J. Dairy Sci.

107:1228–1243
https://doi.org/10.3168/jds.2023-23588
© 2024, The Authors. Published by Elsevier 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/).

Influence of prepartum dietary cation-anion difference

and the magnitude of calcium decline at the onset of lactation
on mineral metabolism and physiological responses
M. K. Connelly,1 R. M. Rodney Harris,2 J. Kuehnl,1 J. P. N. Andrade,1 F. Sonnewend Andrade,1
S. Henschel,1 E. Block,3 I. J. Lean,4 and L. L. Hernandez1*
1
Department of Animal and Dairy Sciences, University of Wisconsin–Madison, Madison, WI 53706
2
Australian National University, Canberra 2601, Australia
3
Arm & Hammer Animal Nutrition, Princeton, NJ 08543
4
Scibus, Camden, NSW 2570, Australia

ABSTRACT and improve blood Ca profiles at the onset of lactation

whereas CaGlc infusion disrupts mineral metabolism.
The onset of lactation is characterized by substan- Key words: calcium, dietary cation-anion difference,
tially altered calcium (Ca) metabolism; recently, em- transition cow
phasis has been placed on understanding the dynamics
of blood Ca in the peripartal cow in response to this
INTRODUCTION
change. Thus, the aim of our study was to delineate
how prepartum dietary cation-anion difference (DCAD) Calcium (Ca) homeostasis is a highly coordinated
diets and the magnitude of Ca decline at the onset of process that maintains blood Ca concentrations through
lactation altered blood Ca dynamics in the peripartu- modulations of kidney Ca reabsorption, intestinal Ca
rient cow. Thirty-two multiparous Holstein cows were transport, and bone resorption (Goff et al., 2002). The
blocked by parity, previous 305-d milk yield and expect- onset of lactation presents an extreme Ca disturbance
ed parturition date, and randomly allocated to either due to the rapid and substantial increase in Ca demand
a positive (+120 mEq/kg) or negative (−120 mEq/kg) by the mammary gland due to irreversible loss of Ca
DCAD diet from 251 d of gestation until parturition to milk (Ramberg et al., 1970). Thus, the periparturi-
(n = 16/diet). Immediately after parturition cows were ent cow often experiences Ca dysregulation as Ca is
continuously infused for 24 h with (1) an intravenous pulled from peripheral tissue and blood pools at a rate
solution of 10% dextrose or (2) Ca gluconate (CaGlc) which cannot be maintained (Goff et al., 2002). Despite
to maintain blood ionized (iCa) concentrations at ~1.2 a wide range of interventions developed to support Ca
mM (normocalcemia) to form 4 treatment groups (n = homeostasis, subclinical hypocalcemia still occurs and
8/treatment). Blood was sampled every 6 h from 102 affects up to 50% of multiparous dairy cows (Reinhardt
h before parturition until 96 h after parturition and et al., 2011). The deleterious effects of hypocalcemia
every 30 min during 24 h continuous infusion. Cows in the peripartal period have been well-characterized
fed a negative DCAD diet prepartum exhibited a less and have clearly demonstrated the increased risks for
pronounced decline in blood iCa approaching parturi- displaced abomasum, ketosis, mastitis, metritis and
tion with lesser magnitude of decline relative to positive decreased neutrophil oxidative burst (Martinez et al.,
DCAD-fed cows. Cows fed a negative DCAD diet pre- 2014; Merriman et al., 2019; McArt and Neves, 2020;
partum required lower rates of CaGlc infusion to main- Venjakob et al., 2021). Thus, prepartum nutritional
tain normocalcemia in the 24 h postpartum relative to intervention strategies are commonly used practices
positive DCAD-fed cows. Infusion of CaGlc disrupted that have been demonstrated to reduce hypocalcemic
blood Ca and P dynamics in the immediate 24 h after incidences and improve cow health and performance
parturition and in the days following infusion. Collec- (Block et al., 1994; Lean et al., 2019).
tively, these data demonstrate that prepartum negative One common dietary intervention strategy currently
DCAD diets facilitate a more transient hypocalcemia used is feeding a negative DCAD diet prepartum (Lean
et al., 2013). Feeding a negative DCAD diet prepartum
improves Ca metabolism in the peripartum cow through
Received April 6, 2023.
Accepted September 4, 2023. instigating a mild metabolic acidosis via feeding ex-
*Corresponding author: llhernandez@​wisc​.edu cess dietary anions (Block, 1984). The corresponding
1228
 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1229

metabolic acidosis elicited allows better maintenance rupt mineral metabolism and that this would be more
in blood Ca concentrations at the onset of lactation by robust in cows fed a positive DCAD diet prepartum.
facilitating increased Ca flux via kidney, rumen, and
bone alterations to increase readily exchangeable Ca MATERIALS AND METHODS
pools allowing labile Ca to be readily partitioned to
meet milk Ca demand at the onset of lactation (Bush- All experimental procedures performed in this study
insky et al., 1993; Block, 1984; Grünberg et al., 2011, were approved by the College of Agriculture and Life
Wilkens et al., 2016). Sciences Animal Care and Use Committee at the Univer-
A transient decline in blood Ca at the onset of lacta- sity of Wisconsin–Madison (protocol number A006054)
tion has been suggested to be pertinent for stimulating and strictly followed. A sample-size calculation for 0.1
critical adaptive responses to allow for a successful mM differences in ionized Ca (iCa) concentrations was
adaptation to lactation. McArt and Neves (2020) es- calculated between negative and positive DCAD groups
tablished that cows that experience a transient hypo- on d 0 of lactation using ionized Ca concentration data
calcemia at the onset of lactation produce more milk collected from cows on negative and positive DCAD
than prolonged-, delayed-, and normo-calcemic cows. diets reported in Slater et al., 2018 and Rodney et al.,
Seely et al. (2021) further supported these findings as 2018 with a type I error risk of 5% and a type II error
cows experiencing a transient hypocalcemia produced risk of 10%, resulting in an n = 8 per treatment group.
more milk despite consuming similar amounts of feed.
These findings suggest thoughtful consideration of the Cows and Housing
classification and timing of hypocalcemia thresholds
and efficacy of commonly used oral and intravenous Ca The experiment was conducted at the Dairy Cattle
intervention strategies to improve blood Ca concentra- Center at the University of Wisconsin–Madison and
tions after calving. Such treatments could disrupt the used 32 pregnant, multiparous Holstein cows rang-
homeostatic and homeorhetic adaptations to control ing from second to fourth lactation, with one positive
blood Ca that may be stimulated by transient hypocal- DCAD-fed cow being removed from analysis due to
cemia. Calcium bolus and intravenous Ca treatments catheter infection (Table 1). Thus, the final experi-
shortly after parturition successfully increase blood mental dataset included 31 multiparous Holstein cows
Ca acutely and provide benefits, but may alter blood with postpartum lactation number averaging 2.81 ±
Ca dynamics resulting in prolonged hypocalcemia and 0.27 (SEM). Prepartum, pregnant cows were brought
increase risks of metritis and morbidity of cows (Blanc to the Dairy Cattle Center from the Blaine Dairy at
et al., 2014; Martinez et al., 2016). the University of Wisconsin–Madison and introduced
Therefore, the aim of our study was to determine to facilities at 241 d of gestation to allow for acclima-
how the prevention of a transient hypocalcemia at par- tion to tiestall housing and fed a standard low-energy,
turition in combination with prepartum dietary DCAD high-fiber, low-DCAD dry cow diet before dietary en-
would affect blood Ca dynamics and influence mineral rollment (diet not shown). From thereon until 60 DIM
metabolism. We hypothesized that preventing Ca de- cows were housed in the enclosed tie-stall facility and
cline in the immediate 24 h after parturition would dis- let out daily prepartum and twice daily postpartum for

Table 1. Means (±SEM) for days on treatment diet, previous lactation 305-d mature-equivalent milk
production, parity, BW, BCS, and ionized Ca (iCa) at enrollment

Prepartum diet1

Negative DCAD Positive DCAD

Variable Ca Dex Ca Dex

Days on diet 21.8 (0.98) 24.1 (1.39) 21.7 (1.14) 25.5 (1.58)
Previous 305 d (kg) 13,426 (294) 13,896 (566) 13,739 (664) 14,087 (847)
Parity2 1.7 (0.30) 1.5 (0.26) 1.8 (0.33) 1.6 (0.25)
BW (kg) 742.7 (14.6) 718.1 (33.1) 725.9 (16.8) 732.8 (21.0)
BCS 3.56 (0.04) 3.55 (0.09) 3.65 (0.05) 3.62 (0.07)
DMI (kg/d) 14.6 (0.54) 13.3 (0.49) 14.3 (0.30) 13.9 (0.33)
iCa (mM) 1.20 (0.02) 1.18 (0.01) 1.17 (0.02) 1.17 (0.01)
1
Ca = calcium gluconate infusion; Dex = 10% dextrose infusion.
2
Parity is before calving.

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1230

exercise. Postpartum cows were milked twice daily and was based upon iCa values previously reported (Rodney
fed the standard herd lactating cow diet. et al., 2018; Slater et al., 2018). Energy equivalencies in
the infusions were matched to account for metabolizable
Treatments and Feeding Management energy on a 100 mL basis for the gluconic acid delivered
when infusing CaGlc. The CaGlc contains 21 g per 100
At 251 d of gestation cows (gestation length of 278 mL of gluconate. The estimated energy supplied by the
d) were blocked by parity, previous 305-d milk yield, Ca gluconate infusion, based on Stetten and Stetten
and expected parturition date and randomly allocated Jr. (1950), and allowing for the conversion of gluconate
to receive either a positive (+120 mEq/kg) or negative to glucose is 0.30 MJ and dextrose infusion is 0.29 MJ.
(−120 mEq/kg) prepartum dietary treatment ad libi- The difference in energy availability between groups is
tum. Urine pH was expected to be in a range of 6.0 to estimated to be 0.008 MJ. Intravenous 10% dextrose
6.5. The negative DCAD was achieved by supplement- was prepared aseptically with 50% Dextrose Solution
ing with an acidogenic protein meal (BIO-CHLOR, (Aspen Veterinary Resources, Greeley, CO) diluted
Church & Dwight Co. Inc.) until parturition (n = 16/ with Sterile Water (Aspen Veterinary Resources, Gree-
diet; Table 2). Upon parturition, a sample was taken ley, CO) to make 10% dextrose for infusion.
within 5 min (represented as 0 h), and then cows were Cows were fed TMR diets once daily pre- and post-
immediately started on infusion. The first cow to calve partum at 0900 h with feed intake recorded daily by
within each block, on each diet, was assigned to 24-h farm staff. Daily feed intake was recorded with refus-
continuous intravenous infusion of 23% Ca gluconate als measured before each morning feeding. Samples
solution (CaGlc; Aspen Veterinary Resources, Greeley, of individual feed ingredients, experimental diets and
CO) with the goal to maintain normocalcemia (~1.2 the lactating herd diet were collected weekly. Samples
mM ionized Ca concentrations). The subsequent pair were dried using a forced-air drying oven as described
cow within each block on the same diet received a con- in Toledo et al. (2017) with forage and grain DM val-
tinuous 24-h intravenous infusion of 10% dextrose solu- ues updated weekly to adjust as-fed inclusion rates of
tion at a rate equivalent to the CaGlc infusion received ration ingredients. Dry matter intake was calculated
by their diet-pair. Hence, the 4 treatments beginning based upon feed offered and refused and the accompa-
at parturition and then maintained throughout the nying weekly DM value for the respective TMR. Pre-
remainder of the experiment were: prepartum positive partum and postpartum TMR samples had minerals
DCAD and postpartum CaGlc infusion (PCa), prepar- analyzed for wet chemistry at a commercial laboratory
tum positive DCAD and postpartum dextrose infusion (Dairyland Labs, Arcadia, Wisconsin). The composi-
(PDex), prepartum negative DCAD and postpartum tion of experimental diets and the lactating herd diet
CaGlc infusion (NCa), and prepartum negative DCAD are presented in Table 2. Milk yield was recorded at
and postpartum dextrose infusion (NDex). each milking during the first 30 d after parturition.
The rate of infusion and energy equivalency were
maintained equally between CaGlc and dextrose groups. Blood and Urine Sample Collection
Infusion volumes were matched within each diet of each
block by an infusion pump (Heska Vet IV, Loveland, Before cows starting experimental diets, baseline
CO) to modulate rate of infusion equally between cows blood samples were collected via the coccygeal vein for
paired by infusion (CaGlc and dextrose). Infusion rates 2 d. Urine was collected daily prepartum starting −21
were adjusted based upon blood iCa concentrations d relative to expected parturition date and at 0, 1, 2,
in CaGlc infused cows on the same diet, with blood 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 after parturition
sampled every 30 min to modulate infusion rate and by gently massaging the perineal area and collecting
maintain blood Ca appropriately. Cows infused with samples mid-stream in a 5-mL Eppendorf tube. Urine
CaGlc all began at an infusion rate of 100 mL/h (2.14 g pH was immediately measured and recorded (B-712
Ca/100 mL) and then adjusted every 30 min to achieve LAQUA twin, Horiba Scientific, Edison, NJ), with dai-
1.2 mM blood iCa concentrations which was deter- ly pH calibrations performed before measurement and
mined by CG8+ cartridges using a cow-side hand-held recording. Ten days before expected parturition date
biochemical analyzer (i-Stat System, Abbott Laborato- gas sterilized, tygon tubing, jugular catheters were in-
ries, Abbott Park, IL). All blood samples collected were serted into both jugular veins with catheter placement,
analyzed within 1 to 2 min of collection. Dextrose cows maintenance and monitoring as previously described
were infused at the same rate as a CaGlc cow on the (Slater et al., 2018) and removed at 4 DIM. During
same diet within the same block, resulting in separate continuous 24-h infusion, one catheter was exclusively
infusion rates for positive DCAD and negative DCAD used for intravenous infusion and the opposing catheter
cows. The selection of 1.2 mM iCa as normocalcemia used for blood sampling. Upon completion of the 24-h
Journal of Dairy Science Vol. 107 No. 2, 2024
 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1231
Table 2. Dietary ingredients and average nutrient composition of diets fed pre- and postpartum

Prepartum diet1

Item Positive DCAD Negative DCAD Lactation2

Ingredient (% DM)
Corn silage 49.2 49.2 27.6
Alfalfa haylage — — 24.5
Straw 28.4 28.4 —
Fuzzy cottonseed — — 5.8
Exceller meal 5.4 6.3 3.2
Soybean meal 9.7 3.0 —
Canola meal 5.2 5.2 9.1
Distillers grain — — 2.7
Soy hulls — — 1.8
Fat — — 0.4
Bio-Chlor3 — 6.2 —
Ground corn — — 22.0
Calcium carbonate 0.7 0.8 1.3
Sodium bicarbonate — — 0.9
Magnesium oxide 0.4 0.2 0.1
Salt 0.2 — —
Urea 0.3 0.3 0.2
Prepartum vitamin/mineral mix4 0.4 0.4 —
Postpartum vitamin/mineral mix5 — — 0.4
Nutrients (% DM)
DM 46.7 46.3 50.3
CP 13.9 14.1 17.6
aNDF6 43.6 43.7 31.7
ADF 28.7 28.4 22.7
Starch 21.3 21.9 25.5
NFC 34.1 33.7 38.4
EE7 2.54 2.61 5.01
Ash 8.41 8.79 9.07
Ca 0.55 0.57 0.99
P 0.31 0.33 0.43
Mg 0.39 0.39 0.35
K 1.07 1.05 1.39
Na 0.10 0.11 0.38
S 0.18 0.32 0.22
Cl 0.26 0.71 0.48
DCAD8 (mEq/kg) 130.2 −85.7 250.7
NEL (Mcal/kg) 1.45 1.45 1.60
1
Prepartum diets were fed ad libitum from 251 d of gestation until parturition and formulated for a positive
(+120 mEq/kg) or negative (−120 mEq/kg) DCAD.
2
Postpartum lactation diet was fed ad libitum from the day of parturition until 60 DIM.
3
Bio-Chlor is a patented prepartum fermentation product containing dried condensed extracted glutamic acid
fermentation product, dried condensed corn fermentation solubles, processed grain by-products, and magne-
sium chloride (Arm & Hammer Animal Nutrition, Princeton, NJ). Analysis on a DM basis consisted of CP =
48.63%, K = 1.22%, S = 3.60%, Na = 1.49%, and Cl = 9.08%.
4
Rumensin (Elanco Animal Health, Greenfield, IN): 16 mg/kg of DM and vitamin D at a rate of 2,695 IU/kg
of DM.
5
Rumensin (Elanco Animal Health, Greenfield, IN): 14.4 mg/kg of DM.
6
aNDF = amylase-treated NDF.
7
EE = ether extract.
8
DCAD = [(mEq of Na + mEq of K) − (mEq of Cl + mEq of S)].

infusion the catheter used for CaGlc or dextrose infu- into 10-mL BD Vacutainer Serum (367820, BD, Frank-
sion was not sampled from and only flushed. lin Lakes, NJ) and 10-mL Lithium Heparin 158 USP
From ~4 d (−102 h) before parturition blood was Units (367880, BD, Franklin Lakes, NJ) blood collec-
collected every 6 h through 4 d postpartum (+96 h). tion tubes and inverted gently. Immediately following
Before blood sampling from catheters 8 mL of whole inversion, whole blood was analyzed within 1 to 3 min
blood were drawn and discarded to remove any residual for iCa, pH, HCO3, base excess and partial pressure of
heparinized saline. Whole blood was sampled from a CO2 (pCO2), K, and Na using a cow-side hand-held
jugular catheter every 6 h from −102 h through +96 h biochemical analyzer (i-Stat System, Abbott Laborato-
Journal of Dairy Science Vol. 107 No. 2, 2024
 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1232

ries, Abbott Park, IL). Serum samples were maintained ness Technologies, Palm City, FL). All standards were
at room temperature and allowed to clot before being within the expected calibrated ranges provided by the
placed at 4°C until processing. Serum and plasma were manufacturer during calibration events (Catachem,
harvested from vacutainer collection tubes by centrifu- Oxford, CT). Samples determined by the autoanalyzer
gation at 3,000 × g for 20 min at 4°C. Samples were were read in cuvettes in duplicate and a reference pool
then divided into multiple aliquots and stored at −80°C sample was included and used for assay quality con-
until analysis. trol (Pralle et al., 2021). Methods for Mg and P are
based on the work of Ratge et al. (1986) using xylidyl
Milk Samples blue and Amador and Urban (1972) using ammonium
molybdate, respectively. Intra-assay CV never exceeded
Composite milk samples were collected for the first 10% for determination of blood metabolites and the
4 milkings after parturition and then during the morn- interassay CV for magnesium were 15% and 8% for Mg
ing and evening milking once weekly throughout the and P, respectively.
first 15 weeks of lactation to be analyzed for milk fat,
protein, lactose, and SCC by a commercial laboratory Statistical Analysis
(AgSource, Verona, WI). A secondary composite milk
sample was also collected during the first 4 milkings Data were analyzed using the MIXED procedure of
after parturition into a 10 mL conical and stored at SAS (version 9.4, SAS Institute Inc., Cary, NC). Fixed
−20°C until analysis of tCa. Colostrum was harvested terms in all prepartum models were diet, time, block,
within 8 h of parturition. and the interaction between time and diet. The SLICE
command was used to evaluate prepartum interactions
BW and BCS when P ≤ 0.10. Fixed terms in all postpartum models
included the prepartum mixed model described with
Cows were weighed before enrollment in the experi- additional fixed effects of infusion, and the interac-
ment and then twice weekly during the prepartum pe- tions between infusion and diet, infusion and time,
riod, in the morning, as they went out to exercise. Body and the 3-way interaction of infusion, diet, and time.
condition score was determined before enrollment and Two separate analyses were used for blood metabolites
once weekly by the same trained evaluators using a 1 to in the postpartum period to analyze response during
5 scale (Ferguson et al., 1994). Cows were weighed twice infusion (6 h through 24 h) and response after infu-
weekly during the postpartum period immediately after sion (30 h through 96 h). Time was considered the
milking located in the return alley. Body condition was repeated measure in all analyses and to account for
scored weekly postpartum as described above. autocorrelated errors the ar(1) structure was used for
all analyses except urine pH postpartum, upon which
Mineral Serum and Milk Laboratory Analyses the spatial power structure was used to account for
unequal spacing of samples. The random statements
Total serum Ca (tCa) concentrations were deter- in all models included cow(treatment). Data were ana-
mined using a colorimetric Ca assay (700550, Cayman lyzed for normality and when that assumption failed,
Chemical, Ann Arbor, MI) per manufacturer’s instruc- data were transformed. Transformations were based on
tions as previously described (Laporta et al., 2015) diagnostic plots and overall model fit with either rank
with serum tCa intra- and interassay coefficient of or log-transformation performed on response variables
variation (CV) of 1.98% and 8.41%, respectively. Milk to improve normality. If transformation was required
tCa concentration was determined by colorimetric as- in one analysis, it was thus transformed in the adjoin-
say (700550, Cayman Chemical, Ann Arbor, MI). Milk ing analysis to maintain consistency. The adjustment
samples were thawed and mixed thoroughly. Then, 500 of Tukey was made for all pairwise comparisons. Least
uL of 0.5 M acetic acid was added to 500 uL of sample, squares means and SEM are reported for each variable
mixed, and centrifuged for 12 min at 13,000 × g for 12 and statistical significance was declared at P ≤ 0.05,
min at 4°C. This allowed for digestion and precipitation with tendencies for differences declared at 0.05 ≤ P ≤
of fat from the milk. After centrifugation, supernatant 0.10.
was pipetted off and diluted to a final concentration of
1:50 with ddH20. The intra- and interassay CV of milk RESULTS
tCa were 11% and 4.09%, respectively.
Serum magnesium (Mg) and phosphorus (P) were Of the 31 cows included in the statistical analyses, 3
determined using Catachem VETSPEC reagents on cows (1 NCa, 1 PCa, and 1 PDex) experienced clini-
the ChemWell-T AutoAnalyzer (ChemWell-T, Aware- cal hypocalcemia (when a cow was unable to rise and
Journal of Dairy Science Vol. 107 No. 2, 2024
 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1233

confirmed by iCa <0.7 mM) and were treated with in- cows had decreased iCa (1.04 vs. 1.15 ± 0.02 mM, re-
travenous CaGlc infusion on d 1 of lactation. Therefore, spectively; P = 0.002) and tCa concentrations (2.01
those 3 cows were removed prematurely from the 6-h vs. 1.86 ± 0.06 mM, respectively; P = 0.04) relative
time analysis postpartum and contributed data from to dextrose infused cows. Further, a tendency for iCa
−102 h up until Ca treatment. Disease incidences were concentrations was observed for a diet by infusion by
recorded throughout the experiment and were: NCa (1 time interaction (P = 0.06), with CaGlc infused cows
retained placenta, 1 clinical hypocalcemia), NDex (2 re- exhibiting a rapid decline in iCa after termination of
tained placenta), PCa (1 clinical hypocalcemia), PDex infusion followed by a corresponding increase once iCa
(2 retained placenta, 3 ketosis, 1 displaced abomasum, concentrations recovered. Positive DCAD CaGlc cows
1 clinical hypocalcemia). Subclinical ketosis was identi- experienced a calcemic nadir 12 h after termination of
fied and diagnosed by barn and veterinary staff via infusion (36 h) and had decreased iCa concentrations
urine Ketostix and a confirmation of serum BHB >1.20 relative to all other treatments (0.89 mM; P < 0.03),
mM. However, these cows were included in the analyses whereas NCa cows experienced their lowest blood iCa
for dry matter intake and milk yield. concentrations 18 h after termination of infusion (42
h) and had decreased iCa concentrations compared
Ionized Ca and tCa Concentrations with dextrose infused cows on both diets (0.91 mM;
P < 0.04). After the calcemic nadir occurred in PCa
During the 4 d before parturition cows fed a prepar- and NCa cows iCa concentrations increased in both
tum negative DCAD diet had higher iCa concentra- groups for the remainder of the experiment. Moreover,
tions relative to cows fed a positive DCAD diet (1.21 PDex and NDex cows both had iCa concentrations that
vs. 1.18 ± 0.01 mM, respectively; P = 0.03; Figure steadily increased from 30 to 96 h postpartum. Further,
1A). An effect of time was observed prepartum with NCa cows had the lowest iCa concentrations at 90 h
all cows, regardless of diet, experiencing a transient de- postpartum relative to all other treatments (P < 0.03).
cline in iCa and tCa concentrations as they approached Infusion of CaGlc increased iCa concentrations
parturition (P < 0.0001 and P < 0.0001, respectively). relative to dextrose infusion (1.21 vs. 1.02 ± 0.02 mM,
Moreover, from 30 h before parturition until parturition respectively; P < 0.0001; Figure 2A). No differences
cows fed a negative DCAD diet prepartum had higher were detected in iCa concentrations between PCa and
iCa concentrations than cows fed a positive DCAD diet NCa cows (1.21 vs. 1.22 mM ± 0.03, respectively; P >
(P < 0.05). As cows approached parturition blood iCa 0.05) across the 24-h infusion period. Negative DCAD
concentrations decreased earlier relative to parturition dextrose infused cows had increased iCa concentrations
in positive DCAD-fed cows (positive DCAD −24 h vs. relative to PDex cows during the 24 h postpartum (1.05
0 h, P = 0.01) compared with negative DCAD-fed cows vs. 0.98 mM ± 0.03, P < 0.0001). Effects of both time
(negative DCAD −18 h vs. 0 h, P < 0.0001). (P < 0.0001) and infusion by time (P = 0.002) were
Immediately after parturition (0 h) and before infu- observed, with iCa concentrations oscillating across the
sion, cows fed a negative DCAD diet had greater iCa 24-h period in all treatments. Depending on the sam-
concentrations than cows fed a positive DCAD diet pling time point, PDex cows ranged from 0.95 to 1.02
(1.06 vs. 0.98 ± 0.02 mM, respectively; P = 0.008) but mM, NDex cows ranged from 1.03 to 1.08 mM, PCa
no differences were detected in tCa concentrations (1.80 ranged from 1.06 to 1.24 mM, and NCa cows ranged
vs. 1.76 ± 0.05 P > 0.05). Infusion of CaGlc robustly from 1.14 to 1.25 mM iCa concentrations. Cows fed a
increased blood iCa concentrations relative to dextrose positive DCAD diet prepartum required higher rates
infused cows (1.22 vs. 1.02 ± 0.02 mM, respectively; of CaGlc infusion (P = 0.03) in the 24 h after parturi-
P < 0.0001) and increased blood tCa concentrations tion to maintain normocalcemia relative to cows fed a
(2.19 vs. 1.77 ± 0.05 mM, respectively; P < 0.0001). negative DCAD diet prepartum (70.36 vs. 54.68 ± 4.59
An effect of time was observed (P = 0.02) with iCa mL/h, respectively; Figure 2B-2C). However, before
concentrations decreasing at 18 h postpartum and then termination of infusion (22 to 24 h) negative DCAD
increasing 6 h later. Positive DCAD dextrose infused cows required similar rates of CaGlc infusion relative to
cows tended to have decreased iCa concentrations positive DCAD cows (negative DCAD = 76.25 mL/h,
compared with NDex cows during the 24 h postpartum positive DCAD 70.63 mL/h). A diet by time interac-
(P = 0.07; Figure 1A), but no differences were detected tion (P = 0.01) was observed with positive DCAD cows
in iCa or tCa concentrations between PCa and NCa requiring greater phasic occurrences in CaGlc infusion
cows during the 24-h infusion period (P > 0.05). across the experimental period. No differences were
No effect of diet was observed from 30 through 96 h observed due to infusion after termination of infusion
after parturition (P > 0.05); however, CaGlc infused (P > 0.05).

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1234

Figure 1. Whole blood ionized Ca (iCa; A) and total Ca (tCA; B) concentrations from 102 h before parturition to 96 h after parturition
of cows fed a negative (-DCAD, −120 mEq/kg) or positive (+DCAD, +120 mEq/kg) DCAD diet and infused with Ca gluconate (NCa: CaGlc
infusion after negative DCAD prepartum diet; and PCa: CaGlc infusion after positive DCAD prepartum diet) or 10% dextrose (NDex: dextrose
infusion after negative DCAD prepartum diet; and PDex: dextrose infusion after positive DCAD prepartum diet) for 24 h after parturition.
Prepartum whole blood iCa concentrations: diet (P = 0.03), time (P < 0.0001). Infusion whole blood iCa concentrations: infusion (P < 0.0001),
time (P < 0.0001). After termination whole blood iCa concentrations: infusion (P = 0.002), time (P < 0.0001), diet × time (P = 0.07), diet ×
infusion × time (P = 0.06). Prepartum serum tCa concentrations: time (P < 0.0001). Infusion serum tCa concentrations: infusion (P < 0.0001),
diet × infusion (P = 0.08). After termination serum tCa concentrations: time (P < 0.0001), infusion × time (P < 0.04). Error bars represent
standard error of the mean.

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1235

Figure 2. Whole blood ionized Ca (iCa) concentrations of cows fed a negative (−120 mEq/kg) or positive (+120 mEq/kg) DCAD diet and
infused with Ca gluconate (NCa: CaGlc infusion after negative DCAD prepartum diet; and PCa: CaGlc infusion after positive DCAD prepar-
tum diet) or 10% dextrose (NDex: dextrose infusion after negative DCAD prepartum diet; and PDex: dextrose infusion after positive DCAD
prepartum diet) for 24 h after parturition (A), infusion rates of cows fed a negative or positive DCAD diet and infused with Ca gluconate or
10% dextrose for 24 h after parturition (B), and mean averages of infusion rates (C) across the infusion period. Whole blood iCa concentrations:
infusion (P < 0.0001), time (P < 0.0001), infusion × time (P < 0.0001). Infusion rate: diet (P = 0.004), time (P < 0.0001), diet × time (P =
0.01). Error bars represent standard error of the mean.

Magnesium and P Concentrations blood P concentrations as they approached parturition

(P < 0.0001). No effect of diet was observed during the
Prepartum serum Mg concentrations did not dif- infusion period. The CaGlc infusion tended to elevate
fer between diets (P > 0.05). A tendency for a diet blood P concentrations relative to dextrose infusion
by time effect was observed (P = 0.10) as negative (P = 0.06; Figure 3B) during the infusion period. As
DCAD-fed cows had a greater oscillation in serum Mg cows progressed through the 24-h infusion period, all
the 102 h before parturition (Figure 3A). No difference cows declined in blood P concentrations, with a nadir
was observed in serum Mg concentrations due to diet reached at 24 h postpartum (Figure 3B). In the period
(P > 0.05) during the 24-h infusion period; however, after termination of infusion CaGlc infused cows tended
cows infused with CaGlc had lower serum Mg concen- to have lower (P = 0.07) serum P concentrations than
trations (P = 0.10) than dextrose infused cows. A time dextrose infused cows. A tendency for a time effect
(P = 0.001) and an infusion by time interaction (P = was observed (P = 0.07) as serum P concentrations
0.10) was observed during the infusion period as all increased steadily from 30 to 96 h postpartum (Figure
cows had a decline in serum Mg concentrations, and 3B).
cows infused with CaGlc exhibited a greater reduction
in serum Mg concentrations during the infusion period Acid-Base Status
(Figure 3A).
Serum P did not differ due to diet prepartum Cows fed a negative DCAD diet had decreased urine
(Figure 3B). Regardless of diet, all cows declined in pH concentrations across the prepartum period (P <
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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1236

Figure 3. Serum magnesium (A) and phosphorus (B) concentrations of cows fed a negative (-DCAD, −120 mEq/kg) or positive (+DCAD,
+120 mEq/kg) dietary cation-anion difference diet and infused with Ca gluconate (NCa: CaGlc infusion after negative DCAD prepartum diet;
and PCa: CaGlc infusion after positive DCAD prepartum diet) or 10% dextrose (NDex: dextrose infusion after negative DCAD prepartum diet;
and PDex: dextrose infusion after positive DCAD prepartum diet) for 24 h after parturition. Prepartum serum magnesium concentrations: diet
× time (P = 0.10). Infusion serum magnesium concentrations: infusion × time (P = 0.10), time (P = 0.001), time (P = 0.008). After termina-
tion serum magnesium concentrations: infusion × time (P = 0.08), diet × infusion × time (P = 0.10). Prepartum serum phosphorus concen-
trations: time (P < 0.0001). Infusion serum phosphorus concentrations: infusion (P = 0.06), time (P < 0.0001). After termination phosphorus
concentrations:​infusion (P = 0.07), time (P = 0.07), diet × infusion × time (P = 0.04). Error bars represent standard error of the mean.

0.0001) relative to cows fed a positive DCAD diet (6.03 4, and 5). A diet by day effect occurred (P = 0.0004)
vs. 8.16 ± 0.07 pH, respectively). No differences in with cows fed a negative DCAD diet prepartum having
urine pH were detected due to diet or day (P > 0.05) lower urine pH concentrations on the immediate day
postpartum, however an interaction of infusion by day postpartum.
was significant (P < 0.0001) as CaGlc infused cows had Cows fed a negative DCAD diet prepartum had de-
lower urine pH on d 0 and d 1 of lactation (Tables 3, creased blood pH relative to cows fed a positive DCAD

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1237
Table 3. Effect of DCAD on acid-base status and blood parameters in relative to dextrose infusion (P = 0.03; Table 4) and
Holstein cows prepartum (LSM ± SEM)
feeding a negative DCAD diet prepartum decreased
Item1 Negative DCAD Positive DCAD P-value blood pH relative to a positive DCAD diet (P = 0.002).
Blood
Similar to blood pH, HCO3 and base excess were in-
pH 7.405 (0.005) 7.439 (0.005) <0.0001 creased (P = 0.02 and P = 0.01, respectively; Table 4)
HCO3 (mM) 25.02 (0.39) 29.00 (0.39) <0.0001 in response to CaGlc infusion. Additionally, cows fed a
BE (mM) 0.36 (0.42) 4.96 (0.44) <0.0001 negative DCAD diet prepartum had decreased blood
pCO2 (mm Hg) 39.99 (0.56) 42.9 (0.50) 0.002
K (mM) 3.90 (0.04) 3.71 (0.04) 0.0004 HCO3 (P = 0.0002), base excess (P = 0.0003) and pCO2
Na (mM) 143.70 (0.24) 144.41 (0.23) 0.06 (P = 0.003) relative to positive DCAD-fed cows (Table
Urine pH 6.03 (0.07) 8.16 (0.07) <0.0001 3). We observed no effect of infusion on pCO2, Na or K
1
HCO3 = bicarbonate; BE = base excess; pCO2 = partial pressure of concentrations (P > 0.05; Table 4). However, negative
CO2.
DCAD-fed cows had elevated Na and K concentrations
(P = 0.03 and P = 0.003; Table 4) relative to positive
diet during the prepartum period (7.405 vs. 7.439 ± DCAD cows. We observed no effect of diet, infusion,
0.005, respectively; P < 0.0001; Table 3). Additionally, or the interaction of the 2 after termination of infusion
blood HCO3 (25.02 vs. 29.00 ± 0.39; P < 0.0001), base (30 h postpartum through 96 h postpartum) on blood
excess (0.36 vs. 4.96 ± 0.44; P < 0.0001), and pCO2 pH, HCO3, base excess, pCO2, K, and Na (P > 0.05;
(39.99 vs. 42.90 ± 0.56; P = 0.002) were decreased in Table 5). However, negative DCAD cows did tend to
cows fed negative DCAD prepartum (Table 3). Blood have higher blood potassium concentrations (P = 0.06;
Na concentrations tended to be decreased (143.70 vs. Table 5).
144.41 ± 0.24; P = 0.06) in cows fed a negative DCAD
prepartum, whereas blood K concentrations were sig- DMI and Production Parameters
nificantly increased (3.90 vs. 3.71 ± 0.04; P = 0.0004;
Table 3) in cows fed a negative DCAD diet. During the Dry matter intake was not different between cows
24-h infusion period CaGlc infusion increased blood pH fed a negative DCAD or positive DCAD diet prepar-

Table 4. Effect of DCAD and infusion1 on acid-base status on blood parameters in Holstein cows during the experimental infusion period (LSM
± SEM)

Negative DCAD Positive DCAD P-value

Item2 Ca Dex Ca Dex DCAD Infusion DCAD × Infusion

Blood
pH 7.449 (0.008) 7.437 (0.008) 7.487 (0.008) 7.458 (0.009) 0.002 0.03 0.41
HCO3 (mM) 29.62 (0.78) 27.39 (0.78) 33.32 (0.86) 31.48 (0.86) 0.0002 0.02 0.85
BE (mM) 5.59 (0.88) 3.25 (0.88) 9.94 (0.97) 7.18 (0.97) 0.0003 0.01 0.82
pCO2 (mm Hg) 42.50 (0.75) 40.67 (0.76) 44.19 (0.81) 44.08 (0.81) 0.003 0.24 0.48
K (mM) 3.93 (0.07) 3.85 (0.07) 3.74 (0.08) 3.66 (0.08) 0.003 0.21 0.78
Na (mM) 144.72 (0.62) 145.12 (0.62) 143.16 (0.68) 144.2 (0.68) 0.03 0.30 0.76
1
Ca = calcium gluconate infusion; Dex = 10% dextrose infusion.
2
HCO3 = bicarbonate; BE = base excess; pCO2 = partial pressure of CO2.

Table 5. Effect of DCAD and infusion1 on acid-base status on blood parameters in Holstein cows after termination of infusion (LSM ± SEM)

Negative DCAD Positive DCAD P-value

Item2 Ca Dex Ca Dex DCAD Infusion DCAD × infusion

Blood
pH 7.465 (0.008) 7.458 (0.007) 7.447 (0.008) 7.446 (0.009) 0.13 0.67 0.91
HCO3 (mM) 30.96 (0.78) 30.37 (0.76) 30.78 (0.78) 30.33 (0.92) 0.91 0.70 0.86
BE (mM) 7.07 (0.79) 6.50 (0.77) 6.61 (0.79) 6.22 (0.92) 0.66 0.56 0.92
pCO2 (mm Hg) 42.45 (1.08) 42.90 (1.05) 44.50 (1.08) 44.26 (1.26) 0.21 0.90 0.87
K (mM) 3.90 (0.05) 3.93 (0.05) 3.73 (0.05) 3.86 (0.05) 0.06 0.13 0.26
Na (mM) 42.67 (0.32) 142.85 (0.31) 143.3 (0.32) 142.50 (0.31) 0.76 0.36 0.20
Urine pH 7.96 (0.05) 8.09 (0.05) 8.16 (0.05) 8.07 (0.06) 0.76 0.51 0.28
1
Ca = calcium gluconate infusion; Dex = 10% dextrose infusion.
2
HCO3 = bicarbonate; BE = base excess; pCO2 = partial pressure of CO2.

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1238

tum (P > 0.05). However, an effect of day was evident Na, pCO2 and HCO3 and an increase in blood K of
(P < 0.0001) with DMI decreasing as cows approached cows. Prepartum negative DCAD diets are a commonly
parturition (Figure 4A). Postpartum dry matter in- used strategy to mitigate hypocalcemic risk at the on-
take tended (P = 0.09) to be greater for cows fed a set of lactation and improve metabolism and lactation
negative DCAD prepartum. Asignificant effect of day performance (Block, 1984; Martinez et al., 2018b; Rod-
(P < 0.0001) was observed as DMI increased as cows ney et al., 2018). Negative DCAD diets elicit their ac-
progressed into lactation and a tendency (P = 0.06) for tions by inducing a mild metabolic acidosis via feeding
higher DMI in CaGlc infused cows (Figure 4A) was also excess anions (Block, 1984), which allows for improved
observed. Milk yield did not differ with diet or infusion Ca metabolism through a variety of mechanisms, some
(P > 0.05); however, an infusion by DIM interaction of which are increased ruminal Ca transport (Wilkens
occurred as dextrose infused cows had greater volatility et al., 2016), increased parathyroid hormone (PTH)
in milk yield during the first 50 DIM (P = 0.004; Fig- secretion (Lopez et al., 2002) and tissue sensitivity to
ure 4B), specifically in the PDex treatment. Milk yield PTH (Goff et al., 2014), and stimulation of osteoclast
increased in all cows (P < 0.0001) as DIM increased. activity (Krieger et al., 1992) to collectively facilitate a
Weekly milk components did not differ due to diet or readily exchangeable Ca pool. Additionally, when cows
infusion for fat content, protein content, lactose con- were challenged with EDTA, a nonspecific Ca chelator,
tent, or SCS (Table 6). A diet by infusion interaction cows fed a reduced DCAD were able to maintain blood
was observed for lactose content (Table 6; P < 0.01). Ca concentrations more readily (Giesy et al., 1997).
Calcium content in milk was not significantly differ- Our data supports this collective action as cows infused
ent due to diet or infusion during the first 6 milkings with CaGlc and fed a negative DCAD diet prepartum
postpartum (Table 6). (NCa) required lower rates of infusion to maintain
Body condition score was not different due to diet normocalcemia relative to PCa cows. Thus, suggest-
pre- or postpartum (P > 0.05). Body condition score ing cows fed a negative DCAD require less support
postpartum was lower (P = 0.04) in dextrose infused (exogenous Ca) to maintain normocalcemia during the
cows (3.27) compared with CaGlc infused cows (3.35). immediate period postpartum due to prepartum acido-
All cows declined in BCS as they progressed into lacta- genic exchangeable Ca pool benefits and increased Ca
tion. Body weight was not different pre- or postpartum flux.
due to diet (P > 0.05). Neither diet or infusion altered Interestingly, phasic increases in infusion rate were
BW postpartum (P > 0.05). observed in both groups of cows at different times
across the 24-h period, with PCa cows exhibiting larger
DISCUSSION changes in infusion rates to maintain 1.2 mM iCa con-
centrations; however, both NCa and PCa exhibited
The onset of lactation results in dynamic changes in similar trends in modulation of required CaGlc infusion
mineral metabolism as the peripartal cow undergoes rate increases to maintain normocalcemia across the
a rapid shift in physiology to adapt to the demands 24-h after parturition. Upon initiation of infusion cows
of lactation. In the dairy cow the shift in Ca metabo- on both diets promptly increased iCa and reached iCa
lism is exacerbated due to the massive increase in Ca maintenance levels, with PCa cows exhibiting a slightly
requirements between late gestation and early lacta- slower increase to iCa maintenance concentrations,
tion. Calcium research has sought to understand and despite higher rates of CaGlc infusion than NCa cows.
characterize the magnitude and duration of Ca decline Moreover, cows on both diets required phasic increases
at the onset of lactation and factors that modulate this in infusion rates beginning at 4 to 5 h and at 18 to 19
decline. The data herein demonstrates prepartum nega- h postpartum. Previous work using the Ca chelators
tive DCAD diets influence the magnitude and duration EDTA and EGTA to induce subclinical hypocalcemia
of Ca decline. Moreover, manipulating the decline in suggested changes in blood Ca concentrations indicate
blood Ca immediately postpartum severely disrupts the timing of adaptive responses to blood Ca which
blood Ca dynamics. are representative of various systems critical for Ca ho-
The present data corroborates the role of negative meostasis (Ramberg et al.,1967; Connelly et al., 2022).
DCAD in improving Ca metabolism as cows fed a Although this study maintained normocalcemia, rather
negative DCAD diet prepartum maintained elevated than inducing hypocalcemia, an interesting overlap
blood tCa and iCa concentrations as they approached of phase characterization in response to the timing of
parturition and had a smaller magnitude of decline in blood Ca changes suggest that similar systems are be-
tCa and iCa. Feeding a negative DCAD diet prepartum ing activated or deactivated in PCa and NCa cows.
induced a mild metabolic acidosis evident by the pre- These similar phasic increases in CaGlc requirements
partum reductions in urine pH, blood pH, base excess, to maintain normocalcemia indicate a Ca draw from
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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1239

Figure 4. Dry matter intake (A) and milk yield (B) of cows fed a negative (-DCAD, −120 mEq/kg) or positive (+DCAD, +120 mEq/kg)
dietary cation-anion difference diet and infused with Ca gluconate (NCa: CaGlc infusion after negative DCAD prepartum diet; and PCa: CaGlc
infusion after positive DCAD prepartum diet) or 10% dextrose (NDex: dextrose infusion after negative DCAD prepartum diet; and PDex:
dextrose infusion after positive DCAD prepartum diet) for 24 h after parturition. Prepartum DMI: time (P < 0.0001). Postpartum DMI: diet
(P = 0.09), time (P < 0.0001), infusion (P = 0.06). Milk yield: time (P < 0.0001), infusion × DIM (P = 0.004). Error bars represent standard
error of the mean.

physiological systems involved in Ca homeostasis at cows at similar time points postpartum, it is a termina-
time points that are relative to parturition itself, or a tion of a particular physiological system or depletion
deactivation process of systems previously stimulated of Ca stores that limit the ability to support blood Ca
by prepartum decline as exogenous CaGlc is supplied. concentrations. Further, despite NCa requiring lower
Although the current study cannot definitively answer rates of CaGlc infusion across the infusion period, ~22
this question, we hypothesize based on the numerical h after parturition during CaGlc infusion, NCa cows
increases or maintenance in iCa in NDex and PDex began to require similar infusion rates of CaGlc as PCa

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1240
Table 6. Effect of DCAD and infusion1 on postpartum daily milk yield, weekly milk composition, and milk Ca during the first 6 milkings (LSM
± SEM)

Negative DCAD Positive DCAD P-value

Variable Ca Dex Ca Dex DCAD Infusion DCAD × infusion

Milk yield (kg/d) 44.83 (3.02) 43.36 (3.01) 48.02 (2.88) 40.66 (3.61) 0.94 0.18 0.36
Milk composition (%)
Fat content 4.25 (0.19) 4.05 (0.17) 4.05 (0.18) 4.12 (0.20) 0.23 0.31 0.77
Protein content 3.51 (0.31) 3.20 (0.30) 3.47 (0.31) 3.07 (0.33) 0.47 0.81 0.31
Lactose content 4.83 (0.07) 4.91 (0.07) 4.82 (0.07) 4.96 (0.08) 0.73 0.26 0.01
SCS 113 (37) 100 (34) 86 (37) 79 (40) 0.35 0.74 0.37
Ca content (g/L) 2.35 (0.08) 2.44 (0.08) 2.08 (0.07) 2.57 (0.08) 0.87 0.17 0.25
1
Ca = calcium gluconate infusion; Dex = 10% dextrose infusion.

cows to maintain normocalcemia (Figure 2B, 2C). This postpartum may characterize the periparturient Ca
observation further suggests calcemic systems activated profile inaccurately. The data herein, Horst and Jor-
prepartum due to metabolic acidosis which allowed for gensen (1982), and Megahed et al. (2018) suggest that
greater endogenous blood Ca maintenance are no lon- blood Ca declines much earlier than previously thought
ger present. Moreover, by 24 h postpartum urine and and may not be properly captured in studies with less
blood pH were increasing and thus metabolic acidosis intensive sampling and standardization.
conditions at which infusions were initiated were no Blood Ca remained decreased across the entire 24-h
longer present to the same extent. postpartum period in dextrose infused cows, with nota-
Most studies examining blood Ca dynamics at the ble increases in iCa concentrations not occurring until
onset of lactation collect samples daily, with few ex- ~36 h postpartum. This supports Megahed et al. (2018)
periments fully capturing the decline in blood Ca con- who found that blood Ca remained decreased for at least
centrations as cows approach parturition. Thus, due to 28 h after parturition. Although timing of the calcemic
sampling technique and time points the entirety and nadir looks relatively similar in 6-h sampling frequency
rate of the magnitude of Ca decline at the onset of in PDex and NDex, 3-min iCa analysis indicated that
lactation may potentially be missed and different Ca the calcemic nadir occurs earlier during the 24 h after
profiles may be captured and classified inaccurately. parturition in PDex relative to NDex cows. However,
Research has suggested that blood Ca concentrations the calcemic nadir is a relative term as blood Ca re-
may start to decline 9 h before parturition (Megahed mains low the entire 24-h period. After termination of
et al., 2018), with the present data corroborating and infusion iCa concentrations rapidly decreased in CaGlc
expanding the time at which blood Ca declines before infused cows with the calcemic nadir being achieved
parturition. Horst and Jorgensen (1982) also observed more rapidly in PCa cows (36 h) than NCa cows (42 h).
similar declines in blood Ca concentrations before par- Hypercalcemia due to intravenous Ca infusion results
turition, with cows that became paretic or borderline in a corresponding hypocalcemia (Blanc et al., 2014).
paretic experiencing a decline in blood Ca up to 24 h Calcium gluconate infusion seemingly delayed the
before parturition, whereas nonparetic cows were nor- transient hypocalcemia experienced in PDex and NDex
mocalcemic 12 h before parturition. Interestingly, blood cows, with the transient hypocalcemia occurring after
Ca concentrations began to decline in positive DCAD termination of infusion in CaGlc infused cows. Interest-
cows, that are more susceptible to hypocalcemia, ~18 h ingly, during the corresponding transient hypocalcemia
before parturition, whereas negative DCAD cows, that in PCa and NCa cows, cows on both diets were able to
are less susceptible to hypocalcemia, exhibited a decline increase iCa concentrations earlier during the transient
in iCa concentrations 12 h before parturition. However, hypocalcemia experienced than PDex and NDex cows.
regardless of timing of initiation of blood Ca declines, This suggests a greater magnitude of Ca decline may
cows on both diets saw further subsequent declines in stimulate more rapid and robust feedback mechanisms
blood Ca concentrations at each successive 6 h time to improve blood Ca concentrations. It is important
point as cows approached parturition. Moreover, this to consider within the discussion of cow response that
poses interesting consideration when discussing blood variability exists between animals on biological Ca set-
Ca profiles around parturition and studies that aim point. Within the current study we assumed a set point
to evaluate the magnitude of Ca decline and rate of of 1.2 mM for eucalcemia to administer infusion treat-
decline as 24-h sampling intervals or sampling times ment, but variation may still exist between cow’s for
not standardized to the anticipated time of parturition response due to our selected eucalcemia set-point being

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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1241

higher or lower than the cow’s biological Ca set-point. concentrations increases renal phosphate loss and in-
A difference in biological set-point (i.e., 1.0–1.1 mM) creases salivary secretion of phosphorus (Wright et al.,
could have resulted in less robust blood Ca responses, 1982), and a reduction in PTH secretion may contribute
with cows experiencing lower blood Ca at the onset of to the elevation in blood P in response to CaGlc infu-
lactation having a reduced ability to maintain blood sion during the 24 h postpartum. Interestingly, in the
Ca and respond to calcemic fluctuations. Urinary Ca after termination period blood P concentrations were
concentrations are not available to determine the excre- reduced due to CaGlc infusion. The opposite effect,
tion rates of Ca in our study, but would be relevant to an increase in renal phosphate and salivary secretion
examine in future studies. due to increased PTH secretion and action could give
When evaluating the 6 h sampling frequency the rise to the reduced blood P concentrations. Serum Mg
present data supports previous studies demonstrat- concentrations were lower in CaGlc infused cows than
ing the benefits of iCa concentration maintenance dextrose cows in the 24-h infusion period. Elevated
at the onset of lactation due to prepartum negative blood Ca decreases renal reabsorption of Mg in the loop
DCAD diets. Cows fed a prepartum negative DCAD of Henle (Quamme, 1989) and could contribute to lower
diet experienced a smaller magnitude of Ca decline serum Mg concentrations. Given that both Na and K
as they approached parturition and immediately after are included in the calculations for the DCAD equation
parturition. Cows fed a positive DCAD diet exhibited [(Na + K) − (Cl + S)], we examined concentrations
a more rapid decline in blood Ca as they approached of Na and K in the blood. Our findings that the nega-
parturition relative to cows fed a negative DCAD diet. tive DCAD diet increased blood K and decreased blood
However, no differences were observed in rate of return Na are consistent with an induced metabolic acidosis.
to normocalcemia postpartum, with PDex and NDex Acidemia shifts K from intracellular stores to extracel-
cows increasing blood Ca concentrations in similar lular space (Aronson and Giebisch, 2011) and the body
fashions beginning at ~30 to 36 h postpartum. From will conserve K at the expense of Na, which supports
when Ca increased postpartum in PDex and NDex our findings of increased blood K and decreased Na
cows and considering the prepartum blood Ca decline, concentrations (Block, 1984).
beginning at 12 h (negative DCAD) and 18 h (positive Dry matter intake was only numerically decreased in
DCAD) postpartum, an approximate 2 to 3 d transient negative DCAD-fed cows prepartum. Negative DCAD
hypocalcemia occurred in all cows. A decline in blood diets reduce dry matter intake due to metabolic acido-
Ca multiple hours before parturition and maintenance sis and corresponding acid-base status changes (Zimpel
for ~48 h supports the physiological events required for et al., 2018). Prepartum urine pH, blood pH, HCO3,
PTH to be able to exert action, as PTH requires ~48 base excess and pCO2 were all significantly reduced in
h of action to activate Ca support mechanisms and cows fed a negative DCAD diet, which is consistent
mobilize Ca reservoirs (Goff et al., 1986; Martín-Tereso with studies demonstrating that a mild metabolic aci-
and Verstegen, 2011) to return blood to normocalce- dosis was induced through feeding supplemental anions
mia. Congruently, cows fed a negative DCAD diet have (Rodney et al., 2018; Zimpel et al., 2018). Postpartum
been suggested to have increased PTH secretion and DMI tended to be higher in cows fed a negative DCAD
action (Lopez et al., 2002; Goff et al., 2014) and a more diet and infused with CaGlc, which may be due to the
labile Ca pool available to support a return more read- larger number of morbidity incidences in the positive
ily to normocalcemia (Lean et al., 2014; Rodney et al., DCAD and dextrose infused cows. This is congruent
2018). Thus, prepartum negative DCAD diets improve with the literature as negative DCAD diets have been
resistance to Ca decline at the onset of lactation and shown to improve DMI postpartum (Lean et al., 2019)
facilitate a more transient hypocalcemia at the onset of and supplemental Ca in the form of boluses has shown
lactation relative to positive DCAD diets. benefit in multiparous cows with low Ca at parturition
Feeding a negative DCAD diet prepartum did not and suggested to improve health (Leno et al., 2018).
alter prepartum blood P concentrations and cows on Milk yield was lowest in PDex cows, which could be due
all diets experienced the classical decline in blood P as to both lower DMI and increased incidences of disease.
cows approach parturition and during the 24-h infu- However, the current study was not powered to analyze
sion period (Goff et al., 2002). Blood P was increased binomial outcomes, the main focus was investigating
in response to CaGlc infusion during the 24-h infusion physiological interactions surrounding the periparturi-
period, which corresponds to Wilms et al. (2022) who ent period. Cows that presented with a postpartum
observed an increase in serum P concentrations as disease in the PDex group were left in the dataset due
blood Ca increased in response to intravenous infusion to all corresponding diseases being potential effects of
of Ca to cows immediately postpartum. Parathyroid prepartum dietary DCAD treatment (Martinez et al.,
hormone, which is secreted in response to low blood Ca 2018a; Venjakob et al., 2021). We did not observe al-
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 Connelly et al.: CALCIUM DECLINE AND MINERAL METABOLISM 1242

terations in milk composition due to dietary treatments ment of subclinical hypocalcemia with oral or intravenous calcium.
J. Dairy Sci. 97:6901–6906. https:​/​/​doi​.org/​10​.3168/​jds​.2014​-7927.
except during the postpartum period where a DCAD × Block, E. 1984. Manipulating dietary anions and cations for prepartum
infusion effect on milk lactose content occurred. Lactose dairy cows to reduce incidence of milk fever. J. Dairy Sci. 67:2939–
content was lowest in CaGlc infused cows, who made 2948. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​-0302(84)81657​-4.
Block, E. 1994. Manipulation of dietary cation-anion difference on nu-
more milk than their dextrose infusion counterparts on tritionally related production diseases, productivity, and metabolic
the same diet. Although significantly different, large responses of dairy cows. J. Dairy Sci. 77:1437–1450. https:​/​/​doi​
variations were observed in milk yield and components .org/​10​.3168/​jds​.S0022​-0302(94)77082​-X.
Bushinsky, D. A., W. Wolbach, N. E. Sessler, R. Mogilevsky, and
and likely of little biological relevance. R. Levi-Setti. 1993. Physicochemical effects of acidosis on bone
calcium flux and surface ion composition. J. Bone Miner. Res.
8:93–102. https:​/​/​doi​.org/​10​.1002/​jbmr​.5650080112.
CONCLUSIONS Connelly, M. K., S. R. Henschel, J. M. Kuehnl, A. A. Cheng, F.
Nashold, and L. L. Hernandez. 2022. Physiological adaptations
Negative DCAD diets improved blood Ca concentra- in early-lactation cows result in differential responses to calcium
tions in the immediate days around parturition. Cows perturbation relative to nonlactating, nonpregnant cows. J. Dairy
Sci. 105:904–920. https:​/​/​doi​.org/​10​.3168/​jds​.2021​-20890.
fed a negative DCAD diet experienced a more transient Ferguson, J. D., D. T. Galligan, and N. Thomsen. 1994. Principle de-
hypocalcemia at the onset of lactation, with a smaller scriptors of body condition score in Holstein cows. J. Dairy Sci.
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ACKNOWLEDGMENTS during induced and spontaneous hypocalcemia in ruminants.
J. Dairy Sci. 65:2332–2337. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​
Financial support for the project was provided by -0302(82)82505​-8.
Krieger, N. S., N. E. Sessler, and D. A. Bushinsky. 1992. Acidosis
Arm & Hammer, Scibus and student support provided inhibits osteoblastic and stimulates osteoclastic activity in vitro.
by the United States Department of Agriculture-Na- Am. J. Physiol. 262:F442–448. https:​/​/​doi​.org/​10​.1152/​a jprenal​
tional Institute of Food and Agriculture (#2016-67015- .1992​.262​.3​.F442.
Laporta, J., S. A. E. Moore, S. R. Weaver, C. M. Cronick, M. Olsen,
34584; Washington, DC). The authors thank all of the A. P. Prichard, B. P. Schnell, T. D. Crenshaw, F. Penagaricano, R.
Dairy Cattle Center Staff at the University of Wiscon- M. Bruckmaier, and L. L. Hernandez. 2015. Increasing serotonin
sin–Madison (Luke Kastenson, Caleb Hamm, Brenda concentrations alter calcium and energy metabolism in dairy cows.
J. Endocrinol. 226:43–55. https:​/​/​doi​.org/​10​.1530/​JOE​-14​-0693.
Jelle; Madison, WI) and the fantastic assistance of the Lean, I. J., P. J. DeGaris, P. Celi, D. M. McNeill, R. M. Rodney, and
RARC veterinary staff (Mike Maroney, Kay Nelson, D. R. Fraser. 2014. Influencing the future: Interactions of skeleton,
Jane Reimann, and Taylor Legried). The authors have energy, protein and calcium during late gestation and early lac-
tation. Anim. Prod. Sci. 54:1177–1189. https:​/​/​doi​.org/​10​.1071/​
not stated any conflicts of interest. AN14479.
Lean, I. J., J. E. Santos, E. Block, and H. M. Golder. 2019. Effects
of prepartum dietary cation-anion difference intake on production
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