J. Dairy Sci.
104
https://doi.org/10.3168/jds.2021-20483
© 2021 American Dairy Science Association®. Published by Elsevier Inc. and Fass Inc. All rights reserved.
Effects of maternal level of dietary cation-anion difference fed to prepartum
nulliparous cows on offspring acid-base balance, metabolism, and growth
R. Zimpel,1,2 M. Nehme Marinho,1 K. V. Almeida,1 A. Revilla Ruiz,1 C. D. Nelson,1 W. W. Thatcher,1,2
and J. E. P. Santos1,2*
1
Department of Animal Sciences, University of Florida, Gainesville, 32611
2
DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, 32611
ABSTRACT
The objectives were to determine the effects of dietary
cation-anion difference (DCAD) fed to pregnant cows
during the last 22 d of gestation on offspring acid-base
balance, metabolism, growth, and health preweaning.
A total of 132 nulliparous Holstein cows were enrolled
at 250 (248 to 253) d of gestation in a randomized
block design. Cows were blocked by genomic merit of
energy-corrected milk yield and assigned randomly to
diets varying in DCAD: +200 (P200, n = 43), −50
(N50, n = 45), or −150 (N150, n = 44) mEq/kg of dry
matter (DM). Newborn calves (15 males and 28 females
in P200, 22 males and 23 females in N50, and 18 males
and 26 females in N150) were followed for the first 7 or
56 d of age if males or females, respectively. Measures
of acid-base balance and concentrations of minerals in
blood were measured in all calves on d 0 before colos-
trum feeding, and on d 1, 3, and 7. Each calf was fed
3.78 L of colostrum from the respective treatment, and
apparent efficiency of IgG absorption was determined.
All calves were weighed at birth, and females were
weighed again at 21, 42, and 56 d of age. Concentra-
tions in serum of total calcium (tCa), total magnesium
(tMg), and total phosphorus (tP) were measured up
to 56 d of age; intakes of milk and starter grain DM
were measured daily from 21 to 56 d of age; and inci-
dence of disease was recorded for the first 56 d of age
in females. Treatment did not affect acid-base balance
measured in all calves. Calves were born with metabolic
and respiratory acidosis, which reversed by 1 d of age.
In the first 24 h after birth, blood pH increased from
7.215 to 7.421 and bicarbonate from 26.2 to 31.7 mM,
whereas partial pressure of CO2 decreased from 64.1 to
48.7 mm of Hg in all treatments. Maternal DCAD did
not affect colostrum IgG content fed to calves (P200
= 95.0 vs. N50 = 91.0 vs. N150 = 97.1 ± 4.1 g/L) or
Received March 18, 2021.
Accepted April 7, 2021.
*Corresponding author: jepsantos@​ufl​.edu
apparent efficiency of IgG absorption (P200 = 33.1 vs.
N50 = 33.1 vs. N150 = 34.2 ± 1.9%). Males were born
heavier than females, but maternal DCAD did not af-
fect birth weight of all calves (P200 = 37.7 vs. N50 =
37.3 vs. N150 = 37.8 ± 0.7 kg) or daily weight gain in
females in the first 56 d of life (P200 = 0.80 vs. N50 =
0.81 vs. N150 = 0.77 ± 0.03 kg/d). Treatment did not
affect intake of milk (P200 = 1.11 vs. N50 = 1.04 vs.
N150 = 1.19 ± 0.06 kg/d) or starter grain DM (P200
= 0.27 vs. N50 = 0.27 vs. N150 = 0.21 ± 0.06 kg/d),
or measures of feed efficiency. Treatment did not af-
fect concentrations of minerals in serum, morbidity, or
age at morbidity. Manipulating the DCAD of pregnant
nulliparous dams during late gestation did not affect
offspring performance in the first 2 mo of age.
Key words: acid-base balance, calf, DCAD,
immunoglobulin
INTRODUCTION
Prepartum dairy cows are often fed acidogenic diets
to reduce the risk of hypocalcemia (NAHMS, 2014).
The reduction in hypocalcemia is mediated by changes
in acid-base balance that increase concentrations of
ionized (iCa) and total (tCa) calcium in the first days
postpartum (Block, 1984; Charbonneau et al., 2006;
Rodney et al., 2018), which is linked with improved
postpartum productive performance and health (Block,
1984; Santos et al., 2019). The mechanisms by which
acidogenic diets improve cows’ mineral metabolism
involve changes in acid-base balance, resulting in meta-
bolic acidosis with reduced blood pH, HCO3−, and base
excess that is followed by compensatory renal and respi-
ratory mechanisms (Rodney et al., 2018). Irrespective
of dam diets, calves are born with respiratory acidosis,
typically observed in the first hours after birth, because
delivery can result in hypoxia with oxygen deprivation
and hypercapnia (Massip, 1980). Disturbances in utero-
placental circulation caused by uterine contractions or
immaturity of the lungs for proper gas exchange can
exacerbate acidosis in newborns (Massip, 1980). The
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
resulting hypercapnia causes a transient acidemia, typi-
cally compensated in the first few hours after birth to
restore proper acid-base balance (Bleul et al., 2007;
Vannucchi et al., 2015).
It is well documented that the maternal environment
can affect the phenotype of the offspring (Rando and
Simmons, 2015). In most species, metabolic acidosis is
an abnormal event, and it is usually associated with
pre-existing diseases. In dairy cattle, diet-induced met-
abolic acidosis in late gestation might affect calves in
early life. Dudenhausen and Milz (2007) demonstrated
increased neonatal morbidity when the pH of blood col-
lected from the uterine artery dropped below 7.20 in
women. Limited research, however, has been conducted
to understand the effects of metabolic acidosis in the
dam during late gestation on the postnatal physiology
and growth of the calf. Tucker et al. (1992) fed nullipa-
rous and parous cows prepartum diets containing +93
or −34 mEq/kg of DM during the last 28 d of gestation.
Those authors observed that prepartum DCAD did not
affect the calves’ blood concentration of HCO3− or par-
tial pressure of CO2 (pCO2) on the day of calving and
in the first 4 wk following calving. Morrill et al. (2010)
fed diets to prepartum cows with either +77 or −100
mEq/kg of DM and did not observe changes in effi-
ciency of IgG absorption by newborn calves. In general,
colostrum yield, nutrient content, and IgG concentra-
tion are not affected by prepartum DCAD (Lopera et
al., 2018; Martinez et al., 2018). Although experiments
have shown that acidogenic diets fed to prepartum
cows do not alter acid-base status or plasma mineral
concentrations in newborn calves, the effects of those
diets on preweaning intake, growth, and health have
not been thoroughly investigated. Understanding the
influence of acidogenic diets fed to dams on offspring
performance is important for proper implementation of
programs for prevention of hypocalcemia that have no
detrimental effect on calf performance.
The hypothesis of the present experiment was that
manipulating the DCAD of diets fed to late-gestating
dairy cows would not affect offspring acid-base balance,
mineral and energy metabolism, growth, and health.
Therefore, objectives of the experiment were to deter-
mine the effects of 3 levels of maternal dietary levels
of DCAD during prepartum on calf acid-base balance,
mineral and energy metabolism, growth, and health.
MATERIALS AND METHODS
The University of Florida Institutional Animal Care
and Use Committee approved all procedures involv-
ing animals in the experiment under protocol number
201810505. The experiment was conducted at the
Journal of Dairy Science Vol. 104 No. 8, 2021
University of Florida dairy and calf unit, Gainesville,
between November 2018 and October 2019.
Sample Size Calculation
Sample size was calculated using the POWER pro-
cedure of SAS (SAS/STAT version 9.4; SAS Institute
Inc.) for responses of dams reported in Zimpel et al.
(2021a,b). The calculation was based on results from
Santos et al. (2019) that reducing DCAD from +200 to
−150 mEq/kg was expected to reduce yield of ECM by
approximately 2.5 kg/d. The standard deviation (SD)
for ECM yield in primiparous cows in the first 105 d
of lactation used was 3.5 kg, based on records from the
University of Florida dairy herd. A 2-sided sample size
calculation was performed. Because 3 treatments were
implemented, the sample size was calculated adjusting
α to 0.017 (0.05/3) to allow for multiple comparisons
adjustment using the Bonferroni method. Therefore,
the sample size calculated with α = 0.017 and β =
0.20 resulted in a minimum of 42 cows per treatment.
It was anticipated that 60% of newborn calves would
be females, because of use of sexed semen when dams
received their first AI. Thus, it was anticipated that 75
to 80 newborn heifers would be available for the experi-
ment for measurements after d 7, because male calves
would be sold by then. Given the observed daily BW
gain in the first 8 wk of life in females at the University
of Florida Dairy Unit of 0.80 kg/d and the expected
SD for daily BW gain in the preweaning period of 0.20
kg, the sample size of 25 calves per treatment would
allow a detection of 0.18 kg/d difference in daily BW
change among treatments (α = 0.017 and β = 0.20).
Because treatments were arranged such that orthogonal
polynomials would be used, the expected power for the
analyses for the difference in daily BW changes would
be greater than 80%, or a smaller difference would be
needed among treatments to reject the null hypothesis.
Cows, Experimental Design, and Maternal
Dietary Treatments
A total of 132 apparently healthy nulliparous Holstein
cows pregnant to timed AI were assigned to the experi-
ment. All dams used in the experiment were genotyped
in the first 4 mo of age. Weekly, nulliparous cows were
moved to the experimental barn at 243 d of gestation
(ranged from 241 to 246 d) to acclimate to the facilities.
Cows were assigned an individual feeding gate (Calan
Broadbent feeding system, American Calan Inc.) and
trained to use the gate for the first 7 d.
The experiment followed a randomized complete
block design. Cohorts of 3 to 13 nulliparous cows were
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
ranked by genomic merit for yield of ECM, and each
3 cows were assigned a block. Within each block, cows
were assigned randomly to 1 of 3 dietary treatments
that were fed as TMR that varied in DCAD. Treat-
ments were as follows: +200 (P200; n = 43), −50 (N50,
n = 45), or −150 (N150, n = 44) mEq/kg of DM. Diets
were formulated to have the same forage-to-concentrate
ratio and have similar contents of NEL, MP, and miner-
als and vitamins, except for the strong anions that were
manipulated by incorporating different amounts of an
acidogenic product to achieve the desired DCAD (Table
1). Treatments were applied to dams starting at 250 d
of gestation until calving, which resulted in a mean of
22 d in the prepartum treatments (Supplemental Table
S1; https:​/​/​figshare​.com/​s/​b412f7b6661e5c3c5cd0).
Additional details of the management of cows prepar-
tum are presented in Zimpel et al. (2021a,b). Authors
were not blind to treatments.
Calving and Calf Management
Dams were observed continuously prepartum and
calved in the experimental barn. Calves were separated
from their dams within the first 30 min of birth. All
cows delivered singleton calves, and 2 calves in P200 (1
female and 1 male), 2 in N50 (2 males), and 2 in N150
(2 females) were stillbirths. All calves were weighed
immediately after birth, including stillbirths, and live
calves had their navels dipped into a 7% iodine solution.
Upon calving, the cow was moved to the parlor and
milked with a mean (±SD) of 124 ± 121 min. Colostrum
yield was measured and a sample collected and frozen
at −20°C for later analyses. Calves were fed 3.78 L
of frozen-thawed colostrum from their respective treat-
ments by esophageal tubing. Briefly, the first 2 calves
born in each treatment were not evaluated for passive
transfer because they were not fed colostrum from cows
in the experiment. The colostrum harvested from those
dams was frozen for subsequent feeding of other calves
in the same treatment. The number of calves born alive
were 14 males and 27 females in P200, 20 males and 23
females in N50, and 18 males and 24 females in N150.
Male calves were followed for the first 7 d, after which
they were sold. Female calves were followed for the first
56 d of life.
All calves were housed in individual sand bedded
hutches for the first 21 d of life, a period in which
they were offered 6 L/d of pasteurized hospital milk
fed using buckets. Milk was split into 2 equal meals of
3 L offered at 0600 and 1400 h. Calves had ad libitum
access to water and starter grain containing 20% CP
and 2% of fat (Ampli-Calf Starter 20, Purina Animal
Nutrition LLC). Intakes of milk or starter grain DM
were not measured in the first 21 d.
Journal of Dairy Science Vol. 104 No. 8, 2021
Starting at 22 d of age, female calves were group-
housed in pens with calves of similar age (±3 d), and
pens were equipped with an automatic feeding system
(DeLaval CF1000X). Calves were trained in the first
days to consume milk replacer and concentrate via the
automatic feeders until the calf accessed the feeder
without any assistance. Calves were fed milk replacer
containing 28% CP and 15% fat (UF Special 28/15 Bova
DFB Medicated, Southeast Milk), which was mixed at
150 g/L. From 22 to 56 d of age, calves had ad libitum
access to starter grain that was offered using an auto-
matic feeding system. From 22 to 53 d of age, calves
had ad libitum access to milk replacer offered using an
automatic feeding system. Starting at 54 d of age, the
allowance of milk replacer was gradually decreased over
the course of 10 d to wean calves. Data on intake of DM
from milk and starter grain was evaluated until 56 d
of age. The chemical compositions of the milk replacer
and starter grain are depicted in Supplemental Table
S2 (https:​/​/​figshare​.com/​s/​b412f7b6661e5c3c5cd0).
Water was supplied ad libitum, although intake was
not recorded. Samples of milk replacer and calf starter
grain were collected twice a week and analyzed for their
DM contents (105°C for 24 h) to calculate DM intake.
The automatic feeding system recorded the daily milk
replacer and starter grain consumed by individual
calves (Forster Technik Software).
Female calves were weighed at birth, before colos-
trum feeding, and on d 21, 42, and 56 of age, using
a calibrated electronic scale. The intakes of DM from
milk replacer and starter grain, and the daily BW gain
in the respective weighing intervals were calculated.
The feed conversion ratio (kg of BW gain/kg of DMI)
was calculated for the period between 21 and 56 d of
age.
Calves were observed daily for incidence of diseases
for the first 56 d of age, and those identified as po-
tentially sick underwent a physical examination by
veterinarians from the University of Florida. Health
treatments and the vaccination program in the first
56 d of age were standardized and applied to all calves
in the experiment according to the standard operating
procedures established by the attending veterinarians.
Analysis of IgG in Colostrum and Serum
and Efficiency of IgG Absorption
Blood was sampled on d 1 from 41 calves in P200
(27 females and 14 males), 43 calves in N50 (23 females
and 20 males), and 42 calves in N150 (24 females and
18 males) by puncture of the jugular vein into serum
separator evacuated tubes (Vacutainer System, Becton
Dickinson Co.). Blood was allowed to clot for approxi-
mately 20 min, and tubes were then placed in ice and
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

transported to the laboratory within 1 h of collection per manufacturer’s protocol (https:​/​/​kentlabs​.com/​jjj/​

for serum separation and storage for subsequent analy-
ses of IgG concentration.
Concentrations of IgG in colostrum fed to each calf
and serum of each calf were quantified by radial immu-
nodiffusion assay (Triple J Farms, Bellingham, WA),
triple​-j​-farms​-product​-information/​). Briefly, colostrum
was diluted 1:6 in 0.9% saline, and serum was diluted
1:1 in 0.9% saline, such that the concentrations of IgG
would fall within the linear range of the standard curve
of the assay. The diluted samples were pipetted into the

Table 1. Ingredient composition and nutrient content of dietary treatments fed to dams

Treatment1

Ingredient, % of diet DM P200 N50 N150

Corn silage 57.3 57.3 57.3
Bermuda hay 12.2 12.2 12.2
Grain mix 30.5 30.5 30.5
Composition of grain mixes, % mix DM      
  Whole cottonseed 22.8 22.8 22.8
  Citrus pulp, dry 17.4 21.4 21.7
  Soybean meal 47%, solvent extract 9.4 17.1 15.1
  Expeller soybean meal2 31.3 5.7 —
  Rumen-protected choline3 1.7 1.7 1.7
  Mycotoxin binder4 1.7 1.7 1.7
  Sodium bicarbonate 2.0 — —
  Magnesium oxide 0.9 0.3 —
  Acidogenic product5 — 16.5 24.2
  Mineral and vitamin6 12.8 12.8 12.8
Nutrient content, DM basis7 (±SD)      
  DM, % 58.4 ± 5.5 58.2 ± 5.9 58.5 ± 5.7
 NEL,8 Mcal/kg 1.52 1.53 1.54
  OM, % 93.6 ± 0.4 93.3 ± 0.6 93.6 ± 0.6
  CP, % 14.8 ± 0.5 15.3 ± 0.7 15.1 ± 0.7
 MP,8 % 10.0 9.8 9.8
  NDF, % 45.2 ± 2.5 44.1 ± 3.0 44.5 ± 2.4
  Forage NDF, % 38.2 ± 2.6 38.2 ± 2.6 38.2 ± 2.6
  ADF, % 24.9 ± 1.4 24.5 ± 1.7 24.8 ± 1.2
  Fatty acids, % 2.8 ± 0.1 3.2 ± 0.1 3.1 ± 0.2
 NFC,9 % 30.8 ± 2.7 30.7 ± 2.7 30.9 ± 2.5
  Ca, % 0.68 ± 0.07 0.63 ± 0.12 0.74 ± 0.09
 P, % 0.36 ± 0.02 0.36 ± 0.02 0.38 ± 0.02
  Mg, % 0.47 ± 0.03 0.45 ± 0.03 0.40 ± 0.03
  K, % 1.32 ± 0.04 1.24 ± 0.06 1.22 ± 0.06
  Na, % 0.21 ± 0.01 0.10 ± 0.01 0.14 ± 0.01
  Cl, % 0.34 ± 0.06 0.71 ± 0.08 0.93 ± 0.07
  S, % 0.18 ± 0.01 0.34 ± 0.01 0.41 ± 0.01
 DCAD,10 mEq/kg 224 ± 12 −46 ± 22 −148 ± 11
1
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, randomly
assigned to receive 1 of 3 prepartum diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/
kg of DM from 250 d of gestation to calving.
2
SoyPlus (Landus Cooperative).
3
ReaShure (28.8% choline chloride, Balchem Corp.).
4
Novasil Plus (calcium montmorillonite clay, BASF Corp.).
5
Bio-Chlor (fermentation product containing dried condensed extracted glutamic acid fermentation product,
dried condensed corn fermentation solubles, processed grain by-products, and magnesium chloride; Arm and
Hammer Animal Nutrition).
6
Contents: 76.0% wheat middlings; 17.1% calcium carbonate; 4.3% magnesium oxide; 0.92% Avalia4 (Zinpro
Corp.); 0.66% dry vitamin E 50%; 0.34% Rumensin 90 (Elanco Animal Health); 0.32% Sel-Plex 3000 (Alltech);
0.22% vitamin and trace minerals (Furst-McNess); 0.05% manganese sulfate; 0.04% vitamin A; 0.02% ethylene-
diamine dihydroiodide. Each kilogram contained 13.6% CP, 7.38% Ca, 0.73% P, 3.02% Mg, 0.84% K, 0.14% Na,
0.03% Cl, 0.01% S, 515 mg of Zn, 180 mg of Cu, 461 mg of Mn, 10 mg of Se, 18 mg of Co, 12 mg of I, 248,500
IU of vitamin A, 72,000 IU of vitamin D, 3,570 IU of vitamin E, and 730 mg of monensin.
7
Means of 4 composite samples analyzed.
8
Calculated using NRC (2001) according to the chemical composition of the dietary ingredients and adjusted
for 10 kg of DMI prepartum.
9
Calculated as follows: NFC = DM − (ash + CP + ether extract + NDF).
10
Calculated as follows: DCAD = [(mEq of K+) + (mEq of Na+)] − [(mEq of Cl−) + (mEq of S2−)].

Journal of Dairy Science Vol. 104 No. 8, 2021

 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
bovine anti-bovine IgG antibody plate and incubated
for 24 h on a flat surface protected from light. The
diameter of the precipitin ring was measured using a
7× scale loupe (no. 1975, Peak Optics, GWJ Company)
and used to calculate IgG concentrations. The CV for
colostrum and serum assays were, respectively, 6.3 and
7.3%.
The apparent efficiency of IgG absorption from co-
lostrum was quantified based on the BW of calves at
birth, the amount of colostrum fed and the respective
IgG concentration in colostrum, and the concentration
of IgG in serum of calves on d 1, according to Quigley
et al. (1998).
Blood Sampling, Processing, and Assays
Blood was sampled by puncture of the jugular vein
into 10-mL evacuated lithium-heparinized tubes or se-
rum separator tubes (Vacutainer System, Becton Dick-
inson Co.) on d 0, immediately after birth and before
colostrum feeding, and between 1500 and 1530 h on d
1, 2, 3, 7, 21, 42, and 56 of age. Blood sampled into
serum separator tubes was allowed to clot for 20 min,
and then tubes were placed in ice and transported to
the laboratory within 1 h and centrifuged for 20 min at
2,000 × g at room temperature for serum separation.
Serum samples were transferred into cryovials with
multiple aliquots of 1.5 mL and stored frozen at −20°C
until analyses.
Whole venous blood sampled on d 0, 1, 3, and 7 from
34 calves in P200 (11 males and 23 females), 33 calves
in N50 (16 males and 17 females), and 34 calves in
N150 (16 males and 18 females) was analyzed within
1 min of sampling, using a hand-held biochemical ana-
lyzer (VetScan i-STAT, Abaxis). Analytes quantified
included blood gases: pCO2 (as mm of Hg, mmHg),
partial pressure of O2 (pO2), total dissolved CO2, and
saturation of O2; minerals: iCa, ionized sodium (iNa),
and ionized potassium; and concentrations of HCO3−,
base excess, hematocrit, and hemoglobin.
Serum harvested on d 0, 1, 2, 3, 7, 21, 42, and 56
was assayed for concentrations of tCa, total magne-
sium (tMg), total phosphorus (tP), and IgG, and
Brix value. Serum sampled on d 0, 7, 21, 42, and 56
was assayed for fatty acids. Concentrations of tCa and
tMg were assayed using a spectrophotometer equipped
with calcium- and magnesium-specific hollow cathode
lamps (AAnalyst 200, PerkinElmer Inc.), as described
by Martinez et al. (2012). Intra- and interassay coef-
ficients of variation (CV) were, respectively, 4.1 and
4.9% for tCa and 3.2 and 7.3% for tMg. Concentrations
of tP were assayed according to Quinlan and DeSesa
(1955). The intra- and interassay CV were 4.4 and
5.1%, respectively. The Brix value was quantified us-
Journal of Dairy Science Vol. 104 No. 8, 2021
ing a digital refractometer (Milwaukee Instruments).
The intra-assay CV was 0.5%. Concentrations of IgG
were assayed concurrently, with samples collected on d
1 as described previously. Concentrations of FA were
quantified using an enzymatic assay (NEFA-C Kit;
Wako Diagnostics Inc.) according to Johnson and Pe-
ters (1993). The intra- and interassay CV were 4.6 and
4.7%, respectively.
Statistical Analyses
Normality of residuals and homogeneity of variance
were examined for each continuous dependent variable
analyzed after fitting the statistical models. Responses
that violated the assumptions of normality were sub-
jected to power transformation according to the Box-
Cox procedure (Box and Cox, 1964) using a macro for
mixed models in SAS (Piepho, 2009). The least squares
means (LSM) and standard error of the mean (SEM)
were back transformed for presentation of results ac-
cording to Jørgensen and Pedersen (1998). Analyses of
starter grain DM intake, blood pO2, and serum fatty
acids were transformed to the natural logarithm before
analyses to correct for heteroscedasticity.
Continuous data were analyzed by ANOVA with
linear mixed-effects models using the MIXED proce-
dure of SAS (SAS version 9.4, SAS/STAT). Responses
with a single measurement per calf were analyzed with
models that included the fixed effect of dam treatment
(P200 vs. N50 vs. N150) and the random effect of
block. Responses with repeated measures within ex-
perimental unit were analyzed with statistical models
that included the fixed effects of dam treatment, age
of calf at measurement, and interaction between treat-
ment and age, and the random effects of block and calf
nested within dam treatment. Age was the term in the
REPEATED statement, and the covariance structure
was selected based on spacing of measurements and
model fit assessed based on the smallest Akaike’s in-
formation criterion. For analyses with unequal spacing,
the spatial power was the covariance structure used.
For responses analyzed with equal spacing between
measurements, the autoregressive 1 and the autore-
gressive heterogeneous 1 were the selected covariance
structures. Models for responses measured in male and
female calves also included the fixed effects of calf sex
and interaction between dam treatment and calf sex.
When an interaction between dam treatment and age
of calf or dam treatment and calf sex resulted in P ≤
0.10, means among treatments at different time points
or within male or female calves were partitioned using
the SLICE command of SAS (SAS/STAT).
Binary data were analyzed by generalized linear
mixed-effects models using logistic regression with the
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
GLIMMIX procedure of SAS (SAS/STAT). The statis-
tical models included the fixed effects of dam treatment
and the dam’s genomic PTA for calf diseases (PTA for
calf scours used for analysis of diarrhea; PTA for calf
respiratory disease used for analysis of respiratory dis-
ease; and calf livability used for analysis of morbidity or
multiple diseases), and the random effect of block. The
LSM and respective SEM for the dam’s genomic PTA
for calf diseases are depicted in Supplemental Table S1
(https:​/​/​figshare​.com/​s/​b412f7b6661e5c3c5cd0).
In all mixed-effects models, the Kenward-Roger
method was used to approximate the denominator
degrees of freedom for the F tests. Orthogonal poly-
nomial contrasts were used to determine linear and
quadratic effects of dam treatments on the responses
analyzed. The interactive matrix language procedure
of SAS (PROC IML, SAS/STAT) was used to gener-
ate the orthogonal polynomial coefficients to adjust for
the unequal spacing of DCAD in dam treatments. The
resulting coefficients for P200, N50, and N150 were,
respectively, 0.787, −0.206, and −0.581 for the linear
contrast, and 0.217, −0.790, and 0.573 for the quadratic
contrast.
Days to morbidity were analyzed with the Cox’s
proportional hazard regression using the PHREG pro-
cedure of SAS (SAS/STAT). The model included the
fixed effects of dam treatment and the dam’s genomic
PTA for calf livability. The adjusted hazard ratios and
the 95% confidence intervals were calculated. The ad-
justed survival curves were generated from the Cox’s
model using the BASELINE and the COVARIATE
statements to adjust for the covariates in the model.
The LIFETEST procedure of SAS (SAS/STAT) was
used to compute the mean and median days to morbid-
ity.
Additional models for analyses of apparent effi-
ciency of absorption of IgG were built to investigate
the association between blood acid-base balance im-
mediately before colostrum feeding on d 0 and IgG
absorption evaluated on d 1. Data were analyzed with
the MIXED procedure of SAS (SAS/STAT), and the
model included the fixed effects of dam treatment, calf
sex, the interaction between dam treatment and calf
sex, blood acid-base measure (pH, pCO2, HCO3−, or
base excess), the interaction between dam treatment
and blood acid-base measure, the interaction between
calf sex and blood acid-base measure, and the interac-
tion between dam treatment and calf sex and blood
acid-base measure, and the random effect of block. The
nonsignificant interactions (P > 0.10) were removed
from the final models. Figures were created using the
predicted apparent efficiency of absorption of IgG plot-
ted against the acid-base measure used as predictor in
the model.
Journal of Dairy Science Vol. 104 No. 8, 2021
A linear mixed-effects model was used to obtain the
predicted DMI for each female calf irrespective of treat-
ment, to understand whether residual DMI differed
among treatments. The statistical model included the
fixed effects of metabolic BW, daily BW change, and
age, and the random effects of block and calf. Age was
the term in the REPEATED statement, and the covari-
ance structure selected based on the smallest Akaike’s
information criterion was the autoregressive 1. The
residuals for each calf according to age were calculated
as the observed minus the predicted intake. Residual
DMI was then analyzed with a model that included
the fixed effects of dam treatment, age of calf at mea-
surement, and interaction between treatment and age,
and the random effects of block and calf nested within
dam treatment, as previously described for continuous
variables with repeated measures.
Treatment differences with P ≤ 0.05 were considered
significant, whereas tendencies for differences were re-
ported if 0.05 < P ≤ 0.10.
RESULTS
From the 132 cows enrolled in the experiment, 132
calves were born, 77 females and 55 males. Treat-
ment did not affect gestation length. The genomic
PTA values of dams for calving ease did not differ
among treatments, although cows assigned to N150
had the smallest (P = 0.04) genomic PTA for still-
birth (Supplemental Table S1; https:​/​/​figshare​.com/​s/​
b412f7b6661e5c3c5cd0). Treatment did not affect the
incidence of dystocia or stillbirth, and 6 calves were
stillborn (Table 2). The BW of calves at birth, includ-
ing stillbirth calves, was greater (P < 0.001) for males
than females (39.2 ± 0.6 vs. 36.0 ± 0.5 kg), but neither
treatment nor the interaction between treatment and
calf sex affected BW at birth. As DCAD decreased, the
dam’s acid-base balance became more acidotic, with
reduced (P < 0.001) concentration of HCO3− (P200 =
28.4 vs. N50 = 26.7 vs. N150 = 24.9 ± 0.4 mM) and
pH (P200 = 7.442 vs. N50 = 7.431 vs. N150 = 7.410 ±
0.004) in blood (Zimpel et al., 2021b).
Colostrum IgG Content and Apparent
Efficiency of Absorption
As intended, the interval between birth to when
colostrum was fed to calves did not differ among treat-
ments, resulting in a mean of 73 min after birth (Table
2). Similar to the mean time to feeding, treatment did
not affect the median intervals from birth to feeding co-
lostrum, which were 65, 72, and 81 min for P200, N50,
and N150, respectively. The interval from colostrum
feeding on d 0 to sampling blood to assess apparent
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

efficiency of IgG absorption did not differ among treat-
ments, with a mean of 27.5 ± 1.1 h (Table 2).
Treatment did not affect the concentration of IgG or
the Brix value in colostrum. Calves consumed a mean
of 353 ± 16 g of IgG or 9.39 ± 0.46 g of IgG/kg of BW,
and treatment did not affect the concentration of IgG
in serum or the apparent efficiency of IgG absorption
(Table 2). Only 2 calves had failure of passive transfer,
based on serum IgG <15 g/L: 1 female P200 with serum
IgG of 12.7 g/L and 1 female N50 with serum IgG of 9.9
g/L. All other calves had serum IgG >15 g/L on d 1.
Blood Gases, Acid-Base Balance, and Associations
with Apparent Efficiency of IgG Absorption
Treatment did not affect calves’ blood gases in the
first 7 d of age (Table 3) and had only a minor effect
on calves’ acid-base balance in the first 7 d of age.
A tendency (P = 0.06) for a quadratic reduction in
concentration of HCO3− with a reduction in DCAD
was detected. At birth, calves were in a state of respi-
ratory and metabolic acidosis, with means for blood
pH and HCO3− of 7.215 ± 0.007 and 26.2 ± 0.4 mM,
respectively, and a mean pCO2 of 64.1 ± 0.7 mmHg.
Nevertheless, between birth and 1 d of age, blood pH
and concentrations of HCO3− and base excess increased
(P < 0.001), whereas blood pCO2 decreased (P <
0.001) (Figures 1A to 1D). Concurrently, the venous
blood pO2 (d 0 = 23.3 vs. d 1 = 27.3 ± 0.3 mmHg)
and O2 saturation (d 0 = 28.9 vs. d 1 = 48.2 ± 1.4%)
increased (P < 0.001) from birth to 1 d of age, after
which they remained unaltered. The temporal changes
in blood pH, HCO3−, and gases in the first 24 h of life
were observed in all 3 treatments without interactions
between treatment and age.
The associations between acid-base balance and ap-
parent efficiency of absorption of IgG were analyzed,
controlling for the effects of treatment and sex of calf.
The interactions between dam treatment and any of the
measures of acid-base balance were not associated (P >
0.14) with IgG absorption. Blood pH, HCO3−, or base
excess were not associated with apparent efficiency of
IgG absorption (Figures 2A, 2C, and 2D). However, a
positive linear association (P = 0.05) between venous
blood pCO2 and IgG absorption was observed in all
3 treatments, and the β coefficient was 0.26 ± 0.13,
indicating that for each 10 mmHg increase in pCO2,
apparent efficiency of IgG absorption increased 2.6
percentage units (Figure 2B). Males had greater (P
= 0.01) apparent efficiency of IgG absorption than
females (36.3 ± 1.7 vs. 30.6 ± 1.5%; Figure 2).
Blood and Serum Minerals and Metabolites
Treatment did not affect calves’ blood concentrations
of iNa, ionized potassium, iCa, or glucose (Table 3; Fig-

Table 2. Effect of prepartum DCAD fed to dams on incidence of dystocia, stillbirth, calf birth weight, colostrum IgG content, and apparent
absorption IgG

Treatment1 P-value2

Item P200 N50 N150 SEM TRT TRT × sex Linear Quadratic
3
Birth BW, kg 37.7 37.3 37.8 0.7 0.86 0.67 0.99 0.58
Dystocia,3 % 14.0 15.6 6.8 — 0.84 0.46 0.79 0.55
Stillbirth,3,4 % 4.7 4.4 4.6 — 0.74 — 0.52 0.64
Colostrum fed to calves                
  Birth to feeding,5 min 62 79 79 8 0.19 0.99 0.09 0.64
  Brix % 26.0 26.0 25.6 0.6 0.80 0.83 0.62 0.66
  IgG, g/L 95.0 91.0 97.1 4.1 0.55 0.58 0.93 0.28
Intake of IgG5                
  Total g 350 343 365 16 0.60 0.63 0.67 0.36
  g/kg of BW 9.30 9.23 9.63 0.46 0.81 0.65 0.70 0.60
Colostrum to blood,6 h 27.4 28.4 26.6 1.1 0.46 0.39 0.80 0.23
Serum IgG on d 1,5 g/L 28.9 30.4 32.3 1.6 0.33 0.46 0.16 0.63
IgG apparent absorption,5,7 % 33.1 33.1 34.2 1.9 0.90 0.95 0.76 0.75
1
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, assigned randomly to receive 1 of 3 prepartum
diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
2
TRT = effect of treatment (P200 vs. N50 vs. N150); TRT × sex = effect of the interaction between TRT and sex of calf; Linear = linear effect
of DCAD; Quadratic = quadratic effect of DCAD.
3
Number of calves in each treatment: P200 = 28 females and 15 males; N50 = 23 females and 22 males; N150 = 26 females and 18 males.
4
The interaction between TRT and calf sex was not estimable because of quasi-complete separation of data points.
5
Colostrum was fed to calves in the first hours after birth, and serum was sampled from calves on d 1, approximately 27 h after feeding. Number
of calves in each treatment: P200 = 27 females and 14 males; N50 = 23 females and 20 males; N150 = 24 females and 18 males.
6
Interval from colostrum feeding on d 0 to blood sampling for IgG measurement on d 1.
7
Calculated according to Quigley et al. (1998).

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 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

ures 3A and 4A). An interaction (P = 0.03) between s/​b412f7b6661e5c3c5cd0). Concentrations of tCa in

treatment and age was observed for hematocrit because
calves from treatment P200 had the greatest hemato-
crit on d 1 of age (P200 = 29.8 vs. N50 = 28.9 vs. N150
= 27.9 ± 0.9%), and then hematocrit decreased (P <
0.001), reaching a nadir on d 3, and then increased
(P < 0.001) on d 7, reaching a maximum for calves in
N50 (P200 = 26.0 vs. N50 = 27.3 vs. N150 = 25.3 ±
0.9%; Supplemental Figure S1E, https:​/​/​figshare​.com/​
s/​b412f7b6661e5c3c5cd0). Blood hemoglobin followed
the same pattern as that of hematocrit, with an inter-
action (P = 0.02) between treatment and age. Calves
born to dams fed P200 had the greatest hemoglobin
on d 1 of age (P200 = 10.4 vs. N50 = 9.8 vs. N150 =
9.5 ± 0.3 g/dL), and then hemoglobin decreased (P <
0.001), reaching a nadir on d 3, and then increased (P
< 0.001) on d 7, with the maximum value for calves in
N50 (P200 = 9.1 vs. N50 = 9.3 vs. N150 = 8.6 ± 0.3 g/
dL; Supplemental Figure S1F, https:​/​/​figshare​.com/​s/​
b412f7b6661e5c3c5cd0).
An interaction (P = 0.006) between treatment and
age affected serum tCa in females because calves in
the first 3 d of age had concentrations that remained
somewhat constant in N150; however, tCa decreased
from 3.13 mM on d 0 to 2.81 mM on d 1, followed by
an increase to 3.19 mM on d 3 in P200 calves (Table
4; Supplemental Figure S1A, https:​/​/​figshare​.com/​
serum in all 3 treatments declined (P < 0.001) over
time in the first 56 d of age, such that by d 56 the
concentration had a mean of 2.81 ± 0.07 mM (Figure
3B). The proportion of tCa represented by iCa in blood
of females in the first 7 d of age decreased linearly (P =
0.04) with a decrease in DCAD (Table 3). At birth, iCa
represented 43.7% of tCa, and this proportion increased
(P = 0.03) to 45.6% on d 7 of age (Supplemental Figure
S1B; https:​/​/​figshare​.com/​s/​b412f7b6661e5c3c5cd0).
Treatment did not affect the concentration of tMg or
tP in serum of calves (Table 4). Serum concentrations
of tMg declined (P < 0.001) from 0.89 ± 0.02 mM
on d 0 to a nadir of 0.76 ± 0.02 mM on d 3 and then
remained mostly stable until d 56 of age (Figure 3C).
By contrast, concentrations of tP in serum initially
decreased (P < 0.001) from 2.01 ± 0.04 on d 0 to 1.86
± 0.04 on d 1 and 2, and then steadily increased (P <
0.001) to reach a plateau by 21 d of age (Figure 3D).
The interaction (P = 0.004) between treatment and
age affected serum concentrations of fatty acids because
at birth concentrations increased (P < 0.01) with a de-
crease in DCAD (Table 4, Figure 4B), whereas after d 0,
concentrations decreased and no longer differed among
treatments. As anticipated, calves were born practically
agammaglobulinemic, with a mean IgG concentration
of only 5.1 ± 1.0 g/L in serum sampled before colos-

Table 3. Effect of prepartum DCAD fed to dams on measures of venous blood acid-base status and chemistry in newborn calves

Treatment3 P-value4

Item1,2 P200 N50 N150 SEM TRT TRT × sex TRT × age Linear Quadratic
Venous blood gases                  
 pCO2, mm of Hg 53.7 52.2 52.8 0.8 0.37 0.12 0.43 0.27 0.36
 pO2, mm of Hg 25.4 25.0 25.4 0.6 0.87 0.70 0.28 0.94 0.60
 tCO2, mM 33.3 32.4 32.3 0.4 0.19 0.30 0.51 0.07 0.67
 sO2, % 44.2 43.4 43.8 1.5 0.93 0.67 0.37 0.80 0.78
Venous blood chemistry                  
 pH 7.378 7.378 7.374 0.008 0.91 0.13 0.43 0.78 0.73
 HCO3−, mM 31.7 30.7 30.7 0.4 0.14 0.13 0.33 0.06 0.63
  Base excess, mM 6.34 5.58 5.49 0.44 0.36 0.20 0.37 0.16 0.79
  Ionized Na, mM 136.7 136.9 136.5 0.4 0.59 0.59 0.29 0.71 0.34
  Ionized K, mM 4.76 4.67 4.71 0.03 0.39 0.08 0.42 0.20 0.63
  Ionized Ca, mM 1.364 1.355 1.362 0.008 0.68 0.93 0.63 0.74 0.41
  Ionized Ca, % total Ca5 45.0 44.2 42.8 0.8 0.09 — 0.11 0.04 0.40
  Glucose, mM 7.11 7.24 7.39 0.19 0.60 0.27 0.12 0.34 0.75
Hematocrit, % 26.4 26.5 25.6 0.8 0.69 0.90 0.03 0.58 0.51
Hemoglobin, g/dL 9.17 8.99 8.70 0.27 0.45 0.98 0.02 0.26 0.62
1
Blood samples collected on d 0, 1, 3, and 7. Number of calves with blood sampled and analyzed in each treatment were: P200 = 23 females and
11 males; N50 = 17 females and 16 males; and N150 = 18 females and 16 males.
2
pCO2 = partial pressure of CO2; pO2 = partial pressure of O2; tCO2 = total dissolved CO2; sO2 = saturation of O2.
3
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, assigned randomly to receive 1 of 3 prepartum
diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
4
TRT = effect of treatment (P200 vs. N50 vs. N150); TRT × sex = effect of the interaction between TRT and sex of calf; TRT × age = effect
of the interaction between TRT and age of calf; Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.
5
Only female calves included because total Ca was not measured in males.

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 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

Figure 1. Venous blood pH (A), partial pressure of CO2 (pCO2, B), concentration of HCO3− (C), and base excess (D) in calves born to
nulliparous cows fed prepartum diets with DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calv-
ing. Number of calves with blood sampled and analyzed in each treatment were as follows: P200 = 23 females and 11 males; N50 = 17 females
and 16 males; and N150 = 18 females and 16 males. Error bars represent SEM. Panel A: effects of treatment (P = 0.91), interaction between
treatment and sex (P = 0.13), interaction between treatment and age (P = 0.43), and linear (P = 0.78) and quadratic (P = 0.73) orthogonal
polynomial contrasts. Panel B: effects of treatment (P = 0.37), interaction between treatment and sex (P = 0.12), interaction between treatment
and age (P = 0.43), and linear (P = 0.36) and quadratic (P = 0.19) orthogonal polynomial contrasts. Panel C: effects of treatment (P = 0.14),
interaction between treatment and sex (P = 0.13), interaction between treatment and age (P = 0.33), and linear (P = 0.06) and quadratic (P =
0.63) orthogonal polynomial contrasts. Panel D: effects of treatment (P = 0.36), interaction between treatment and sex (P = 0.20), interaction
between treatment and age (P = 0.37), and linear (P = 0.16) and quadratic (P = 0.79) orthogonal polynomial contrasts.

trum feeding. Concentrations of IgG increased (P <
0.001) 6-fold on d 1 and then declined until d 21 and
plateaued at 18.7 ± 0.9 g/L (Figure 4C). Treatment or
the interaction between treatment and age did not af-
fect serum IgG concentrations. The Brix value of serum
followed the same temporal pattern observed for IgG,
although the interaction between treatment and age
affected (P = 0.03) serum Brix (Figure 4D). Calves in
N150 had greater (P = 0.006) serum Brix on d 1 than
those born to dams fed P200 or N50 (P200 = 9.44 vs.
N50 = 9.87 vs. N150 = 10.26 ± 0.20%), whereas calves
in N50 had greater (P = 0.008) serum Brix on d 3 than
those born to dams fed P200 or N150 (P200 = 9.09 vs.
N50 = 9.89 vs. N150 = 9.58 ± 0.19%). Treatment did
not affect serum Brix after 3 d of age.
Intake of DM, Growth, and Feed Efficiency
Female calves had ad libitum access to starter grain
throughout the experiment and to milk from 22 to
53 d of age, after which milk allowance was gradu-
ally decreased. Treatment or the interaction between
treatment and age did not affect intake of DM either
from milk or from starter grain (Table 5). Intake of

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Figure 2. Apparent efficiency of IgG absorption in male (M) and female (F) calves measured on d 1 according to venous blood pH (A),
partial pressure of CO2 (pCO2, B), concentration of HCO3− (C), and base excess (D) measured on d 0, before colostrum feeding. Calves were
born to nulliparous cows fed prepartum diets with DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation
to calving. Panel A: association between blood pH and efficiency of IgG absorption (P = 0.70). Panel B: association between blood pCO2 and
efficiency of IgG absorption (P = 0.05). Panel C: association between blood pH and efficiency of IgG absorption (P = 0.63). Panel D: association
between blood pH and efficiency of IgG absorption (P = 0.82).

milk and starter grain DM increased (P < 0.001) with
age in all 3 treatments (Figures 5A and 5B) and, in
the eighth week of age, calves had mean intakes of
1.11 ± 0.05 kg of milk replacer DM and 0.53 ± 0.04
kg of starter grain DM. The decline in DM intake
from milk in wk 8 of age was caused by the gradual
decrease in milk allowance starting on d 54, to begin
the weaning process. The mean BW and the daily BW
gain during the 56-d experiment did not differ among
treatments (Table 5). Calves gained a mean of 0.58
± 0.02 kg/d in the first 21 d of age, after which the
daily BW gain increased (P < 0.001) to 0.90 ± 0.02
kg/d until 56 d of age (Figure 5C). Calves reached 56
d of age with continuous growth and weighing a mean
of 80.0 ± 1.7 kg (Table 4, Figure 5D). Efficiency of
feed utilization based on BW gain per kilogram of
DMI did not differ among treatments, with a mean
of 0.666 ± 0.025 kg of BW per kg of DMI. Similar to
feed conversion ratio, the residual DMI did not differ
among treatments (Table 5).
Morbidity and Mortality
Treatment did not affect morbidity in female calves
in the first 56 d of age (Table 6). Diarrhea and respira-
tory disease affected 56.8 and 13.5% of the calves in
the experiment, and 10.8% of the calves were diagnosed
with multiple diseases. Treatment did not affect the
rate of morbidity (Figure 6). Of the 74 female calves, 2
died before 56 d of age, 1 P200 that died on d 20 and
1 N50 that died on d 8. Both calves died of septicemia
associated with diarrhea.

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DISCUSSION influence offspring performance during the preweaning

Feeding acidogenic diets aims to reduce the risk of
hypocalcemia in periparturient dairy cows. Although
research is extensive on the effects of manipulating pre-
partum diets to influence the acid-base balance and risk
of hypocalcemia in parous cows, less is known about
the effects of such diets on nulliparous cows or on the
offspring. The objective of the current experiment was
to evaluate whether altering the level of DCAD fed to
nulliparous cows starting at 250 d of gestation would
period. Reducing the DCAD did not affect gestation
length, BW of calves at birth, colostrum IgG content,
or apparent efficiency of IgG absorption. Treatment
did not affect blood gases and measures of measures
of acid-base balance in all calves in the first days of
life, or DM intake from milk or starter grain, daily BW
gain, and weaning BW in female calves. Indeed, risk
and rate of morbidity were unaffected by treatments.
Collectively, the present results corroborate findings
from others showing that inducing metabolic acidosis

Figure 3. Concentrations of blood ionized Ca (iCa, A), and serum total Ca (tCa, B), total Mg (tMg, C), and total P (tP, D) in calves born to
nulliparous cows fed prepartum diets with DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
Number of calves with blood sampled and analyzed and for iCa: P200 = 23 females and 11 males; N50 = 17 females and 16 males; and N150
= 18 females and 16 males. Number of female calves with blood sampled and serum analyzed for tCa, tMg, and tP: P200 = 27; N50 = 23; and
N150 = 24. Error bars represent SEM. Panel A: effects of treatment (P = 0.68), interaction between treatment and age (P = 0.63), interaction
between treatment and calf sex (P = 0.93), and linear (P = 0.74) and quadratic (P = 0.41) orthogonal polynomial contrasts. Panel B: effects of
treatment (P = 0.55), interaction between treatment and age (P = 0.006), and linear (P = 0.30) and quadratic (P = 0.70) orthogonal polynomial
contrasts. Panel C: effects of treatment (P = 0.65), interaction between treatment and age (P = 0.37), and linear (P = 0.36) and quadratic (P
= 0.96) orthogonal polynomial contrasts. Panel D: effects of treatment (P = 0.31), interaction between treatment and age (P = 0.21), and linear
(P = 0.43) and quadratic (P = 0.20) orthogonal polynomial contrasts. *Within day, treatments differ (P < 0.05).

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 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

in dams in late gestation to control hypocalcemia does
not seem to have implications to offspring growth and
health in the first 2 mo of life.
Blood pH depends on the relative concentrations of
bases, acids, and buffers, and acidosis can result from
either excessive production or insufficient removal of
acid from blood (Engelking, 2014). Metabolic acidosis
is characterized by imbalances in the HCO3− buffer
system, whereas respiratory acidosis is characterized
by imbalances in pCO2, primarily because of impaired
lung function. Both imbalances can result in acidemia
(Engelking, 2014). Acidosis is common in newborns in
the first hours after birth (Bleul et al., 2007), irrespec-
tive of prepartum diet or acid-base balance of dams
(Collazos et al., 2017; Diehl et al., 2018). Respiratory
acidosis in newborns occurs because parturition can
result in hypoxia, hypercapnia, and disturbances in
utero-placental circulation. During delivery, immatu-
rity of the lungs for proper gas exchange can result
in hypercapnia and respiratory acidosis in newborn
calves (Massip, 1980). Difficult delivery can exacer-
bate acidosis in calves (Vannucchi et al., 2015). Calves
in all 3 treatments were born in a state of metabolic
and respiratory acidosis with reduced blood HCO3−,
acidemia with a blood pH of 7.21, and hypercapnia,
hallmarks of uncompensated metabolic acidosis associ-

Figure 4. Blood concentrations of glucose (A) and serum concentrations of fatty acids (B), IgG (C), and Brix (D) in calves born to nul-
liparous cows fed prepartum diets with DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
Number of calves with blood sampled and analyzed for glucose: P200 = 23 females and 11 males; N50 = 17 females and 16 males; and N150
= 18 females and 16 males. Number of female calves with blood sampled and serum analyzed for fatty acids, IgG, and Brix: P200 = 27; N50
= 23; and N150 = 24. Error bars represent SEM. Panel A: effects of treatment (P = 0.60), interaction between treatment and sex (P = 0.27),
interaction between treatment and age (P = 0.12), and linear (P = 0.34) and quadratic (P = 0.75) orthogonal polynomial contrasts. Panel B:
effects of treatment (P = 0.12), interaction between treatment and age (P = 0.004), and linear (P = 0.06) and quadratic (P = 0.43) orthogonal
polynomial contrasts. Panel C: effects of treatment (P = 0.36), interaction between treatment and age (P = 0.28), and linear (P = 0.16) and
quadratic (P = 0.99) orthogonal polynomial contrasts. Panel D: effects of treatment (P = 0.13), interaction between treatment and age (P =
0.03), and linear (P = 0.05) and quadratic (P = 0.82) orthogonal polynomial contrasts. *Within day, treatments differ (P < 0.05).

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Table 4. Effect of prepartum DCAD fed to dams on serum concentrations of minerals, immunoglobulins, and fatty acids in female calves

Treatment2 P-value3

Item1 P200 N50 N150 SEM TRT TRT × age Linear Quadratic
Total Ca, mM 2.97 2.99 3.02 0.04 0.55 0.006 0.30 0.70
Total Mg, mM 0.80 0.81 0.81 0.02 0.65 0.37 0.36 0.96
Total P, mM 2.11 2.04 2.09 0.04 0.31 0.21 0.43 0.20
Fatty acids, mM 0.27 0.28 0.32 0.02 0.12 0.004 0.06 0.43
Serum IgG, g/L 19.8 21.0 21.5 1.0 0.36 0.28 0.16 0.99
Serum Brix, % 8.81 9.02 9.05 0.10 0.13 0.03 0.05 0.82
1
Serum samples from females collected on d 0, 1, 2, 3, 7, 21, 42, and 56 were analyzed for concentrations of minerals, IgG, and Brix; and those
collected on d 0, 7, 21, 42, and 56 were analyzed for fatty acids. Number of female calves sampled in each treatment were: P200 = 27, N50 =
23, and N150 = 24.
2
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, assigned randomly to receive 1 of 3 prepartum
diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
3
TRT = effect of treatment (P200 vs. N50 vs. N150); TRT × age = effect of the interaction between TRT and age of calf; Linear = linear effect
of DCAD; Quadratic = quadratic effect of DCAD.

ated with respiratory acidosis. This state of acidosis
was mostly recovered by 1 d of age. At birth, the lung
tissue contains fluid that undergoes clearance required
for proper gas exchange, and both pO2 and saturation
of O2 increased from birth to d 1 of age, supporting
the concept of improved gas exchange after birth in
these calves correcting the respiratory and metabolic
acidosis.
As expected, cows fed P200, N50, and N150 had
marked differences in blood gases and acid-base bal-
ance prepartum, which paralleled the level of DCAD
fed (reported in Zimpel et al., 2021b). Dams fed N50
and N150 received diets with more chlorine to induce
metabolic acidosis, compared with those fed P200, and
thus we speculated that potential changes in chloride
ion content in the dam’s blood could be transferred to
the fetus and affect acid-base balance prenatally. Pla-
cental transfer of chloride ions has been measured in
pregnant ewes and found to be comparable to the rate
of transfer of iNa (Thornburg et al., 1979). If the cow
placenta behaves similarly to that of the ewe, then it is
possible that the placenta controls the transfer of ions
and maintains a somewhat equal transfer of chloride
ions and iNa and, therefore, prevents acidification of
the fetal blood. That might explain the lack of effects
of maternal diets on acid-base balance in calves in the
first hours after birth observed in the present experi-
ment and by others (Collazos et al., 2017; Diehl et al.,
2018). The observed responses in blood chemistry and
gases in the first days of age were consistent with find-
ings by others (Collazos et al., 2017; Diehl et al., 2018),
a reduction in pCO2 and increases in pH, HCO3−, and
base excess. Furthermore, neither treatment nor the
interaction between treatment and calf sex affected

Table 5. Effect of prepartum DCAD fed to dams on DMI, BW, daily weight gain, and feed efficiency in female calves in the first 56 d of life

Treatment2 P-value3

Item1 P200 N50 N150 SEM TRT TRT × age Linear Quadratic
4
DMI, kg/d                
 Milk 1.11 1.04 1.19 0.06 0.19 0.64 0.58 0.08
  Starter grain 0.27 0.27 0.21 0.06 0.66 0.23 0.51 0.52
 Total 1.36 1.33 1.39 0.05 0.64 0.68 0.78 0.37
BW d 0, kg 36.0 35.6 36.8 0.9 0.58 — 0.61 0.37
BW d 56, kg 79.6 80.5 79.8 1.7 0.91 — 0.85 0.71
Mean BW (0 to 56), kg 57.5 58.1 58.3 1.1 0.88 0.82 0.61 0.99
BW gain, kg/d 0.799 0.810 0.773 0.027 0.61 0.63 0.63 0.39
BW gain/DMI,4 kg/kg 0.676 0.681 0.640 0.026 0.48 0.68 0.43 0.35
Residual DMI,4 kg/d 0.003 −0.042 0.039 0.040 0.37 0.95 0.77 0.17
1
Number of female calves in each treatment were P200 = 27, N50 = 23, and N150 = 24.
2
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, assigned randomly to receive 1 of 3 prepartum
diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
3
TRT = effect of treatment (P200 vs. N50 vs. N150); TRT × age = interaction between TRT and age of calves; Linear = linear effect of DCAD;
Quadratic = quadratic effect of DCAD.
4
Intake and feed efficiency measured from 21 to 56 d of age. Residual DMI calculated as observed minus predicted DMI, with predicted DMI cal-
culated following a statistical model that included metabolic BW, age at measurement, daily BW gain, and the random effects of block and calf.

Journal of Dairy Science Vol. 104 No. 8, 2021

 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

Figure 5. Milk DMI (A), starter grain DMI (B), daily BW gain (C), and BW (D) in female calves born to nulliparous cows fed prepartum
diets with a DCAD of +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving. Number of female calves
per treatment: P200 = 27; N50 = 23; and N150 = 24. Error bars represent SEM. Panel A: effects of treatment (P = 0.19), interaction between
treatment and age (P = 0.64), and linear (P = 0.58) and quadratic (P = 0.08) orthogonal polynomial contrasts. Panel B: effects of treatment
(P = 0.66), interaction between treatment and age (P = 0.23), and linear (P = 0.51) and quadratic (P = 0.52) orthogonal polynomial contrasts.
Panel C: effects of treatment (P = 0.61), interaction between treatment and age (P = 0.63), and linear (P = 0.63) and quadratic (P = 0.39)
orthogonal polynomial contrasts. Panel D: effects of treatment (P = 0.88), interaction between treatment and age (P = 0.82), and linear (P =
0.61) and quadratic (P = 0.99) orthogonal polynomial contrasts.

Table 6. Effect of prepartum DCAD fed to dams on the incidence of diseases in female calves in the first 56 d of life

Treatment2 P-value3
Incidence, %
Disease,1 % (n/n) P200 N50 N150   TRT Linear Quadratic
Diarrhea 56.8 (42/74) 63.0 56.5 50.0   0.56 0.30 0.78
Respiratory 13.5 (10/74) 11.1 13.0 16.7   0.84 0.67 0.84
Morbidity 59.5 (44/74) 66.7 60.9 50.0   0.50 0.33 0.54
Multiple diseases 10.8 (8/74) 7.4 8.7 16.7   0.68 0.55 0.69
1
Only female calves were evaluated for disease incidence. Number of females in each treatment were P200 = 27, N50 = 23, and N150 = 24.
2
Nulliparous cows were blocked by genomic PTA for energy-corrected milk yield and, within block, assigned randomly to receive 1 of 3 prepartum
diets varying in DCAD as +200 (P200), −50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving.
3
TRT = effect of treatment (P200 vs. N50 vs. N150); Linear = linear effect of DCAD; Quadratic = quadratic effect of DCAD.

Journal of Dairy Science Vol. 104 No. 8, 2021

 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH

Figure 6. Survival curves for age at morbidity in female calves born to nulliparous cows fed prepartum diets with a DCAD of +200 (P200),
−50 (N50), or −150 (N150) mEq/kg of DM from 250 d of gestation to calving. Number of female calves per treatment: P200 = 27; N50 = 23; and
N150 = 24. Effects of treatment (P = 0.43) and linear (P = 0.50) and quadratic (P = 0.24) orthogonal polynomial contrasts. The adjusted haz-
ard ratios were 1.18 (95% CI = 0.58 to 2.40) and 0.70 (95% CI = 0.34 to 1.46) for treatments N50 and N150, respectively, compared with P200.

acid-base status of calves. It is important to note that,
although we did not evaluate the duration of stage 2 of
calving, the incidence of dystocia and stillbirth did not
differ among treatments, thereby suggesting that calves
in all treatments were exposed to the same conditions
that induce respiratory acidosis in newborns.
It is well established that efficiency of IgG absorption
declines rapidly between 6 and 12 h after birth (Bush
and Staley, 1980), and intake of total IgG per unit of
BW immediately after birth is the most important fac-
tor determining IgG concentration in serum (Bush and
Staley, 1980). Treatment did not affect the content of
IgG in colostrum from dams used to feed the calves,
thus resulting in no effect of treatment on the amount
of IgG consumed per kilogram of BW. Also, the time
between birth and colostrum feeding had a mean of
just over 1 h, and only 1 calf received colostrum later
than 6 h after birth. Thus, the present timely colos-
trum feeding probably facilitated absorption of IgG,
resulting in adequate passive transfer in all but 2 calves
that had serum IgG <15 g/L. Altogether, the degree
of perturbation in acid-base balance observed in dams
induced by the acidogenic diets fed prepartum did not
affect the acid-base status of calves in the first week of
life or transfer of passive immunity.
Fetal metabolism results in production of organic ac-
ids that can be exacerbated by hypoxia (Omo-Aghoja,
2014; Matsell and Hiatt, 2017). Concentrations of
lactate in blood of calves increase with difficult calv-
ing (Murray et al., 2015) but decrease in the day after
birth (Diehl et al., 2018), suggesting less anaerobic
metabolism, compatible with normalized lung function.
Respiratory acidosis has been associated with reduced
adequate passive transfer of IgG (Besser et al., 1990),
although this link has not consistently been observed
(Murray et al., 2015; Collazos et al., 2017). Tyler and
Ramsey (1991) assigned 12 calves at birth to treat-
ments that resulted in normoxia or hypoxia and then
subjected the calves to different colostrum feeding regi-
mens. Absorption of colostral IgG in the first 18 h of life
was delayed in calves with hypoxia (Tyler and Ramsey,
1991). Respiratory acidosis was observed after birth in
the present experiment and, thus, could possibly affect
IgG absorption (Besser et al., 1990; Tyler and Ramsey,
1991). However, no association was observed between
measures of acid-base balance in calves immediately
before colostrum feeding and apparent efficiency of IgG
absorption. The only association observed was between
blood pCO2 and IgG absorption, which was positive.
Others have also showed no association between blood

Journal of Dairy Science Vol. 104 No. 8, 2021

 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
pH and apparent efficiency of IgG absorption (Collazos
et al., 2017), even when 21% of the 48 calves analyzed
were delivered with dystocia (Murray et al., 2015). Nev-
ertheless, Murray et al. (2015) also found that apparent
efficiency of IgG absorption increased 6.3 percentage
units in calves that first attained sternal recumbency
in the first 15 min after birth, compared with those
with delayed sternal recumbency. It is possible that
the association between acidosis and IgG absorption
observed by some might be related to the degree of
depression of calves, rather than only to measures of
acid-base balance. Because we did not measure blood
gases or acid-base balance multiple times between birth
and d 1, we do not know whether the readjustment to
normal blood chemistry occurred in the first few hours
after birth, which might have benefited IgG absorption.
Very likely, initiation of normal lung and renal function
in the newborns reduced hypercapnia and facilitated
the normalization of blood acid-base balance before d 1
(Vannucchi et al., 2015).
Treatment did not affect BW of calves at birth. The
same lack of effect of prepartum DCAD on birth BW
has been previously shown in cows fed different levels
of negative DCAD (Collazos et al., 2017; Diehl et al.,
2018) or when comparing positive with negative DCAD
(Weich et al., 2013; Rajaeerad et al., 2020). Dams in
all 3 treatments received the prepartum diet for a mean
of 22 d, and treatment did not affect intake of milk or
starter grain DM, nor growth performance of calves in
the preweaning period. Calves were fed milk ad libitum,
which explains the limited starter grain intake in the
first 56 d of life. Furthermore, treatment did not affect
the risk and rate of morbidity in the preweaning period.
Both nutrient intake and morbidity affect growth in
calves, and the fact that treatment had no effect in
either explains the lack of differences in daily BW gain
and final BW of calves on d 56. Rajaeerad et al. (2020)
showed that feeding an acidogenic diet prepartum did
not affect daily BW or feed conversion ratio in calves in
the first 70 d of life; however, calves born to dams fed
the acidogenic compared with the alkalogenic diet had
lower mean BW in the first 55 d because of differences
in wk 1 and 2 after birth. After weaning on d 55 of age,
BW on d 70 did not differ between calves born to dams
fed the acidogenic or the alkalogenic diet (Rajaeerad
et al., 2020). Skeletal growth, based on hip height, in
the first 62 d of life remained unaffected in calves born
to dams fed diets containing −70 or −180 mEq/kg for
the last 21 or 42 d of gestation (Collazos et al., 2017).
The same authors detected the effect of an interaction
between duration of feeding the acidogenic diets (21 vs.
42 d) and age of calves on BW. Calves born to dams fed
the acidogenic diet for the last 42 d of gestation were
lighter at birth, in part because of shorter gestation in
Journal of Dairy Science Vol. 104 No. 8, 2021
those cows, and the difference in BW maintained until
62 d of age. Nevertheless, the same experiment showed
that BW at 3 or 6 mo of age and daily BW gain did not
differ among treatments. Collectively, when cows are
fed acidogenic diets for the last 3 wk of gestation, as in
the present experiment, calf growth in the preweaning
period is not affected.
Treatment had minor effects on concentrations of
minerals, fatty acids, glucose, or IgG in blood or serum
of calves. The interaction between treatment and age
observed for concentrations of fatty acids occurred be-
cause of the linear increase observed in the first hour
after birth with the decrease in DCAD. One could pos-
tulate that some of the fatty acids detected in the calf’s
blood in the first hour of life might have originated from
the dams through placental supply (Perazzolo et al.,
2017). Acidogenic diets reduce DMI in prepartum cows
(Zimpel et al., 2018), which was observed in the pres-
ent experiment reported by Zimpel et al. (2021a), and
metabolic acidosis induced by acidogenic diets is known
to reduce tissue insulin signaling in cows (Vieira-Neto
et al., 2021). Perhaps the increased serum fatty acids in
calves reflect increased maternal transfer of fatty acids.
Also, it is possible that calves born to cows in N150 had
increased concentrations of cortisol or catecholamines
during delivery, which would induce more lipolysis,
although treatment did not affect dystocia. Other than
the day of calving, concentrations of fatty acids did not
differ among treatments, likely because treatment did
not affect nutrient intake or morbidity.
Concentrations of tCa and iCa are greater in calves
than in cows, likely because the set point for controlling
iCa concentration in blood changes as the animal ages.
Control of iCa concentrations depends on calcium-sens-
ing receptors (McGehee et al., 1997; Conigrave, 2016).
In rats, calcium-sensing receptor mRNA and protein
expression in tissues have been found to change with
age (Autry et al., 1997). Young animals are continu-
ously accreting bone as they grow, and bone density
increases with age (Jones et al., 1978), requiring cal-
cium and phosphorus deposition in hydroxyapatite.
Therefore, the greater concentrations of tCa and iCa
in blood of calves than in that of adult cows reflect the
needs of calcium for bone formation.
The placenta expresses TRPV6 and calbindin-D9k
needed for active transport of iCa in bovines (Sprekeler
et al., 2012), and Delivoria-Papadopoulos et al. (1967)
showed that fetal plasma proteins in sheep have greater
calcium binding per gram of protein than maternal
plasma proteins. Indeed, in spite of the low blood pH
of calves in the first day, iCa represented less than 45%
of the tCa in blood, perhaps because of increased bind-
ing capacity of albumin and other plasma proteins to
iCa, thereby allowing increased tCa concentration in
 Zimpel et al.: PREPARTUM DCAD AND PREWEANING CALF METABOLISM AND GROWTH
blood without the same increment in iCa. Dams fed
the acidogenic diets had increased iCa in blood (Zimpel
et al., 2021b), but this effect was not observed in the
calves. Nevertheless, iCa concentration at 1 d of age
decreased compared with iCa at birth or on d 3 of age.
This seems to be a common characteristic in mammals,
in which the decrease in iCa concentrations occurs be-
cause of the rise in blood pH and the concurrent loss of
placental flux of calcium (Kovacs, 2014).
Concentrations of tMg did not differ with treatment;
however, they decreased in the first 3 d of life, after
which they remained stable. The decrease in concentra-
tions of serum tMg coincided with an increase in iCa
and tCa. Increased concentrations of iCa might induce
activation of calcium-sensing receptors in the Henle
loop and distal convoluted tubules inhibiting the Na+/
K+/2Cl− cotransporter system, which is important for
paracellular reabsorption of calcium and magnesium in
the kidneys (Dai et al., 2001). Such changes cause a
reduction in the lumen positive voltage, which reduces
paracellular mineral reabsorption in the kidney (Hebert
et al., 1997) and, therefore, might increase magnesium
excretion in urine and explain the decrease in serum
tMg. A temporal pattern also was observed for tP con-
centrations in serum of calves. Concentrations initially
declined, which is consistent with the loss of placental
phosphorus supply (Kovacs, 2014) and reliance on gas-
trointestinal absorption. Nonetheless, concentrations of
tP increased starting on d 3 of age until d 21, after
which they remained mainly stable. This increase in
serum tP has been observed in newborns of numerous
species (Garel and Barlet, 1976), and the increase likely
persists until full activation of the parathyroid gland,
concurrent with secretion of fibroblast growth factor
23 by osteocytes to avoid hyperphosphatemia by in-
creasing excretion of phosphorus in urine (Kovacs and
Kronenberg, 1997). The greater concentration of phos-
phate in blood of calves compared with that of cows
probably reflects the needs of young animals to accrete
bone mass and to increase bone density as they grow.
Corroborating our results, others have also showed no
effect of maternal DCAD on concentrations of miner-
als in blood of calves (Collazos et al., 2017; Rajaeerad
et al., 2020). Altogether our results demonstrate that
the differences in acid-base balance and iCa in dams
induced by acidogenic diets does not affect mineral
metabolism of calves in the first 2 mo of life.
CONCLUSIONS
Manipulating the maternal diet fed to nulliparous
Holstein cows in the last 22 d of gestation by feeding
diets with DCAD ranging from +200 to −150 mEq/kg
of DM had no influence on offspring acid-base balance,
Journal of Dairy Science Vol. 104 No. 8, 2021
passive transfer of immunity, or mineral metabolism in
the first 56 d of life. A potential concern that metabolic
acidosis in dams would influence acid-base balance of
calves was not observed, and dam treatment had no
effect on intake of DM from milk or starter grain, daily
BW gain, or risk of diseases. Either the overwhelming
effect of respiratory acidosis that calves are born with
on acid-base balance or placental control of ion trans-
port prevents maternal acid-base balance perturbations
from further affecting the newborn acid-base balance.
Collectively, the data from the present experiment indi-
cate that diet-induced metabolic acidosis in nulliparous
dams during late gestation, through manipulating the
DCAD, does not compromise offspring passive immu-
nity, metabolism, growth, or health in the first 2 mo
of life.
ACKNOWLEDGMENTS
The authors thank Michael B. Poindexter, Achilles
Vieira-Neto, Ali Husnain, Usman Arshad, Ana C. M.
da Silva, Milerky C. Perdomo, Iago M. R. Leão, Jés-
sica G. Prim, Karla Ferreira, Félix Welter, Ingrid N.
F. Edelhoff, Vitória F. S. de Camargo, Túlio H. Souza,
Gustavo Avelin, Silvia Rodrigues, Gabriel Godoy, and
Xun Cao (University of Florida) for their help ensuring
proper care of cows and calves and collection of samples.
The help of the staff of the University of Florida Dairy
and Calf Units (Gainesville) is greatly appreciated. The
authors thank Elliot Block of Arm and Hammer Animal
and Food Production for providing Bio-Chlor, sodium
bicarbonate, and potassium carbonate; Terry Creel
and Tim Brown of Landus Cooperative for providing
SoyPlus; and Barbara Barton and Marcos Zenobi of
Balchem Animal Nutrition and Health for providing
ReaShure for this experiment. Partial funding for this
project was provided by a grant from the Southeast
Milk Check-Off Program (Southeast Milk Inc.) and by
Arm and Hammer Animal and Food Production. The
authors have not stated any other conflicts of interest.
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