EFFECT OF ORAL CALCIUM BOLUS SUPPLEMENTATION ON RUMINATION AND

ACTIVITY PATTERNS IN EARLY LACTATION DAIRY COWS

By

MYRIAM BERENICE JIMENEZ MEDRANO

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER IN SCIENCE

UNIVERSITY OF FLORIDA

2017

© 2017 Myriam Berenice Jimenez Medrano

To my husband and family, who have never stopped loving and being a support system in my

life

4

ACKNOWLEDGMENTS

Primarily I thank my Lord and Savior, Jesus for every blessing He has given me. I thank

my parents Hugo and Myriam and siblings Sarai and Jacob for always pushing us to achieve our

dreams. I thank my husband who was always there through thick and thin, loving me. I thank my

grandpa Benito Medrano and grandma Nohemi Vizuet for being always there for me. My uncles

Josue, Noe and aunts Jael and Mirella all from whom I have a love of science and animals. I

would not be who I am today without the influence and guidance of my family.

I also thank my friends and colleagues who have helped keep me sane. My wonderful

advisor Dr. Fiona Maunsell who has pushed and guided me above and beyond. I thank my

committee members Dr. Risco, Dr. Santos and Dr. Galvao who drove me to learn and grow. I

deeply thank the help of Dr. Johanny Perez, Dr. Ricardo Chebel, Dr. Rafael Bisinotto, Dr.

Andersen Veronese and Xiaojie. For the support of my resident mates Dr. Judd Sims and Dr.

Gabriel Gomes. I thank the FARMS family Dr. Rae, Dr. Donovan, Dr. Rae, Dr. Irsik, Delores,

Laura and Anita.

5

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................7

LIST OF FIGURES .........................................................................................................................8

LIST OF ABBREVIATIONS ..........................................................................................................9

ABSTRACT ...................................................................................................................................11

CHAPTER

1 INTRODUCTION ..................................................................................................................12

2 LITERATURE REVIEW .......................................................................................................14

Calcium Metabolism ...............................................................................................................14

Hypocalcemia .........................................................................................................................19 Physiology of Hypocalcemia ...........................................................................................19

Epidemiology of Hypocalcemia ......................................................................................21 Consequences of Subclinical Hypocalcemia ...................................................................23

Management Strategies to Reduce the Risk of Hypocalcemia ........................................24 Prepartum dietary management ................................................................................24

Calcium administration at calving ............................................................................26 Calcium formulations used in the treatment of hypocalcemia: ................................26

Research on the Oral Ca Bolus Supplement Bovikalc®: ................................................28

Energy Balance during the Transition Period .........................................................................31 Energy Balance in the Transition Period and its Effects on Cow Health and

Performance .................................................................................................................31 NEFA ...............................................................................................................................32 BHBA ..............................................................................................................................33 Glucose ............................................................................................................................35 Monitoring of Blood NEFA, BHBA and Glucose in Transition Cows ...........................36

Rumination .............................................................................................................................36 Rumination Measurement ...............................................................................................37

Association between Rumination and Hypocalcemia .....................................................39 Objectives and Hypothesis .....................................................................................................40

3 MATERIALS AND METHODS ...........................................................................................42

Cows, Housing and Herd Management ..................................................................................42 Experimental Design and Treatments .....................................................................................43 Milk Yield and Health Data ....................................................................................................44

6

Rumination and Activity Data Recording ..............................................................................45

Blood Collection and Analyses ..............................................................................................45 Statistical Analyses .................................................................................................................46

4 RESULTS ...............................................................................................................................50

Exclusions and Missing Data..................................................................................................50 Baseline Values ......................................................................................................................50 Effect of Treatment on Serum Concentrations of Total Calcium ...........................................51 Effect of Treatment on Serum Concentrations of NEFA, BHBA and Glucose .....................51

Correlation between Serum Calcium Concentration and Rumination over the First 24

Hours Post Calving .............................................................................................................52 Effect of Treatment on Rumination during the First 24 Hours Post Calving .........................52

Effect of Treatment on Daily Rumination during the First 30 DIM .......................................53 Effect of Treatment on Daily Activity during the First 30 DIM ............................................53 Effect of Treatment on Milk Yield during the First 30 DIM ..................................................54

Association of Subclinical Hypocalcemia at Calving with Rumination during the First

24 Hours Post Calving ........................................................................................................54

5 DISCUSSION .........................................................................................................................66

6 CONCLUSIONS ....................................................................................................................70

LIST OF REFERENCES ...............................................................................................................71

BIOGRAPHICAL SKETCH .........................................................................................................80

7

LIST OF TABLES

Table page

2-1 Cud chewing times for dairy cows fed a TMR. Adapted from (Lindgren, 2009) .............41

3-1 Ingredient and nutrient composition of pre- and postpartum diets (DM basis). ................49

4-1 Baseline values for least squares means (LSM) +/- std. error of the mean (SEM) for

control cows (n = 36) and cows supplemented with oral Ca boluses (n = 39). .................55

4-2 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of serum tCa concentration (mg/dL) over the first 24 hours after calving

between control cows and cows supplemented with oral Ca boluses. ...............................55

4-3 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of serum NEFA concentration (mEq/L) over the first 24 hours after

calving between control cows and cows supplemented with oral Ca boluses.. .................55

4-4 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of serum BHBA concentration (mmol/L) over the first 24 hours after

calving between control cows and cows supplemented with oral Ca boluses.. .................56

4-5 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of serum glucose concentration (mmol/L) over the first 24 hours after

calving, between control cows and cows supplemented with oral Ca boluses.. ................56

4-6 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of daily rumination in minutes, between control and supplemented cows

for the first 24 hours postpartum.. ......................................................................................56

4-7 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of daily rumination in minutes, between control and supplemented cows

for the first 30 DIM.. ..........................................................................................................57

4-8 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of daily activity in minutes, between control and supplemented cows for

the first 30 DIM.. ...............................................................................................................58

4-9 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of daily milk yield in kg between control and supplemented cows for the

first 30 DIM.. .....................................................................................................................59

4-10 Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of rumination (minutes) between cows with normocalcemia (NC) and

subclinical hypocalcemia (SCH) for the first 24 hours after calving.................................60

8

LIST OF FIGURES

Figure page

2-1 The association of hypocalcemia with a wide variety of post-partum diseases and

performance in the dairy cow(DMI=dry matter intake; DA=displaced abomasum) .........41

2-2 The effect of blood pH and plasma Mg concentrations on PTH receptor

conformation. Taken from (Goff 2008, p. 51) ...................................................................41

4-1 The percentage change in serum total Ca concentrations from pretreatment to 0.5

hours after administration of an oral Ca bolus containing 43 g of calcium.. .....................60

4-2 Characterization of serum concentrations of total Ca (tCa) during the first 24 hours

after calving for control cows and cows supplemented with oral Ca boluses. ..................61

4-3 Characterization of serum values for NEFA concentrations during the first 24 hours

after calving for control cows and cows supplemented with oral Ca boluses. ..................61

4-4 Characterization of serum concentrations of BHBA during the first 24 hours after

calving for control cows and cows supplemented with oral Ca boluses.. ..........................62

4-5 Characterization of serum concentrations of glucose during the first 24 hours after

calving for control cows and cows supplemented with oral Ca boluses.. ..........................62

4-6 Correlation between the average blood total Ca concentration during the first 24

hours after calving and rumination during the first 24 hours after calving. Data

includes both control cows and cows that were supplemented with oral Ca boluses. .......63

4-7 Characterization of rumination minutes in the first 24 hours after calving per 2 hour

blocks for control and cows supplemented with oral Ca boluses.. ....................................63

4-8 Characterization of daily rumination minutes in the first 30 days in milk (DIM), for

control and cows supplemented with oral Ca boluses. ......................................................64

4-9 Characterization of daily activity (minutes of activity per day) in the first 30 days in

milk (DIM), for control and cows supplemented with oral Ca boluses.. ...........................64

4-10 Characterization of daily milk yield in kg for the first 30 DIM after calving, for

control and cows supplemented with oral Ca boluses.. .....................................................65

4-11 Characterization of rumination (minutes per 2-hour block) during the first 24 hours

after calving, for cows with normocalcemia or SCH (tCa < 8.59 mg/dL).. ......................65

9

LIST OF ABBREVIATIONS

305ME Mature equivalent adjusted for 305 DIM

BCS Body condition score

BHBA Beta hydroxybutyrate

BW Body weight

Ca Calcium

Ca0 Calcium within 2 hours after calving, previous to treatment

Ca2+ Ionized calcium

CaCl2 Calcium chloride

CaR Calcium ion sensing receptor

CaR Calcium ion sensing receptors

CaSO4 Calcium sulfate

Cl Chloride

DA Displaced abomasum

DCAD Dietary cation anion difference

DHIA Dairy Herd Improvement Association

DIM Days in milk

DM Dry matter

DMI Dry matter intake

DU Dairy Unit

IP3 Phosphatidynositol triphosphate

IV Intravenous

K Potassium

kg Kilograms

LDA Left displaced abomasum

10

LS Lameness score

LSM Least squares means

mEq Milliequivalents

Mg Magnesium

Na Sodium

NC Normocalcemia

NEB Negative energy balance

NEFA Nonsterified fatty acids

PO4 Phosphate

PTH Parathyroid hormone

RFM Retained fetal membranes

SCH Subclinical hypocalcemia

SEM Standard error means

SO4 Sulfate

tCa Total calcium.

TMR Total mixed ration

NDCAD Negative dietary cation anion difference

UF University of Florida

11

Abstract of Thesis Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Master in Science

EFFECT OF ORAL CALCIUM BOLUS SUPPLEMENTATION ON RUMINATION AND

ACTIVITY PATTERNS IN EARLY LACTATION DAIRY COWS

By

Myriam Berenice Jimenez Medrano

August 2017

Chair: Fiona P. Maunsell

Major: Veterinary Medical Sciences

Subclinical hypocalcemia occurs frequently in parous dairy cattle during early lactation.

Calcium supplements are widely used in an attempt to mitigate the substantial negative effects of

subclinical hypocalcemia on health and production. The experiment objective was to determine

whether oral supplementation of calcium was associated with changes in rumination or activity

in multiparous Holstein cows. Parous cows (n=76) were fitted with rumination and activity-

monitoring collars 21 days prior to expected calving date. Cows were randomly assigned to a

treatment or control group (no supplementation) at calving. Treatment was1 oral Ca bolus

containing 43 g of available Ca administered within 2 h after calving and repeated 12 ± 2 h after.

Blood samples were collected prior to and 30 min after each treatment or at equivalent times for

control cows, and at 24 h after calving to determine serum tCa, NEFA, BHBA and glucose

concentrations. Calcium supplementation at calving and 12 h after calving had no effect on the

time spent ruminating in the 24 h after calving (12.45 ± 1.11 and 11.87 ± 1.13 min/2h for control

and treatment, respectively). There was no effect of treatment on rumination during the first 30

DIM (334.81 ± 11.72 and 330.47 ± 11.72 min/day for control and treatment, respectively). There

was also no effect of treatment on activity and milk production during the first 30 DIM. Treated

cows had higher serum tCa for the first 24 hours after calving than control cows. Serum NEFA,

BHBA and glucose did not differ significantly between groups.

12

CHAPTER 1

INTRODUCTION

As cows transition from pregnant nonlactating to lactating nonpregnant, numerous

physical, physiological and endocrine changes occur and such changes can impact subsequent

health, production and reproductive performance (Drackley, 1999). For the purpose of this

experiment, the transition period is defined as by Grummer in 1995 (Grummer, 1995) as lasting

from 3 weeks before calving until 3 weeks after calving. Other researchers, such as Grant and

Albright (Grant and Albright, 1995) define the transition period as two stages, one from 5 to 7

days prepartum and the second one from calving to 21 days in milk (DIM).

The transition period places large demands on the cow for calories and nutrients such as

calcium (Ca). This is because of rapid fetal growth during the last trimester of gestation,

especially during the last 3 weeks before calving (Bell, 1995). In the instance of Ca, even more

important than fetal growth is the increased demands because of sequestration in the mammary

gland for synthesis of colostrum and then milk at the onset of lactation. In addition, the cow also

undergoes drastic hormonal and physiological changes, including immune dysregulation with

reduced immune cell function, and increased inflammatory responses (Bertoni et al., 2008).

These changes invariably result in at least some degree of negative nutrient balance, which

causes negative energy balance (NEB), with the nadir typically observed during the first week

post-partum (Grummer, 1995). Excessive NEB postpartum, quantified by increased blood

plasma concentrations of nonsterified fatty acids (NEFA), is associated with increased risk of

diseases in early lactation as well as increased risk of early culling (Roberts et al., 2012).

Subclinical hypocalcemia (SCH) is a common condition during the transition period

because of the inability of many cows to quickly cope with the increased demands for Ca for

colostrum and milk synthesis. Calcium is essential for many body functions, including

13

gastrointestinal motility; SCH is associated with reduced rumen motility (Huber et al., 1981) and

reduced dry matter intake (DMI) (Hansen et al., 2003) in dairy cows as well as an increased risk

of many postpartum diseases, such as dystocia, retained fetal membranes (RFM), ketosis,

mastitis and metritis (Curtis et al., 1983, Martinez et al., 2012). The importance of SCH in post-

partum dairy cow health has driven development of commercial products intended to supplement

Ca in cows at high risk of SCH. There is little data available on the efficacy of these products for

improving rumen function, including rumination, or health in post-partum dairy cattle.

14

CHAPTER 2

LITERATURE REVIEW

Calcium Metabolism

Calcium is an essential mineral for any mammal to perform numerous body functions. It

is a mineral that not only forms part of the bone structure, but functions as a second messenger

during cell signaling, and it is an essential mediator for diverse functions such as

gluconeogenesis, glycolysis, growth, membrane stabilization, reproduction, blood clotting, nerve

signal transmission, immune cell responses, and, as discovered by Sidney Ringer (Ringer, 1882,

1883), for muscle contraction. Calcium is also important for lipid metabolism in adipocytes and

hepatocytes. Because Ca plays an essential role in body functions, mechanisms have evolved to

control the absorption, resorption, storage, and excretion of Ca.

In mammals, bone and diet are the main sources of Ca in the body (Horst, 1994). Almost

all of the Ca in bones exists in the form of hydroxyl-apatite crystals (Nussey and Whitehead,

2001). The rest of the Ca is present in the blood at a range between 8.5 to 10 mg/dL (Goff,

2014), with a very small proportion of the total body Ca present in the intracellular compartment

(Nussey and Whitehead, 2001). The blood Ca is either protein bound (50%), ionized (42 to 48%)

or in solution as salts of Ca (Goff, 2014). The sum of salts of Ca, ionized (Ca2+) and protein

bound Ca is known as total calcium (tCa). Ionized Ca is the portion of extracellular Ca that is

regulated by hormones and is responsible for determining the balance of Ca in the body (Nussey

and Whitehead, 2001). This Ca2+ is available for utilization in bodily functions. Calcium in bone

is relatively inaccessible, but there is a small (6 to 15 g) solubilized Ca pool in bone fluids that

can be quickly mobilized (Vagg and Payne, 1970). Calcium from the diet is absorbed in the

15

rumen via active and passive transport (Schröder et al., 2015). Calcium concentration in the

serosal layer of the rumen wall has been shown to increase immediately after an increase in Ca in

the mucosal layer, following the administration of a bolus of 40 g of Ca into a rumen of 100 L in

volume (Schröder et al., 2015). The molecular mechanisms involved in Ca absorption in the

rumen, however, are yet to be well characterized. Calcium is also absorbed in the intestinal tract;

this is explained below. Calcium is excreted in major amounts via milk and colostrum (20 to 50

g/d) and in small amounts via urine (0.5 to 2 g/d) and feces (5-7 g/d), although the latter is

dependent on the amount ingested and its source (Goff, 2000).

There are three main hormones that control Ca metabolism throughout the body;

parathyroid hormone (PTH), 1, 25-dihydroxyvitamin D (calcitriol) and calcitonin (Nussey and

Whitehead, 2001). Parathyroid hormone is responsible for the homeostatic control of increasing

Ca2+ in the blood. A decrease in the circulating blood Ca2+ concentration is sensed by Ca2+

receptors known as extracellular Ca ion sensing receptors (CaR), located on the epithelium cells

in the kidney and plasma membrane of parathyroid chief cells (Brown et al., 1993). This

increases PTH secretion, whereas a rise in blood Ca2+ concentration reduces PTH secretion

(Nussey and Whitehead, 2001). A rise in Ca2+ will stimulate phospholipase C through receptor

binding, which will inhibit adenylate cyclase, causing a rise in phosphatidynositol triphosphate

(IP3), which will reduce cAMP concentrations and PTH secretion. On the other hand, low Ca2+

will decrease the stimulation of the CaR, which will diminish the production of IP3, leading to an

increase in cAMP that will lead to a rise in PTH secretion (Nussey and Whitehead, 2001).

The receptors for PTH are PTH1R and PTH2R. Parathyroid hormone receptors PTH1R

are found mainly in the kidneys and within the bone on osteoblasts, but PTH2R are present in

various other tissues (Clemens et al., 2001). In the kidneys, binding of PTH to its receptor

16

increases the renal tubular reabsorption of Ca in the ascending loop of Henle and the distal

convoluted tubule (Nussey and Whitehead, 2001, Goff, 2014). Another very important function

of PTH in the kidney is to cause an increase of the enzyme 25-hydroxyvitamin D 1α-

hydroxylase, which converts 25-hydroxycholecalciferol into its activated form, as 1, 25-

dihydroxyvitamin D. This activated vitamin D causes increased active absorption of Ca from the

diet (Horst, 1994).

Calcium uptake in the intestinal tract occurs via passive and active absorption (Bronner,

1987). Passive uptake of Ca from the brush border of the luminal surface is also called the

paracellular pathway. In this process, Ca passes between the cells and there is an exchange with

sodium (McCarthy, 2004). Passive absorption is dependent upon a large gradient of Ca (>1 mM)

from the lumen of the gut to extracellular fluid, allowing passive diffusion across the intestinal

wall (Bronner et al., 1986).

Active transport of Ca across the intestinal epithelium is dependent on Vitamin D

(Bronner, 1987). The hormone 1, 25-dihydroxyvitamin D binds to its receptor within intestinal

epithelial cells. These receptors dimerize with other receptors, such as the retinoic acid receptor.

After this, the dimerized receptor will bind to a region in the DNA. Via gene expression,

activated vitamin D causes upregulation of proteins required for Ca transport, including

calbindin, apical membrane Ca channel proteins and the basolateral membrane Ca-ATPase pump

(Gardner and Shoback, 2011). As activated vitamin D functions as a steroidal hormone, it can

also act via a non-genomic pathway through membrane receptors (Nussey and Whitehead, 2001).

Calcium can also be transported across intestinal epithelial cells via lysozymal vesicles, which

requires 1,25-dihydroxyvitamin D-dependent Ca binding proteins (Nussey and Whitehead,

17

2001). The full upregulation of proteins required for active transport of Ca across the intestinal

epithelium requires 24-48 hours after activation of vitamin D (Goff et al., 1986).

Another function of 1, 25-dihydroxyvitamin D is to increase Ca resorption from bone,

along with PTH. When the receptors in bone cells bind with PTH and 1, 25-dihydroxyvitamin D,

mobilization of Ca begins within 1-2 hours. Solubilized Ca, located in the small fluid filled

conduits known as canaliculi or lacunae, is gripped by a syncytial process of activated osteocytes

and moved to the cortex of the bone and from there into the extracellular matrix; this pool of Ca

is relatively labile (Nussey and Whitehead, 2001). A few hours after stimulation with PTH, the

resorption of mineralized bone also begins, in which both phosphorus and Ca are released into

the extracellular matrix. This is achieved through enzymes released on the bone surface, which

cause a drop in pH, which aids the bone mineral dissolution (Nussey and Whitehead, 2001).

The inhibition of Ca absorption and resorption is achieved through calcitonin, which is

secreted by the C-cells of the thyroid gland. Increased calcitonin secretion is activated by

elevated blood Ca2+ concentrations sensed by CaR (Gardner and Shoback, 2011). Once secreted,

calcitonin is sensed by calcitonin receptors on osteoclasts and on renal tubule cells. Calcitonin

then causes increased Ca loss through urinary excretion, but it’s most important role is inhibiting

osteoclastic Ca resorption from the bones (Nussey and Whitehead, 2001, Gardner and Shoback,

2011). Calcitonin achieves this by changing the morphology of the osteoclast, making them

withdraw from the ruffled border of the bone surface, which is where bone resorption occurs

(Gardner and Shoback, 2011).

As mentioned before, in dairy cows blood tCa is maintained within a range of 8.5 to 10

mg/dL (2.13 to 2.50 mmol/L) (Rosol et al., 1995). At parturition when there is greatly increased

demand for Ca for the production of colostrum, blood Ca often undergoes a transient decrease in

18

the dairy cow. The nadir of blood Ca typically occurs within the first 48 hours postpartum

(Kimura et al., 2006, Goff, 2008).

The Ca requirements of an adult dairy cow are comprised of three main areas of need:

1. Maintenance requirements: Maintenance Ca requirements for a non-lactating cow in

accordance to Visek and colleagues (Visek et al., 1953) are 0.0154 g of absorbed Ca/kg BW. For

a 650 kg adult Holstein dairy cow, this equates to approximately 10 g of Ca per day. Compared

with needs for fetal growth and milk production, this is the smallest contributor to the Ca

requirements of a dairy cow during the transition period.

2. Fetal growth: Converse (Converse, 1954) discovered that the minimum concentration

of Ca needed for gestation was 0.16% Ca on a dry matter (DM) basis of the total ration.

According to House and Bell (House and Bell, 1993), losses of Ca to fetal growth in a 714 kg

dairy cow account for approximately 10 to 11 g/d at the end of pregnancy (280 days), which is

when the calf grows the most.

3. Lactation: Milk and colostrum synthesis require large amounts of Ca, particularly in

high-producing cows. According to Martz and colleagues (Martz et al., 1990), dairy cows need

to meet their maintenance requirements plus 1.22 g of absorbed Ca/kg milk produced. A

lactating Holstein cow at peak production may need 80 g of Ca/day for milk production.

Colostrum is even richer in Ca than milk; according to the NRC (2001) (NRC, 2001), a cow will

need 2.1 g of absorbed Ca/kg of colostrum produced.

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Hypocalcemia

Physiology of Hypocalcemia

Hypocalcemia is a metabolic problem in many mammals, occurring when the

mechanisms of Ca homeostasis fail to cope with a relatively sudden change in the demand for

Ca. For example, in dogs and cats hypocalcemia is known as eclampsia and presents as tetany,

muscle fasciculation, no loss of consciousness and hyperthermia (Groman, 2012). Eclampsia

occurs within the first 4 weeks post-partum associated with the Ca demands of milk production

or, more commonly, in the last few weeks pre-partum associated with the Ca demands for rapid

fetal growth (Davidson, 2012).

Dairy cattle are particularly vulnerable to hypocalcemia. As described in the previous

section, there is a remarkable difference among the amount of Ca needed by a dry dairy cow with

a growing late-term fetus versus a periparturient cow coping with colostrum production and the

onset of lactation. Cows have an extracellular reserve of Ca, of around 0.1% of the total amount

in the body, which translates to 8-10 g in the extracellular pool. At calving there is a sudden

demand for 20-30 g of Ca in colostrum/day, in some instances even reaching 50g/day, greatly

exceeding Ca available in the extracellular pool (Goff, 2000, NRC, 2001, Tsioulpas et al., 2007).

This demand drives the removal of Ca from bone storage and increased efficiency of absorption

of Ca from the diet through the actions of PTH and 1, 25-dihydroxyvitamin D (Nussey and

Whitehead, 2001). During the dry period when Ca needs are relatively low, the mechanisms for

Ca mobilization from bone and absorption from the diet are downregulated. When lactation

begins, hypocalcemia occurs if the homeostatic mechanisms cannot upregulate Ca mobilization

20

and absorption quickly enough to keep up with the sudden increase in demand (Horst et al.,

2005).

Hypocalcemia can be clinical or subclinical. Subclinical hypocalcemia occurs when

blood Ca falls to a concentration that is associated with an increased risk of associated health

disorders, but there are no outward clinical signs of hypocalcemia (Oetzel, 2011). According to

Reinhardt and colleagues (Reinhardt et al., 2011), subclinical hypocalcemia is considered as

blood Ca concentrations from 5.5 to 8 mg/dL. Oetzel (Oetzel, 2004), does not make a separation

between SCH and clinical hypocalcemia and considers hypocalcemia to be a tCa below 8.0

mg/dL, with or without clinical signs. The work of Martinez and colleagues (Martinez et al.,

2012), showed that the threshold at which SCH was associated with an increased risk of metritis

was < 8.59mg/dL of tCa.

When blood Ca continues to fall, clinical hypocalcemia, also known as milk fever, can

occur (Horst et al., 2005, Goff, 2008, Martinez et al., 2012). During clinical hypocalcemia, a cow

will follow three stages as her symptoms worsen:

Stage 1: The cow will show nervousness (repeated weight shifting), weakness or

excitability without recumbency. This stage can easily be overlooked because the signs are often

subtle and it does not last very long (Oetzel, 2011).

Stage 2: The cow will be in sternal recumbency, most likely with her neck and head bent

towards her hind legs, with muscle weakness and progressive flaccid paralysis as well as central

nervous system depression. This is the cow that won’t get up to be milked and who shows an ‘S’

shaped neck (Oetzel, 2011).

Stage 3: The cow will be prostrated in lateral recumbency, with severe depression, bloat

because of rumen hypomotility, flaccid limb paralysis, cold extremities, and her heart will be

21

barely audible, but with marked tachycardia. There is an inability to thermoregulate and cows

may be hypothermic or hyperthermic depending on the external environment. A cow in stage 3

of hypocalcemia will most likely die if not attended promptly (Oetzel, 2011).

Epidemiology of Hypocalcemia

In modern dairy production systems in North America, clinical hypocalcemia affects

around 5% of cows (USDA, 2007). In contrast, SCH often affects around half of multiparous

cows, even in herds that add anionic salts to the prepartum diet to reduce the risk of clinical

hypocalcemia (Horst et al., 2003, Reinhardt et al., 2011). Factors associated with hypocalcemia

after calving include dietary factors such as a high dietary cation-anion difference (DCAD) in the

prepartum diet (Leclerc and Block, 1989, Goff, 2008) and hypomagnesemia (Lean et al., 2006),

and non-dietary factors such as breed, age, and other health challenges like dystocia, twins and

stillbirth (Martinez et al., 2012, Benzaquen et al., 2015), as well as lameness (Neves et al., 2016).

Subclinical hypocalcemia may begin before calving, as multiparous cows with low serum tCa

concentrations (<2.4 mmol/L) measured within 7 days before the predicted calving date were

more likely to have SCH at calving (relative risk = 1.7) (Neves et al., 2016).

A high DCAD, when there is an excess of the strong cations potassium (K), sodium (Na),

Ca and magnesium (Mg) in the diet relative to the strong anions chloride (Cl), sulfate (SO4) and

phosphate (PO4), is common in forage-based rations fed to dairy cows. The effect of the dietary

DCAD on Ca metabolism is discussed below under management strategies.

Hypomagnesemia is well documented as a risk factor for clinical hypocalcemia (Lean et

al., 2006). Magnesium is an essential element required for the secretion of PTH and for

downstream signaling to target tissues after binding of PTH to its receptor (van de Braak et al.,

22

1987, Fatemi et al., 1991). Low plasma Mg concentrations impair the ability of the cow to

increase mobilization and absorption of Ca at parturition. Plasma Mg is maintained at normal

concentrations through dietary absorption, so when dietary Mg is low or conditions exist that

impair its absorption, subclinical or clinical hypomagnesemia can occur. Inclusion of adequate

Mg in prepartum and early lactating cow diets is therefore important in reducing the risk of

hypocalcemia; Goff (Goff, 2008) recommends Mg be included at 0.35 to 0.4% of the diet.

Although there is strong evidence that low plasma Mg concentrations are associated with

increased risk of clinical hypocalcemia (Lean et al., 2006), little has been published on its

association with SCH. In a recent experiment, Neves and colleagues (Neves et al., 2016)

attempted to investigate the association of prepartum plasma Mg concentrations with SCH at

calving, but a very low prevalence of low plasma Mg concentrations in their study population

meant they had insufficient power to draw conclusions.

The risk of hypocalcemia increases with age (Horst et al., 1997, Moore et al., 2000). As

cows age, the number of active bone cells decrease, which means there are fewer cells available

to respond to PTH to mobilize Ca from bone; primiparous animals, who are still growing, have

greater numbers of active osteoblasts than older animals (Goff, 2000). In addition, primiparous

animals generally produce a smaller volume of colostrum than multiparous cows, and therefore

have a smaller demand for Ca at the onset of lactation. Cows of greater production potential may

be more likely to develop SCH (Jawor et al., 2012).

Breed has an effect on the risk of hypocalcemia at calving, with Jersey cows having a

greater incidence of milk fever compared with Holstein cows (Lean et al., 2006). Factors that

may be associated with increased risk of hypocalcemia in Jerseys include greater concentrations

of Ca in milk and colostrum compared with Holsteins, as well as fewer intestinal receptors for 1,

23

25-dihydroxyvitamin D (Goff, 2014). In addition to age and breed, recent work suggests that

genetics plays a direct or indirect (via production potential) role in the risk for hypocalcemia

(Tsiamadis et al., 2016).

Consequences of Subclinical Hypocalcemia

Subclinical hypocalcemia is associated with increased risk of a wide variety of health

disorders during early lactation. SCH is really a silent enemy, affecting proper nervous function

as well as rumination and digestion via muscle contraction in the rumen and abomasum (Hara et

al., 2003). Figure 1 illustrates the association of hypocalcemia with a wide variety of postpartum

diseases as well as performance of a dairy cow.

Subclinical hypocalcemia reduces DMI, gastrointestinal motility, and milk yields and

increases the risk of contracting infectious or metabolic diseases (Curtis et al., 1983, Hara et al.,

2003, Chapinal et al., 2012). Low Ca concentrations impair muscle motility, which reduces

rumen, abomasum and intestinal contractions (Jørgensen et al., 1998, Hara et al., 2003).

Immunity is also compromised, as shown by Martinez and colleagues, as low Ca impairs the

phagocytic abilities of neutrophils (Chapinal et al., 2011, Martinez et al., 2014). Impaired

immune function and/or low muscle motility increase the risk of diseases such as RFM (Risco et

al., 1994), uterine prolapse (Risco et al., 1984), metritis (Martinez et al., 2012), DA (Massey et

al., 2003) and ketosis (Curtis et al., 1983, Goff, 2014). Subclinical hypocalcemia in the

transition period is associated with reduced milk production and with impaired reproductive

performance in early lactation (Chapinal et al., 2012, Ribeiro et al., 2013). Because of all these,

SCH can have a severe effect on the finances of a dairy farm. There is low milk production to

24

account for, treatment costs and even early culling, impairing the cows potential to fulfill their

productive life (Oetzel, 2013a).

Management Strategies to Reduce the Risk of Hypocalcemia

Prepartum dietary management

Prepartum dietary management strategies to reduce the risk of hypocalcemia aim to

promote Ca mobilization and absorption in the late dry period so that these mechanisms are

already upregulated when the cow is faced with the sudden demand for Ca at calving. Feeding

strategies include feeding a diet high in anions or feeding a low Ca diet.

i) Altering the DCAD of the diet: Feeding a prepartum diet high in anions. The

influence of the dietary cation anion difference (DCAD) was first investigated by Norwegian

researchers who noticed that when prepartum diets were high in Na and K, and were low in Cl

and SO4, the incidence of clinical hypocalcemia increased (Dishington, 1975). When the

opposite happened and a diet was low in Na and K, but high in Cl and SO4, the incidence of

clinical hypocalcemia decreased. These observations led scientists to experiment with dietary

cations and anions, confirming that a diet relatively high in anions was effective in lowering the

risk of clinical hypocalcemia (Dishington, 1975). Although other cations, such as Ca and Mg and

anions, such as PO4, contribute to DCAD, it is usually estimated using the formula (Na+ + K+ ) -

(Cl- + SO4), with all concentrations in milliequivalents (mEq) per kg. Recommendations are to

feed a prepartum diet with a DCAD of approximately -100 mEq/kg of dry matter in the diet

(Horst et al., 1997).

25

Diets with an excess of strong anions (low DCAD) are acidogenic, and the equilibrium

between Ca2+ and protein-bound Ca is affected by pH (Craige and Stoll, 1947). Metabolic

alkalosis is known to predispose cows to hypocalcemia, whereas acidosis increases the ionized

fraction of Ca, helping in the prevention of hypocalcemia (Craige and Stoll, 1947). An ideal

prepartum DCAD will promote aciduria with a target urine pH between 5.5 and 7.0, which is

optimal to prevent clinical hypocalcemia (Jardon, 1995). A urine pH greater than 7.35 indicates

that acidification is ineffective to prevent clinical hypocalcemia (Goff, 2000, 2014), whereas one

under 5.5 can indicate severe metabolic acidosis, reduced DMI and subsequent health problems

for the cow (Goff, 2000, 2014). Under the compensated metabolic acidosis induced by a diet

with a negative DCAD, there is a tight lock-key interaction between the PTH receptor and PTH

in the target tissues (Figure 2). Under more alkaline conditions, however, this receptor

conformation changes, which might impair the proper coupling with PTH, thus impairing Ca

absorption and resorption (Bushinsky, 1996). Also, metabolic acidosis increases intestinal

absorption of Ca because it favors the production of 1, 25-dihydroxyvitamin D under the

influence of PTH in the kidney (Goff and Horst, 1993). According to Goff, the key to clinical

hypocalcemia prevention and a reduction in the risk of SCH is to maintain Na and K as near to

the dietary requirement of the cow as possible and then to add Cl (or other strong anions) to

counteract the effects of K on the alkalinity of the blood (Goff, 2000).

ii) Feeding a prepartum diet low in Ca: Another prepartum feeding strategy is to

restrict the amount of Ca in the diet. When cows are fed a very low amount of Ca, they enter a

negative Ca balance, which subsequently upregulates the secretion of PTH (Goings et al., 1974).

This will start a homeostatic response to put the cow into normocalcemic status through the

routes discussed previously. In this way, mechanisms of Ca mobilization and absorption are

26

already upregulated when the cow calves and are able to cope with the sudden increase in Ca

demand. To be effective, however, a low Ca diet needs to supply less than 20 g of Ca a day

(Goings et al., 1974, Goff, 2000), which is very difficult to achieve using the forages typically

fed to dairy cows. Hence, this approach is not widely used.

Calcium administration at calving

Supplemental Ca can be administered to cows at calving to treat clinical hypocalcemia or

in an attempt to reduce the risk or severity of SCH. Solutions containing Ca for intravenous or

subcutaneous injection are available and are mainly used for the treatment of clinical

hypocalcemia (Oetzel, 2013a). Ca can also be delivered orally through various methods, such as

water-soluble powder for drenching, as well as gels and boluses. Cows that are suspected of SCH

or are in stage 1 of clinical hypocalcemia can be treated with oral Ca (Horst et al., 1997). Most

commercially-available oral Ca products take approximately half an hour to be absorbed and

provide between 4 to 6 hours of an increase in blood Ca (Goff and Horst, 1993, 1994).

Commercially available oral Ca supplements are typically formulated with more than one type of

Ca in an attempt to provide a product that is available for both rapid and sustained absorption

from the gastrointestinal tract.

Calcium formulations used in the treatment of hypocalcemia:

Calcium can be provided orally as Ca chloride, Ca propionate, Ca carbonate or Ca

sulfate. Calcium chloride is the preferred Ca source when giving an oral drench, paste or bolus

(Goff and Horst, 1993). This is because Ca chloride has the best bioavailability, mainly because

27

of its high solubility and concentration of available Ca (36%) (Goff et al., 1996). According to

Goff and colleagues, a 50g dose of Ca chloride is enough to achieve adequate Ca absorption

comparable to 4 grams of the same Ca given intravenously (Goff and Horst, 1993). Calcium

chloride is very caustic if aspirated into the upper airways when delivered in water or gel

solutions, or if it remains in the esophagus if the cow fails to swallow properly (Goff and Horst,

1993). Also, if a cow is administered a high dose of Ca chloride, then an uncompensated

metabolic acidosis can occur, which will cause a decrease in DMI (Goff and Horst, 1993, Goff et

al., 1996). When Ca is given orally, toxicity can occur when 250g of soluble Ca or higher are

administered, so precaution is advised (Goff, 1999).

Unlike Ca chloride, Ca propionate does not have the ability to decrease the blood pH and

it is absorbed more slowly, but this gives it the ability to cause a more sustained increase in

plasma Ca (Goff and Horst, 1993). It also has a smaller concentration of available Ca (21.5%)

compared with Ca chloride, and a dose of 75 to 125 g is recommended for treatment of cows

with SCH (Goff and Horst, 1993). In addition, the propionate component of this product is a

glucose precursor, which is an advantage in early lactation when the cow is in negative energy

balance (Goff et al., 1996).

Calcium sulfate is often included in commercial oral Ca supplements with Ca chloride

because Ca sulfate is theoretically less soluble in the rumen and provides Ca over a more

sustained period. A product containing both Ca chloride and Ca sulfate should therefore provide

a rapid but sustained increase in blood Ca (Oetzel and Miller, 2012). There is little data on the

oral availability of Ca sulfate in dairy cattle.

28

Calcium carbonate is the least bioavailable of the Ca formulations used in oral

supplements. In an experiment performed by Goff and colleagues, oral Ca carbonate was unable

to raise plasma Ca (Goff and Horst, 1993).

Calcium can also be administered through intravenous (IV) and/or subcutaneous routes to

treat cows with clinical hypocalcemia (Goff and Horst, 1993). Ca borogluconate is obtained

through the reaction of one part boric acid and five parts calcium gluconate mixed in an aqueous

solution (Mcpherson and Stewart, 1938). This solution has an acidic pH of 3.5 and is only

recommended for IV or subcutaneous use. Calcium borogluconate is typically administered to

provide 8-10g of Ca into the bloodstream to immediately raise blood Ca (Goff and Horst, 1993).

The subcutaneous approach is usually attempted to prevent clinical hypocalcemia or to

supplement intravenous Ca (Goff, 1999).

Research on the Oral Ca Bolus Supplement Bovikalc®:

Bovikalc® (Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO) is an oral bolus

containing Ca chloride (CaCl2) and Ca sulfate (CaSO4) (71% Ca chloride and 29% Ca sulfate)

totaling 43g of Ca, covered in a coating of fat to protect the oral and esophageal mucosa. The

manufacturer recommends that the bolus is delivered twice, first immediately after calving and a

second bolus 12 hours later. The goal with this administration protocol is to provide a sustained

increase in blood Ca over the first 24 hours postpartum (Sampson et al., 2009).

Oetzel and Miller (Oetzel and Miller, 2012) performed an experiment to evaluate the

effects of the bolus on the health and milk yield of parous cows (n=927) on 2 large Wisconsin

dairies. These cows were fed a diet with a low DCAD during the prepartum period. Cows were

randomly assigned to a treatment or control group at calving. Control cows did not receive any

29

Ca bolus, whereas treated cows were administered 2 boluses. The first bolus was given within

the first 2 hours post-calving, and the second one within 8 to 36 hours after calving. Blood

samples were collected just before the second bolus was administered to the cows, and were

analyzed to measure Ca2+. During this experiment, just 14% of the animals were hypocalcemic

(Ca2+ < 1.0 mmol/L) and just 0.6% of calvings developed clinical hypocalcemia. Concentrations

of Ca2+ did not differ between treated and untreated animals. The investigators found that lame

cows supplemented with the Ca bolus, however, had reduced risk of health problems during the

first 30 DIM than unsupplemented lame cows. They also found a 2.9 kg increase in milk yield at

the first postpartum DHIA test for cows that were greater producers in their previous lactation

(305ME milk production >105% of herd rank) and supplemented with the bolus, when compared

with comparable unsupplemented cows. The investigators concluded that some subpopulations

of cows, such as lame or high producing animals, could benefit from oral Ca supplementation at

calving (Oetzel and Miller, 2012). According to Sampson and colleagues (Sampson et al., 2009),

administration of Bovikalc® oral Ca boluses as recommended by the company to hypocalcemic

cows results in more sustained improvements in blood Ca concentrations than observed in

previous studies with oral Ca chloride alone or Ca propionate in water. Their experiment was

performed on 20 parous Holstein cows, 10 per group. These cows were not fed a low DCAD diet

during the prepartum period, and were only enrolled if they presented with a blood Ca2+ of ≤ 1.1

mmol/L at calving, assuring all the cows were hypocalcemic or approaching hypocalcemia.

Control cows received no Ca boluses, and treated cows received 2 boluses, one within 2 hours

from calving and a second one 12 hours later. Supplemented cows had an 8.2% rise in Ca2+

concentrations, with an average of 1.03 mmol/L within 1 hour after calving and at 24 hours,

whereas control cows remainedhypocalcemic, with an average Ca2+ of 0.94 mmol/L.

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Martinez and colleagues (Martinez et al., 2016a, Martinez et al., 2016b) found more

complex interactions. The authors conducted two experiments. In experiment one, 9 primiparous

and 9 multiparous Holstein cows were randomly assigned on the day of calving to receive 0, 1 or

2 Ca boluses. Cows supplemented with 1 or 2 boluses had an increase in plasma Ca2+, but this

effect was of short duration (2 hours for 1 bolus and less than 8 hours for 2 boluses). The

increase in Ca2+ for the cows supplemented with 2 boluses was 50% greater in nulliparous cows

than in parous cows (Martinez et al., 2016a). The second experiment consisted of 450 Holstein

cows (nulliparous and parous), that were divided into low risk of metritis (normal calving) or

high risk of metritis (dystocia, twins, stillbirth, RFM, lacerations in the vulva and/or vagina, or a

combination of these factors). These cows were blocked by parity on their calving date, and

randomly assigned to 3 groups: 1) Control, with no bolus supplementation; 2) CaS1 86 g of Ca

on d 0 and 1 postpartum, 3) CaS4 2 boluses at calving, and 2 boluses on day 1 postpartum and

then 1 bolus on days 2 to 4 postpartum. As shown by Oetzel and Miller (Oetzel and Miller,

2012), Martinez and colleagues (Martinez et al., 2016b) also reported that for multiparous cows,

oral Ca bolus supplementation was associated with an increase in milk yield in the first 30 DIM

in cows of greater production potential, but was associated with reduced milk yield in

multiparous cows with below average production potential. In addition, primiparous cows treated

with the oral Ca boluses had worse reproductive performance than untreated cows, with fewer

pregnancies per artificial insemination (control = 48.5%, CaS1 = 34.6%, CaS4 = 38.5%). For

multiparous cows, treatment was associated with increased reproductive performance, with

greater pregnancies per artificial insemination (control = 28.1%, CaS1 = 35.3%, CaS4 = 40.5%).

Primiparous cows supplemented with oral Ca boluses had a greater risk of metritis than

31

unsupplemented cows, whereas parous cows supplemented with oral Ca boluses had a reduced

incidence of diseases other than metritis and ketosis.

From these experiments, we can conclude that oral Ca supplementation using Bovikalc®

Ca boluses can be beneficial for specific cohorts of multiparous animals, but not for nulliparous

cows.

Energy Balance during the Transition Period

Energy Balance in the Transition Period and its Effects on Cow Health and Performance

High producing dairy cows undoubtedly fall into negative energy balance (NEB) during

early lactation. This is mainly because of their inability to meet the increased energy demands

associated with the onset of milk production, from feed intake alone. Cows can have up to 30%

of DMI decrease during the days prior to parturition (Hayirli et al., 2002, Roberts et al., 2012).

Other factors that contribute to NEB are the smaller capacity of the rumen at calving because of

the physical space-occupying effects of the heavily gravid uterus, as well as a period of relative

insulin resistance at calving (Hayirli et al., 2002). At parturition, the increased demand for

energy associated with the onset of colostrum and milk production results in rapid fat

mobilization (Wathes et al., 2009). Concurrent disease in the transition period may further reduce

DMI and exacerbate the NEB experienced by the cow (Herdt, 2000).

Homeostatic mechanisms should prevent excessive fat mobilization and excessive ketone

body production. If these mechanisms fail, then high concentrations of non-esterified fatty acids

(NEFA) and ketone bodies are produced during fat mobilization and incomplete lipid oxidation.

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Metabolic diseases such as ketosis and the accumulation of triacylglycerols in the liver, known

as fatty liver, can result from failure of energy homeostasis in the transition cow (Herdt, 2000).

The liver is responsible for gluconeogenesis, fatty acid oxidation, and the synthesis of

triacylglycerol (Herdt, 2000). If the rate of fat accumulation in the liver is excessive, then the

liver function will be compromised, affecting gluconeogenesis as well as numerous other liver

functions (Wathes et al., 2009). Cows with excessive NEB as indicated by elevated

concentrations of NEFA and ketone bodies such as beta hydroxybutyrate (BHBA) have a greater

risk of disease in early lactation, as well as impaired reproductive performance (Chapinal et al.,

2012). According to LeBlanc (LeBlanc, 2010) as many as 50% of dairy cows can be affected

with metabolic or infectious diseases during the transition period, especially metritis, RFM, DA,

ketosis and hypocalcemia.

NEFA

Fat mobilization is known as lipolysis, and it is achieved through the breakdown of the

triglycerides that are stored inside adipocytes. Triglycerides are broken down by the cleavage of

their ester bond, which releases three non-esterified fatty acids known as NEFAs (Bauman and

Currie, 1980, Herdt, 2000, Chapinal et al., 2011). Non-esterified fatty acids can then enter the

blood and be used as an energy source by tissues throughout the body when dietary uptake is

unable to meet the energy needs of the cow. When the NEFA release exceeds the ability of the

tissues to use them or of the hepatocytes to restore them back as triglycerides through the

renovation of the ester bond in a process known as lipogenesis, then blood NEFA concentrations

are elevated (Herdt, 2000). Non esterified fatty acids release from adipose tissue is also related to

norepinephrine and epinephrine secretion. This is important because epinephrine is released

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during stress and is a strong stimulant for lipolysis. This means that a cow going through the

stress of the transition period can have NEFA production stimulated via epinephrine secretion

(Herdt, 2000). All these factors together mean that the liver can be presented with high

concentrations of circulating NEFAs in the transition cow.

The liver, if functioning properly, will metabolize NEFAs into ketone bodies (BHBA,

acetone and acetoacetate) or re-esterify them into triglycerides then export them back to the

bloodstream. If the liver is unable to cope with the amount of NEFAs produced during rapid fat

mobilization there will be a toxic accumulation of NEFAs in the body. If hepatic triglyceride

production exceeds the capacity of the liver to export the triglycerides out of the cell,

triglycerides will accumulate and cause fatty liver. Elevated NEFAs during the transition period

have been related to increased disease risk of DA, RFM, uterine diseases and fatty liver

(LeBlanc, 2010, Chapinal et al., 2011). According to Le Blanc (LeBlanc, 2010), cows with

elevated blood NEFA concentrations were up to 80% more likely to have concurrent RFM than

cows with low NEFA concentrations. Transition cows with elevated NEFA concentrations have

reduced reproductive performance in the subsequent lactation. According to Ospina (Ospina et

al., 2010), cows that have elevated BHBA and NEFA concentrations within two weeks pre-

calving up to two weeks post-calving have a smaller chance of becoming pregnant within the

first 70 days after the end of the voluntary waiting period.

BHBA

Beta hydroxybutyrate is one of the ketone bodies formed during the incomplete oxidation

of NEFAs in the liver, along with acetone and acetoacetate. Non esterified fatty acids are

metabolized into Acetyl CoA in the hepatocyte mitochondria; it is this Acetyl CoA that becomes

34

the precursor of ketone bodies. Acetyl CoA is metabolized for the most part into acetoacetate.

Acetoacetate is transported into the cytosol, where it is partly transformed into BHBA. Because

NEFAs are direct precursors of ketone bodies, an elevated concentration of BHBA is highly

correlated with a high NEFA concentration. All three ketone bodies are used by muscle as

energy sources. Nevertheless, if there is an over production and accumulation of ketone bodies,

then a metabolic disease known as ketosis occurs. Ketosis can be type I (primary) or type II

(secondary). In type I ketosis there is a low concentration of glucose and insulin in the blood; this

stimulates rapid transport of NEFAs into hepatocyte mitochondria (Pethick et al., 2005). This

will cause production of a large amount of ketone bodies, and only a small proportion of NEFAs

will be re-esterified into triglycerides, hence avoiding fatty liver. This will cause a ketotic

condition in the absence of fatty liver and is most commonly found during peak lactation.

In contrast, type II ketosis is typically accompanied by fatty liver. In this situation there

are high concentrations of NEFAs taken up by hepatocytes, but the transport into the

mitochondria is somewhat impaired (Pethick et al., 2005). In this instance, a proportion of

NEFAs are esterified in the cytosol. In order for triglycerides to be used in other tissues, they

need to be transported out of the liver as low-density lipoprotein particles. The bovine liver,

however, has an inherently low production rate of these particles, which gets even poorer during

early lactation. Then triglycerides accumulate rapidly in the liver, impairing liver function and

causing fatty liver. In the more severe instances, fatty liver prevents gluconeogenesis, and the

resulting hypoglycemia increases fat mobilization further, setting the cow into a more severe

state of fatty liver that can lead to liver dysfunction (Herdt, 2000, Pethick et al., 2005).

Various studies have associated SCH, elevated BHBA and elevated NEFA concentrations

with low production and greater risk of disease (Chapinal et al., 2011, Martinez et al., 2012).

35

According to Martinez and colleagues (Martinez et al., 2012), cows affected with SCH within

their first 3 days in lactation had greater BHBA and NEFA concentrations, as well as a greater

incidence of reproductive problems such as metritis, longer birth to conception interval and

reduced pregnancy rates. Blood Ca concentrations in transition cows are negatively associated

with blood NEFA concentrations. For example, Reinhardt and colleagues (Reinhardt et al., 2011)

reported that cows with blood Ca over 8.4 mg/dL (2.1 mmol/L) had reduced blood

concentrations of NEFAs compared with cows with Ca < 8.4 mg/dL. Transition cows with SCH

accumulate more lipid within hepatocytes than normocalcemic cows (Chamberlin et al., 2013).

Together these data provide strong evidence that SCH is associated with increased blood NEFA

and BHBA concentrations and increased rates of metabolic disease (Oetzel, 2004, Martinez et

al., 2012).

Glucose

When there is sufficient blood glucose to meet energy demands, the body will store

energy through lipogenesis. In this state of abundance, NEFAs are not needed as an alternate

source of energy, and their concentrations will be low. For the most part, the regulation of

lipogenesis while there is high glucose available is mediated by insulin. Insulin is the primary

hormone of energy metabolism and is directly influenced by the availability of glucose and

glucose precursors in the blood (Pethick et al., 2005). In the adipocytes, insulin suppresses

lipolysis and stimulates lipogenesis. As a result, NEFA mobilization is inhibited. In a period of

NEB, as is the transition period, the body moves to lipolysis and utilizes NEFAs as an alternate

source of energy. During this NEB period, the cow will also use muscle protein for

gluconeogenesis (Herdt, 2000). The liver is the organ where most of the gluconeogenesis occurs,

36

so if it becomes impaired because of excessive accumulation of triglycerides (fatty liver),

glucose synthesis and blood glucose to supply the tissues will be impaired.

Monitoring of Blood NEFA, BHBA and Glucose in Transition Cows

Glucose, NEFA and BHBA can be monitored in blood either in a laboratory setting or on

farm through hand held meters. Glucose and ketone bodies (acetoacetate) can also easily be

monitored through urine using a variety of strips that change in color based on the concentration

present in urine. Beta hydroxybutyrate can also be monitored through milk using similar strips

(Geishauser et al., 2000). In blood, NEFA concentrations are considered to be elevated if they

exceed 400 μmol/L in the early lactation dairy cow (González et al., 2011). The threshold most

commonly used to define subclinical ketosis is BHBA in between 1.2 and 3.0 mmol/L, and the

clinical ketosis threshold is often considered as BHBA > 3.0 mmol/L (Oetzel, 2013b).

Hypoglycemia in the early lactation dairy cow is defined as blood glucose of ＜ 2.5 mmol/L

(González et al., 2011). Together, blood NEFA, BHBA, and, to a lesser extent, blood glucose

can be used to evaluate the energy status of the dairy cow (Geishauser et al., 2000, González et

al., 2011).

Rumination

It is a common saying that a happy healthy cow is the one chewing her cud. When cattle

eat, they do not spend much time chewing the feed before swallowing it. This is mainly because

the real chewing is performed during rumination, which consists of regurgitation of a food bolus,

37

mastication, and re-swallowing the bolus (Hastings, 1944). Rumination allows ruminants to

digest forages more efficiently than non-ruminants, and is one of the most important parts of the

digestive function in ruminants. It has the function of breaking down forage particles to make

them more accessible for digestion by rumen microbes and to facilitate passage out of the

forestomachs (McDonald et al., 2002). Rumination also increases saliva production, which

contains high amounts of bicarbonate and phosphate buffers which are necessary to prevent

rumen acidosis (McDonald et al., 2002). During rumen movements, rough and hard feed

stimulate nerve endings situated around the cardia to promote the regurgitation of the cud, which

is a semi-digested feed bolus (Sjaastad et al., 2003). After chewing of the cud, it is swallowed

again to complete its path through the rumen. This is followed by regurgitation and chewing of a

new bolus (Sjaastad et al., 2003). Cows spend a substantial amount of their day ruminating;

approximately 350-500 minutes for dairy cows fed a total mixed ration (Table 2-1).

Rumination Measurement

As soon as it was noticed that rumen function was highly related with the cow’s health,

scientists became interested in developing ways to measure ingestion and rumination in a non-

invasive fashion. At first, visual observations, either directly or via recorded video were

performed, and this method has been used by several researchers (Krause et al., 1998, Lindström

et al., 2001). Given that visual observation is a very labor intensive and time consuming method

there has been an intensive effort to develop other methodologies (Kononoff et al., 2002).

Various prototypes were developed to measure jaw movements and bites, such as the micro

mercury switch attached to a halter to record the total bites numbers and grazing bites developed

by Stobbs and Cowper (Stobbs and Cowper, 1972). Then there was the stretchable noseband by

38

Penning (Penning, 1983) which created electrical signals when there were jaw movements. This

was accompanied by a computer software that analyzed the data and gave a total movement

number. Later on Penning (Penning et al., 1984) created an algorithm that could differentiate

between rumination chews and grazing bites. This eventually developed into the IGER Behavior

Recorder and Graze jaw movement analysis software that is commercially available (Ungar and

Rutter, 2006).

The use of acoustic biotelemetry to monitor rumination was suggested by Alkon, (Alkon,

1989). A small sound microphone recorded chewing sounds and used radio transmission to send

the information to a remote device. In trials to validate this method Laca and colleagues (Laca et

al., 1992) positioned a microphone facing inward on the forehead of steers grazing. Through that

trial it was discovered that bite sounds were distinguishable from grinding chewing sounds and

therefore sound could replace visual observation for jaw movement measurements. The acoustic

method was then compared by Ungar and Rutter (Ungar and Rutter, 2006) to the previously

described IGER jaw movement monitor using visual observation as the gold standard. The

results were that the acoustic monitoring had a 1% error rate, while the IGER had a 22% error

rate. Laca and colleagues (Laca et al., 1994) determined that the acoustic method could

appropriately differentiate between the chew-bites when a cow is eating fresh feed versus the

sounds associated with cud chewing. The SCR rumination collars (SCR Engineers Ltd., Netanya,

Israel) allow automated monitoring of eating and ruminating behavior in dairy cows and have

been validated through a series of studies. Both Schirman and colleagues (Schirmann et al.,

2009) and Lindgren (Lindgren, 2009), concluded that the SCR rumination collars were of value

to research and in commercial dairy production to monitor rumination with excellent accuracy in

a minimally invasive manner.

39

The SCR company developed the RuminAct™ (HR-Tag rumination monitoring system,

SCR Engineers Ltd., Netanya, Israel) monitoring system, which is the method used in this trial.

The HR-Tag rumination monitoring system consists of a collar with a weight attached to its

bottom and a recording device attached to the left side of the collar. This recording device

records the specific sounds associated with regurgitation of a food bolus and thencud chewing

and converts these into rumination through computer software. The HR-Tag rumination

monitoring system records data for 2 hours and then sends it remotely via infrared signal to an

antenna connected to the SCR software in a computer, summarizing data as raw rumination data

every 2 hours and time spent ruminating per 24 hours (absolute values and a rolling average).

Association between Rumination and Hypocalcemia

Hypocalcemia negatively affects gastrointestinal motility, including rumen motility.

When hypocalcemia is induced experimentally, a decrease in the frequency and strength of

rumen contractions is observed, with total cessation of rumen motility in animals with severe

hypocalcemia (Huber et al., 1981, Daniel, 1983, Jørgensen et al., 1998). Rumen function is also

affected during SCH. Huber and colleagues (Huber et al., 1981) reported that rumen motility was

impaired before the appearance of other clinical signs of hypocalcemia. Martinez and colleagues

(Martinez et al., 2014) found that both DMI and the number of rumen contractions per 2 min

were reduced during experimentally-induced SCH. These studies did not evaluate cud chewing

behavior.

There is limited data on the effect of SCH on the time spent ruminating. Sterret and

colleagues (Sterret et al., 2014) studied rumination in 120 dairy cows (Holstein = 90, crossbred =

19, Jersey = 11) fitted with HR-Tag rumination collars. They measured blood tCa on days 3, 7

40

and 14 postpartum and did not find any statistical difference in rumination between cows with

SCH (≤ 1.8 mmol/L) and normocalcemic cows (316.99 ± 8.35 and 299.90 ± 5.12 min/d for SCH

and non- SCH cows respectively). Liboreiro and colleagues (Liboreiro et al., 2015) studied

rumination in 296 Holstein cows and reported that cows with SCH (tCa < 8.55 mg/dl within 72

hours after calving) had significantly reduced rumination on the day of calving (P < 0.01) and

tended to have reduced rumination at 3 days after calving (P = 0.08) compared with

normocalcemic cows.

Objectives and Hypothesis

The objective of this experiment was to determine if the oral administration of a calcium

bolus (Bovikalc ®) providing 43g of calcium within 2 hours after calving and again 12 hours

later affects rumination in multiparous Holstein cows. A secondary objective was to compare the

serum tCa, BHBA, NEFA and glucose concentrations in the first 24 hours after calving in

supplemented and unsupplemented cows. Lastly, we aimed to evaluate activity and milk yield in

supplemented and unsupplemented cows.

We hypothesized that oral supplementation of calcium at calving would improve rumen

function in multiparous cows, and therefore lead to more time spent cud chewing in the first few

days in milk. We expected that cows treated with the oral calcium boluses would have higher

blood tCa concentrations, and lower NEFA and BHBA concentrations compared to the untreated

group.

41

Table 2-1. Cud chewing times for dairy cows fed a TMR. Adapted from (Lindgren, 2009)

Activity Minutes/day

Eating chews 185 - 350

Cud chewing 344 - 496

Figure 2-1. The association of hypocalcemia with a wide variety of post-partum diseases and

performance in the dairy cow(DMI=dry matter intake; DA=displaced abomasum)

Figure 2-2. The effect of blood pH and plasma Mg concentrations on PTH receptor

conformation. Lock-key PTH receptor at a blood pH of 7.35 and when concentrations

of Mg in blood are adequate (normal Mg) or during hypomagnesemia, compared with

blood pH of 7.45 during normal Mg conditions. Taken from (Goff 2008, p. 51)

42

CHAPTER 3

MATERIALS AND METHODS

All animal procedures were approved by the University of Florida (UF) Institutional

Animal Care and Use Committee (IACUC protocol 201508970).

Cows, Housing and Herd Management

The experiment was conducted at the UF Institute of Food and Agricultural Sciences

Dairy Unit (DU) in Gainesville, FL, from December 2015 to December 2016. The DU herd has

approximately 500 lactating Holstein cows housed in free stall barns. The yearly rolling herd

average milk yield was approximately 10,000 kg/cow.

Healthy dry cows were moved from an early dry period grassed lot to a prepartum free

stall pen 21 to 25 days before expected calving date. The diets were fed as total mixed rations

(TMR), and the pre- and postpartum diets fed during the experiment are described in Table 3-1.

The prepartum diet was formulated to have a negative dietary cation-anion difference by feeding

an acidogenic product. Fresh feed was delivered once a day at approximately 0830 hours and

was pushed up four times a day between feedings. The prepartum pen was equipped with

sprinklers over the feed bunk and fan ventilators atop of sand bedded free stalls. Within 3 hours

after calving, cows were moved to an early lactation pen, which contained sand-bedded free

stalls and similar cooling to the prepartum pen. Sprinklers and fans were activated when the

environmental temperature rose above 15C. Early lactation cows were milked 4 times a day at

approximately 0600, 1200, 1800 and midnight. Cows were fed for ad libitum intake, twice daily,

with feed pushed up to 4 times a day. The TMR fed to the early lactation cows was formulated to

43

meet or surpass the guidelines recommended by the National Research Council. Potable fresh

water was available ad libitum at all times to all cows.

Experimental Design and Treatments

The experiment was a randomized block design. Any potentially eligible parous healthy

dry cow was fitted with a rumination and activity collar (HR-Tag rumination monitoring system,

SCR Engineers Ltd., Netanya, Israel) on days 259-263 of gestation. Because of variation in

gestation length, this corresponded to -25 to -7 days prepartum in the experimental population.

Collars were removed at 30 DIM. At the time of collar placement, lameness score (LS) and body

condition score (BCS) were evaluated and recorded, and lactation number and previous lactation

health data was obtained from herd records. At calving, cows that had spent at least 7 days with

the collar on were assigned to a treatment or a control using a randomized block design.

Computer software was used to create a randomized list consisting of number zero (control) or

one (treatment) for 76 cows (Research Randomizer Version 4.0, 2015, Urbaniak and Plous,

http://www.randomizer.org/). Exclusion criteria at calving included all cows that calved less than

7 days after the collar was fitted as well as cows that were lame (locomotion score of 3 or more;

see below) or cows that developed clinical illness during the prepartum period, including cows

that developed clinical hypocalcemia (milk fever) at calving.

Treated cows received one oral Ca bolus (Bovikalc®, Boehringer Ingelheim Vetmedica

Inc., St. Joseph, MO) within 2 hours after calving, and a second one approximately 12 hours

after the first was administered, according to the manufacturer’s protocol. Bovikalc® is an oral

bolus containing 71% Ca chloride and 29% Ca sulfate covered in a coating of fat to protect the

44

oral and esophageal mucosa, and providing 43 g of Ca per bolus. Control cows received no

placebo, but were submitted to the same health and sampling protocols as treated cows.

Milk Yield and Health Data

Milk yield data from each milking was measured using Afimilk meters (SAE Kibbutz

Afikim, Israel) and recorded by the software system used at the DU (Afikim; SAE Kibbutz

Afikim, Israel). Total daily milk yield data was downloaded from the farm management database

for each cow until 30 DIM.

Body condition score and LS were assessed when the collar was placed and at 24 hours

after calving. Cows were examined for any signs of illness at calving and at 12 and 24 hours

after calving by trained research personnel. Data recorded at these examinations included rectal

temperature, urine ketone concentrations (Ketostix, Bayer Corporation Elkhart, IN, USA), rumen

contraction rate, heart rate and respiratory rate. Milk was examined for evidence of clinical

mastitis. The reproductive tract was evaluated by rectal palpation at 12 hours after calving. Body

condition score was assessed by trained personnel on a scale of 1 to 5 with 0.25 increments using

standard dairy production methodology (Body Condition Scoring Dairy Cattle, Elanco Animal

Health, Indianapolis, IN). Body condition score was assessed at the time of collar placement and

at 24 hours after calving. Locomotion score was assessed by trained personnel on a scale of 0 to

4 using the LS system in place at the DU (Zinpro Corporation, Eden Prairie, MN). In addition to

the examinations by trained research personnel over the first 24 hours after calving, the farm

technicians or UF veterinarians performed full physical examinations as cows were sorted after

the first morning milking on days 4, 7 and 12 after calving and at any day there was a negative

deviation from expected milk yield, (calculated by the Afifarm software) for 2 or more milkings.

45

These postpartum health evaluations included attitude, rectal temperature, rectal palpation, udder

check, urinalysis to detect ketone bodies and assessment of rumen function and auscultation and

percussion to detect a displaced abomasum. Health events during the first 30 DIM were recorded

by farm personnel in the DU farm database (Afifarm; SAE Kibbutz Afikim, Israel) and were

downloaded into excel spreadsheets for later analyses.

Rumination and Activity Data Recording

Cows were fixed with a rumination collar (Hr-Tag Rumination Monitoring System, SCR

Engineers Ltd., Netanya, Israel) at 259 to 263 days of gestation. This collar is equipped with a

tag containing a microphone that records the sounds associated with rumination and an

accelerometer that records movement associated with walking activity. The device sends data to

a receiver via radio frequency and this is captured by long-distance antennas on the milking

parlor. The associated computer software provides output data for rumination (minutes) and

activity (steps) as raw data per 2-hour block. Data were downloaded from the software every 24

hours into Excel spreadsheets for later analyses.

Blood Collection and Analyses

Blood (serum) samples were obtained from the coccygeal vessels. Samples were

collected from all cows within 2 hours after calving (0 hours), which was immediately before the

first bolus for treated cows, then at 0.5 hours, 12 hours (immediately before the second bolus for

treated cows), 12.5 hours, and 24 hours after calving. After clotting, samples were refrigerated

and then centrifuged within 12 hours after collection (Eppendorf Multi-purpose Centrifuge 5810)

46

for 10 min at 3000 rpm. Serum was then harvested and 3 identical aliquots were stored at -57 °C

until analyses. Before analyses, serum samples were thawed at room temperature. Samples were

coded so that the research personnel performing analyses were blinded to treatments.

Concentrations of tCa, BHBA, NEFA and glucose in serum were measured by

photometry using a clinical chemistry analyzer (RANDOX Daytona © Randox Laboratories

Ltd). Each time the analyzer was operated it was calibrated to obtain a data curve, and then

measurements were taken at 20-second intervals for a 10-minute period per sample. A water

blank was used to correct minor variations on all samples, then the machine measured the

standard samples and calculated the absorbance differences from the reactions based on the

calibration curve data to obtain a concentration value.

Statistical Analyses

For all data presented, calving day was defined as day 0. Data were analyzed utilizing

ANOVA with the GLM and MIXED procedures of SAS (SAS/STAT ver. 9.4, SAS Institute Inc.,

Cary, NC). For outcome variables with no repeated measures such as the baseline values, PROC

GLM was used. The MIXED procedure was used to evaluate the outcome variables with

repeated measures, which included rumination during the first 24 hours, daily rumination,

activity and milk yield for the first 30 DIM, and serum tCa, NEFA, BHBA and glucose

concentrations. Associations between treatment and covariates were determined using a SLICE

option in the MIXED procedure. Data were tested for normality of residuals and no

transformation was needed before analyses. For the mixed models F tests, the method of

Kenward-Roger was used to determine the denominator degrees of freedom (DF).

47

To analyze the effect of treatment on concentrations of metabolites in serum (tCa, NEFA,

BHBA and glucose), an ANOVA was performed for each metabolite. The fixed effect for each

model was treatment (control or bolus). Baseline metabolite values measured in the serum

samples collected before the first treatment (Ca0, NEFA0, BHBA0 or Glucose0 for the relevant

model) were evaluated as covariates. Other variables assessed for inclusion in the model were

BCS, parity (2 or ≥ 3), sample number as well as treatment interactions with each variable. Cow

was the random term nested within treatment. For the independent variables, a stepwise

backward elimination was performed in accordance to Wald-statistics criterion when P > 0.05.

The models to evaluate the effect of treatment with 2-hourly rumination and activity

within the first 24 hours after calving included the fixed effect of treatment (control or

treatment). The variables parity (2 or ≥ 3), hours after calving (2, 4, 6, 8, 10, 12, 14, 16, 18, 20,

22, 24, 26), BCS and Ca0 were evaluated for inclusion in the model, as well as treatment

interactions with each variable. Cow nested within treatment was a random term in the model. A

stepwise backward elimination analysis was then performed to remove all independent variables

with a P > 0.05.

A correlation analysis was performed using PROC CORR on SAS to evaluate the

possible correlation between the concentration of serum tCa at calving (Ca0) and rumination.

The correlation values were taken as weak when r < 0.3, moderate when r = 0.31 - 0.5 and strong

when r > 0.5.

The models to evaluate the effect of treatment with daily rumination, daily activity and

milk yield for the first 30 DIM, include the fixed effect of treatment (control or treatment). The

variables parity (2 or ≥ 3), DIM, BCS, and Ca0 were evaluated for inclusion in the model, as

well as treatment interactions for each variable. For the outcome variable of daily rumination,

48

prepartum rumination level (average daily rumination in minutes for that cow from the time of

collar placement until day -5) was included as a fixed effect. For the outcome variable of daily

activity, pre-partum activity (average daily activity in minutes from the time of collar placement

until day-5) was included as a fixed effect. Cow ID was the random term nested within

treatment. A stepwise backward elimination analysis was then performed to remove all variables

with a P > 0.05.

To evaluate the association of SCH on rumination, cows were classified into SCH or

normocalcemic, irrespective of treatment status. A cow was considered to have SCH if the

average serum tCa (average Ca value for all samples except Ca0) was < 8.59 mg/dL. The models

to evaluate the association of SCH with rumination at 2-h intervals within the first 24 hours after

calving included the fixed effect of Ca status (SCH or normocalcemic). The variables hours (2, 4,

6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26), BCS, parity (2 or ≥ 3), and Ca0 as well as the interactions

of SCH status with each variable were evaluated for inclusion in the model. Cow nested within

treatment was a random parameter in the model. A stepwise backward elimination analysis was

then performed to remove all variables with a P > 0.05.

49

Table 3-1. Ingredient and nutrient composition of pre- and postpartum diets (DM basis). Diet

Item Prepartum Postpartum

Ingredient %

Corn silage 43.8 36.7

Bermuda grass hay 24.2 -

Alfalfa hay - 11.1

Brewer’s grains 14.6 10.0

Citrus pulp 4.2 7.1

Corn grain - 11.1

Soybean hulls - 5.6

Soybean meal, 47% CP 3.3 11.8

Energy Booster Mag1 - 1.8

Postpartum mineral supplement - 4.9

Prepartum mineral supplement 4.2 -

SoyChlor2 5.8 -

Nutrient profile

Net energy,3 Mcal/kg 1.54 1.78

Crude protein, % 13.6 17.2

Neutral detergent fiber, % 43.6 29.9

Acid detergent fiber, % 27.1 20.3

Starch, % 18.4 23.2

Ether extract, % 4.2 5.4

Ca, % 0.75 0.78

P, % 0.34 0.35

Mg, % 0.56 0.41

K, % 1.02 1.60

Na, % 0.06 0.56

Cl, % 0.87 0.54

S, % 0.27 0.19

DCAD,4 mEq/100g -12.29 +38.27

Fe, ppm 137.4 154.5

Zn, ppm 69.3 63.3

Cu, ppm 12.6 14.1

Mn, ppm 63.3 43.5

Se, ppm 0.44 0.42

Co, ppm 0.75 0.31

I, ppm 0.68 0.70

Vitamin A, IU/kg 9,680 7,304

Vitamin D, IU/kg 2,794 1,254

Vitamin E, IU/kg 139.7 63.8 1 Energy Booster Mag contains hydrolyzed animal and vegetable fat and magnesium oxide; Milk Specialties Global,

Eden Prairie, MN)

2SoyChlorTM (Dairy Nutrition Plus, Princeton, NJ) contains the following (DM basis): 20.6% CP, 4.1% starch,

2.84% Mg, 0.48% K, 0.04% Na, 0.35% S, 4.5% Ca and 10.3% Cl

3Calculated at 11 and 19 kg of DM/d for the pre- and postpartum diets (CPM-Dairy version 3.0.8.1;

https://cahpwww.vet.upenn.edu/doku.php/software:cpm:start)

4 DCAD = dietary cation-anion difference calculated as mEq of K and Na minus mEq of Cl and S.

50

CHAPTER 4

RESULTS

Exclusions and Missing Data

Of the 76 cows enrolled, there was 1 cow removed from all analyses because she calved

too early and had been enrolled by mistake. Of the remaining 75 cows, rumination data for the

first 24 hours after calving was excluded from analysis from 2 cows, because the first day of data

was missing due to technical problems (power outage) on the farm. Therefore, 73 cows remained

to perform the 24 hour rumination analysis. For the metabolite analyses, 1 cow was removed

because of extreme outlying values associated with severe fatty liver, leaving 74 cows in these

analyses. For the daily rumination, activity and milk yield during the first 30 DIM, 3 cows were

removed from the analyses because of treatment with other forms of Ca on ≥ 2DIM (1 developed

clinical hypocalcemia after 24 hours postpartum, 2 developed LDA), 1 cow was removed due to

early culling (neuropathy), and 1 cow died (severe fatty liver). Therefore, these analyses were

performed with data from 70 cows.

Baseline Values

Baseline values for treated and control cows are shown in table 4-1. Baseline values did not

differ between treated and control cows for metabolites at calving (tCa, BHBA, NEFA and

glucose). There was no difference between treated and control cows in BCS, parity, pre-partum

rumination minutes, as well as partum rumination minutes and peri-partum activity. There was a

51

difference in average pre-partum activity minutes/day (P = 0.03) between treated and control

cows.

Effect of Treatment on Serum Concentrations of Total Calcium

Descriptive data illustrating the change in serum tCa concentrations from baseline to 0.5

hours after each bolus (or equivalent time for control cows) is shown in figure 4-1. The effect of

treatment on tCa concentrations is shown in figure 4-2 and table 4-2. The average tCa over the

first 24 hours after calving was less (P < 0.01) in control cows compared with treated cows (8.14

± 0.08 vs. 8.44 ± 0.07 mg/dL). There was no interaction between treatment groups and sample

number (P = 0.4). At 0.5 hours after bolus administration (treated group) or equivalent time for

control cows, tCa was less (P = 0.04) in control compared with treated cows (7.94 ± 0.10 vs.

8.21 ± 0.09 mg/dL). At 12 hours, immediately before the second bolus for treated cows, or

equivalent time for control cows, however, there was no difference (P = 0.19) between control

and treated cows (8.33 ± 0.10 vs. 8.50 ± 0.09 mg/dL). At 12.5 hours (4th sample) tCa was less (P

< 0.01) in control compared with treated cows (8.24 ± 0.10 for controls and 8.64 ± 0.09 mg/dL),

and similarly at 24 hours there was a difference (P = 0.02) between tCa for control and treated

cows (8.07 ± 0.10 and 8.39 ± 0.09 mg/dL).

Effect of Treatment on Serum Concentrations of NEFA, BHBA and Glucose

The effect of treatment on serum NEFA concentrations is shown in figure 4-3 and table

4-3. There was no difference in overall serum NEFA concentrations (P = 0.7) between control

52

and treated cows (0.68 ± 0.05 vs. 0.71 ± 0.05 mEq/L). There was no interaction between

treatment group and sample (P = 0.8).

The effect of treatment on serum BHBA is shown in figure 4-4 and table 4-4. There was

no difference in overall serum BHBA concentrations (P = 0.06) between control and treated

cows (0.8 ± 0.03 vs. 0.7 ± 0.03 mmol/L). There was no interaction between treatment group and

sample (P = 0.9).

The effect of treatment on serum glucose concentrations is shown in figure 4-5 and table

4-5. There was no difference in overall serum glucose concentrations (P = 0.6) between control

and treated cows (4.35 ± 0.11 vs. 4.43 ± 0.10 mmol/L). There was no interaction between

treatment group and sample (P = 0.4).

Correlation between Serum Calcium Concentration and Rumination over the First 24

Hours Post Calving

There was a moderate correlation (Corr = 0.4; P <0.01) between the average serum tCa

and rumination during the first 24 hours after calving. The scatterplot illustrating this association

is shown in figure 4-6.

Effect of Treatment on Rumination during the First 24 Hours Post Calving

The effect of treatment on rumination during the first 24 hours after calving is shown in

figure 4-7 and table 4-6. There was no difference in average rumination (minutes per 2 hour

block) over the first 24 hours postpartum (P = 0.7) between control and treated cows (12.45 ±

1.11 vs. 11.87 ± 1.13 min/2hrs). There was no difference for the interaction between treatment

53

group and time after calving (P = 0.6). The variables hours postpartum and parity had significant

positive associations with rumination, with (P < 0.01) and (P = 0.01), respectively.

Effect of Treatment on Daily Rumination during the First 30 DIM

The effect of treatment on daily rumination during the first 30 DIM is shown in figure 4-8

and table 4-7. There was no difference (P = 0.8) in daily rumination (minutes) for the first 30

DIM between control and treated cows (334.81 ± 11.72 vs. 330.47 ± 11.72 min/day). There was

also no interaction (P = 0.5) between treatment group and DIM. The variables DIM (P < 0.01)

and pre-partum rumination (P < 0.01) were positively associated with daily rumination during

the first 30 DIM.

Effect of Treatment on Daily Activity during the First 30 DIM

The effect of treatment on daily activity (minutes) during the first 30 DIM is shown in

figure 4-9 and table 4-8. There was no difference (P = 0.3) in overall daily activity (minutes) for

the first 30 DIM between control and treated cows (486.67 ± 8.41 vs. 474.50 ± 8.40 min/day).

There was no difference (P = 0.7) for the interaction between treatment group and DIM. The

variables DIM (P < 0.01) and pre-partum activity (P < 0.01) were positively associated with

daily activity level during the first 30 DIM.

54

Effect of Treatment on Milk Yield during the First 30 DIM

The effect of treatment on daily milk yield during the first 30 DIM is shown in figure 4-

10 and table 4-9. There was no difference (P = 0.8) in overall daily milk yield for the first 30

DIM between control and treated cows (81.67 ± 2.28 vs. 81.02 ± 2.28 Lbs/day). There was no

difference (P = 0.2) for the interaction between treatment group and DIM. The variables DIM (P

< 0.01) and parity (P < 0.01) were positively associated with daily milk yield.

Association of Subclinical Hypocalcemia at Calving with Rumination during the First 24

Hours Post Calving

The association between SCH over the first 24 hours after calving and rumination

(minutes per 2 hour block) is shown in figure 4-11 and table 4-10. Using the average tCa value

over the first 24 hours after calving (excluding Ca0), irrespective of treatment group, 45% of the

cows were classified as normocalcemic and 55% were classified as SCH (tCa <8.59 mg/dL).

Cows with SCH had spent less time ruminating (minutes/2 hour block) compared with

normocalcemic cows (9.83 ± 1.05 and 14.16 ± 1.17 minutes for SCH and normocalcemic cows,

respectively; P < 0.01). There was no interaction between tCa status and time (P = 0.08).

55

Table 4-1. Baseline values for least squares means (LSM) +/- std. error of the mean (SEM) for

control cows (n = 36) and cows supplemented with oral Ca boluses (n = 39). Control Treatment

Variable name LSM SEM LSM SEM

Calcium at calving mg/dL 8.04 0.15 7.88 0.15

BHBA at calving mmol/L 0.70 0.05 0.62 0.05

NEFAs at calving mEq/L 0.80 0.08 0.73 0.08

Glucose at calving mmol/L *5.79 0.29 6.81 0.28

Cows with SCH at calving (%) 83 81

Body condition score 3.40 0.35 3.38 0.37

Lactation number 3.00 1.24 3.14 1.28 aRumination minutes prepartum 353.85 31.62 369.42 31.23 bRumination minutes peri-partum 302.40 29.79 338.82 29.60 cActivity pre-partum *500.19 14.52 454.62 14.59 dActivty peri-partum 511.52 32.13 485.39 31.92

aRumination min prepartum = from day -25 to -5 bRumination min peri-partum = from day -4 to 0 cActivity prepartum = from day -25 to -5 dActivity peri-partum = from day -4 to 0

*P = 0.03

SCH= < 8.59 mg/dL tCa within the first 2 hours after calving, before colostrum milking.

Table 4-2. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of serum tCa concentration (mg/dL) over the first 24

hours after calving between control cows and cows supplemented with

oral Ca boluses. Data used for figure 4-2. Control Treatment

Hour LSM SEM LSM SEM

0.5 7.94 0.10 8.21 0.09

12 8.33 0.10 8.50 0.09

12.5 8.24 0.10 8.64 0.09

24 8.07 0.10 8.39 0.09

Table 4-3. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of serum NEFA concentration (mEq/L) over the first 24

hours after calving between control cows and cows supplemented with

oral Ca boluses. Data used for figure 4-3. Control Treatment

Hour LSM SEM LSM SEM

0.5 0.73 0.07 0.73 0.07

12 0.60 0.07 0.69 0.07

12.5 0.66 0.07 0.71 0.07

24 0.71 0.07 0.72 0.07

56

Table 4-4. Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of serum BHBA concentration (mmol/L) over the first 24

hours after calving between control cows and cows supplemented with

oral Ca boluses. Data used for figure 4-4. Control Treatment

Hour LSM SEM LSM SEM

0.5 0.73 0.04 0.68 0.04

12 0.78 0.04 0.69 0.04

12.5 0.78 0.04 0.72 0.04

24 0.79 0.04 0.71 0.04

Table 4-5. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of serum glucose concentration (mmol/L) over the first

24 hours after calving, between control cows and cows supplemented

with oral Ca boluses. Data used for figure 4-5. Control Treatment

Hour LSM SEM LSM SEM

0.5 6.32 0.21 6.78 0.20

12 3.64 0.21 3.69 0.20

12.5 3.84 0.22 3.88 0.20

24 3.61 0.21 3.37 0.20

Table 4-6. Least squares means (LSM) and standard error of the mean (SEM) for the

comparison of daily rumination in minutes, between control and

supplemented cows for the first 24 hours postpartum. Data used for

figure 4-7. Control Treatment

Hours LSM SEM LSM SEM

2 5.70 2.18 4.46 2.20

4 6.81 2.18 4.10 2.20

6 8.68 2.18 9.29 2.20

8 9.15 2.18 13.18 2.20

10 11.12 2.18 14.32 2.20

12 14.43 2.18 12.10 2.20

14 11.65 2.18 12.01 2.20

16 15.65 2.18 11.43 2.20

18 14.37 2.18 12.29 2.20

20 12.98 2.18 16.21 2.20

22 16.23 2.18 16.79 2.20

24 15.87 2.18 16.07 2.20

26 17.59 2.18 15.71 2.20

57

Table 4-7. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of daily rumination in minutes, between control and

supplemented cows for the first 30 DIM. Data used for figure 4-8. Control Treatment

DIM LSM SEM LSM SEM

0 196.14 17.52 196.08 17.52

1 201.58 17.52 179.22 17.64

2 273.64 17.64 236.78 17.52

3 278.43 17.52 299.76 17.64

4 311.11 17.94 331.52 17.80

5 345.94 17.80 346.96 17.80

6 358.26 17.52 351.02 17.52

7 351.26 17.52 349.10 17.52

8 344.83 17.64 361.55 17.79

9 353.17 17.52 375.31 17.79

10 335.64 17.52 368.52 17.52

11 338.02 17.64 360.31 17.52

12 339.96 17.52 371.49 17.52

13 336.70 17.52 344.87 17.52

14 352.51 17.66 331.75 17.52

15 350.56 17.66 335.58 17.52

16 379.52 17.52 350.87 17.66

17 359.11 17.66 349.62 17.66

18 343.76 17.66 333.81 17.52

19 366.27 17.66 334.05 17.52

20 363.02 17.68 331.10 17.52

21 352.40 17.83 318.08 17.52

22 359.50 17.83 332.09 17.81

23 337.28 17.68 334.64 17.98

24 342.56 17.68 304.56 17.83

25 353.62 17.79 339.24 17.99

26 365.20 17.52 355.91 17.99

27 348.37 17.52 355.16 17.69

28 345.79 17.66 364.56 17.69

29 350.06 17.66 341.14 17.69

30 353.61 17.52 360.87 17.99

58

Table 4-8. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of daily activity in minutes, between control and

supplemented cows for the first 30 DIM. Data used for figure 4-9. Control Treatment

DIM LSM SEM LSM SEM

0 541.00 12.50 529.14 12.45

1 533.68 12.50 501.25 12.53

2 479.67 12.57 467.96 12.45

3 470.09 12.50 473.53 12.53

4 496.40 12.77 479.72 12.64

5 474.16 12.68 463.43 12.64

6 450.47 12.50 451.96 12.45

7 472.09 12.50 470.05 12.45

8 471.18 12.57 475.68 12.62

9 483.32 12.50 469.20 12.62

10 466.85 12.50 465.90 12.45

11 486.79 12.58 475.99 12.45

12 473.18 12.50 479.20 12.45

13 468.21 12.50 470.70 12.45

14 476.18 12.59 464.46 12.45

15 474.81 12.59 472.96 12.45

16 477.00 12.50 463.67 12.54

17 473.17 12.59 473.72 12.54

18 476.57 12.59 465.96 12.45

19 478.42 12.59 476.52 12.45

20 474.15 12.61 477.08 12.45

21 476.41 12.71 473.52 12.45

22 476.14 12.71 481.82 12.64

23 466.51 12.62 470.04 12.77

24 494.78 12.61 472.12 12.67

25 495.38 12.67 454.90 12.77

26 484.77 12.50 453.34 12.77

27 489.44 12.50 460.35 12.57

28 491.80 12.59 465.45 12.57

29 496.22 12.59 462.97 12.57

30 476.53 12.50 470.92 12.78

59

Table 4-9. Least squares means (LSM) and standard error of the mean (SEM)

for the comparison of daily milk yield in kg between control and

supplemented cows for the first 30 DIM. Data used for figure 4-10. Control Treatment

DIM LSM SEM LSM SEM

0 9.28 1.58 9.06 1.55

1 17.17 1.55 19.54 1.54

2 23.36 1.55 24.48 1.54

3 26.24 1.56 26.79 1.54

4 32.54 1.57 29.25 1.55

5 30.98 1.56 32.07 1.55

6 33.33 1.55 33.49 1.54

7 33.98 1.55 34.03 1.54

8 34.21 1.55 36.90 1.54

9 36.49 1.55 35.38 1.54

10 35.90 1.55 36.21 1.54

11 38.13 1.55 36.35 1.54

12 36.14 1.55 39.65 1.54

13 40.14 1.55 37.89 1.54

14 40.28 1.56 39.20 1.54

15 39.55 1.56 37.30 1.54

16 40.68 1.55 37.52 1.54

17 40.98 1.56 39.95 1.55

18 40.12 1.58 42.05 1.57

19 41.21 1.56 39.23 1.55

20 43.57 1.55 41.32 1.55

21 41.02 1.55 42.02 1.55

22 41.10 1.55 42.20 1.55

23 42.36 1.55 43.66 1.55

24 44.27 1.55 42.34 1.55

25 43.64 1.55 42.74 1.55

26 44.74 1.55 44.19 1.55

27 42.98 1.55 42.70 1.55

28 43.16 1.55 42.16 1.54

29 45.26 1.56 44.13 1.55

30 45.56 1.57 45.39 1.55

60

Table 4-10. Least squares means (LSM) and standard error of the mean (SEM) for

the comparison of rumination (minutes) between cows with

normocalcemia (NC) and subclinical hypocalcemia (SCH) for the first

24 hours after calving. Data used for figure 4-11. SCH NC

HRS LSM SEM LSM SEM

2 4.4 2.1 4.9 2.3

4 5.4 2.1 4.4 2.3

6 9.8 2.1 6.8 2.3

8 8.6 2.1 13.3 2.3

10 9.4 2.1 15.8 2.3

12 11.0 2.1 15.0 2.3

14 10.5 2.1 12.4 2.3

16 11.4 2.1 15.1 2.3

18 9.3 2.1 17.3 2.3

20 9.9 2.1 19.3 2.3

22 13.5 2.1 19.2 2.3

24 10.9 2.1 21.3 2.3

26 13.7 2.1 19.3 2.3

Figure 4-1. The percentage change in serum total Ca concentrations from pretreatment to 0.5

hours after administration of an oral Ca bolus containing 43 g of calcium. The first Ca

bolus was administered within 2 hours after calving and the second bolus 12 hours

later. Control cows received no supplemental oral Ca.

Treatment Treatment

61

Figure 4-2. Characterization of serum concentrations of total Ca (tCa) during the first 24 hours

after calving for control cows and cows supplemented with oral Ca boluses. There

was an effect of treatment (P < 0.01) on serum concentrations of tCa. There was an

interaction between treatment and time postpartum (P = 0.40), and an association of

time with serum tCa concentration (P < 0.01).

Figure 4-3. Characterization of serum values for NEFA concentrations during the first 24 hours

after calving for control cows and cows supplemented with oral Ca boluses. There

was no effect of treatment (P = 0.7), time (P = 0.32) or treatment by time interaction

(P = 0.8) on serum NEFA concentrations

Ca boluses

Ca boluses

62

Figure 4-4. Characterization of serum concentrations of BHBA during the first 24 hours after

calving for control cows and cows supplemented with oral Ca boluses. Treatment

tended (P = 0.06) to reduce serum concentrations of BHBA. . There was no effect of

time (P = 0.50) or treatment by time interaction (P = 0.90) on serum BHBA.

Figure 4-5. Characterization of serum concentrations of glucose during the first 24 hours after

calving for control cows and cows supplemented with oral Ca boluses. There was no

effect of treatment (P = 0.60) or treatment by time interaction (P = 0.40) on

concentrations of glucose in serum. There was an association with time with glucose

serum concentrations (P < 0.01).

Ca boluses

Ca boluses

63

Figure 4-6. Correlation between the average blood total Ca concentration during the first 24

hours after calving and rumination during the first 24 hours after calving. Data

includes both control cows and cows that were supplemented with oral Ca boluses.

Figure 4-7. Characterization of rumination minutes in the first 24 hours after calving per 2 hour

blocks for control and cows supplemented with oral Ca boluses. There was no effect

of treatment (P = 0.70) on the average rumination during the first 24 hours

postpartum (control = 12.45 ± 1.11 vs. Bovikalc = 11.87 ± 1.13 min/2 hours). There

was no association between treatment and time after calving (P = 0.60). The variables

hours postpartum and parity had significant positive associations with rumination,

with P < 0.01 and P = 0.01, respectively.

Ca boluses

64

Figure 4-8. Characterization of daily rumination minutes in the first 30 days in milk (DIM), for

control and cows supplemented with oral Ca boluses (Bovikalc®). There was no

effect of treatment (P = 0.80) on daily rumination minutes during the first 30 DIM,

which averaged 334.81 ± 11.72 and 330.47 ± 11.72 min/day for control and treated

cows, respectively. There was no association between treatment and DIM (P = 0.50).

The variables DIM (P < 0.01) and pre-partum rumination (P < 0.01) were positively

associated with daily rumination during the first 30 DIM.

Figure 4-9. Characterization of daily activity (minutes of activity per day) in the first 30 days in

milk (DIM), for control and cows supplemented with oral Ca boluses (Bovikalc®).

There was no effect of treatment (P = 0.30) on daily activity during the first 30 DIM,

which averaged 486.67 ± 8.41 and 474.50 ± 8.40 min/day for control and treated

cows, respectively. There was no association between treatment and DIM (P = 0.7).

The variables DIM (P < 0.01) and pre-partum activity (P < 0.01) were positively

associated with daily activity during the first 30 DIM.

Ca boluses

Ca boluses

65

Figure 4-10. Characterization of daily milk yield in kg for the first 30 DIM after calving, for

control and cows supplemented with oral Ca boluses (Bovikalc®). There was no

effect of treatment (P = 0.80) on daily milk yield during the first 30 days in milk

(DIM). There was no association between treatment group and DIM (P = 0.2). The

variables DIM (P < 0.01) and parity (P < 0.01) were positively associated with daily

milk yield.

Figure 4-11. Characterization of rumination (minutes per 2-hour block) during the first 24 hours

after calving, for cows with normocalcemia or SCH (tCa < 8.59 mg/dL). There was

an association between tCa status and rumination (9.83 ± 1.05 and 14.16 ± 1.17 for

SCH and normocalcemic cows, respectively; P < 0.01). There was no association

between tCa status and time (P = 0.08).

Ca boluses

66

CHAPTER 5

DISCUSSION

The primary objective of this experiment was to determine whether oral Ca

supplementation on the day of calving affects rumination in multiparous cows. A secondary

objective was to characterize the serum Ca, BHBA, NEFA and glucose concentrations in the first

24 hours after calving in treated and untreated cows. Lastly, we aimed to evaluate activity and

milk yield in treated and untreated cows.

In the current experiment, administration of 43 g of oral Ca within 2 hours after calving

and again approximately 12 hours later resulted in increased tCa over the 24-hour observation

period compared with untreated cows. Similar results were observed by Sampson and colleagues

(Sampson et al., 2009), who reported an increase in the percentage of Ca in blood of 8.2% within

24 hours for hypocalcemic cows treated with Bovikalc®. In that small experiment (n=10 cows

per group), supplemented cows had significantly higher serum tCa levels at 13 hours after

calving, compared with control cows. In this experiment there was an average increase in tCa of

3.7% in the 30 minutes after the first bolus administration, an increase of 1.8% in the 30 minutes

after the second bolus administration and an overall increase of 7.1% at 24 hours after the first

bolus administration.

Other investigators have also reported on the effect of oral Ca bolus supplementation on

blood Ca concentrations at the doses used in this experiment (Sampson et al., 2009, Martinez et

al., 2016a). Martinez and colleagues (Martinez et al., 2016a) reported that supplementing 43g of

Ca using Bovikalc® boluses only raised blood Ca concentration for approximately 2 hours; in

the current experiment we found that tCa was increased 30 min after each bolus and also at 24

hours after calving, but was not different from control cows at 12 hours after calving. Because

we did not collect samples at 2 hours after the first bolus, we cannot directly compare our results

67

to that of Martinez and colleagues. We had a high incidence of SCH (< 8.59 mg/dL) at calving in

our experiment population (81%), and mean tCa was only increased above the SCH threshold in

treated cows at 30 min after the second bolus before decreasing below the threshold again by 24

hours after calving. This high incidence of SCH occurred despite the fact that our experimental

population was fed a diet supplemented with anionic salts before calving to reduce the risk of

hypocalcemia; prepartum urine pH was not monitored as part of this experiment so we are

unable to evaluate the effectiveness of the anionic salt feeding program. We had a large

proportion of older cows in our experiment population, which may account for the high overall

incidence of SCH.

In this experiment we found a moderate correlation between average tCa and rumination

(correlation r = 0.4; P < 0.01). This is not surprising, given that SCH negatively affects DMI and

rumen motility (Huber et al., 1981; Martinez et al., 2014). Our results are in agreement with

those of Liboreiro and colleagues (Liboreiro et al., 2015), who reported that cows with SCH (tCa

< 8.55 mg/dl within 72 hours after calving) had significantly reduced rumination on the day of

calving (P < 0.01) and tended to have reduced rumination at 3 days after calving (P = 0.08)

compared with normocalcemic cows. The correlation between Ca concentration and rumination

(r = 0.14; P = 0.03) reported by Liboreiro (Liboreiro et al., 2015) was weaker than the

correlation we observed, but overall our results are consistent with their findings..

No other studies that we are aware of have evaluated the effect of Ca supplementation at

calving on rumination. Our underlying hypothesis was that cows with SCH would ruminate less

than normocalcemic cows, and that supplementation with Ca would improve blood Ca

concentrations and thereby result in improved rumination. This hypothesis was based on research

showing that rumen motility and DMI (Martinez et al., 2014) as well as rumination (Liboreiro et

68

al., 2015) are reduced in cows with SCH. However, we did not observe a significant effect of

treatment with oral Ca boluses on rumination, despite an increase in tCa in treated cows. This

may be because treatment failed to increase tCa above the threshold for SCH (> 8.59 mg/dL) in

the majority of treated cows. Supplementation of oral Ca chloride at calving has recently been

reported to have some negative effects on health and production in certain subsets of multiparous

cows (Martinez et al., 2016b) despite an increase in blood Ca concentrations, including cows of

lower production potential. The reason for these negative effects is unknown, but could include

negative effects of rebound hypercalcemia following supplementation in some cows (Martinez et

al., 2016a). We did not evaluate production potential in our study, but it is plausible that any

benefit to rumination caused by increased blood Ca in supplemented cows could have been offset

by other (as yet undefined), negative effects of bolus supplementation.

We did not observe a difference in blood NEFA, BHBA or glucose concentrations

between cows supplemented with Ca boluses and untreated cows. Rumination is correlated with

DMI; given that we did not observe improved rumination in treated cows, it is reasonable to infer

that DMI and energy balance was similar in both groups.

We found no difference in activity in cows supplemented with Ca boluses at calving,

compared with untreated cows, although we did not compare activity levels with respect to Ca

status. Other researchers have reported that activity levels are similar in cows with SCH

compared with normocalcemic cows. Jawor and colleagues (Jawor et al., 2012) found that cows

with SCH within the first 24 hours postpartum visited the water trough and feed bins less daily,

but their overall activity and DMI was similar to normocalcemic cows. Similarly, Liboreiro and

colleagues (Liboreiro et al., 2015) did not find any association between overall activity and tCa

in early lactation.

69

We did not find any difference between treated and untreated cows in milk production in

the first 30 DIM. Other investigators (Oetzel and Miller, 2012, Martinez et al., 2016b) have

reported that milk production was increased in certain subgroups of cows supplemented with oral

Ca at calving, specifically, parous cows with high milk production potential. We did not

subdivide our experiment cows into high and low producers to review the effect of

supplementation on high and low producing cows. DIM and parity did have a positive

association with milk yield, with 3+ lactation cows having a larger milk yield than 2nd lactation

cows, and as DIM progressed the cow produced more milk, with is in accordance with the

literature.

The current experiment was not designed to evaluate the effect of supplementation with

Ca on health outcomes in early lactation. It is possible that the increase in blood tCa that we

observed in treated cows may be beneficial to health. Future studies on larger populations could

be performed to assess if there is benefit of treatment on health outcomes. In addition, other

researchers have reported that lame cows may benefit from Ca supplementation at calving

(Oetzel and Miller, 2012); future research on subsets of cows with specific health disorders could

be conducted to determine if there is a benefit of supplementation in those populations.

70

CHAPTER 6

CONCLUSIONS

Supplementation of multiparous dairy cows with oral boluses containing CaCl2 and

CaSO4 (Bovikalc®) at calving and approximately 12 hours later did not affect rumination when

evaluated as rumination minutes per 2 hours for the first 24 hours after calving, or as daily

rumination minutes for the first 30 DIM. Cows with SCH, however, had reduced rumination on

the day of calving, compared to normocalcemic cows. Although supplemented cows had greater

serum tCa over the first 24 hours after calving than untreated controls, treatment failed to

increase tCa above the threshold for SCH which may account for the lack of differences

observed in rumination between treatment groups.

Cows supplemented with Bovikalc® had similar blood NEFA, BHBA and glucose

concentrations during the first 24 hours after calving, when compared with control cows.

Similarly, there was no effect of treatment on daily activity levels within the first 30 DIM or

activity per 2-hour block within the first 24 hours after calving. Treatment had no significant

effect on milk yield.

Overall, although supplementation of parous cows with an oral Ca bolus (Bovikalc®) at

calving and approximately 12 hours later resulted in a modest increase in total blood Ca

concentrations, and there was no beneficial nor adverse effect of supplementation on rumination,

activity, milk yield or NEFA, BHBA or blood glucose concentrations.

71

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BIOGRAPHICAL SKETCH

Myriam Berenice Jimenez Medrano was born in Mexico City, Mexico. She is the oldest

of three siblings of an architect father and dentist mother. Since her very early childhood and

influenced by her uncle Josue Medrano, a veterinarian, Myriam was interested in veterinary

medicine. Her family also owned a small sheep farm on the state of Puebla in Mexico.This led

Myriam to pursue a degree in Veterinary Medicine and Husbandry from the National

Autonomous University of Mexico in 2003-2008. Throughout vet school, Myriam was very

interested in surgery and small animal medicine. By her 6th semester, the situation in which some

owners kept their animals disheartened her, and she went looking for a change in path. At the

start of her clinical rotations, Myriam did a rotation on bovine reproduction, her professor Dr.

Hector Basurto Camberos bet her that “if she didn’t love cows by the end of the 2 weeks, he

would buy her dinner anywhere”. It was during that time that Myriam found love in food animal

medicine and reproduction. She then went to do as many food animal classes as she could and

did her mandatory social service in dairy grazing NZ Holstein cattle on CEIEPAA,

Tequisquiapan, Qro. Mex. She then graduated veterinary school in 2008. Myriam performed her

thesis in Limousin beef cattle, under the supervision of Drs. Kunio Yabuta Osorio and Dr.

Vicente Lemus at CEIEPAA. In January 2010, a nearly 5,000 animal dairy farm in the small

town of Pedro Escobedo, hired her as the deputy director, position she held until December 2012.

In this position, Myriam was able to learn to manage the administrative side of dairy farming,

while broadening her clinical knowledge from well-known Drs. Roberto Ruiz Diaz and Alberto

Quintana Erdozain. After that, Myriam finished her thesis and quit her position in order to have

time to study for her defense and apply for a MSc degree in another country. Myriam defended

her thesis with honors. Myriam applied to a residency with a MSs at the University of Florida

College of Veterinary Medicine at the end of 2013, for which she was accepted. She then went to

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work for six months at a feeding and laboratory company in the state of Qro. She then came to

the USA on July 2014 to start her residency and master’s. She is expecting to graduate in

summer 2017 and complete her residency program in July of the same year. Myriam’s upcoming

goals are to pursue a doctoral degree in Veterinary Medical Sciences at the same University.