP in terms of fat content, whereas the HL and HP treatments aimed to represent formulation strategies currently proposed in Europe and North America, respectively.To define these formulations, fat, lactose, and protein were exchanged on a weight-to-weight basis (wt/wt).The fat concentrate used was based on spray-dried fat kernels, with 65% derived from palm oil and 35% fat from coconut oil.In all 3 MR formulas, the percentage of solids was 13.5% to standardize the ash percentage across MR formulations and to remain close to industrial conditions.In addition, this allowed isonitrogenous comparisons between HL and HF, which would not be possible if the concentrations of MR were adjusted to metabolizable energy (ME) density.Milk replacer and WP were prepared using a milk shuttle (Urban MS100 Wüsting Germany) reconstituted with water at a concentration of 135 g/L and supplied in a teat bucket at 40°C.The concentration was chosen to be close to the percentage of solids of bovine WM.Treatments were administered daily through teat buck-ets in 3 equal meals of 3.0 L at 0600, 1200, and 1800 h, and calves were allowed to drink their milk meal for 15 min.Water was available ad libitum through plain buckets and no solid feeds were offered during throughout the experimental period.Calves were purchased in successive batches and incorporated into the study as the blocks were set, and measurement periods were staggered accordingly.

Housing
Calves were housed indoors in individual pens (2.34 m × 1.16 m) separated by galvanized bar fences and equipped with rubber-slatted floor in the front area (50% of the total pen area) and a laying area in the back, which contained a mattress covered with flax straw.During total urine and fecal collection, calves were tethered to the front of the pen and an elevated plateau covered with rubber was placed at the front of the pen to elevate the animals and facilitate urine collection.The temperature in the calf pen was maintained at a minimum of 12°C and a maximum of 28°C.Relative humidity was maintained between 60 and 85%.Calves were exposed to daylight and artificial light from 0530 to 2130 h.

Measurements
Milk and water intake were recorded daily throughout the study period by weighing the unconsumed volumes.Body weight was measured weekly starting at arrival, on d 7, 14, 21 and 27 (day before the postprandial response) at 1100 h with a custom scale (W2000; Welvaarts Weegsystemen, Hertogenbosch, the Netherlands).This means that blood parameters were likely affected by the morning meal, however, the time of weekly sampling was standardized relative to the morning meal.Fecal scoring was performed over the first 21 d after arrival through visual assessment of photos of feces taken daily after the morning meal.The scoring was performed by only one examiner using a 3-level scoring system as follows: normal feces (score 0), wet feces (score 1), watery feces (score 2).Weekly blood samples for general health evaluation were obtained by venipuncture from the jugular vein at the same time than BW measurements.Blood samples were collected in 2 9-mL lithium-heparin tubes (BD Vacutainer ® , Becton Dickinson, Vianen, the Netherlands), 2 9-mL serum/gel tubes, and one 5.0 mL NaF tube.
On d 21, gut permeability was assessed by measuring urinary recovery of indigestible markers.Lactulose (0.2 g/kg BW; Sigma-Aldrich ® , Zwijndrecht, the Netherlands) and D-mannitol (0.12 g/kg BW; Sigma-Aldrich ® ) were dissolved separately in 100 mL of warm water.The volume of liquid Cr-EDTA (179 mM Cr-EDTA solution; Masterlab, Boxmeer, the Netherlands) was adjusted for each calf to provide a dose of 0.1 g/ kg BW of Cr-EDTA.Both solutions were orally pulse dosed to the calves separately using 100-mL syringes (BD Plastipak, Merkala, Alkmaar, the Netherlands) instead of the morning meal at 0630 h to avoid interference between the dynamics of marker uptake and the macronutrient composition of the liquid feed (Amado et al., 2019;Welboren et al., 2021b).Following marker administration, urine was quantitatively collected over 6 and 24 h on d 21 using urine collection bags attached with medical glue, as described in Wilms et al. (2020).Two subsequent samples were taken from the 24 h urine collection: 0 to 6 h and 6 to 24 h.Feces were quantita-tively collected over 24 h at the same time as the urine collection using fecal collection bags, as described in Wilms et al. (2020), to evaluate apparent total-tract digestibility.This means that from 0630 h on d 21 to 0630 h on d 22 after arrival, calves were fed only 2 milk meals, which were accounted for when evaluating totaltract apparent digestibility of treatments.
To investigate abomasal emptying rate and postprandial kinetics, all calves were sedated by intramuscular (IM) injection of an anesthetic (Sedamun; Xylazine 2%/20 mg; 23.3 mg xylazine hydrochloride) into the neck at 1400 h on d 27 after arrival to alleviate the stress associated with catheter placement in the jugular vein.Water was withdrawn until the end of the procedure.Throughout the procedure, the calf was kept in the sternal position, and the effect of sedation was reversed with an IM injection of an antisedative (Antisedan; atipamezole hydrochloride; 5 g/mL).Intakes of MR from the subsequent evening meal were not affected by the procedure.On d 28, sequential blood sampling was performed to evaluate postprandial dynamics.To assess abomasal emptying, a dose of acetaminophen (Ac; 150 mg Ac/kg BW) was mixed into the morning milk meal at 0600 h.Notwithstanding, postprandial responses have been successfully performed in previous experiments with lower doses of acetaminophen (0.13 g/kg of BW 0.75 ; MacPherson et al., 2016; Stahel  et al., 2016; Welboren et al., 2021a).On the day of postprandial sampling, calves received their second milk meal at 1300 h rather than 1200 h to avoid affecting postprandial dynamics.Blood samples were collected in one 9-mL lithium heparin monovette, one 5-L NaF monovette, and one 9-mL monovette with gel (for serum) at −30 min and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, and 420 min relative to the morning meal offered at 0600 h.Tubes with lithium heparin and NaF tubes were placed on ice and were centrifuged within 5 min at 1,500 × g for 15 min at 4°C (Rotina 380R, Hettich, Tuttlingen, Germany).Serum tubes were set for 15 min at ambient temperature and centrifuged at 1,500 × g for 15 min at ambient temperature.Plasma, serum, and NaF aliquots were stored in 1.5 mL cryotubes at −18°C.All samples were transported in boxes with cooling elements and stored at −18°C.

Chemical Analysis
Feed, urine, and fecal samples were processed and analyzed at MasterLab (Boxmeer, the Netherlands).Analyses of crude fat, crude protein, crude ash, minerals, lactose and DM in MR and WP samples are described in Wilms et al. (2022).Similarly, the whey protein nitrogen index (WPNI) which reflects protein quality was analyzed as described in Wilms et al. (2022).Fecal samples were analyzed for DM, pH, crude ash, crude fat, crude protein, and macro-minerals.Dry matter content was determined by drying in a 103°C oven for 4 h to a constant weight (EC 152/2009;EC, 2009).Crude ash was analyzed by incineration in a muffle furnace by combustion for 4 h at 550°C (EC 152/2009;EC, 2009).Crude fat was determined by treating the sample with hydrochloric acid followed by extraction with petroleum (EC 152/2009;EC, 2009).Crude protein content was analyzed by combustion using the Dumas method (Etheridge et al., 1998;ISO 16634-1, 2008).Macro-minerals were analyzed by inductively coupled plasma mass spectrometry (PerkinElmer ICP-MS 300D) according to NEN-EN 2017(2017).Chloride was analyzed as described by Wilms et al. (2019).
Blood and urine samples were processed and analyzed at the University of Nottingham (Nottingham, UK).Elemental minerals (Cr, Na, K, Ca, P, and Mg) in urine were determined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher XSeriesII, Thermo Fisher Scientific, Waltham, MA, USA).Analysis of lactulose and D-mannitol is described in Mellors et al. (2023).Weekly serum samples were analyzed for urea, total protein, nonesterified fatty acids (NEFA), alkaline phosphatase (ALP), and total triglyceride (TG) using colorimetric Kits according to manufacturer's instructions using the RX IMOLA (Randox Laboratories Ltd., Crumlin, UK).Postprandial serum samples were analyzed for acetaminophen using the Paracetamol (Acetaminophen) Assay Kit-K8002 (Cambridge Life Sciences Ltd., Ely, UK; MacPherson et al., 2016) and for insulin using the Mercodia Bovine Insulin ELISA Kit (Mercodia, Uppsala, Sweden; Bach et al., 2013).Postprandial NaF samples were analyzed for glucose using the EnzyChrom Glucose Assay Kit (Bio-Assay Systems, Hayward, CA; Zebeli et al., 2012).The concentration of 2 adipokines, leptin and adiponectin, were analyzed in serum at the University of Bonn, as described in Sauerwein et al. (2004) and Mielenz et al. (2013), respectively.
Model Parameterization Using Postprandial Data.A mechanistic model incorporating glucoseinsulin dynamics with intermittent gastric emptying developed by Stahel et al. (2016), allowing for the estimation of pancreatic responsiveness, whole-body insulin sensitivity, and glucose effectiveness, was used to parametrize glycemic responses.In brief, the model included pools for Ac and glucose content in the abomasum, as well as Ac, glucose, and insulin in circulation.Differential equations translating input and output fluxes for each plasma pool were then solved using a fourth order Runge-Kutta algorithm in acslX (AEgis Technologies Group Inc., Huntsville, AL).For each calf, the differential equations describing the disappearance of Ac from the abomasum and serum Ac concentrations were solved using Wolframaplha (Wolfram Research, 2010) and then were entered in Microsoft® Office Ex-cel® (2007) to predict values at each sampling time point.The Z value representing the occurrence of abomasal emptying, and its speed (Z = 0 for no gastric emptying occurring, Z = 1 being slow, and Z = 2 being fast), was determined based on the slope of the observed successive Ac postprandial concentrations.The best-fit parameters of Ac kinetics (slow emptying rate, fast emptying rate, and utilization rate) were estimated with the Solver function of Microsoft Excel to minimize the residual sum of squares between observed and predicted serum Ac.Parameters for glucose-insulin kinetics presented in Table 6 were estimated with a differential evolution algorithm, as previously described (Stahel et al., 2016).Finally, the root mean squared prediction error expressed as a percentage of the observed mean for serum Ac, serum insulin, and plasma glucose dynamics were calculated to indicate the goodness of fit.

Calculations and Statistical Analysis
The apparent total-tract digestibility of DM and nutrients (fat, protein, ash, minerals) was calculated using MR intakes and fecal output over a 24 h period on d 21 as follows: digestibility = 100 -in feces (g) / in feed ingested (g) × 100 Continuous variables were analyzed using mixed-effects model with PROC MIXED in SAS (SAS 9.4M6, SAS Institute Inc., Cary, NC).The calf was the experimental unit, and the statistical model was as follows: where: Y ijkl is the dependent variable; µ is the overall mean; T i is the fixed effect of the ith treatment; V j is the random effect of the jth block; W k is the fixed effect of the kth time point entering the model as a repeated measure; TW ik is the fixed effect interaction between the ith treatment and the kth time point; and ε ijk is the random error associated with the jth block at the kth day with the ith treatment.In the case of BW and ADG, arrival BW was used as a baseline covariate (µ 0 ).For variables with equally spaced time points, the covariance structure (autoregressive covariance [AR(1)] or the heterogeneous autoregressive covariance [ARH(1)]) with the lowest corrected Akaike Information Criterion was used.The heterogeneous Toeplitz (TOEPH) covariance structure was used for variables with unequally spaced time points.Data that did not meet the assumptions of normality of residuals had to be log-transformed (base 10).After the log-transformation, the data distribution was tested again to ensure a normal distribution.Significant effect of treatment was explored with the LSMEANS statement using the PDIFF option of the MIXED procedure of SAS.Significant interactions between treatment and time were explored with the SLICE option of the LSMEANS statement using the PDIFF option of the MIXED procedure of SAS.Results in tables and figures are presented as least squares means (LSM) with the standard error of the means (SEM).Discrete variables (e.g., therapeutic interventions, fecal scores) were analyzed using mixed effects logistic regressions.These analyses were conducted with PROC GENMOD in SAS.Significance was declared at P ≤ 0.05, and the trend threshold was set at 0.05 < P ≤ 0.10.

Correlations, Principal Components Analysis, and Hierarchical Clustering
Heat maps for Pearson correlations were used to determine relationships between variables using an online platform for multivariate analysis (MVApp; Julkowska et al., 2019).Only variables measured in wk 3 were considered for this analysis as urinary and fecal collection took place in wk 3.For macronutrient intakes, the total intake expressed in kg DM over the first 28 d was considered.Correlations were defined as weakly positive (r = |0.1| to |0.3|), moderately positive (r = |0.3| to |0.7|), and strongly positive (r = |0.7| to |1.0|).Furthermore, the correlation structure was plotted as a principal component analysis (PCA) to determine correlation patterns among the individual traits using the MVApp online platform.The significance of correlations was declared at P ≤ 0.05.

Intakes and Weekly Blood Parameters
Four calves from which one belonged to WP and 3 to HF were removed after admission due to diarrhea and dehydration.Despite adequate randomization, the concentration of IgG measured between 48 and 72 h after birth was lower in HF calves than in the other treatment groups (P = 0.03; Table 2).All 4 calves removed post-inclusion from the experiment had blood IgG at arrival lower than 1800 mg/mL.In the first week after arrival, the percentage of days with diarrhea (defined as a fecal score of 2) was lower in HP calves (28%) than in the other groups (44%; P < 0.01).In wk 2, there were no differences across treatment groups, but in wk 3, the percentage of days with diarrhea was higher in WP calves (13%) than in the other groups (4%; P = 0.02).Despite these differences, the number of calves receiving therapeutic interventions for diarrhea and respiratory diseases did not differ across treatment groups.Daily milk intakes expressed as volume fed is presented in Supplemental material S1.Milk intakes (as volume fed) throughout the experimental period of 28 d were not analyzed due to infinite likelihood in wk 3 and 4 as calves would consume the full 9.0 L. When considering only the first 14 d after arrival, milk intakes were lower for WP than all other treatment groups (P < 0.01) and lower in HP than HL (P < 0.01).Milk replacer DM intake over the first 28 d was lower in WP than all other treatment groups and higher in HL than HP (P < 0.01).As expected from MR composition, fat intake was higher in HF than in the other treatments and in WP than in HL and HP (P < 0.01).Protein intake was higher in HP than in the other treatment groups (P < 0.01).Lactose intake was the highest in HL, followed by HP, HF, and at last WP (P < 0.01).Total ME intake over the first 28 d was higher in HF calves than in the other treatment groups (P < 0.01), whereas no differences were observed among other groups.Body weight and ADG did not differ across treatment groups when considering the entire experimental period of 28 d.Serum urea concentration was greater in WP calves than in HF and HL calves, and greater in HP than in HL calves (P < 0.01).Serum NEFA concentrations were greater in WP and HF calves than in HL and greater in HF calves than in HP calves (P < 0.01).The enzymatic activity of alkaline phosphatase (ALP) was lower in WP calves compared with the other treatments (P < 0.01).Serum concentrations of triglycerides (TG) were higher in calves fed WP compared with HP (P = 0.02).

Urine Chemistry, Digestibility, and Gut Permeability
Results for urine chemistry and total apparent tract digestibility are shown in Table 3. Urinary urea output was lower in WP calves than in the other groups (P < 0.01), whereas creatinine did not differ among treatments.Urinary sodium content was lower in calves fed WP than in the other groups (P < 0.01).Urinary potassium content was higher in HL than in WP and HF and higher in HP than in WP fed calves (P < 0.01).This resulted in lower urine osmolality in WP fed calves than in the other treatments for the urine sample collected between 6 and 24 h after initiation of total collection (P = 0.03).Apparent total-tract digestibility of DM was lower in WP fed calves than in the other groups (P = 0.02).During the first 6 h of urine collection, urinary lactulose recovery was lower in HL than in the other treatments (P = 0.02; Table 4).Recovery of D-mannitol was lower in HL than in WP and HP calves (P = 0.01), and recovery of Cr-EDTA was higher in WP than in the other treatments in the first 6 h of urine collection (P = 0.01).When considering the total 24 h urine collection, urinary D-mannitol recovery was lower in HL than in the other treatments and higher in HP than in HF (P < 0.01).The 24 h urinary recovery of Cr-EDTA was lower in HL than in WP and HP and higher in WP than in HF (P < 0.01).For 6-and 24-h urine collection, the intestinal permeability (IP) index, defined as the ratio of urinary recovery (%) of lactulose to D-mannitol, did not differ between treatment groups.

Correlations and Principal Component Analysis
Heatmap for Pearson correlations matrix between macronutrient intakes, blood metabolites, urinary parameters, and total apparent tract nutrient and DM digestibility measured in wk 3 are presented in Supplemental Figure S2.Serum urea was positively correlated with total serum protein (r = 0.51, P < 0.01) and with protein intakes (R 2 = 0.43, P = 0.01).Urinary lactulose recovery over 24 h collection was positively correlated with Cr-EDTA recovery (r = 0.78; P < 0.01) and with urine osmolality over 6 h collection (r = 0.36; P = 0.04).Supplemental Figure S3A shows the biplot of the PCA analysis with the contributions of different variables including macronutrient intakes, blood metabolites, urinary parameters, and total apparent tract nutrient and DM digestibility measured in wk 3. PCA 1 explained 20.5% of the observed variation, corresponding mainly to fecal volume and nutrient digestibility (Supplemental Figure S3B).Moreover, PCA 2 explained 14.3% of the observed variation, corresponding primarily to gut permeability marker recovery (Supplemental Figure S3C).

Postprandial Dynamics
The results for postprandial dynamics are shown in Table 5. Serum postprandial NEFA concentrations were higher in HF than other treatment groups (P = 0.04; Supplemental Figure S4A).Consistently, the maximum concentration (C max ) was greater in HF than in HL calves (P = 0.02).Basal TG concentration was lower in HP as compared with all other treatment groups and higher in HF as compared with WP and HL (P < 0.01).Accordingly, TG C max was greater in WP and HF calves than in HP calves (P = 0.01).When considering only time points from 210 to 420 min post-meal, WP was significantly higher than all other treatment groups including HF (P < 0.01; Figure 1A).The baseline concentration of adiponectin was greater in HL than in the other treatments (P < 0.01).In addition, there was an interaction between treatment and time for the postprandial adiponectin concentration (P = 0.05; Figure 1B), in which adiponectin concentration was greater in HL than in calves fed WP between 90-and 210-min post meal.At 60-, 90-, 150-, and 420-min post meal, adiponectin concentrations were greater in HL than in HP calves.Finally, at 90-, 150-, and 420-min post meal, adiponectin concentrations were greater in HL than in HF calves.No differences were observed in the time to reach the maximum concentration (T max ) and in C max .Baseline leptin concentrations tended to be lower in HL than in the other treatments (P = 0.06; Supplemental Figure S4B).The ratio between adiponectin and leptin was consistently higher in HL calves than in other treatment groups in the postprandial samples (P < 0.01; Supplemental Figure S4C).There was an interaction between treatment and time for postprandial Ac (P < 0.01; Figure 1C), with serum Ac lower in WP fed calves between 240-and 420-min post meal than in the other treatment groups.At 360 min post meal, serum Ac was lower in HP than HL.At 420-min post meal, serum Ac was lower in HP than HL and HF.The area under the curve (AUC) for postprandial serum Ac was lower in WP than HF and HL (P = 0.02), whereas HP did not differ with other treatment groups.The T max of Ac was shorter in WP than in other treatment groups (P = 0.01), whereas C max was lower in WP than in HF and HL and lower in HP than in HF (P = 0.02).For postprandial glucose dynamics (Figure 1D), the interaction between treatment and time tended to be significant (P = 0.08).Between 150-and 180 min post meal, glucose concentration was higher in HF than WP and HP (P ≤ 0.01).At 210 min post meal, glucose was higher in HF than HL (P = 0.04) and HP (P = 0.03), whereas at 240 min post meal glucose was higher in HF than WP (P = 0.02) and HL (P = 0.04).Finally, between 360-and 420 min post meal, glucose concentration was higher in WP than HF (P ≤ 0.05).In addition, the AUC for glucose was lower for HL than for WP and HF and lower for HP than for WP (P = 0.03).There was an interaction between treatment and time for postprandial insulin concentrations (P < 0.01; Figure 1E).Serum insulin concentrations were greater in WP fed calves at 60-min post meal than in calves fed HF and HL (P = 0.05).At 180-min post meal, serum insulin was lower in WP than HP and HL calves, whereas at 210-min post meal serum insulin was lower in WP than HP (P = 0.02) and HL (P = 0.05).At 240-min post meal, serum insulin was lower in WP than all other treatments (P = 0.01).At 360-and 420-min post meal, serum insulin was slightly greater in WP fed calves than HF (P < 0.02).The delta insulin concentration, defined as the insulin concentration at a given time minus the basal insulin concentration, was lower in HF than in WP and HP fed calves (P = 0.05).The AUC for insulin was greater

Modeling of Insulin-Glucose Kinetics
The results of estimated insulin glucose kinetics are shown in Table 6.The results of estimated insulin glucose kinetics are shown in Table 6.The first-order, slow gastric emptying rate constant (k SP,2 ), and fast gastric emptying rate constant (k SP,3 ) did not differ across treatment groups.In contrast, there was a statistical trend toward a greater first-order Ac utilization rate constant in WP than HF and HP (k Ac,UAc ; P = 0.07).The time in which abomasal emptying was fast (Z = 2, time fast) was shorter in WP than in HF and HL fed calves (P = 0.03), whereas HP did not differ from the other groups.In contrast, the parameters for which abomasal emptying was slow (Z = 1, time slow) or off (Z = 0, time off) did not differ among treatments.Postprandial glucose kinetics parameters showed that the initial rate of endogenous glucose production (iPGl end ) was lower in WP than in other treatment groups (P < 0.01).Similarly, the absorption lag time from stomach to plasma (T lag,SP ) was lower in WP calves than in MR fed calves (P < 0.01).Similarly, the Hill coefficient for glucose-dependent insulin secretion (exp PIn ) tended to be greater in HP than in other treatment groups (P = 0.08).Finally, the root mean squared prediction error for insulin (rMSPE ln ) was greater in HL than in the other treatment groups (P < 0.01).

DISCUSSION
This experiment examined the effect of macronutrient profile in MR on gastrointestinal health and postprandial responses in male dairy calves fed 3 times daily. 2Total of number of calves treated for diarrhea and respiratory diseases.Values in brackets represents the number of calves getting a treatment out of the total number of calves in that treatment group.These values were used to calculate the percentage of calves treated.
3 Total milk intakes, expressed in kg DM, was calculated by adding the daily consumption of calves over a 28-d period. 4Metabolizable energy (ME) content of the MR was calculated according to NRC (2001). 5Water intake refers to the plain water that was offered ad libitum to calves next to their milk meals. 6BW and ADG were measured weekly from wk 1 to 4. Arrival BW entered the statistical model as baseline covariate.Expressed in % oral dose.Markers were orally pulse dosed instead of the morning meal in wk 3. Lactulose (0.2 g/kg BW; Sigma-Aldrich®, Zwijndrecht, the Netherlands) and D-mannitol (0.12 g/kg BW; Sigma-Aldrich®, Zwijndrecht, the Netherlands) were dissolved separately in 100 mL of warm water.The amount of liquid Cr-EDTA (concentration; Masterlab, Boxmeer, the Netherlands) was adjusted for each calf to provide an individual dose of 0.1 g/kg BW.Despite the greater ME intake in HF calves throughout the experimental period, growth within the first 28 d after arrival did not differ across treatment groups.In the current study, gut permeability was the highest in calves fed WP followed by calves fed HP, and the lowest in calves fed HL.Postprandial responses were largely influenced by macronutrient composition, with WP and HP exhibiting slower abomasal emptying rates, likely due to higher casein content of the meals.Finally, the AUC for insulin was greater in calves fed HP than in calves fed WP and HF, but the ratio of insulin to glucose did not differ across treatment groups.

Gastrointestinal Health
The percentage of days with diarrhea was lower in wk 1 for HP calves and higher in wk 3 for WP calves than other treatment groups.The number of therapeutic interventions for diarrhea and respiratory diseases and calf removal was numerically higher in calves fed HF than in the other treatment groups.These results are inconsistent with those of Wilms et al. (2022), in which calves fed ad libitum levels of the same HF MR did not exhibit more health disorders than other treatment groups.In the current study, despite adequate randomization, blood IgG measured upon arrival at the facility was significantly lower in HF calves.This may explain the numerically higher removal rate, as IgG below 1500 mg/dL has been associated with higher morbidity and mortality in calves (Weaver et al., 2000).
Indigestible permeability markers are commonly used to assess the mucosal integrity of the gastrointestinal tract (GIT) (Wilms et al., 2019;Welboren et al., 2021b).In the current study, urinary recovery of Cr-EDTA and lactulose measured in wk 3 after arrival were greater in calves fed WP and HP compared with calves fed HF.Transport of molecules such as Cr-EDTA (344 g/mol) and lactulose (342 g/mol) through the intestinal epithelium is paracellular and occurs via tight junction proteins (Linsalata et al., 2020).The greater recovery of these markers in urine may indicate decreased intestinal barrier function associated with structural changes such as the opening of tight junction proteins.Consistent with other markers, urinary recovery of D-mannitol, a small size marker (182 g/ mol) that is absorbed both transcellularly and paracellularly (Linsalata et al., 2020), was higher in WP and HP calves than in the other treatment groups.However, the absence of differences across treatment groups for the intestinal permeability (IP) index, defined as the ratio of urinary recovery (%) of lactulose to Dmannitol, does not indicate damage to the intestinal mucosa, but rather an overall increase in paracellular permeability.Dietary proteins are largely digested in the small intestine; however, a high protein intake may result in more proteins entering the large intestine and colon.Because Cr-EDTA passes through the intestinal wall in the small and the large intestines (Maxton et al., 1986;Elia et al., 1987), protein fermentation in the large intestine may influence Cr-EDTA recovery.
The amount of protein that reaches the large intestine is influenced not only by protein intake but also by protein source and quality.Highly digestible proteins such as casein are digested in the proximal intestine, resulting in less microbial fermentation than with plant proteins (Ma et al., 2017).In the current study, only highly digestible milk proteins were used (Mellors et al., 2023), but DM digestibility was lower in the WP treatment.However, it is important to highlight that the total collection period in the current experiment might have been too short to accurately determine the apparent total-tract digestibility.This lower DM digestibility is consistent with the lower whey protein nitrogen index in WP compared with other treatment groups.This likely indicates denaturation of the milk proteins during the pasteurization or the evaporation process (Wilms et al., 2022;Mellors et al., 2023).These results suggest that a greater quantity of undigested proteins may have reached the large intestine, which may explain the higher intestinal permeability in WP fed calves.This also aligns with the higher percentage of days with diarrhea in wk 3 in WP calves as compared with other treatment groups.Denaturation of the protein fraction leads to a change in the tertiary structure of the protein and other chemical modifications that negatively affect the accessibility of AA to proteolytic enzymes, thus reducing digestibility (van Lieshout et al., 2019).Alternatively, Mellors et al. (2023) showed that the fatty acid composition of the proximal jejunum and ileum differed between WP and HF and that the fatty acid profile of gut tissues largely mirrors that of the fat fraction of the milk diets.The higher ratio of n-6 to n-3 PUFA in the gut tissue of calves fed HF as compared with calves fed WP may have modulated gut permeability (Khajuria et al., 2002).

Blood Metabolites Measured Weekly
Urea is the major end product of protein metabolism and the major solute in urine (Bankir et al., 1996).Dietary AA are either used for anabolic functions or broken down in the liver and converted into urea (Weiner et al., 2015).The lower urinary urea and electrolyte content of WP fed calves resulted in lower urine osmolality in this group in the 6 h after initiation of urine collection, but not in the 6 to 24-h collection period.Serum urea was greater in HP than in HL and higher in WP than in HF and HL.These results align with those of Wilms et al. (2022), in which serum urea was higher in WP and HP calves over a 12-wk rearing period.The authors attributed the higher serum urea in HP calves to higher protein intake, which is consistent with the positive correlation between these parameters observed in the current study.A higher protein intake results in more protein being degraded to AA, which in turn is converted to urea and released into the bloodstream.The higher serum urea concentration in WP fed calves could be attributed to protein denaturation from the WP treatment, which may affect protein biological value and activity.However, the lower urea urinary output in WP calves could be due to better protein utilization than in calves fed other treatments.
In the current study, calves fed HF had greater serum NEFA concentration measured weekly than calves fed HL and HP, which aligns with results from Wilms et al. (2022).In addition, calves fed WP had higher serum NEFA concentrations than HL calves.Calves fed HF and WP MR also had the highest fat intake; however, the correlation between fat intake and serum NEFA was low.Serum TG measured weekly was lower in HP  5. APostprandial dynamics measured at 4 wk of age in calves fed milk replacers differing in macronutrient profile or whole milk powder three times daily (n = 64).Blood was collected at −30 min and at 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 330, and   The adiponectin to leptin ratio was calculated using basal concentrations. 3 The Delta insulin concentration was defined as the maximum change from baseline and was calculated by subtracting basal insulin concentrations. a,b,c Means with a different superscript are significantly different (P ≤ 0.05).

A, B
Means with a different superscript includes a trend (0.05 < P ≤ 0.10).*SEM expressed as log.
than in WP calves, while HF and HL did not differ from the other treatment groups.These results contrast Wilms et al. (2022), where no differences in serum TG were found among treatment groups.This could be related to the timing of blood collection relative to the milk meal, which was controlled in the current study but not in Wilms et al. (2022) where calves were fed ad libitum.Common assays for serum ALP enzymatic activity do not differentiate between placental ALP, intestinal ALP, and liver, bone, kidney ALP, making it difficult to interpret differences across treatments.In the current experiment, WP fed calves had lower serum ALP enzymatic activity as compared with the other treatment groups.Overall, the enzymatic activity of ALP in blood correlated negatively with serum urea and recovery of Cr-EDTA over a 24-h urine collection.Previous studies have shown that the activity of ALP in the gut is significantly reduced when gut integrity is compromised (Pearce et al., 2013).Nevertheless, the correlation between intestinal and blood ALP enzymatic activity remains to be evaluated.

Abomasal Emptying and Postprandial Dynamics
Dietary fat from liquid feed is ingested as TG, which needs to be hydrolyzed into fatty acids and monoglycerides by digestive enzymes in the upper GIT and in the intestines before absorption (Noble, 1980;Lambert and Parks, 2012).After a meal, a fraction of dietary fatty acids is not incorporated into adipose tissue and increases serum NEFA concentrations (Frayn, 2003).In the current study, postprandial TG dynamics increased from 180 min post meal ingestion onwards.Although the time to reach the maximum postprandial concentration (T max ) did not differ for NEFA and TG, the maximum postprandial concentration (C max ) for NEFA was greater in HF than in HL calves, and TG C max was greater in WP and HF than in HP calves.The magnitude of the response of TG depends on the amount and composition of dietary fats (Lambert and Parks, 2012).This is consistent with the current study, in which overall postprandial TG concentrations were greater in WP and HF calves than in HL and HP.In addition, calves fed WP had higher postprandial TG concentrations than calves fed HF when considering the serum TG concentrations from 180 min post meal onwards (Supplemental Figure S4B).This may indicate that milk fat leads to different fat absorption dynamics and utilization than the same amount of vegetable oils.
Leptin and adiponectin are adipokines whose circulating concentrations are positively and negatively related to body fat content, respectively, in cattle (Häussler et al., 2022), though the association of adiponectin with body fat is limited to a study in dry cows (De Koster et al., 2017).Leptin is known to reduce appetite, enhance  6. Parameter values (least squares means) derived from parametrization of a mechanistic model of abomasal emptying and glucoseinsulin kinetics.The model uses postprandial acetaminophen, glucose, and insulin in calves fed milk replacers differing in macronutrient profile or whole milk powder three times daily (n = 64).Blood was collected at −30 min and at 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, and   Means with a different superscript are significantly different (P ≤ 0.05).

A, B
Means with a different superscript includes a trend (0.05 < P ≤ 0.10).*SEM expressed as log.fatty acid oxidation, decrease glucose, and thus reduce body fat, whereas adiponectin is regarded as insulin sensitizer; however, the situation in cattle, in particular calves, is less clear (Sauerwein and Häussler, 2016;Kesser et al., 2017).From human studies, the postprandial concentration of both adipokines hardly changed during 120 min after oral glucose or fat challenge tests in normal-weight adults (Larsen et al., 2019).In 4 mo old children, no difference in the plasma concentrations of leptin and adiponectin before and 1 h after a meal was observed (Tomasik et al., 2011).This aligns with Blum et al. (2005) in which postprandial plasma leptin measured in young calves remain stable which suggest that plasma leptin is not involved in the immediate partitioning of nutrients following a meal.When assessing the response to high-fat, high protein, or high carbohydrate and medium protein diets for 9 h in overweight dogs, Blees et al. (2020) did not observe any change in adiponectin, but leptin increased until 6 h.The type of diet affected the baseline values of adiponectin but not leptin; the area under the response curve of leptin (AUC) was the lowest in the high protein diet; for the adiponectin AUC, all tested diets yielded lower values than the basal maintenance diet (Blees et al., 2020).For cattle, to the best of our knowledge, the present study is the first to report the postprandial time course of adiponectin.In the current experiment, the basal concentration of adiponectin was greater, whereas the basal leptin concentration tended to be lower in HL fed calves than in other treatment groups.Postprandial dynamics also consistently showed higher adiponectin concentrations in HL calves leading to a greater postprandial adiponectin to leptin ratio.This is likely due to the higher lactose intake in this group, resulting in an increased need for glucose homeostasis regulation in HL calves.Interestingly, when considering the first 120 min post meal ingestion, HL seemed having the least glucose response with the smallest insulin concentrations.This supports a greater insulin sensitivity (Frühbeck et al., 2018) in this group and is consistent with the greater postprandial adiponectin concentration which is thought to enhance the body sensitivity to insulin.Nevertheless, when considering the entire sampling period of 420 min relative to the milk meal, the AUC for insulin and the glucose to insulin ratio in HL calves were not different from other treatment groups.Low leptin concentrations associated with high adiponectin concentrations could also indicate increased hunger or decreased satiety in calves fed MR with high lactose content.These findings may partially explain why these animals consistently consume more high-lactose milk when fed ad libitum than other groups (Berends et al., 2020;Echeverry-Munera et al., 2021;Wilms et al., 2022).
Gastric emptying depends on several characteristics of the meal, such as meal size (McPherson et al., 2016), caloric density (Calbet and MacLean, 1997), and protein content (Burn-Murdoch et al., 1978).In the current study, gastric emptying was slower in WP and HP fed calves.Consistently, the time in which gastric emptying was fast was lower in calves fed WP than other treatment groups throughout the 420 min of postprandial sampling.As described in Stahel et al. (2016), serum Ac arises from the emptying of the abomasum and disappears according to the first-order elimination constant (k Ac,UAc ; Stahel et al., 2016).The k Ac,UAc constant tended to be greater in WP than in calves fed HF and HP, whereas HL showed no differences from the other treatment groups.This slower abomasal emptying in WP calves is likely related to differences in protein characteristics between treatments.The protein fraction of the WP treatment consisted of 82% casein proteins and 18% whey proteins, equivalent to a ratio of 4.6 to 1.In contrast, MR treatments (HF, HL, and HP) contained a greater proportion of whey proteins than WP, resulting in a casein to whey ratio of 2.15 for HF, 2.06 for HL, and 1.47 for HP.For the same protein intake, a lower ratio of casein to whey protein in the total protein fraction resulted in less curd formation in the abomasum and, thus, faster passage rate through the GIT (Longenbach and Heinrichs, 1998).In addition to protein composition, a higher protein intake may also decrease gastric emptying rate because the end products of protein hydrolysis affect duodenal receptors that regulate gastric emptying (Burn-Murdoch et al., 1978).
Plasma glucose dynamics are determined by exogenous appearance and endogenous glucose production and utilization as described in the model developed by Stahel et al. (2016).Interestingly, the absorption lag time from the GIT to the plasma (refer to as movement from the stomach to the plasma in the model) was slower in WP than in MR fed calves.As mentioned in the paragraph above, curd formation in the abomasum was likely greater in WP than in MR fed calves due to the greater proportion of casein in the protein fraction.While casein remains longer in the abomasum, the liquid phase containing lactose, whey proteins, and the fat separates from the curd and flows rapidly into the intestines.This is consistent with the greater insulin concentrations observed in WP calves 60 min post-meal compared with HF and HL, followed by lower insulin concentrations between 180 and 240 min compared with HP and HF.The higher root mean squared prediction error for insulin (rMSPE in ) in HL calves than other treatment groups suggests that the model developed by Stahel et al. (2016) may not predict postprandial insulin dynamics as accurately in calves fed HL.

Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
The AUC for insulin was greater in HP than in WP and HF calves, whereas HL did not differ from the other treatment groups.This is consistent with previous studies showing that high protein intake leads to increased concentrations of insulin-releasing AA, which stimulates insulin secretion and insulin-like growth factor I (Ketelslegers et al., 1996;Michaelsen and Greer, 2014;Luque et al., 2015).Consistently, Wilms et al. (2022) showed that ad libitum feeding of MR high in protein and lactose resulted in an increase in serum IGF-I concentrations during a 12-wk rearing period compared with WP fed calves.Because the ratio of postprandial glucose to insulin did not differ between groups, conclusions regarding insulin sensitivity cannot be drawn in this study.Further work is needed to determine the effects of a high-protein MR formulation on long-term glucose-to-insulin homeostasis in calves.
Results from the current study indicate that postprandial nutrient signaling differs greatly depending on the macronutrient inclusion and the macronutrient composition.The higher curd formation in WP fed calves resulted in slower gastric emptying, which affected postprandial glucose and insulin dynamics.In addition, the postprandial TG response was increased in calves fed WP, which could be due to different digestion and absorption dynamics of dietary fats.Nevertheless, calves fed WP may not be representative of calves fed fresh WM because processing steps disrupted fat globule membranes and decreased protein quality.Although colostrum management at birth was standardized, differences in blood IgG at arrival may have negatively affected gastrointestinal tract development, and thus intestinal permeability and nutrient digestibility in calves fed HF.

CONCLUSION
Despite its optimal nutrient composition, the WP treatment resulted in a lower DM digestibility and increased gut permeability.Differences in the macronutrient profile and composition of liquid feed led to distinct metabolic and endocrine profiles in calves.The HP treatment resulted in a higher area under the curve for insulin than WP and HF, although the glucose to insulin ratio did not differ from other treatment groups.Calves fed HL had an increased postprandial ratio of adiponectin to leptin, indicating an increased need for glucose homeostasis regulation.Although it is unclear whether HP and HL diets negatively affected insulin sensitivity in calves, the absence of differences between HF and WP in the area under the curve for insulin, as well as in postprandial leptin and adiponectin, suggest that high-fat MR are preferable for maintaining hormonal balance in calves.
Wilms et al.: MACRONUTRIENTS IN MILK REPLACER after arrival.The randomization was performed using the random function [RANDBETWEEN (0,100000)] in Microsoft® Excel® (Microsoft 365 MSO, Version Wilms et al.: MACRONUTRIENTS IN MILK REPLACER Wilms et al.: MACRONUTRIENTS IN MILK REPLACER Wilms et al.: MACRONUTRIENTS IN MILK REPLACER Wilms et al.: MACRONUTRIENTS IN MILK REPLACERin HP than in WP and HF calves, whereas HL did not differ from the other treatments (P = 0.05).
a,b,c Means with a different superscript are significantly different (P ≤ 0.05).A,B Means with a different superscript includes a trend (0.05 < P ≤ 0.10).*SEM expressed as log.Journal of Dairy Science Vol.TBC No. TBC, TBC Wilms et al.: MACRONUTRIENTS IN MILK REPLACER TABLE 3. Urine chemistry and total apparent tract digestibility measured at 21 d after arrival quantitative collection of urine and feces over 24 h in calves fed milk replacers differing in macronutrient profile or a whole milk powder 3.0 L three times daily (n = 64) Item 1

2
Treatments (Treat) included a whole milk powder (26% fat; WP; n = 16), and three milk replacers (MR) including a MR with high fat (25% fat; HF; n = 14), a MR with high lactose (44% lactose; HL; n = 17), and a MR with high protein (26% protein; HP; n = 17) fed at 135 g/L. 3 The intestinal permeability (IP) index was determined as the ratio between urinary recovery (%) of lactulose and D-mannitol.a,b,c Means with a different superscript are significantly different (P ≤ 0.05).*SEM expressed as log.
Wilms et al.: MACRONUTRIENTS IN MILK REPLACER Wilms et al.: MACRONUTRIENTS IN MILK REPLACERTABLE 420 min relative to the morning milk meal of 3.0 L Item 1,2
Wilms et al.: MACRONUTRIENTS IN MILK REPLACERTABLE 420 min relative to the morning meal of 3.0 L Item 1,2

TABLE 1 .
Ingredient and analyzed nutrient composition of milk replacers and whole milk powder fed to calves 3.0 L three times daily

TABLE 2 .
Wilms et al.: MACRONUTRIENTS IN MILK REPLACER Parameters describing calves before and after treatment initiation.Calves were fed milk replacers differing in macronutrient profile or a whole milk powder 3.0 L three times daily (n = 64).Blood samples were collected on d 7, d 14, and d 21

TABLE 4 .
Gut permeability assessed by fractional urinary recovery of lactulose, D-mannitol, and Cr-EDTA measured at 21 d after arrival in calves fed milk replacers differing in macronutrient profile or whole milk powder 3.0 L three times daily (n = 64) 1Total urine and fecal collection were initiated at the same time than administration of indigestible markers at 0630 h.Calves received their milk meals at 1230 h and total collection ended at 0630 h on the following day. 2 Treatments (Treat) included a whole milk powder (26% fat; WP; n = 16), and three milk replacers (MR) including a MR with high fat (25% fat; HF; n = 14), a MR with high lactose (44% lactose; HL; n = 17), and a MR with high protein (26% protein; HP; n = 17) fed at 135 g/L.a,b,c Means with a different superscript are significantly different (P ≤ 0.05).*SEM expressed as log.
Ac, acetaminophen; k SP,2 , first-order, slow gastric emptying rate constant; k SP,3 , first-order, fast gastric emptying rate constant; k Ac,UAc , first-order, Ac utilization rate constant; iPGl end , initial rate of endogenous glucose production; k Gl,UGl , first-order, glucose-dependent glucose utilization rate constant; k Is,UGl , first-order, insulin-dependent glucose utilization rate constant; T lag,SP , absorption lag time from stomach to plasma; rMSPE Gl (% of mean), root mean squared prediction error for glucose; V PIn , maximal rate of insulin secretion; k Gl,PIn , glucose-dependent insulin secretion Michaelis constant; exp PIn , Hill coefficient for glucose-dependent insulin secretion; iIs, basal insulin signal mass before meal; T lag,IS , signaling lag time for insulin; k In,UIn , first-order insulin utilization rate constant; rMSPE In , root mean squared prediction error for insulin.