J. Dairy Sci.
105:6670–6692
https://doi.org/10.3168/jds.2022-21870
© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Macronutrient profile in milk replacer or a whole milk powder
modulates growth performance, feeding behavior,
and blood metabolites in ad libitum-fed calves
J. N. Wilms,1,2* M. H. Ghaffari,3 M. A. Steele,2 H. Sauerwein,3 J. Martín-Tereso,1 and L. N. Leal1
1
Trouw Nutrition R&D, P.O. Box 299, 3800 AG, Amersfoort, the Netherlands
2
Department of Animal Biosciences, Animal Science and Nutrition, University of Guelph, Guelph, ON, Canada N1G 1Y2
3
Institute of Animal Science, University of Bonn, 53111 Bonn, Germany
ABSTRACT
Milk replacers (MR) for calves usually contain more
lactose and less fat than bovine whole milk (WM).
There are insufficient data to determine whether these
MR formulations are optimal for calves fed at high
planes of nutrition. Thus, the effect of 3 MR formula-
tions and a WM powder were evaluated on growth,
feeding behavior, and blood metabolites in 96 male
Holstein calves fed ad libitum and with 45.5 ± 4.30
kg (mean ± SD) BW at arrival. Calves were blocked
based on arrival sequence, and randomly assigned
within block to one of the 4 treatments (n = 24 calves/
group): a high-fat MR (25.0% fat, dry matter basis;
22.5% protein, 38.6% lactose; 21.3 MJ/kg; HF), a high
lactose MR (44.6% lactose, 22.5% protein, 18.0% fat;
19.7 MJ/kg; HL), a high protein MR (26.0% protein,
18.0% fat, 41.5% lactose; 20.0 MJ/kg; HP), and a WM
powder (26.0% fat; 24.5% protein, 38.0% lactose; 21.6
MJ/kg; WP). In the first 2 wk after arrival, calves were
individually housed and were fed 3.0 L of their respec-
tive liquid feed 3 times daily at 135 g/L. They were
then moved to group housing and fed ad libitum until
d 42 after arrival. Weaning was gradual and took place
between d 43 and 70 after arrival; thereafter, calves
were fed solids only. Concentrates, chopped straw, and
water were available ad libitum throughout the study.
Body weight was measured, and blood was collected
at arrival and then weekly thereafter from wk 1 to 12.
Weight gain and height were greater in HL than WP
calves. In the preweaning phase, HL and HP-fed calves
consumed more milk than WP, and HL-fed calves con-
sumed more milk than HF calves. In wk 10, starter
feed intakes were lower in HF calves than in the other
groups. In the preweaning phase, ME intakes were the
same for all treatments. This suggests that milk intakes
Received January 26, 2022.
Accepted March 26, 2022.
*Corresponding author: juliette.wilms@​trouwnutrition​.com
6670
were regulated by the energy density of the milk sup-
plied. The percentage of calves requiring therapeutic
interventions related to diarrhea was greater in WP-fed
calves (29%) than HF and HL calves (4%), whereas
HP (13%) did not differ with other groups. This was
coupled with lower blood acid–base, blood gas, and
blood sodium in WP than in MR-fed calves. Calves fed
HF had greater serum nonesterified fatty acids com-
pared with other groups, and greater serum amyloid A
compared with WP and HL calves. Among the serum
parameters, insulin-like growth factor-1 and lactate
dehydrogenase correlated positively with ME intake
and average daily gain. The high lactose and protein
intakes in HL and HP calves led to greater insulin-like
growth factor-1 concentrations than in WP-fed calves.
Although growth differences were limited among MR
groups, the metabolic profile largely differed and these
differences require further investigation.
Key words: calf, macronutrient profile, milk replacer,
feeding behavior
INTRODUCTION
In current calf nutrition trends, replacement heif-
ers are fed elevated planes of milk (~20% birth BW;
MacPherson et al., 2016; Welboren et al., 2019; Leal
et al., 2021). The effectiveness of milk replacer (MR)
formulations in supporting development and health of
calves fed large amounts of liquid feed is not well char-
acterized (Diaz et al., 2001; van Niekerk et al., 2021b).
Milk replacers for calves contain higher levels of lactose
(40–50 vs. ~35% DM basis; Gilbert et al., 2016; Amado
et al., 2019; Wilms et al., 2020a) and lower levels of
fat (16–20% vs. ~30%) than bovine whole milk (WM)
from Holstein dairy cows. Although protein levels are
rather comparable between MR and WM (20–24%
vs. ~24%), high protein MR formulations (≥26%) are
also available but not in wide use (Daniels et al., 2008;
USDA, 2012). For the same percentage of solids, low
levels of fat in MR (≤20%) result in a lower ME density
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
and a higher dietary protein to energy ratio compared
with high-fat MR and WM. For a same percentage of
solids, calves fed ad libitum levels of MR with high lac-
tose have greater liquid feed intakes and slightly greater
preweaning growth compared with MR with high fat
(≥23% fat; Berends et al., 2020; Echeverry-Munera
et al., 2021). Additionally, feeding isocaloric MR with
higher protein content at 10 to 14% of BW daily to
dairy calves improved growth rates and gain-to-feed
ratios (Bartlett et al., 2006).
Although performance in early life remains a main
focus in calf nutrition, the metabolic implications re-
lated to dietary macronutrient imbalances need to be
addressed in calves fed elevated planes of MR. In infant
nutrition, excessive protein intake from infant formula
(15–20% energy) in early in life (<4 mo of age) drives
early programming of hormonal mechanisms by pro-
moting growth acceleration, whereas slower growth in
breast-fed and low-protein formula-fed infants reduces
the incidence of cardiovascular disease and associated
risks later in life (Leunissen et al., 2009; Socha et al.,
2011; Oddy et al., 2014). This was attributed to in-
creased concentrations of insulin-releasing AA stimu-
lating insulin and insulin-like growth factor I (IGF-I;
Rolland-Cachera et al., 1984; Rolland-Cachera and
Peneau, 2010; Michaelsen and Greer, 2014). Similarly,
feeding MR with high lactose increases postprandial
insulin concentrations (Welboren et al., 2019; Yohe
et al., 2021); which in turn modulates hepatic IGF-I
production through a direct regulation of IGF-I tran-
script levels (Boni-Schnetzler et al., 1991). Thus, feed-
ing high-lactose and high-protein MR at high planes of
nutrition (~20% birth BW) could result in increased
IGF-I signals in calves.
Beyond the metabolic effects linked with excessive
intakes of lactose and protein from liquid feeds, higher
fat inclusion in MR (≥23%) has been associated with
decreased mortality (Urie et al., 2018), improved health
in terms of fecal consistency (Amado et al., 2019), re-
duced therapeutic interventions (Berends et al., 2020),
and greater gastrointestinal weight (Welboren et al.,
2021) in dairy calves. Dietary fat from WM provides
about 50% of the total dietary energy, as well as es-
sential fatty acids, presenting important structural and
metabolic functions to newborn animals (Delplanque
et al., 2015; Grote et al., 2016; Wilms et al., 2022). Fat
stores are mobilized during infections and potentially
explain the association between nutritional status and
mortality in infants (Kuzawa, 1998).
The present study tested the hypothesis that high
fat intakes from liquid feeds would result in lower milk
intakes, slower growth, improved health, and distinct
metabolic profiles than calves fed high-lactose and
high-protein MR. The objective was to evaluate how
Journal of Dairy Science Vol. 105 No. 8, 2022
6671
the macronutrient profile of MR would affect growth
performance, feeding behavior, and blood metabolites
in male dairy calves fed ad libitum. In addition, a group
of calves fed a WM powder served as a biological ref-
erence for growth and development expected from its
natural macronutrient and micronutrient profile.
MATERIALS AND METHODS
This study was conducted at the Calf Research Facil-
ity of Trouw Nutrition Research and Development (Sint
Anthonis, the Netherlands) between April 2018 and July
2019. All procedures described in this article complied
with the Dutch Law on Experimental Animals, which
meets European guidelines defined by ETS123 (Council
of Europe 1985 and the 86/609/EEC Directive) and
were approved by the animal welfare authority (Cen-
trale Commissie Dierproeven, CCD, the Netherlands).
The project application code is AVD2040020173425.
Animals and Experimental Design
A total of 96 male Holstein Friesian calves with an
arrival BW of 45.5 ± 4.30 kg (mean ± SD) were pur-
chased from neighboring dairy farms. At the farm of ori-
gin, a standardized protocol for colostrum management
was applied, 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 brix values of
colostrum fed were recorded. Thereafter, meals of 3.0
L of MR at 135 g/L (Sprayfo Excellent, Trouw Nutri-
tion) were offered twice daily until the day of collection.
Calves were brought to the research facility between 0
and 3 d after birth, and blood IgG concentrations were
measured within 48 to 72 h after birth using a por-
table Multi-Test Analyzer (DVM Rapid Test II, Vetlab
Supply Inc.). Calves were assigned to 1 of 24 blocks
based on age and arrival date to minimize age differ-
ences within blocks, and within each block, calves were
randomly assigned to 1 of 4 dietary treatments. The 4
experimental diets were supplied from arrival onward
up to 84 d after arrival to the research facility. The ex-
perimental period was divided into 3 phases, including
preweaning, weaning transition, and postweaning. In
the preweaning phase, all calves were housed individu-
ally and fed 3.0 L (135 g/L) 3 times daily for the first
2 wk after arrival. Subsequently, calves were moved to
a group housing department and fed ad libitum from d
15 to 42 after arrival. Each group pen included 8 calves
(2 blocks of 4 calves) for a total of 4 pens in the depart-
ment. In the second phase, weaning was gradual and
took place between d 43 and 70 after arrival. Finally,
in the postweaning phase, calves were exclusively fed
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
solid feeds from d 70 to 84. The experiment included 3
successive batches of 32 calves each.
Treatments and Feeding
Treatments consisted of 4 experimental diets (n =
24 per treatment): a high-fat MR (25.0% fat; 22.5%
protein, 38.6% lactose; 21.3 MJ/kg; HF), a high lactose
MR (44.6% lactose, 22.5% protein, 18.0% fat; 19.7 MJ/
kg; HL), a high protein MR (26.0% protein, 18.0%
fat, 41.5% lactose; 20.0 MJ/kg; HP), and dehydrated
WM powder (26.0% fat, 24.5% protein, 38% lactose;
21.6 MJ/kg; WP). Treatments were blinded to animal
caretakers by randomly assigning a letter (A, B, C, or
D) to each treatment. Ingredients and nutrient compo-
sition of MR (Sloten B.V., Trouw Nutrition) and WP
(WMP STD 26%, Arla Foods) are presented in Table
1. The HF treatment was designed to be close to the
fat content of WM, whereas the HL and HP treatments
aimed to represent formulation strategies currently pro-
posed 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 35% fat from coconut oil and 65% from palm oil.
Milk replacer concentration was 135.0 g/L of the milk
fed to get closer to the solid percentage of bovine WM.
Having the same percentage of solids allowed leveling
the percentage of ash among the 3 MR formulas and
is likely closer to industry practices. Additionally, this
allowed for isonitrogenous comparisons between HL
and HF, which would not be possible if MR concentra-
tions were adjusted for ME density. Starter feed and
water were available ad libitum throughout the ex-
perimental period. Chopped wheat straw was available
from d 14 onward. Milk replacers were fed ad libitum
via automated milk dispensers (CF1000+, DeLaval),
starter feed via automated feeders (CRFI, Biocontrol),
chopped straw via automated feeders (CRFI, Biocon-
trol), and water via automated bowls (Förster-Tech-
nik). All 4 dietary treatments were available in each of
the 4 pens and were offered individually to the calves
via electronic recognition. This means that each pen
included 2 blocks of 4 calves each and one automated
milk feeder able to dispense all 4 milk treatments (WP
or MR) through the same teat. The minimum time
interval between 2 milk meals was set at 1 h, meal size
was set between a minimum of 0.5 L and a maximum of
2.0 L, and the maximum drinking speed of 0.3 L/min.
In the preweaning phase, calves were linearly weaned
from a maximum daily consumption of 8.0 L on d 43
to 2.0 L on d 70 with a minimum time interval of 1.5 h
between visits to the MR feeder. On d 70, calves were
Journal of Dairy Science Vol. 105 No. 8, 2022
6672
fully weaned but had access to all other feeds, and they
were observed until 84 d.
Housing
For the first 2 wk after arrival, calves were housed
indoors in individual pens (1.22 m × 2.13 m) separated
by galvanized bar fences and equipped with 50% rubber
slatted floors in the front and 50% lying area including
a mattress covered with flax straw in the rear. From d
15 after arrival onward, calves were housed in groups
indoors with 2 blocks per pen (8 calves). The group
pens were 5.0 × 5.9 m and had a resting area (5.0 × 2.8
m) covered with flax straw and rubber slatted floors in
front of the feeders. The temperature in the calf facility
was at a minimum of 12°C and a maximum of 31°C
and the relative humidity between 60 and 80%. Calves
were exposed to daylight and artificial light from 0600
to 2200 h. Environmental data were not used for data
processing but only for animal welfare monitoring.
Measurements
Milk replacers were sampled for analysis at the
beginning of the study. During the first 2 wk after
arrival, intakes of MR, water, and starter feed were
recorded by weighing leftovers. Thereafter, feed and
water intakes were recorded daily by the automated
feeders. The number of rewarded visits, defined as the
number of times a calf visited the automated milk
feeders and received a milk portion, was recorded.
Similarly, the number of unrewarded visits, defined
as the number of times a calf visited the feeders and
did not receive a milk portion, was also registered.
Fecal scores were assessed daily in the first 14 d after
the morning meal at 0900 h according to Renaud et
al. (2020). Body weights were measured by weigh-
ing calves with a custom scale (W2000; Welvaarts
Weegsystemen) at arrival, and then weekly on d 7,
14, 21, 28, 42, 56, 70, and 84 after arrival. At the
same time, body dimensions including withers and hip
height, chest girth, and body barrel were measured
according to Echeverry-Munera et al. (2021). Blood
samples were collected by jugular venipuncture at the
same time as BW measurements. Blood samples were
collected into two 9-mL serum tubes (BD Vacutainer,
Becton Dickinson) for all time points, and into one
9-mL tube with anticoagulant (lithium heparin, BD
Vacutainer, Becton Dickinson) on d 7, 14, 21, and 28.
For blood acid–base (pH, base excess, and HCO3−),
blood gas determination (total carbon dioxide [tCO2],
partial pressure of carbon dioxide [pCO2]), glucose,
electrolytes (sodium, potassium, ionized calcium), and
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6673
Table 1. Ingredient and analyzed nutrient composition of milk replacers, whole milk powder, and starter feed

Treatment1

Item WP HF HL HP Starter feed

Ingredient (% product)
Skim milk powder — 50.0 50.0 50.0 —
Fat blend2 — 25.0 18.0 18.0 —
Whey powder — 12.4 23.4 10.5 —
Whey protein concentrate — 6.8 2.8 15.7 —
Hydrolyzed wheat protein — 2.0 2.0 2.0 —
Maltodextrin — 2.0 2.0 2.0 —
Maize — — — — 30.0
Soybean meal — — — — 25.1
Soy hulls — — — — 18.0
Beet pulp — — — — 8.5
Barley — — — — 5.0
Sugar cane molasses — — — — 4.5
Maize gluten meal — — — — 2.1
Linseed (full fat) — — — — 1.1
Premix3 — 1.8 1.8 1.8 5.8
Nutrients (DM basis)4
DM 960 968 968 966 891
CP 245 225 225 260 196
Crude fat 260 250 180 180 30
Crude ash 65 63 69 66 75
Crude fiber — — — — 98.5
Lactose 380 386 446 415 —
Glucose 0 5 5 5 —
Starch 0 14 14 14 219
Sodium 5.4 4.5 4.9 4.8 4.7
Potassium 14 12 13 13 11.2
Chloride 12 8.3 9.7 9.7 7.8
Calcium 14 8 9 8.3 11.2
Phosphorus 9.8 6.6 7.1 7.1 5.1
Magnesium 1.3 1.2 1.3 1.2 —
CP:ME ratio 48 44 48 54 —
WPNI5 (mg/g) 1.9 6.9 7.1 8.2 —
Osmolality6 (mOsm/kg) 314 326 370 350 —
SID7 (mEq/L) 33 44 45 40 —
NDF — — — — 198
ADF — — — — 123
ADL — — — — 8
ME8 (MJ/kg) 21.6 21.3 19.7 20.0 12.5
1
Treatments included a whole milk powder (WP; n = 24), and 3 milk replacers (MR) including a MR with high
fat (HF; n = 24), a MR with high lactose (HL; n = 24), and a MR with high protein (HP; n = 24).
2
Blend of palm and coconut oils, 2:1.
3
Milk replacers contain vitamins, minerals, probiotics and prebiotics, stabilizers, amino acids, as well as flavor-
ing and stability agents.
4
Expressed in g/kg DM unless specified otherwise.
5
WPNI = whey protein nitrogen index
6
Osmolality (in moles/kg of solvent and expressed in mOsm/kg) was calculated by adding osmolality of carbo-
hydrates and minerals as described in (Wilms et al. 2020a).
7
Strong ion difference (SID), expressed in mEq/L = sodium (mmol/L) + potassium (mmol/L) − chloride
(mmol/L).
8
Starter feed ME value was calculated based on NRC (2001).

hematocrit, one drop of whole blood from the lithium
heparin tube was inserted into CG8+ cartridges and
analyzed immediately using a blood gas analyzer
(VetScan I-STAT 1, ref: 600-7015, Abbott Point of
Care Inc.). The serum tubes were fixed for 15 min
and centrifuged at 1,500 × g for 15 min at 20°C (Ro-
tina 380 R, Hettich). Serum samples were stored in
1.5-mL cryotubes at −18°C and were transported in
Journal of Dairy Science Vol. 105 No. 8, 2022
boxes with cooling elements and stored at −18°C until
analyses were performed.
Chemical Analyses
Feed samples were processed and analyzed at Mas-
terLab (Boxmeer, the Netherlands). Milk replacer and
WP samples were analyzed for DM, crude ash, crude
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
fat, CP, macrominerals, and carbohydrates (lactose and
glucose), whereas starter feed was analyzed for DM, CP,
crude fat, crude ash, crude fiber, starch, and macro-
minerals. In addition, the whey protein nitrogen index
(WPNI), which reflects protein quality, was measured
in MR and WP samples. Dry matter content was deter-
mined by drying to a constant weight in a 103°C oven
for 4 h (EC 152/2009; EC, 2009). Crude ash was ana-
lyzed 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 hydro-
chloric acid followed by extraction with petroleum (EC
152/2009; EC, 2009). Crude protein content was ana-
lyzed by combustion using the Dumas method (Ether-
idge et al., 1998; ISO, 2008). Starter feed was analyzed
for starch content, as described by Rijnen et al. (2001)
and crude fiber was analyzed via intermediate filtration
(NEN-EN-ISO 6865:2000; NEN, 2000). Macrominer-
als were analyzed by inductively coupled plasma mass
spectrometry (PerkinElmer ICP-MS 300D) according
to NEN-EN 15510 (2017). Chloride was analyzed as
described in Wilms et al. (2019). Carbohydrates in MR
and WP were determined by the titrimetric method
according to 71/250/EEG (EEG, 1971) for lactose and
EC 152/2009 (EC, 2009) for glucose.
The WPNI was determined by mixing 5 g of MR or
WP with 50 mL of distilled water, placed in a water
bath at 37°C for 30 min and shaken 6 times. Afterward,
20 g of NaCl was added, and the solution remained in
the water bath for an extra 30 min. After settling at
room temperature for 15 min, the solution was filtered
(Whatman grade 3, 5123613, Cytiva), and 0.24 mL
of 10% HCl was added to 8 mL of the filtrate. After
settling for 30 min, the solution was again filtered
(Whatman S&S Blue Band, Grade 589/3, Cytiva),
and the absorbance was determined at 275 nm using
a standard curve including 2 standard samples with
known WPNI.
Only complete blocks (n = 22) of animals who com-
pleted the entire experimental phase were considered for
blood sample analyses. Serum samples were processed
at the University of Nottingham (Nottingham, UK).
Albumin, aspartate transaminase (AST), gamma-
glutamyl transferase (GGT), BHB, chloride, cerulo-
plasmin, haptoglobin, lactate dehydrogenase, bilirubin,
total protein, triglyceride, and urea were measured
using Randox Kits in the RX IMOLA (Randox Labo-
ratories Ltd.) according to the manufacturer’s instruc-
tions. Nonesterified fatty acids (NEFA) were analyzed
using a commercial kit (NEFA-HR(2) kit, Fujifilm
Wako Chemicals Europe GmbH). The mean interassay
coefficients of variation (CV) was 3.5% for albumin,
3.1% for AST, 1.2% for BHB, 1.2% for chloride, 6.4%
for ceruloplasmin, 2.6% for GGT, 6.3% for haptoglo-
Journal of Dairy Science Vol. 105 No. 8, 2022
6674
bin, 4.0% for lactate dehydrogenase, 2.9% for NEFA,
3.8% for total bilirubin, 1.3% for total protein, 2.2%
for triglyceride, and 1.8% for urea. In addition, serum
immunoglobulins (IgA, IgM, and IgG) were measured
on d 56 and 84 using a kit from Bethyl Laboratories
(Uden, the Netherlands) according to manufacturer’s
instructions.
Serum adiponectin, leptin, serum amyloid A (SAA),
and IGF-I were measured at the University of Bonn
(Bonn, Germany). Serum concentrations of adiponectin
and leptin were determined using in-house developed
ELISA (Sauerwein et al., 2004; Mielenz et al., 2013).
For the adiponectin and leptin ELISA, the intra- and
interassay CV were 8.3, 11.2, 8.0, and 10.9%, respec-
tively. The concentrations of SAA were determined
with commercial sandwich ELISA (Life Diagnostics
Ltd.; SAA −11) with intra- and interassay CV of 4.11
and 9.96%, respectively. The concentration of IGF-I
was determined using the commercial IGF-I ELISA
E20 kit (Mediagnost) with intra- and interassay CV of
2.24 and 4.15%, respectively.
Calculations and Statistical Analysis
Power analysis for sample size estimation was per-
formed for growth performance based on previously
published values (Echeverry-Munera et al., 2021) ob-
tained in the same research facility as the present study.
From the power test analysis, using the α-level = 0.05
and power (1 − β) = 0.80, the projected sample size was
22 calves per treatment group for growth data. Thus, 24
calves per treatment group were included in the study.
The ME content of the MR was calculated as follows:
ME (Mcal/kg) = [0.057 × CP (%) + 0.092 × crude fat
(%) + 0.0395 × lactose (%)] × 0.93 (NRC, 2001). The
ME content of the starter feed was calculated based on
the NRC (2001) ME table values for the raw materials
and applying the equation for calf starter feed. Total
ME intake included MR and starter feed, whereas straw
was not considered. Feed conversion was calculated by
dividing the daily total ME intake by ADG. Continuous
variables (e.g., BW, intakes, blood parameters) were an-
alyzed using mixed-model analysis with PROC MIXED
in SAS (SAS 9.4M6, SAS Institute Inc.). All the data
were screened for normality of distribution using the
UNIVARIATE procedure of SAS (the Shapiro-Wilk
test) before analysis. The calf was the experimental
unit, and the statistical model was as follows:
Yijk = µ + Ti + Vj + Wk + TWik + εijk,
where Yijk is the dependent variable; μ is the overall
mean; Ti is the fixed effect of the ith treatment; Vj is
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
the random effect of the jth block; Wk is the fixed effect
of the kth week entering the model as a repeated mea-
sure; TWik is the fixed effect interaction between the
ith treatment and the kth week; and εijk is the random
error associated with the jth block at the kth week with
the ith treatment. In the case of BW and ADG, arrival
BW was included as a baseline covariate (µ0). Simi-
larly, for body dimension measurements, initial body
dimensions were also used as baseline covariates. For
variables with equally spaced time points (e.g., BW,
intakes, blood acid–base), the covariance structure (au-
toregressive covariance [AR(1)] or the heterogeneous
autoregressive covariance [ARH(1)]) with the lowest
corrected Akaike information criterion was used. For
variables with unequally spaced time points (e.g., blood
parameters), the heterogeneous Toeplitz covariance
structure was used. Non-normally distributed values
were scaled using a log10 transformation to correct for
heteroscedasticity and meet assumptions of normality.
After the log-transformation, the distribution of the
data was tested again, and the data were normally dis-
tributed. Significant effect of treatment was explored
with the LSMEANS statement using the PDIFF option
of the MIXED procedure of SAS. Significant interac-
tions between treatment and time were explored with
the SLICE option of the LSMEANS statement using
the PDIFF option of the MIXED procedure of SAS. In
addition, the PDIFF option included a Tukey adjust-
ment statement to correct for multiple comparisons.
Results in tables and figures are presented as least
squares means with the standard error of the means
(SEM). In case of log-transformed data, the SEM was
not transformed back to the original scale (Bland and
Altman, 1996). Discrete variables (e.g., therapeutic
interventions, mortality) were analyzed using mixed-
effects logistic regressions. These analyses were con-
ducted 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
Pearson correlations were used to determine relation-
ships between variables using an online platform for
multivariate analysis (MVApp; Julkowska et al., 2019).
Correlations were defined as weak (r = 0.1 to 0.3),
moderate (r = 0.3 to 0.7), or strong (r = 0.7 to 1.0).
Furthermore, the correlation structure was plotted as
a principal component analysis (PCA) to determine
patterns of correlation among the individual traits
using the MVApp online platform. The significance
of the correlations was declared at P ≤ 0.05. A heat
Journal of Dairy Science Vol. 105 No. 8, 2022
6675
map was generated by hierarchical clustering of the
blood parameters obtained from the treatment groups.
The data were transformed using the generalized log-
transformation and then Pareto scaled to correct for
heteroscedasticity and to reduce the skewness of the
data. Hierarchical clustering (shown as a heat map)
was performed by Euclidean distance and Ward’s mini-
mum variance method (ward.D) using the web-based
metabolomic data processing tool MetaboAnalyst 5.0
(http:​/​/​www​.metaboanalyst​.ca; Pang et al., 2021).
RESULTS
General Health
Results for general health are presented in Table
2. The colostrum Brix score (25.3%), as well as IgG
concentrations measured between 48 to 72 h after ar-
rival did not differ across treatment groups (2,189 ±
158 mg/dL; LSM ± SD), nor arrival BW was different.
One calf from the WP group and one calf from the HP
group were removed postinclusion because of diarrhea
and dehydration during the third week after arrival.
Data from these calves were not used for performance,
intake, and blood metabolites. The percentage of days
with scours within the first 2 wk was 50% and did not
differ across treatment groups. Diarrhea occurred ex-
clusively in the first 3 wk after arrival, and the number
of calves receiving therapeutic interventions because of
diarrhea was greater in the WP group (29%) than in
the HF and HL groups (4%; P = 0.03), whereas the HP
group (13%) did not differ from the other treatments.
In contrast, the number of calves receiving therapeutic
interventions for respiratory disease, antibiotics, and
the total number of therapeutic interventions did not
differ across treatment groups.
Growth Performance, Intakes, and Feeding Behavior
Body weight (P = 0.03) and ADG (P = 0.02) were
greater in HL than WP calves throughout the whole
experimental phase (Figure 1A and 1B; Table 3).
Calves fed HP tended (P = 0.07) to be heavier than
WP calves and had a greater ADG than WP calves
(P = 0.05). When considering the rearing phases, dif-
ferences in BW and ADG were present only in the
preweaning phase where BW was 5% and ADG was
15% greater in calves fed HL than calves fed WP and
HF (P ≤ 0.03). Milk replacer intakes throughout the
different rearing phases (Figure 2A) present an effect
of treatment (P = 0.01), showing differences between
HL and WP calves. However, milk intakes only differed
during the preweaning phase and were 13% greater in
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6676
Table 2. Parameters describing calves at arrival and number of therapeutic interventions (n = 96)

Treatment1
P-value,
Item WP HF HL HP Pooled SEM treatment
Colostrum Brix score (%) 25.3 25.7 24.6 25.4 0.63 0.63
Blood IgG2 (g/L) 22.5 22.8 21.2 21.2 1.8 0.83
Arrival BW (kg) 44.8 45.0 46.3 45.9 0.89 0.53
Scours3 (% days) 49 49 46 55 — 0.31
Therapeutic interventions (%)
Diarrhea4 29 (7/24)a 4 (1/24)b 4 (1/24)b 13 (3/24)ab — 0.03
Respiratory5 21 (5/24) 33 (8/24) 17 (4/24) 17 (4/24) — 0.48
Antibiotics 38 (9/24) 38 (9/24) 21 (5/24) 21 (5/24) — 0.35
Total calves treated6 42 (10/24) 42 (9/24) 29 (5/24) 42 (7/24) — 0.41
Mortality (%) 4 (1/24) 0 (0/24) 0 (0/24) 4 (1/24) — 0.42
a,b
Means with a different superscript are significantly different (P ≤ 0.05).
1
Treatments included whole milk powder (26% fat; WP; n = 24), and 3 milk replacers (MR) including a MR with high fat (25% fat; HF; n =
24), a MR with high lactose (44% lactose; HL; n = 24), and a MR with high protein (26% protein; HP; n = 24).
2
Blood IgG was measured between 48 and 72 h after arrival.
3
Percentage of days with a fecal score of 2 (watery feces) within the first 14 d after arrival.
4
Therapeutic interventions for diarrhea include oral rehydration solutions, intravenous fluids, and medicines. All diarrhea cases occurred in the
first 3 wk after arrival.
5
Therapeutic interventions for respiratory disease; cases occurred between wk 2 and 12 after arrival.
6
Total of therapeutic interventions was limited to diarrhea and respiratory diseases. Specific disorders, such as navel infection, that could not be
related to nutrition were not considered.

HL than WP and HF calves, and 9% higher in HP than
WP calves (P < 0.01). Table 4 summarizes the main
MR feeding behavior parameters. In the weaning phase,
WP calves were drinking 9% faster than other groups
(P < 0.01). During the preweaning phase, the number
of rewarded visits was 14% greater in HL and HP than
HF (P = 0.02). Then in the weaning phase, the num-
ber of rewarded visits was 9% higher in MR-fed calves
than WP calves (P < 0.01). In contrast, the number
of unrewarded visits did not differ. The total number
of visits to the feeders was 15% higher for HL and HP
than that of HF calves during the preweaning phase (P
= 0.03). There was a treatment by time interaction for
overall starter feed intakes (P = 0.01; Figure 2B) due
to different dynamics through time. Straw intakes were
not affected by treatments. Water intakes tended to be
greater in WP-fed calves in the preweaning phase (P =
0.06) and was greater in the weaning phase (P = 0.05)
than the other treatment groups.
Fat intakes (Figure 3A) were by design consistently
higher for WP and HF calves than HL and HP calves (P
< 0.01), differences that leveled off in the postweaning
phase. By design, lactose intakes (P < 0.01; Figure 3B)
were the highest for HL calves, followed by HP calves,
and then WP and HF calves. Differences in lactose in-
takes also disappeared in the weaning phase as calves
were then fed restricted amounts of milk. Similarly, CP
intakes (Figure 3C) were different among treatments
(P < 0.01), with higher protein intakes in HP calves
compared with other groups, as a direct consequence
of MR composition. In addition, HL calves had higher
protein intakes than HF calves, whereas WP did not
differ from HF and HL. Finally, a treatment by time in-
teraction was also observed for total ME intakes in the
weaning phase (P = 0.02; Figure 3D), and throughout
all rearing phases (P = 0.04). This translated into lower
ME intakes for HF calves than the other groups in wk
10. By design, there was no variation in CP-to-ME ra-
tio in the weaning and postweaning phases. During the
preweaning phase, the CP-to-ME ratio was the highest
in HP and the lowest in HF calves. The feed conversion
ratio was 11% higher in WP than HL calves (P = 0.04)
throughout the experimental period due to differences
present in the preweaning phase (P = 0.01).
In addition to growth performance, differences in
body dimensions are presented in Table 5. In the wean-
ing phase, withers height was 2.3% higher in HL and
HP than in WP calves (P < 0.01). In addition, withers
height was 1.5% greater in HF than WP calves (P <
0.01). In the postweaning phase, withers height was
2.8% higher in HL than WP calves (P < 0.01). Overall,
withers height was 1.6% greater in MR-fed calves com-
pared with WP (P < 0.01). In all rearing phases, hip
height was consistently higher in HL than WP calves
(P < 0.05). Overall, hip height was 1.8% higher in
MR-fed calves than WP (P < 0.01). Throughout all
phases, chest girth was 2.2% higher in HL and HP
than WP calves and 1.8% higher in HL than HF calves
(P < 0.01). No differences were detected for the body
barrel during the different rearing phases. However,
overall body barrel was 1.8% greater in HL than HF
calves (P = 0.03).

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 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6677

Figure 1. Performance including BW (A) and ADG (B) measured weekly at 1000 h in calves fed ad libitum from d 15 after arrival onward.
Treatments included whole milk powder (WP, n = 23) and 3 milk replacers: high fat (HF, n = 24), high lactose (HL, n = 24), and high protein
(HP, n = 23). a,b: Treatments with different superscript letters are significantly different (P ≤ 0.05).

Blood Metabolites HCO3− were 36 and 8% lower, respectively, in WP than

Table 6 summarizes the results from blood param-
eters evaluated weekly in the first 4 wk of life. Blood
glucose was not affected by treatments (P = 0.24),
whereas hematocrit was 13% lower in WP calves than
MR-fed calves (P < 0.01). Blood base excess and
MR-fed calves (P < 0.01). Consistently, blood tCO2,
and pCO2 were 8 and 5% lower, respectively, in WP
calves (P < 0.01). Among the main blood minerals,
only blood sodium was affected by treatment (P <
0.01), with lower concentrations in WP and HF calves
compared with HL and HP calves. Table 7 summarizes

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 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6678
Table 3. Weekly milk, solids, and water intakes (LSM) measured daily in calves fed ad libitum from d 15 after arrival onward (n = 94)

Treatment2 P-value

Item1 WP HF HL HP Pooled SEM Treat Week Treat × Week

Milk intake (L/d)
Preweaning3 8.29c 8.72bc 9.63a 9.05ab 0.029 <0.01 <0.01 0.28
Weaning3 4.28 4.46 4.36 4.34 0.015 0.24 <0.01 0.67
Postweaning — — — — — — — —
Overall3 6.37b 6.67ab 7.02a 6.74ab 0.021 0.01 <0.01 0.35
Starter feed intakes (kg/d)
Preweaning4 0.39 0.33 0.35 0.38 0.068 0.21 <0.01 0.09
Weaning 1.22 1.12 1.21 1.21 0.084 0.82 <0.01 0.19
Postweaning3 3.45 3.41 3.50 3.68 0.041 0.58 <0.01 0.41
Overall3,4 0.70 0.66 0.68 0.68 0.071 0.96 <0.01 0.01
Straw intakes (g/d)
Preweaning — — — — — — — —
Weaning3 179 178 172 232 0.13 0.32 <0.01 0.20
Postweaning3 307 358 345 349 0.10 0.64 0.13 0.81
Overall3 214 224 217 266 0.11 0.37 <0.01 0.30
ME (MJ/d)
Preweaning 24.6 25.6 25.9 24.9 0.69 0.42 <0.01 0.42
Weaning 28.0 26.6 27.2 26.9 0.99 0.75 <0.01 0.02
Postweaning 42.7 42.3 45.2 47.3 1.73 0.17 <0.01 0.65
Overall 28.8 28.7 29.6 29.2 0.70 0.80 <0.01 0.04
Water intakes (L/d)
Preweaning3,5 1.26A 0.63B 0.65B 0.71B 0.228 0.06 <0.01 0.91
Weaning3 5.10a 3.04b 3.27b 3.65ab 0.145 0.05 <0.01 0.37
Postweaning3 14.8 13.2 13.7 15.3 0.061 0.29 <0.01 0.95
Overall3,5 3.64A 2.18B 2.29B 2.49B 0.148 0.06 <0.01 0.74
CP:ME (g/MJ)
Preweaning 11.5 10.6 11.5 13.1 0.23 — — —
Weaning3 13.1b 12.6c 13.2b 14.0a 0.07 <0.01 <0.01 <0.01
Postweaning 15.0 15.0 15.0 15.0 — — — —
Overall 12.6 12.0 12.6 13.7 1.35 — — —
BW (kg)
Preweaning 64.9b 66.3b 69.0a 67.3ab 0.98 0.03 <0.01 0.14
Weaning 97.2 100 104 101 1.82 0.07 <0.01 0.91
Postweaning 125 128 134 130 2.37 0.08 <0.01 0.20
Overall3 77.8b 79.9ab 82.3a 80.7ab 0.013 0.03 <0.01 0.63
ADG (g/d)
Preweaning 889b 955b 1057a 973ab 36.0 <0.01 <0.01 0.78
Weaning 970 955 933 957 42.6 0.94 <0.01 0.89
Postweaning 1341 1465 1569 1518 71.2 0.14 <0.01 0.86
Overall 993b 1040ab 1101a 1066a 25.2 0.02 <0.01 0.80
Feed conversion (MJ/kg gain)
Preweaning3 30.7a 29.9ab 26.5c 27.3bc 0.036 0.01 <0.01 0.68
Weaning3 30.8 29.7 31.4 30.7 0.047 0.96 <0.01 0.91
Postweaning3 33.0A 29.0BC 28.4C 32.0AB 0.051 0.06 <0.01 0.73
Overall3 31.1a 29.8ab 28.0b 29.3ab 0.026 0.04 <0.01 0.80
a–c
Means with a different superscript are significantly different (P ≤ 0.05).
A–C
Means with a different superscript include a trend (0.05 < P ≤ 0.10).
1
In the preweaning phase, calves were first individually housed and fed 3.0 L 3 times daily (135 g/L) in the first 2 wk after arrival. Calves were
then moved to a group housing department and were fed ad libitum from wk 3 to 6 after arrival. Weaning was gradual and took place between
wk 7 and 10 after arrival. In wk 11 and 12, calves were fed solids only.
2
Treatments (Treat) included whole milk powder (26% fat; 21.6 MJ/kg; WP; n = 23) and 3 milk replacers (MR), including a MR with high fat
(25% fat; 21.3 MJ/kg; HF; n = 24), a MR with high lactose (44% lactose; 19.7 MJ/kg; HL; n = 24), and a MR with high protein (26% protein;
20.0 MJ/kg; HP; n = 23).
3
SEM expressed as log.
4
Only available from 28 d after arrival onward due to low intakes within the first 28 d after arrival.
5
Only available from 15 d after arrival onward.

the results of the blood metabolites evaluated weekly in fer, whereas serum NEFA was 27% higher in HF calves
the first 4 wk of life and at d 56, and d 84. Serum urea (P < 0.01) than other groups. The treatment by time
was 23% lower in HF and HL calves than in WP and interaction for serum urea (P = 0.01; Figure 4A) and
HP calves (P < 0.01). Serum triglyceride did not dif- serum NEFA (P = 0.02; Figure 4C) likely shows that

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 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6679

Figure 2. Intakes including milk replacer (A) and starter feed (B), measured daily in calves fed ad libitum from d 15 after arrival onward.
Treatments included whole milk powder (WP, n = 23) and 3 milk replacers: high fat (HF, n = 24), high lactose (HL, n = 24), and high protein
(HP, n = 23). a,b: Treatments with different superscript letters are significantly different (P ≤ 0.05).

treatment differences faded away in the postweaning
phase. Additionally, serum albumin was 3.2% higher in
WP calves than HF and HL calves, and 2.5% higher in
HP than HL calves (P < 0.01). A treatment by time
interaction was present for serum bilirubin (P < 0.01;
Figure 4B), showing lower concentrations in the first
2 wk and higher concentrations from wk 3 onward in
WP-fed calves compared with other treatment groups.
Overall, BHB was lower in HL calves than in the other
Journal of Dairy Science Vol. 105 No. 8, 2022
groups (P < 0.01; Figure 4D). Regarding organ func-
tions, no differences were detected for serum GGT and
lactase dehydrogenase, whereas serum AST was lower
in WP calves in the preweaning phase (P < 0.01). A
treatment by time interaction was also present (P <
0.01; Figure 4E) showing that differences faded away
in the postweaning phase. No differences in serum IgG,
IgM, and IgA-to-IgM ratio were observed. However, a
treatment by time interaction for blood IgA concentra-
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6680
Table 4. Feeding behavior (LSM) measured daily in calves fed ad libitum from d 15 after arrival onward (n = 94)

Treatment2 P-value

Item1 WP HF HL HP Pooled SEM Treat Week Treat × Week

Drinking speed (mL/min)
Preweaning3 310 305 305 295 6.0 0.31 <0.01 0.20
Weaning 383a 345b 349b 357b 6.5 <0.01 0.14 0.38
Overall 347a 326b 327b 326b 5.6 0.01 <0.01 <0.01
Rewarded visits (n/d)
Preweaning3,4 6.56ab 6.20b 7.24a 6.90a 0.038 0.02 <0.01 0.22
Weaning 6.29b 7.03a 6.81a 6.77a 0.136 <0.01 <0.01 0.02
Overall 6.51B 6.67AB 7.01A 6.84AB 0.147 0.10 <0.01 <0.01
Unrewarded visits (n/d)
Preweaning — — — — — — — —
Weaning 10.2 11.0 11.0 12.1 0.87 0.46 <0.01 0.28
Overall — — — — — — — —
Total visits (n/d)
Preweaning3,4 6.93ab 6.46b 7.67a 7.24a 0.042 0.03 <0.01 0.25
Weaning4 15.2 17.2 17.4 18.1 0.06 0.13 <0.01 0.20
Overall4 10.3B 10.5AB 11.6A 11.5A 0.04 0.10 <0.01 0.11
a,b
Means with a different superscript are significantly different (P ≤ 0.05).
A,B
Means with a different superscript include a trend (0.05 < P ≤ 0.10).
1
In the preweaning phase, calves were first individually housed and fed 3.0 L 3 times daily (135 g/L) in the first 2 wk after arrival. Calves were
then moved to a group housing department and were fed ad libitum from wk 3 to 6 after arrival. Weaning was gradual and took place between
wk 7 and 10 after arrival. In wk 11 and 12, calves were fed solids only.
2
Treatments (Treat) included whole milk powder (26% fat; WP; n = 23) and 3 milk replacers (MR), including a MR with high fat (25% fat; HF;
n = 24), a MR with high lactose (44% lactose; HL; n = 24), and a MR with high protein (26% protein; HP; n = 23).
3
Only available from 15 d after arrival onward.
4
SEM expressed as log.

tions was present (P = 0.04), with greater concentra- rum parameters such as IGF-1, lactate dehydrogenase,
tions in HF (0.099 mg/mL) than WP calves (0.076 mg/ and blood gas parameters. Moreover, PCA 2 explained
mL) at d 56. Acute phase proteins did not differ in a much smaller portion of observed variation (11.5%),
case of haptoglobin and ceruloplasmin, whereas serum corresponding mainly to some serum parameters such
SAA was 39% higher in HF than WP and HL calves (P as urea, albumin, and Hp.
= 0.03; Figure 4F). Although adiponectin did not dif- The liquid diets substantially affected the blood pa-
fer across treatments, a treatment by time interaction rameters, as shown on the heatmap plot in Figure 6. The
was present for serum leptin concentrations (P = 0.05) column dendrogram shows the distance or similarity
showing different dynamics through time (Figure 4G). between the treatment groups, allowing clustering. As
Serum insulin-like growth factor-1 (IGF-1) was 21% expected, MR treatments clustered together, whereas
lower in WP than HL and HP calves (P < 0.01). In WP differed the most with MR groups. Among the 3
addition, IGF-1 tented (P = 0.06) to be 14% lower for MR, HL and HP clustered together, whereas treatment
HF than HP calves (Figure 4H). groups with high fat intakes were closer. The row den-
drogram identifies blood metabolite clusters, whereas
Correlations, PCA, and Heatmap the color reflects the relative changes in concentrations
based on the average for each blood parameter. Blood
Serum IGF-1 was positively correlated with ME in- metabolites clustered in 2 groups, with the first one
take (r = 0.74; Figure 5C) and ADG (r = 0.71; Figure including acid–base balance parameters, IGF-1, SAA,
5B). Serum lactase dehydrogenase was positively cor- AST, and GGT. The blue colors in WP calves show the
related with ME intake (r = 0.49; Figure 5A) and ADG lower concentrations in blood acid–base parameters, as
(r = 0.47; Figure 5D). Supplemental Figure S1 (https:​ well as the lower serum IGF-1 as compared with the
/​/​doi​.org/​10​.6084/​m9​.figshare​.20055281​.v2) shows the MR-fed calves. The second cluster included blood pa-
biplot of the PCA analysis with the contributions of rameters related to macronutrient intakes, such as tri-
different variables including intakes, growth, and blood glyceride, NEFA, lactose, urea, albumin, and BHB. In
parameters. The PCA 1 explained 29.3% of the observed this second cluster, the lowest concentrations for blood
variation, corresponding mainly to intakes (ME, fat, parameters were observed in HL and HP as compared
lactose, protein), growth (ADG and BW), and some se- with WP and HF.

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 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6681

Figure 3. Fat (A), lactose (B), protein (C), and ME (D) intakes measured daily in calves fed ad libitum from d 15 after arrival onward.
Treatments included whole milk powder (WP, n = 23) and 3 milk replacers: high fat (HF, n = 24), high lactose (HL, n = 24), and high protein
(HP, n = 23). a,b: Treatments with different superscript letters are significantly different (P ≤ 0.05).

DISCUSSION usually associated with metabolic acidosis in calves

This study evaluated how the macronutrient profile
in MR and WM powder affected growth performance,
feeding behavior, and blood metabolites of ad libitum-
fed dairy calves. Liquid feed intakes were greater in HL
(9.6 L/d) than in WP and HF calves (8.5 L/d) and
greater in HP (9.1 L/d) than in WP calves (8.3 L/d)
during the preweaning period. No differences were ob-
served in intakes of starter feed, straw, and ultimately
total ME. Growth was greater in HL and HP than WP,
whereas HF-fed calves did not differ from other groups
throughout the whole experimental phase. Overall,
MR-fed calves had greater body dimensions than WP-
fed calves.
Although the percentage of days with scours was
similar in the different treatment groups during the first
2 wk after arrival, the WP calves had a higher severity
of diarrhea. This was reflected by the higher percentage
of calves that received therapeutic interventions for di-
arrhea in this group (29%). In addition, WP calves also
had lower blood acid–base, gas, and Na+ concentrations,
Journal of Dairy Science Vol. 105 No. 8, 2022
with diarrhea (Trefz et al., 2012; Wilms et al., 2020b).
The lower blood acid–base values in WP calves may
also be related to the lower strong ion difference (SID)
of the WP treatment compared with MR solutions, as
high SID is known to alkalinize blood of calves (Wilms
et al., 2020b). However, the association between low
blood acid–base and Na+ concentrations and increased
diarrhea treatment does not allow to solely attribute
these differences to MR SID. Although it was intended
to represent a standard WM diet, the dehydrated WM
powder (WP treatment) used in the current experiment
was found to differ substantially from fresh WM, as the
processing steps include homogenization and drying,
which disrupt the fat globule membrane and damage
bioactive components. It is likely that denaturation of
milk proteins occurred, as indicated by the lower nitro-
gen index of whey proteins in the WP treatment (1.9
mg/g) compared with MR (7.4 mg/g). This parameter
is expressed as milligrams of undenatured whey protein
nitrogen per gram of nonfat MR (Zhao et al., 2019) and
is used as a quality indicator for MR. This indicates
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6682
Table 5. Weekly body measurements (LSM), measured at 1000 h in calves fed ad libitum from d 15 after arrival onward (n = 94)

Treatment3 P-value

Item1,2 WP HF HL HP Pooled SEM Treat Week Treat × Week

Withers height
Preweaning 82.9b 83.5ab 84.1a 83.7ab 0.38 0.05 <0.01 0.33
Weaning 91.1c 92.5b 93.7a 92.6ab 0.46 <0.01 <0.01 0.08
Postweaning 96.2b 97.6ab 98.9a 97.5ab 0.57 <0.01 <0.01 0.26
Overall 87.8b 88.8a 89.8a 88.9a 0.35 <0.01 <0.01 0.08
Hip height
Preweaning 87.6b 88.5a 89.2a 88.5a 0.30 <0.01 <0.01 0.61
Weaning 96.3b 97.9ab 99.0a 98.1a 0.49 <0.01 <0.01 0.78
Postweaning 101.6b 103.3a 103.8a 102.7ab 0.57 0.03 <0.01 0.67
Overall 92.7b 94.1a 94.9a 94.1a 0.31 <0.01 <0.01 0.97
Chest girth
Preweaning 90.8c 91.4bc 93.1a 92.4ab 0.50 <0.01 <0.01 0.71
Weaning 102.6c 104.9b 106.4a 105.4ab 0.56 <0.01 <0.01 0.67
Postweaning 110.8b 112.3ab 114.4a 113.5a 0.75 <0.01 <0.01 0.30
Overall 97.9c 99.4b 101.1a 100.3ab 0.46 <0.01 <0.01 0.71
Body barrel
Preweaning 98.2AB 97.0B 98.9A 98.5A 0.58 0.07 <0.01 0.67
Weaning 117.9 116.6 118.7 117.7 1.00 0.45 <0.01 0.77
Postweaning 135.8 134.3 137.8 135.8 1.15 0.20 <0.01 0.34
Overall 111.0ab 109.8b 112.0a 111.1ab 0.58 0.03 <0.01 0.83
a,b
Means with a different superscript are significantly different (P ≤ 0.05).
A,B
Means with a different superscript include a trend (0.05 < P ≤ 0.10).
1
In the preweaning phase, calves were first individually housed and fed 3.0 L 3 times daily (135 g/L) in the first 2 wk after arrival. Calves were
then moved to a group housing department and were fed ad libitum from wk 3 to 6 after arrival. Weaning was gradual and took place between
wk 7 and 10 after arrival. In wk 11 and 12, calves were fed solids only.
2
Expressed in cm.
3
Treatments (Treat) included whole milk powder (26% fat; WP; n = 23) and 3 milk replacers (MR), including a MR with high fat (25% fat; HF;
n = 24), a MR with high lactose (44% lactose; HL; n = 24), and a MR with high protein (26% protein; HP; n = 23).

Table 6. Blood hematology, blood acid–base balance, and blood gas (LSM) measured at 1000 h on d 7, 14, 21, and 28 after arrival in calves fed
ad libitum from d 15 after arrival onward (n = 88)

Treatment2 P-value

Item1 WP HF HL HP Pooled SEM Treat Week Treat × Week

Glucose 6.27 6.38 6.55 6.27 0.122 0.24 <0.01 0.13
Blood hematology
Htc (%) 22.0b 25.9a 25.6a 24.6a 0.85 <0.01 <0.01 0.27
Blood acid–base balance
pH3 7.418 7.430 7.432 7.435 0.001 0.12 <0.01 0.13
BE 5.17b 7.41a 8.28a 8.44a 0.64 <0.01 <0.01 0.18
HCO3− 29.8b 31.7a 32.6a 33.0a 0.57 <0.01 <0.01 0.44
Blood gas
tCO2 31.2b 33.3a 34.0a 34.2a 0.57 <0.01 <0.01 0.29
pCO2 45.8b 48.2a 48.7a 48.5a 0.63 <0.01 0.24 0.07
Blood minerals
Na+ 3 133b 134b 136a 135a 0.004 <0.01 <0.01 0.52
K+ 4.62 4.63 4.74 4.71 0.05 0.14 <0.01 0.36
Cl− 91.0 91.6 92.2 91.5 0.58 0.38 <0.01 0.59
iCa 1.37 1.38 1.36 1.37 0.01 0.23 <0.01 0.50
a,b
Means with a different superscript are significantly different (P ≤ 0.05).
1
Expressed in mmol/L unless specified otherwise. Htc = hematocrit; iCa = ionized calcium; BE = base excess; HCO3− = bicarbonate; tCO2 =
total carbon dioxide; pCO2 = partial pressure of carbon dioxide.
2
Treatments (Treat, n = 22 per treatment group) included whole milk powder (26% fat; WP) and 3 milk replacers (MR), including a MR with
high fat (25% fat; HF), a MR with high lactose (44% lactose; HL), and a MR with high protein (26% protein; HP).
3
SEM expressed as log.

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 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6683
Table 7. Blood metabolites and hormones (LSM) measured at 1000 h on d 7, 14, 21, 28, 56, and 84 in calves fed ad libitum from d 15 after
arrival onward (n = 88)

Treatment2 P-value

Item1 WP HF HL HP Pooled SEM Treat Week Treat × Week

Blood chemistry
Urea (mmol/L)3 3.36a 2.60b 2.66b 3.43a 0.037 <0.01 <0.01 <0.01
Bilirubin (µmol/L)3 2.66 2.80 2.56 2.45 0.157 0.29 <0.01 <0.01
TG (mmol/L)3 0.239 0.250 0.217 0.213 0.0676 0.24 <0.01 0.22
Albumin (g/L) 32.7a 31.9bc 31.5c 32.3ab 0.23 <0.01 <0.01 0.95
TP (g/L) 59.4 59.6 57.7 59.4 0.78 0.22 <0.01 0.27
NEFA (mmol/L)3 0.155b 0.191a 0.143b 0.150b 0.0538 <0.01 <0.01 0.02
BHB (mmol/L)3 0.120a 0.110a 0.097b 0.111a 0.0296 <0.01 <0.01 <0.01
Organ function
GGT (IU/L)3 43.6 47.2 48.0 48.1 0.11 0.89 <0.01 0.64
AST (IU/L)3 38.8b 43.4a 44.9a 45.2a 0.03 <0.01 <0.01 <0.01
LDH (IU/L)3 1,254 1,273 1,295 1,294 0.0170 0.48 <0.01 0.84
Immune function (mg/mL)4
IgG 29.4 30.3 29.2 30.1 1.76 0.95 0.05 0.22
IgM 0.83 0.71 0.75 0.71 0.066 0.49 <0.01 0.32
IgA3 0.115 0.128 0.125 0.125 0.1062 0.77 <0.01 0.04
IgA/IgM 0.156 0.203 0.179 0.191 0.1128 0.28 <0.01 0.17
Acute phase proteins
SAA (mg/L)3 1.32b 1.79a 1.26b 1.49ab 0.097 0.03 <0.01 0.24
Haptoglobin (mg/mL)3 0.160 0.139 0.144 0.147 0.0558 0.29 <0.01 0.88
Ceruloplasmin (mg/mL)3 0.206 0.208 0.192 0.198 0.0539 0.62 <0.01 0.39
Hormones
Adiponectin (µg/mL) 12.5 12.3 12.4 13.0 0.40 0.61 <0.01 0.48
Leptin (ng/mL)3 2.39 2.31 2.26 2.26 0.037 0.65 <0.01 0.05
IGF-1 (ng/mL)3 164b 185ab 199a 214a 0.06 <0.01 <0.01 0.29
a–c
Means with a different superscript are significantly different (P ≤ 0.05).
1
Expressed in mmol/L unless specified otherwise. TG = triglyceride; TP = total protein; NEFA = nonesterified fatty acids; GGT = gamma-glu-
tamyl transferase; AST = aspartate transaminase; LDH = lactate dehydrogenase; SAA = serum amyloid A; IGF-I = insulin-like growth factor.
2
Treatments (Treat, n = 22 per treatment group) included whole milk powder (26% fat; WP) and 3 milk replacers (MR), including a MR with
high fat (25% fat; HF), a MR with high lactose (44% lactose; HL), and a MR with high protein (26% protein; HP).
3
SEM expressed as log.
4
Only includes d 56 and 84.

that the quality of protein fraction and functional prop-
erties of WP treatment were compromised, which could
possibly have affected the performance and health of
WP calves. It cannot be ruled out that the presence of
functional additives such as pre- and probiotics present
in the MR treatments could have mitigated diarrhea
severity in MR-fed calves as earlier studies demon-
strated the health benefits of such components (Ren-
aud et al., 2019; Cangiano et al., 2020; Kayasaki et al.,
2021). However, previous research clearly showed that
high heat treatment of MR components or WM led to
reduced curd formation, reduced digestibility, and in-
creased diarrhea severity in calves (Shillam et al., 1962;
Tagari and Roy, 1969; Laksesvela et al., 1978). Despite
large differences in health outcomes, only marginal
differences in antiboby production were detected, with
slightly lower IgA concentrations in blood of WP than
HF calves. This may be related to the different timing
between diarrhea occurrence and IgA measurement.
During the preweaning period, the number of re-
warded visits per day to the automatic milk feeders
differed among treatment groups. Calves fed HF had
Journal of Dairy Science Vol. 105 No. 8, 2022
fewer rewarded visits to the automated milk feeders
than calves fed HL and HP, although liquid feeds were
offered ad libitum in the preweaning phase, which re-
flects the higher MR intakes in HL and HP calves. In
contrast, WP calves had the lowest milk intakes but
no differences in the number of rewarded visits were
detected with other treatment groups. This suggests
that WP-fed calves may have consumed smaller meals
at each visit than HL and HP-fed calves. A hypotheti-
cal explanation could be a different nutrient absorption
dynamic between WP- and MR-fed calves affecting
nutrient signaling in WP-fed calves. The lower energy
density in the HL and HP diets compared with WP
and HF may also explain the higher MR intakes and
the greater number of rewarded visits to the automated
feeders. The similar ME intakes seem to indicate that
the calves regulate their intakes to meet a certain en-
ergy level when milk is fed ad libitum. These results
are consistent with similar studies investigating the
replacement of lactose with fat in MR in calves fed ad
libitum (Berends et al., 2020; Echeverry-Munera et al.,
2021). Echeverry-Munera et al. (2021) also showed that
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6684

Figure 4. Serum urea (A), bilirubin (B), nonesterified fatty acids (NEFA; C), BHB (D), aspartate transaminase (AST; E), serum amyloid
A (SAA; F), leptin (G), and insulin-like growth factor (IGF-I; H) concentrations measured at 1000 h on d 7, 14, 21, 28, 56, and 84 in calves fed
ad libitum from d 15 after arrival onward. Treatments (n = 22 per treatment group) included whole milk powder (WP) and 3 milk replacers:
high fat (HF), high lactose (HL), and high protein (HP). a,b: Treatments with different superscript letters are significantly different (P ≤ 0.05).

Journal of Dairy Science Vol. 105 No. 8, 2022

 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6685

Figure 5. Pearson correlations between (A) serum insulin-like growth factor (IGF-1) and ME intake, (B) serum IGF-1 and ADG, (C) serum
lactate dehydrogenase (LDH) and ME intake, and (D) serum LDH and ADG across the treatments (n = 88).

ad libitum-fed calves receiving MR with high lactose
(44% lactose) displayed hunger-related behaviors based
on a higher number of unrewarded visits compared
with calves fed a MR with high fat (23% fat) during
the weaning period. However, as no differences were
detected in the number of unrewarded visits during the
weaning phase in the present study, no conclusion can
be drawn regarding hunger stress in HL calves. The
discrepancy between the current study and the study
by Echeverry-Munera et al. (2021) could be explained
by differences in weaning protocols. As seen in the
present study, the calves were weaned gradually, from
d 43 to 70, whereas in an earlier study by Echeverry-
Munera et al. (2021), the calves were weaned from d 36
to 57. Early weaning is known to be more stressful for
Journal of Dairy Science Vol. 105 No. 8, 2022
calves (Hulbert et al., 2011; Meale et al., 2017), which
may have accentuated stress in calves fed the MR with
high lactose in the study from Echeverry-Munera et al.
(2021).
Interestingly, WP-fed calves had a higher drinking
speed than MR-fed calves. This could be related to the
taste, as well as the presence of olfactory components
from milk fat, which are known to increase sucking
activity in infants (Gutiérrez-García et al., 2017).
Whether this is also relevant for sucking activity in
calves has not yet been studied. Furthermore, calves fed
WP had increased water intakes during the preweaning
and weaning phases compared with other groups. The
greater water and the lower milk intakes in WP-fed
calves could be related to the higher diarrhea severity
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER 6686

Figure 6. Heatmap of blood parameters among the treatments (n = 88). The colors in the heatmap reflect the blood parameters abundance
(mean centered and divided by the range of each variable). The data were transformed using the generalized log-transformation and then Pareto
scaled. IGF-I = insulin-like growth factor; GGT = gamma-glutamyl transferase; AST = aspartate transaminase; HCO3− = bicarbonate; tCO2
= total carbon dioxide; Htc = hematocrit; SAA = serum amyloid A; pCO2 = partial pressure of carbon dioxide; CP = ceruloplasmin; LDH =
lactate dehydrogenase; Cl− = chloride; Glu = glucose; TG = triglyceride; TP = total protein; NEFA = nonesterified fatty acids; Na+ = sodium;
K+ = potassium; iCa = ionized calcium; BE = base deficit; Hp = haptoglobin. Treatments (n = 22 per treatment group) included whole milk
powder (WP) and 3 milk replacers: high fat (HF), high lactose (HL), and high protein (HP). *Indicates a significant difference among the treat-
ment groups.

Journal of Dairy Science Vol. 105 No. 8, 2022

 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
in that group. However, differences were present during
the whole preweaning and weaning phases for water
intakes and during the whole preweaning phase for milk
intakes, whereas increased diarrhea severity could only
be detected in the first 3 wk after arrival. This does
not suggest that diarrhea was the main driver of the
observed differences but rather that different nutrient
absorption dynamics or the protein denaturation of the
WP treatment may have triggered the calves to drink
more water.
The differences in milk intakes and treatment com-
position resulted in differences in fat, lactose, and pro-
tein intakes. As expected by diet design, WP and HF
calves consumed more fat than HL and HP calves, and
protein intake was the highest in HP followed by HL,
whereas lactose intake was the highest in HL followed
by HP. Differences in nutrient intakes were not present
anymore during the postweaning phase. However, HF
calves consumed slightly less starter feed than other
groups in wk 10, resulting in a trend toward lower total
ME intake but these differences did not persist in the
postweaning phase. Interestingly, starter feed intakes
surged from d 70 to 77 (+1.1 kg/d) and slowed down
from d 77 to 84 (+0.4 kg/d). This resulted in a de-
crease in ADG from d 77 to 84 (−229 g/d), whereas
ADG increased by 234 g/d from d 70 to 77. These
starter feed intake patterns were previously observed
in other experiments feeding high volumes of milk
(Miller-Cushon et al., 2013; Rosenberger et al., 2017;
Echeverry-Munera et al., 2021). Thus, calves may have
consumed large amounts of solids once milk was no lon-
ger available, resulting in accumulation of dry feed and
overestimation of the BW measured at d 77. The feed
conversion ratio was lower for HL than for WP calves
throughout the experimental period, mainly during the
preweaning phase. This indicates that HL calves were
more efficient because for the same ME intakes, growth
was greater, or that WP calves may have retained more
energy in the form of adipose tissue.
Although ME intakes were the same in all treatment
groups throughout the experimental period, growth
and body dimensions were affected by the treatments.
Growth was greater in HL and HP than in WP calves,
whereas HF did not differ from other groups. At d 84
after arrival, the difference between HL and WP was as
large as 10 kg BW. Although BW is a good indicator
of animal performance and health, it does not provide
insight into body composition, which can be affected by
large differences in dietary fat, lactose, and protein in-
takes. Calves fed HL and HP had higher withers height,
hip height, and chest girth than WP calves. In addi-
tion, chest girth and body barrel were greater in calves
fed HL than calves fed the HF MR. Calves fed HF also
had greater withers height, hip height, and chest girth
Journal of Dairy Science Vol. 105 No. 8, 2022
6687
than WP calves. Thus, similar ME intakes but different
type of liquid feed resulted in taller calves in the MR
groups. Measuring BCS and body mass index could
provide additional information in future experiments.
Stimulation of the somatotropic axis depends on
nutrient availability, and increased protein and energy
intakes from elevated MR feedings may influence blood
and tissue levels of IGF-I in dairy calves (Schäff et
al., 2016; Frieten et al., 2017; Ghaffari et al., 2021).
The results of the current study confirmed that, across
treatment groups, serum IGF-1 was positively corre-
lated with ME intake and ADG. Serum IGF-1 concen-
trations were higher in HL and HP than in WP calves,
and tended to be greater in HP than in HF calves.
These results are consistent with Bartlett et al. (2006)
showing that high circulating IGF-I was mostly driven
by high dietary protein intake rather than ME intake
in preweaning calves. Infant nutrition research showed
that early protein intake (<4 mo) in excess of metabolic
requirements leads to increased weight gain in infancy
(Koletzko et al., 2005). This has been attributed to
an increase in insulin-releasing AA in the bloodstream,
which stimulate the secretion of insulin and IGF-1,
both factors involved in the mammalian growth sig-
naling network (Rolland-Cachera et al., 1984; Rolland-
Cachera and Peneau, 2010). These factors have been
linked to the regulation of early growth, adipose tissue
differentiation, and early adipogenesis (Smith et al.,
1988; Nouguès et al., 1993; Ketelslegers et al., 1995). In
contrast, slower growth in infancy, in conjunction with
optimal daily protein intake, reduces the incidence of
cardiovascular disease and its risk factors in adulthood
(Leunissen et al., 2009; Socha et al., 2011; Oddy et
al., 2014). Although the concept of early, rapid growth
may be attractive in rearing systems where animals are
raised for meat production; it can be hypothesized that
this may negatively affect the metabolic homeostasis of
replacement dairy animals and long-term effects should
be evaluated in future experiments.
In addition to high protein and lactose intakes, feed-
ing low levels of fat (≤18% DM) to young calves may
be detrimental to health (Urie et al., 2018; Berends
et al., 2020) and development (Welboren et al., 2021).
The current study found no beneficial effect of higher
fat content in MR on the percentage of calves treated
for diarrhea and respiratory diseases, nor on fecal scores
during the first 2 wk of life. These results are not con-
sistent with those of Berends et al. (2020), in which ad
libitum-fed calves fed a high-fat MR (23%, DM basis)
received fewer therapeutic interventions than calves
fed a high lactose MR (44%). In the current study,
serum NEFA concentrations were greater in HF calves
than in the other treatment groups. Previous authors
investigating the replacement of lactose with fat in MR
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
attributed the greater serum NEFA concentrations
in HF calves to the higher fat intake (Berends et al.,
2020; Yohe et al., 2021; Echeverry-Munera et al., 2021).
However, in the present study, serum NEFA concentra-
tions were also greater in HF calves than in WP calves
despite similar fat intakes. In contrast, no differences
in serum concentration of triglycerides were observed.
Acute phase protein was also evaluated to provide
additional insights on calf health. The results showed
that serum concentrations of SAA were greater in HF
than in WP and HL calves. Serum amyloid A is a posi-
tive acute phase protein synthesized predominantly by
the liver (Eklund et al., 2012) and increases in response
to inflammation. In contrast, serum albumin is a nega-
tive acute phase protein whose concentration decreases
in case of inflammation. Albumin was 3% lower in
calves fed HF and HL than WP, however, the differ-
ence appeared marginal as some authors evaluated that
the difference should be at least 25% to be indicative
of an acute phase response (Ackermann, 2017). The
presence of metabolic inflammation in HF-fed calves, as
shown by the 39% greater serum SAA, may be related
to the fat composition of MR, which is a mixture of
palm and coconut oils and has an n-6 to n-3 polyun-
saturated fatty acid ratio of 40:1 compared with 6:1 in
WP. Consequently, for the same fat intake, HF calves
consumed larger amounts of n-6 PUFA relative to n-3
PUFA than WP. The total intake of n-6 PUFA was
also greater in HF than HL and HP because calves
consumed greater amounts of fat. As a result, HF
calves were provided with a larger pool of precursors
for arachidonic acid (ARA) than for eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA; Noble
et al., 1978; Sardesai, 1992). Whereas EPA and DHA
are precursors of anti-inflammatory eicosanoids, ARA
is the precursor of pro-inflammatory eicosanoids, which
may explain the higher SAA in HF calves (Noble et al.,
1978; Sardesai, 1992; Calder, 2010). However, a higher
serum SAA concentration does not necessarily mean
that HF calves were in a state of systemic inflamma-
tion. Indeed, recent studies have shown that occurrence
of metabolic disturbances associated with high serum
SAA concentrations in transgenic mice fed a high-fat
diet only occurred with prolonged consumption of 52
wk (Jang et al., 2016), which may not be relevant in
calves fed HF MR for 10 wk. In addition, serum SAA
concentrations remained within normal ranges for
calves (Joshi et al., 2018; van Niekerk et al., 2021b).
Adiponectin and leptin are both adipokines that
regulate lipid and glucose metabolism and insulin sen-
sitivity (Kadowaki et al., 2006). In the current study,
adiponectin concentration was not affected by dietary
treatment. There was an interaction between treatment
and time for serum leptin concentrations, which prob-
Journal of Dairy Science Vol. 105 No. 8, 2022
6688
ably reflected different dynamics at different rearing
stages. Serum leptin concentration increased from d 14
to 28 in calves fed HL, HF, and HP, corresponding
to the ad libitum feeding phase. Later, a decrease was
observed in the weaning phase (d 56) before increasing
again in the postweaning phase (d 84). The postwean-
ing increase in leptin concentration was more marked
in HF-fed calves. In contrast, leptin concentrations in
WP-fed calves peaked on d 21, followed by a decline
from d 21 to 56, and an increase between d 56 and
84. Experimentally increasing circulating leptin con-
centrations resulted in lower feed intakes in rodents
and sheep (Houseknecht et al., 1998; Ahima and Flier,
2000; Morrison et al., 2001). In contrast low leptin in
underfed animals can stimulate feed intakes and modu-
late metabolic responses (Ahima et al., 1996; Friedman
and Halaas 1998; Heiman et al., 1998). In calves, Lee
et al. (2006) showed that leptin concentrations reflect
changes in nutritional status in calves of 5 mo, whereas
leptin decreases with feed restriction, it increases with
refeeding. This could mean that lower leptin concentra-
tions are coupled with hunger, which is consistent with
the lower concentrations at weaning (d 56) compared
with the ad libitum phase (d 28) in the current study.
Therefore, the higher serum leptin concentrations in
HF calves during weaning (d 56) and after weaning (d
84) could indicate increased satiety in this group.
In addition to the differences in hormones and mark-
ers of inflammations, other blood metabolites were
affected by the dietary treatments. Serum urea concen-
trations were elevated in WP and HP calves compared
with HF and HL throughout the experimental phase.
Urea is generated when amino acids are broken down
in the body and is affected by the diet. In this regard,
the greater serum urea in HP calves is likely the result
of higher protein intakes in that group. The higher
urea concentrations in the WP group may be related
to protein denaturation of the WP treatment, which
can affect the tertiary protein structure and thus, their
biological value and activity or increased protein break-
down.
Milk replacer-fed calves had increased concentrations
of serum AST compared with WP calves (45 vs. 39 IU/L,
respectively). Aspartate aminotransferase is used as an
indicator of liver function (Gorman, 2011). In adults,
elevated AST activities (>40 IU/L) can be an indicator
of renal or pancreatic inflammation, however, MR-fed
calves were in good general health. This may not be re-
lated to a specific macronutrient composition, as there
was no difference across MR treatments and therefore
may be linked with other nutrients such as fatty acids
or minerals. Similarly, serum bilirubin concentrations
were higher in WP-fed calves than in HL and HP at
d 28, and higher than in HF and HP calves at d 56
 Wilms et al.: MACRONUTRIENTS IN MILK REPLACER
after arrival. Fat intake did not appear to affect serum
bilirubin, as HF and WP had similar fat intakes but
different bilirubin dynamics. Tennant (1997) described
that greater bilirubin concentrations can be consequent
to bilirubin clearance reduction, which is a usual meta-
bolic function of the liver. Thus, these results may in-
dicate that WP calves did not eliminate bilirubin from
the blood for bile acid production to the same extent as
MR-fed calves. Results also showed that calves fed HL
had lower serum BHB concentrations than the other
treatment groups. This could be related to the lower
fat intake in this group, as BHB is produced by the ru-
men wall and synthesized in the liver from dietary fatty
acids (Newman and Verdin, 2017). However, this is not
consistent with the fact that HP calves also had higher
BHB concentrations than HL calves, despite having the
lowest fat intake. Earlier research showed that increas-
ing serum BHB concentrations is a good indicator of
rumen development, and thus, of a shift in nutrient
source from liquid to solid feed (Coverdale et al., 2004).
Nevertheless, it is unlikely that the slightly lower serum
BHB concentrations in HL calves translated to slower
rumen development, as uptake of solid feeds was similar
across treatment groups. Calves fed the WP treatment
also had lower blood hematocrit levels during the first
4 wk of life compared with other groups. Hematocrit of
WP calves (22%) were lower than the normal range for
healthy male calves between 11 to 30 d of age (23–40%;
Dillane et al., 2018), although normal ranges for calves
fed bovine WM has not been characterized. This could
be related to the substantially greater iron levels in
MR treatments (83 mg/kg) than in bovine WM (~3
mg/kg), which led to a greater iron intake in MR-fed
calves. Indeed, previous research showed the link be-
tween dietary iron supplementation and blood hemato-
crit (Brigety and Pearson, 1970). The greater supply of
iron in MR-fed calves likely promotes the formation of
hemoglobin and thus of erythrocytes, ultimately lead-
ing to increased blood hematocrit in MR-fed calves.
An important finding of this study was that despite
an optimal nutrient profile in terms of macronutri-
ent composition and fatty acids, the dehydrated WM
powder was detrimental to calf health during the first
3 wk of life. This could also explain the lower serum
IGF-1 concentrations and slower growth in this group.
In contrast, all 3 formulations of MR appeared to
better maintain gastrointestinal health and acid–base
balance during the first 4 wk after arrival, regardless
of macronutrient profile. Therefore, the current exper-
iment missed the intended positive control of calves
fed WM, which would be considered the biological
reference for growth and development. Further work
is needed to evaluate the potential negative effects of
excessive protein and lactose intakes in early life on
Journal of Dairy Science Vol. 105 No. 8, 2022
6689
productive life span and future performance of dairy
cows.
CONCLUSIONS
Calves fed the HL and HP diets, having a lower
energy density, had greater MR intakes than WP-fed
calves in the preweaning phase. Despite the same total
ME intake, WP-fed calves were lighter and smaller
than HL and HP calves. Although limited growth dif-
ferences were present between HF and other treatment
groups throughout the experimental period, the meta-
bolic profile largely differed. Feeding HF MR did not
result in high serum IGF-I concentration, as compared
with HL and HP MR. Calves fed the WP treatment
had an increased number of therapeutic interventions
related to diarrhea, as well as indications of increased
diarrhea severity. The dehydrated WM powder used in
this experiment may therefore not be representative of
fresh WM due to the heat treatment during process-
ing, which resulted in protein denaturation. Calves fed
MR with high fat presented increased NEFA and SAA
concentrations. Whether the observed differences are
biologically relevant for future dairy cattle health and
production requires further investigation.
ACKNOWLEDGMENTS
This study was funded by Trouw Nutrition (Amers-
foort, the Netherlands). The authors thank Natasja
Boots, Hanneke de Cock, and the personnel of the Calf
Research Facility of Trouw Nutrition for their techni-
cal assistance. Several of the authors (J. N. Wilms, J.
Martín-Tereso, and L. N. Leal) are employed by Trouw
Nutrition, a company with commercial interests in milk
replacers for calves. Trouw Nutrition Research and De-
velopment adheres to the principles of the European
Code of Conduct for Research Integrity (Drenth et al.,
2011). The authors have not stated any other conflicts
of interest.
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