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EFFECTS OF SUPPLEMENTAL 25-HYDROXYVITAMIN D3 AND VITAMIN D3 ON MINERAL CONCENTRATIONS AND MASTITIS RESISTANCE IN LACTATING DAIRY

COWS

By

MICHAEL B. POINDEXTER

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

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© 2017 Michael B. Poindexter

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To my mother and father for their overwhelming support and love

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ACKNOWLEDGMENTS

I would like to first express my appreciation to my major professor and advisor,

Dr. Corwin Nelson, for offering me the opportunity to come to the University of Florida to

work towards obtaining my Master of Science degree. His knowledge, attitude, and

vision helped guide me through these last two years of my continuing education.

I would also like to thank my other committee members, Dr. José Santos and Dr.

Charles Staples, for their continued interest in my education. Special thanks to Dr. José

Santos for his time working with me through the statistics required to properly analyze

my data and for his advice and guidance that helped me make decisions about my

future. I would also like to thanks Dr. William Thatcher for his time, advice, and always

trying to include me in discussions and meetings.

I would also like to extend my thanks to Dr. John Driver, Dr. Kwang Cheol Jeong,

Dr. Stephanie Wohlgemuth, Dr. Geoffrey Dahl, and Dr. Peter Hansen for allowing me to

use their laboratories and facilities.

I want to thank my labmates Mercedes Kweh and Leslie Blakely for their

assistance throughout the last two years, especially during the data collection period. I

want to thank all the graduate students and interns who have assisted me at some point

throughout the last two years, Roney Zimpel, Francisco Lopes, Achilles Vieira Neto,

Camilo Lopera, Marcos Zenobi, Jorge Zuniga, Sossi Iacovides, Carolina Collazos,

Rafael Moreira, Gian Carlo Negro, Murilo Rômulo, and William Ortiz.

I extend my sincere appreciations to the staff of the University of Florida Dairy

Research Unit, Todd Pritchard, Eryck Lockyer, Patricia Best, and Travis Fulchur.

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My thanks goes to the Department of Animal Sciences staff, particularly to Renee

Parks-James, Joyce Hayden, and Pam Krueger for their assistance.

Finally, I would like to thank my family, particularly my mother and father, Beth

and Lynn Poindexter, and my girlfriend Amy Doherty for their love, support, and infinite

encouragement.

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TABLE OF CONTENTS

Page

ACKNOWLEGMENTS .................................................................................................... 4

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

LIST OF ABBREVIATIONS ........................................................................................... 10

ABSTRACT ................................................................................................................... 12

CHAPTER

1 INTRODUCTION .................................................................................................... 14

2 LITERATURE REVIEW .......................................................................................... 17

Economic Impact of Cattle Diseases ...................................................................... 17 Vitamin D Pathway ................................................................................................. 18 Vitamin D and Mineral Metabolism ......................................................................... 24

Function of -Defensins ................................................................................... 30 Function of Nitric Oxide .................................................................................... 33 Vitamin D and Disease ........................................................................................... 34 Dietary Sources of Vitamin D.................................................................................. 39 Vitamin D Requirements in Cattle ........................................................................... 40 Use and Absorption of 25(OH)D ............................................................................. 44 Summary ................................................................................................................ 46

3 EFFECT OF 25(OH)D AND VITAMIN D3 ON MINERAL METABOLISM AND

MASTITIS RESISTANCE IN LATE LACTATION DAIRY COWS ............................ 47 Introductory Remarks ............................................................................................. 50 Materials and Methods ........................................................................................... 51

Cows and Housing ........................................................................................... 51 Experimental Design and Treatments .............................................................. 52 Milk Yield, Body Weight, and Feed Intake ....................................................... 53 Bacteria Preparation ........................................................................................ 54 Experimental Mastitis Challenge ...................................................................... 54 Serum Minerals and Metabolites ..................................................................... 55 Milk Somatic Cells for Gene Expression .......................................................... 57 Statistical Analysis ........................................................................................... 58 Results ................................................................................................................... 59 Concentrations of Vitamin D Metabolites in Serum .......................................... 59

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Concentrations of Minerals in Serum ............................................................... 62 Concentrations of Energy Metabolites in Serum .............................................. 62 Milk Yield, DMI, BW, and Milk Components ..................................................... 63 Gene Expression in Total Milk Cells from Healthy Milk .................................... 63 Mastitis Challenge............................................................................................ 64 Challenge Gene Expression ............................................................................ 65

Discussion .............................................................................................................. 66 Vitamin D Nutrition and Physiology .................................................................. 68 Mineral Homeostasis ....................................................................................... 70 Vitamin D Immunity and Mastitis Resistance ................................................... 72

4 CONCLUSION AND FUTURE DIRECTIONS......................................................... 92

REFERENCE LIST........................................................................................................ 95

BIOGRAPHICAL SKETCH .......................................................................................... 114

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LIST OF TABLES

Table page

2-1 Criteria considered when blocking and differences between blocks ................... 76

2-2 Ingredient composition and nutrient content of postpartum diets in experiment . 77

2-3 qPCR primer sequences ............................................................................................... 79

2-4 Minerals and energy metabolites ........................................................................ 80

2-5 Milk components ................................................................................................. 81

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LIST OF FIGURES

Figure page

2-1 General vitamin D3 pathway ............................................................................... 48

2-2 Serum 25(OH)D in cows ..................................................................................... 82 2-3 Serum 24,25(OH)2D, serum 1,25(OH)2D, serum vitamin D, and serum free

25(OH)D in cows ................................................................................................ 84 2-4 Serum calcium, phosphorus, and magnesium in cows ....................................... 85 2-5 Serum BHBA, glucose, NEFA, and ALP in cows ................................................ 86 2-6 Milk yield, feed intake, and body weight in cows ................................................ 87 2-7 Gene expression of VDR, Cyp27B1, Cyp24A1, DEFB7, and iNOS in milk somatic

cells .................................................................................................................... 88 2-8 Mastitis challenge ............................................................................................... 89 2-9 Feed intake and milk yield from challenged cows ............................................... 90 2-10 Gene expression of VDR, Cyp27B1, Cyp24A1, DEFB7, and iNOS in milk somatic

cells from challenged quarters ............................................................................ 95

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LIST OF ABBREVIATIONS

1,25(OH)2D2 1,25-dihydroxyvitamin D2

1,25(OH)2D3 1α,25-dihydroxyvitamin D3

1,25(OH)2D Refers to mixture of 1,25(OH)2D2 and 1,25(OH)2D3

25(OH)D2 25-hydroxyvitamin D2

25(OH)D3 25-hydroxyvitamin D3

25(OH)D Refers to mixture of 25(OH)D2 and 25(OH)D3

AMP Antimicrobial peptide

BW Body weight

CAMP Cathelicidin antimicrobial peptide

CFU Colony forming units

Ct Threshold cycle

CV Coefficient of variation

CYP24A1 24-Hydroxylase

CYP27B1 1α-Hydroxylase

DEFB β-defensin

DIM Days in milk

DM Dry matter

DMI Dry matter intake

DPB Vitamin D binding protein

EDTA Ethylenediaminetetraacetic acid

FACS Fluorescence-activated cell sorting

FGF23 Fibroblast growth factor 23

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IFNγ Interferon-gamma

IL Interleukin

mM Milimolar

mRNA Messenger ribonucleic acid

NEFA Non-esterified fatty acid

iNOS Inducible nitric oxide synthase

LAP Lingual antimicrobial peptide

LPS Lipopolysaccharide

P-value Probability value

PAMP Pathogen associated molecular pattern

PBMC Peripheral blood mononuclear cell

PBS Phosphate buffered saline

PTH Parathyroid hormone

RANKL Receptor activator of nuclear factor kappa-B ligand

RNA Ribonucleic acid

SCC Somatic cell count

SCS Somatic cell score

SEM Standard error of the mean

TAP Tracheal antimicrobial peptide

TLR Toll-like receptor

TVPR6 Transient receptor potential vanilloid channel membrane 6

VDR Vitamin D receptor

VDRE Vitamin D response element

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EFFECTS OF SUPPLEMENTAL 25-HYDROXYVITAMIN D3 AND VITAMIN D3 ON MINERAL CONCENTRATIONS AND

MASTITIS RESISTANCE IN LACTATING DAIRY COWS

By

Michael B. Poindexter

May 2017

Chair: Corwin D. Nelson Major: Animal Molecular and Cellular Biology

Objectives in the experiment presented in Chapter 3 were to determine the

effects of feeding supplemental 25-hydroxyvitamin D3 on concentrations of 25-

hydroxyvitamin D [25(OH)D] and minerals in serum, lactation performance, and mastitis

resistance in dairy cows. Treatments containing 25(OH)D3 were successful at

increasing serum concentrations of 25(OH)D concentrations at increased rates and to

greater concentrations than vitamin D3 treatments. Cows fed 25(OH)D3 had increased

serum concentrations of 24,25(OH)2D compared with vitamin D3-treated cows. Vitamin

D3-treated cows had increased concentrations of total vitamin D in serum compared

with 25(OH)D3-treated cows. Cows receiving 3 mg of 25(OH)D3 had increased

concentrations of total calcium and phosphorus in serum after 21 d of supplementation.

Total magnesium, BHBA, NEFA, ALP, and glucose concentrations in serum at 21 d

were not different among treatments. Body weight, feed intake, milk yield and milk

components also were not different among treatments. Expression of vitamin D pathway

genes and select host-defense genes was not different among treatments during the

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first 21 d of treatment. During a mastitis challenge where cows receiving 1 mg vitamin

D3 and cows receiving 3 mg 25(OH)D3 received an intramammary infusion of

Streptococcus uberis, somatic cell scores or bacterial counts were increased but not

affected by treatment. Cows receiving the 3mg 25(OH)D3 treatment had less severe

mastitis severity compared with cows receiving vitamin D at 60 and 72 h post challenge.

The 3mg 25(OH)3 -fed cows also tended to have lower rectal temperatures compared

with 1mg vitamin D3-fed cows during the challenge. Expression of the gene for inducible

nitric oxide synthase, when adjusted for expression of gene for the vitamin D 1-

hydroxylase, was greater in cows fed 3 mg 25(OH)D3 compared with cows fed 1 mg

vitamin D during induced mastitis. Feeding 3 mg of 25(OH)D3 moderately increased

concentrations of Ca, P, and Mg and reduced severity of mastitis in dairy cows.

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CHAPTER 1 INTRODUCTION

In the last three decades, global milk production from cows has increased by

over fifty percent, from 500 million metric tons in 1983 to 769 million metric tons in 2013

(FAO 2013). Dairy cattle were responsible for 656 million metric tons in 2014 and global

milk production has been increasing every year since 2001 (FAO 2014). Total value of

milk produced by cattle in 2013 was 5.8 billion dollars U.S. (FAO 2014). As the

population grows, efforts have been made to improve efficiency and increase dairy

production to feed the population. Improvements have been made, increasing average

production per cow from 7 kg/d 60 years ago to over 32 kg/d by the modern dairy cow in

the U.S. and other developed countries. However, susceptibility to some infectious and

metabolic diseases has been associated with the increased metabolic demands of the

modern dairy cow. Diseases in dairy cattle result in major losses of product output per

animal and increased labor. The major diseases in dairy cattle are ketosis,

hypocalcemia, mastitis, metritis, and respiratory disease. Losses and cost estimates

caused by the incidence of these metabolic and infectious diseases vary greatly in the

literature and depend on several variables including location, production system, breed,

stage of lactation, pathogen, parity, and age. Incidence of these diseases can incur

costs associated with losses in production, replacing culled animals, reproduction, and

cost of treatment. Although much is known about incidence, cause, effects, and

treatment, the dairy industry still has a long way to go to develop prophylactic strategies

in order to combat these diseases.

Vitamin D has been studied for many years and is well known in the dairy

industry to assist with calcium homeostasis. In more recent years, vitamin D has

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warranted attention for its role in the immune system (Adams et al. 2007, Aranow 2011,

DeLuca 2008). Recently, dairy cows experienced improved neutrophil function,

increased milk yield, and reduced incidence of retained fetal membranes when being

fed 25(OH)D during the prepartum period (Martinez 2014). Concentrations of 25(OH)D

reached upwards of 250 ng/mL in this experiment for cows receiving 3 mg of 25(OH)D3

daily. It is suspected that concentrations this high for sustained periods of time could

lead to hypercalcemia and organ calcification. An appropriate dose for cattle must be

determined. Therefore, the experiment presented in this thesis is aimed at

understanding the role of vitamin D on metabolism and immune function of the dairy

cow, gaining a better understanding of proper vitamin D dosage, and exploring

strategies to improve cow health through dietary interventions.

The recently discovered actions of vitamin D in the bovine immune system

provide exciting opportunities to improve disease resistance of cattle through better

nutrition. The contribution of vitamin D to activation of innate antimicrobial defenses,

and evidence that vitamin D signaling is activated in innate immune cells during mastitis

in dairy cows have guided my overall hypothesis that bacterial diseases of cattle can be

mitigated through appropriate vitamin D supplementation. However, there are numerous

important unanswered questions that remain in regards to the relationship between

supplemental vitamin D and vitamin D metabolism. For example, level of vitamin D,

source of vitamin D, vitamin D toxicity, and relationship between serum 25(OH)D and

antimicrobial gene responses. More recent data indicate that vitamin D also plays a role

in host innate immune function. I hypothesize that low serum 25(OH)D puts the animal

at risk for infection by not maximizing the potential of the innate immune system to fight

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pathogens. Supplementation of the diets of dairy cows with 25(OH)D3 bypasses

enzymatic conversion of vitamin D to 25(OH)D in the liver and may increase total

25(OH)D availability in cows giving them a better chance to avoid common diseases

such as mastitis, metritis, respiratory infections, and hypocalcemia. My hypotheses are

multifactorial. First, we aim to determine a justifiable intake of supplemental vitamin D3

and direct 25(OH)D3 to increase serum 25(OH)D concentrations to adequate

concentrations for late lactation dairy cows. Second, to determine if directly providing

25(OH)D3 to the animal will give the innate defense of the host a greater potential to

deter pathogens. Finally, to detect the effects of directly providing 25(OH)D3 on mineral

metabolism of the late lactation dairy cow.

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CHAPTER 2 LITERATURE REVIEW

Economic Impact of Cattle Diseases

Losses and cost estimates due to the incidence of mastitis vary greatly in the

literature and depend on a number of variables including stage of lactation, pathogen

causing mastitis, breed, level of production, value of milk, and parity (Dürr et al. 2008).

Incidence of mastitis can incur costs associated with losses in income bonus for low

somatic cell count, replacing culled animals, pregnancy losses, and cost of treatment.

The majority of monetary losses, approximately 70%, due to the inflammation come

directly from reduced milk yield (Ott 1999). In the United States (U.S.), mastitis has

been estimated to cause a daily loss of 1.17 kg/day of milk per cow (Bartlett et al. 1990).

Total milk losses for first lactation cows averaged 247 kg and increased after the first

lactation to approximately 348 kg, which is expected as most animals will increase

production potential from first to second lactations. Costs for dealing with clinical

mastitis averaged $179 according to Bar et al. (2008), of which $115 was incurred from

lost milk and $50 related to treatment-associated costs including cost of antibiotic,

additional labor costs, and discarded milk due to antibiotic contamination. These costs

can be inflated approximately 18% when milk prices are high. Ultimately the total cost of

clinical mastitis was roughly $71 per cow in the herd per year (Bar et al. 2008). More

recent data from the University of Georgia show even greater costs incurred from

mastitis (Rollin, Dhuyvetter and Overton 2015). According to Rollin et al. (2015), clinical

mastitis within the first 30 DIM costs approximately $444 with $128 of the total coming

from direct costs such as treatment and the remaining $316 coming from indirect costs

such as future milk production losses. Additionally, costs are associated with subclinical

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mastitis but they are difficult to quantify, as the disease is often not acknowledged until it

reaches clinical significance. But it is generally accepted that subclinical mastitis

accounts for a substantial amount of economic losses. When subclinical cost estimates

are included, economic losses are substantially greater at $184 per cow reaching a total

national mastitis cost of $1.8 to $2.7 billion in 2016 dollars annually (Wells and Ott

1998).

The only current treatment for clinical mastitis is antibiotics such as beta-lactams,

including cephalosporins, but antibiotic use has multiple problems. First, milk of treated

cows must be discarded as Federal Pasteurized Milk Ordinance prohibits the sale of

milk containing antibiotics. For some pathogens, antimicrobials have limited efficacy

such as in the case of Staphylococcus aureus and Mycoplasma spp. In addition,

antimicrobial resistance, overall, is continually getting worse (Landers et al. 2012). All

these lead to lost production, revenue, perceptions of reduced food safety and quality,

and poor animal welfare. Therefore, a great need exists to increase disease resistance

of cattle through better management, nutrition, development of alternative disease

prevention and therapeutic measures. Recent discoveries indicate that vitamin D

promotes innate defenses of cattle against infectious diseases and warrants further

investigation of the effects of dietary vitamin D on disease resistance of cattle.

Vitamin D Pathway

Vitamin D3 or cholecalciferol is a seco-steroid that is naturally produced in the

skin of mammals including cattle through the conversion of the zoosterol, 7-

dehydrocholesterol, in the presence of the ultraviolet light provided by the sun. The 7-

dehydrocholesterol is normally present within the skin. An animal will endogenously

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produce vitamin D from 7-dehydrocholesterol as it is exposed to ultraviolet light from the

sun (Holick 2008, Hymoller and Jensen 2010). Vitamin D also can be ingested through

the diet as D3 or D2 (Horst et al. 1981, Sommerfeldt et al. 1983). Vitamin D2 or

ergocalciferol is produced in fungi and protozoa from the conversion from ergosterol by

ultraviolet radiation (Eliot and Park 1938).

Both vitamin D2 and vitamin D3 must undergo two hydroxylation step to become

an active hormone. Cows are able to utilize both forms but vitamin D3 has been shown

to be more efficient at increasing 25(OH)D levels in serum than vitamin D2 (Horst et al.

1981, Sommerfeldt et al. 1983, Horst, Reinhardt and Reddy 2005, Heaney et al. 2010).

First, vitamin D is converted to 25(OH)D by 25-hydroxylases, a class of cytochrome

P450 enzymes that are present in the liver (Horsting and DeLuca 1969). This step is

generally considered to be unregulated, but single nucleotide polymorphisms of the

CYP2J2 gene have been associated with serum 25(OH)D concentrations of cattle

(Casas et al. 2013).This hydroxylation step creates 25(OH)D which is, for the most part,

inactive but has been shown to bind to the vitamin D receptor (VDR) (Blunt, DeLuca and

Schnoes 1968, Beckman et al. 1989, Bergadà et al. 2014). The vitamin D synthesized

by the animal or taken in through the diet is readily converted to 25(OH)D (Norman

1998, Norman, Roth and Orci 1982, NRC 2001). After the 25(OH)D is formed, it must

be hydroxylated again by 1α-hydroxylases in order to become the active hormone,

1,25(OH)2D or calcitriol. The 1α-hydroxylase enzyme is encoded by CYP27B1, and is

another member of the cytochrome P450 family. The majority of 1α-hydroxylase activity

occurs in the kidney, where it is tightly regulated in response to serum calcium and

phosphorus. The 1α-hydroxylase also has been detected in other tissues, including the

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skin, mammary gland, and pancreatic islet cells (Huang et al. 2002, Segersten et al.

2005, Townsend et al. 2005, Norman 2008). It also is expressed in immune cells upon

activation, such as macrophages in response to toll-like receptor activation (Liu et al.

2006b).

The active form, 1,25(OH)2D, is the ligand for the VDR, a nuclear hormone

receptor that is present in most tissues of the body. In order to effect immune function

and mineral metabolism, the active hormone must bind to the VDR. In order to bind to

the VDR, 1,25(OH)2D must be free from the vitamin D binding protein (DBP) that carries

the metabolite through the circulatory system. Once the DBP is in equilibrium with the

1,25(OH)2D at the target cell, the hormone is free to bind to the VDR. The VDR can be

located in the cytoplasm of the cell and moves to the nucleus once bound but also has

been shown to be predominantly a nuclear protein, even in an unoccupied state

(Hunziker et al. 1982). In order to interact with transcriptional factors and effect gene

transcription, the VDR/1,25(OH)2D complex must form a heterodimer with the retinoid X

receptor. This complex can then bind to selective or promoter sites that target cell DNA

(Haussler et al. 2010).

The metabolic pathway of vitamin D includes a critical inactivation step in the

oxidation of the sidechain carbons of vitamin D beginning with the C24 carbon. The C24

hydroxylation is catalyzed by the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1),

another cytochrome P450 that is encoded by the CYP24A1 gene. The CYP24A1

enzyme converts both 25(OH)D and 1,25(OH)2D metabolites to 24-hydroxymetabolites,

which is generally considered to render them inactive (Jones, Prosser and Kaufmann

2012). The CYP24A1 transcription is strongly induced by 1,25(OH)2D as part of a

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stringent feedback loop that controls vitamin D signaling activity (Engstrom et al. 1984).

The strong response of CYP24A1 to 1,25(OH)2D is attributed to the presence of

multiple vitamin D response elements (VDRE) located in promoter and enhancer

regions of the CYP24A1 gene.

In the classic vitamin D endocrine system, the balance of CYP27B1 and

CYP24A1 activity are critical for control of calcium and phosphorus and, as such, are

tightly linked to calcium and phosphorus concentrations. Renal CYP27B1, the activating

enzyme, is induced by parathyroid hormone (PTH) in order to stimulate calcium

resorption pathways to increase serum calcium (DeLuca 1977). This increase in

CYP27B1 results in higher concentrations of 1,25(OH)2D which cause an increase in

osteoclastic activity and intestinal calcium absorption (Fraser and Kodicek 1970). The

increased concentrations of 1,25(OH)2D induce CYP24A1 as a negative feedback

mechanism (Jones, Strugnell and DeLuca 1998). However, 1,25(OH)2D mediated

induction of CYP24A1 is attenuated by PTH in the kidney (Reinhardt and Horst 1990,

Shinki et al. 1992). Current understanding of this attenuation effect is thought to be

caused by a posttranscriptional mechanism that affects the stability of mRNA (Zierold,

Mings and DeLuca 2001). The CYP24A1 is not suppressed in other cells such as

osteoblasts by PTH like it is in the kidney (Armbrecht et al. 1998, Yang, Hyllner and

Christakos 2001). In the osteoblast, PTH enhances 1,25(OH)2D mediated induction of

CYP24A1 (Armbrecht et al. 1998, Yang et al. 2001). It was hypothesized originally that

CYP24A1 was present in the kidney in order to convert 25(OH)D to 24,25-

dihydroxyvitamin D (Jones et al. 1998). It has been shown that CYP24A1 is expressed

in most, if not all, target cells containing the VDR (Jones et al. 1998). This led to the

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hypothesis that the catabolizing enzyme, CYP24A1, also can use the active form of

vitamin D to control the concentration of 1,25(OH)2D and prevent cells from

experiencing excess VDR pathway activation. The CYP24A1 can utilize both 25(OH)D

and 1,25(OH)2D as substrates to create compounds destined for excretion as calcitroic

acid (Jones et al. 1998). The CYP24A1 has greater affinity for the active hormone than

for 25(OH)D which adds to the hypothesis that this enzyme is present to act as a

controlling enzyme to prevent extensive or over expression of genes due to 1,25(OH)2D

(Omdahl et al. 2004).

The protein fibroblast growth factor 23 (FGF23) is involved in the vitamin D

pathway as well and is primarily involved in phosphorus metabolism (Larkins et al.

1973). The protein is secreted by osteocytes in response to elevated 1,25(OH)2D and

phosphate and in a negative feedback loop will suppress activity of CYP27B1, lowering

vitamin D activation, as well as induce expression of CYP24A1 in the kidney, increasing

the catabolism of 1,25(OH)2D (Kolek et al. 2005, Perwad et al. 2007).

In dairy cows, normal serum concentrations of vitamin D, both vitamin D2 and D3

together, range from < 1 to 2.5 ng/mL (Horst et al. 1981, Goff et al. 1982, NRC 2001).

This value normally remains low and not reflective of vitamin D intake because, as

previously mentioned, vitamin D will be quickly converted to 25(OH)D under normal

conditions. In humans, vitamin D3 is converted quite rapidly to 25(OH)D3 (Sommerfeldt

et al. 1983). When orally supplemented with 50,000 IU/week of vitamin D3, 25(OH)D3

concentrations in serum increased approximately 5 ng/mL per week in humans (Heaney

et al. 2010). In the non-pregnant, non-lactating cow, a single intramuscular injection of

15 x 106 IU of vitamin D3 increased serum vitamin D3 concentrations to approximately 35

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ng/mL at peak around 10 days after the injection. This led to a peak in 25(OH)D3

concentrations of approximately 95 ng/mL. Another important note is that serum vitamin

D3 concentrations quickly dropped after day 10 post injection and returned to fairly

normal concentrations by day 40. Concentrations of 25(OH)D3 peaked around day 40

and remained elevated, above 75 ng/mL, for over 90 days after the administration of the

vitamin D3 injection (Littledike and Horst 1982). This indicates two things. The first is that

25(OH)D has a much longer half-life than vitamin D and the second, that vitamin D is

being stored in tissues and released slowly in order to optimize vitamin D availability.

The half-life of vitamin D is approximately 24 hours in humans (Clemens et al. 1982).

The half-life of the hormone, 1,25(OH)2D is even shorter, approximately 4 hours in

humans (Gray et al. 1978). The most stable of the metabolites is 25(OH)D, as

previously mentioned, and carries a half-life of approximately 21 days (Barragry et al.

1978, Clemens et al. 1986, Reid et al. 2011, Zerwekh 2008). In humans, supra-

physiological concentrations of vitamin D usually acquired through a bolus dose lead to

slightly increased 25(OH)D concentrations. The concentration of vitamin D drops quickly

back to normal concentrations within the first 2 weeks but 25(OH)D concentrations

remain elevated for several months again indicating a storage mechanism (Heaney et

al. 2008). For daily doses of vitamin D3, the amount taken is proportional to the

25(OH)D concentrations at higher doses but moderate 25(OH)D concentrations are still

observable when serum vitamin D3 is nearly undetectable creating an overall

logarithmic looking curve (Heaney et al. 2008).

The 25(OH)D concentration in serum is the best indicator of the vitamin D status

of the animal (Horst, Goff and Reinhardt 1994b). Normal serum concentrations of

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25(OH)D have been reported to range from 20 to 50 ng/mL but more recent data may

indicate that concentrations below 30 ng/mL are not sufficient to support immunity

(Horst et al. 1994b, Nelson et al. 2016b). A recent survey of vitamin D status of dairy

cattle in the U.S. showed that the serum concentration of 25(OH)D in most mature dairy

cows lie between 40 and 100 ng/mL, with an average concentration of approximately 68

ng/mL (Nelson et al. 2016c). However, most of the surveyed animals were in mid to late

lactation and were receiving between 30,000 and 50,000 IU of vitamin D3 daily.

Sampled animals were surveyed receiving 20,000 IU daily at the same point in lactation

had an average serum 25(OH)D concentration of only 42 ng/mL. In this same study,

early lactation cows with fewer than 30 days in milk (DIM) had an average serum

concentration of 57 ng/mL compared with mid or late lactation cows at 71 ng/mL

receiving the same 30,000 to 50,000 IU per day. Like most bioactive hormones,

1,25(OH)2D is highly regulated. Therefore, normal concentrations of 1,25(OH)2D are

much less, ranging between 5 and 20 pg/mL. As this hormone plays a role in calcium

homeostasis, it is not uncommon to see serum concentrations rise over 300 pg/mL in

hypocalcemic cows (Goff and Horst 1996). A general overview of the vitamin D3

pathway can be observed in figure 2-1.

Vitamin D and Mineral Metabolism

Most of what is known about vitamin D in cattle originates from its role in calcium

metabolism, specifically relating to the periparturient dairy cow. Vitamin D has been

shown to play a critical role in increasing the entry of calcium into the plasma to

maintain plasma calcium homeostasis (Horst et al. 1994b). The modern dairy cow faces

its greatest metabolic challenge just before parturition and some weeks after due to the

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large calcium demands of the mammary gland to provide milk for the offspring. The

animal also has a less challenging period during gestation to provide calcium for the

growing skeletal structure of the fetus (Horst, Goff and Reinhardt 1997). Plasma calcium

will drop after parturition, as calcium will be drained by the mammary gland to

synthesize milk. Parathyroid glands will release PTH in response to the hypocalcemic

state resulting in bone resorption and an increase of calcium in the plasma calcium

pool. Parathyroid hormone will also induce CYP27B1 in the kidneys causing an

increase in 1,25(OH)2D. The 1,25(OH)2D acting through the VDR will stimulate

osteoblasts to produce receptor activator nuclear factor-КB ligand (RANKL). The

RANKL will stimulate formation of osteoclasts and activation of resting osteoclasts

leading to bone resorption (Suda et al. 2003). In monogastric mammals, calcium

absorption occurs throughout the small intestine in the duodenum, jejunum, and ileum

(Marcus and Lengemann 1962). Increased uptake of calcium, due to 1,25(OH)2D

influence, effects mostly the duodenum and upper jejunum (Bronner 2003, Pansu et al.

1983). After 1,25(OH)2D binds the VDR, an increase in the transcription of specific

genes that encode for calcium channel proteins will increase. The influence of

1,25(OH)2D will induce a calcium channel known as transient receptor potential vanilloid

channel member 6 (TRPV6) to usher calcium into the enterocyte of the brush border

membrane in the duodenum (Jones et al. 1998, Van Cromphaut et al. 2001, Peng,

Brown and Hediger 2003, Song et al. 2003). However, evidence suggests that TRPV6

is not necessary for calcium uptake in the intestine (Kutuzova et al. 2008). Regardless

of the exact mechanism responsible for the increase in calcium uptake by the duodenal

brush border membrane stimulated by to 1,25(OH)2D, there is also evidence that

26

1,25(OH)2D increases the expression of specific genes encoding for the calcium binding

protein calbindin-D9k (Li, Pirro and Demay 1998, Wasserman 1977). This protein binds

calcium with high affinity and diffuses into the cytosol where the protein is able to usher

calcium to the basolateral membrane in order to present the mineral to the calcium

transporter plasma membrane Ca2+ ATPase (PMCA1b) (Bredderman and Wasserman

1974, Cai et al. 1993, Kumar 1995). There is evidence that expression of genes

encoding for PMCA1b is increased by 1,25(OH)2D (van Abel et al. 2003). However,

calcium absorption in the ruminant is not identical to that of the monogastric. Schröder

et al (2015) conducted an experiment using cattle rumen epithelial samples to show

calcium is actively and passively transported across the rumen epithelium (Schröder et

al. 2015). The group also showed the presence of TRPV6 in the rumen epithelium but

concluded that it does not play a major role in calcium transport in the rumen. The exact

mechanisms of calcium transport and absorption in the cow is not clearly defined yet but

there is strong evidence that vitamin D plays a role in increasing calcium absorption in

the cow giving it an important role in combating hypocalcemia.

The 1,25(OH)2D also plays a role in phosphorus metabolism. In the same way

intestinal absorption of calcium is increased due to 1,25(OH)2D, intestinal absorption of

phosphate is also increased (DeLuca 2004). Low serum phosphate will stimulate activity

of CYP27B1 in the kidneys as 1,25(OH)2D has a positive net effect on serum

phosphate. In response to the increase in serum 1,25(OH)2D and phosphate, FGF23

will be secreted by osteocytes. This protein inhibits the activity of CYP27B1 in order to

prevent hyperphosphatemia and hypercalcemia. The FGF23 also will reduce

27

reabsorption by inhibiting a sodium/phosphate cotransporter in the kidneys and increase

the secretion of phosphate to prevent hyperphosphatemia (Jüppner 2011).

Another mineral, magnesium, has been shown to have increased intestinal

absorption in humans with increasing 1,25(OH)2D serum concentrations (Wilz et al.

1979). This does not directly correlate to plasma concentrations of magnesium in cattle

as magnesium is absorbed mostly in the rumen. Magnesium concentrations have been

shown to decrease with increased 1,25(OH)2D3 (Vieira-Neto et al. Under Review).

Vieira-Neto et al. (2017) observed an increase in urinary magnesium loss with high

concentrations of 1,25(OH)2D in plasma, which reduced concentrations of magnesium in

serum for approximately 7 days. Nevertheless, the increased urinary loss of magnesium

was likely the influence of ionized calcium on renal reabsorption of magnesium.

Activation of calcium sensing receptors induced by increases in ionized calcium in the

thick ascending limb of the Henle loop and distal convoluted tubules inhibits the sodium-

potassium- chloride cotransporter system, which is needed for paracellular reabsorption

of calcium and magnesium (Dai et al. 2001). Inhibition of sodium-potassium-chloride

cotransporter system by the increased ionized calcium in cows treated with 1,25(OH)2D3

likely suppressed renal reabsorption of magnesium, which resulted in increased urinary

loss of magnesium.

Many experiments have evaluated the use of active vitamin D metabolites to

prevent hypocalcemia. This strategy has proven to have a positive effect on combating

classical hypocalcemia but often creates some hypocalcemic and hypophosphatemic

tendencies for the animal at a later stage as the exogenous administration of the active

form can downregulate the production of endogenous 1,25(OH)2D3 (Goff and Horst

28

1996, Vieira-Neto et al. Under Review). This strategy may still be substantially beneficial

to the cow because the hormone has been shown to influence measures of immunity, to

increase gene expression for β-defensins and inducible nitric oxide synthase (iNOS),

and to a chemokine known as CCL5 or RANTES (Nelson et al. 2010b, Merriman et al.

2015).

Vitamin D Signaling in Immune Cells

Vitamin D can affect both the innate and adaptive immune responses (Bhalla et

al. 1984, Mattner et al. 2000, Liu et al. 2006a, Nelson et al. 2010b). The VDR is present

in activated lymphocytes (Veldman, Cantorna and DeLuca 2000). In cattle, humans,

and mice 1,25(OH)2D3 has similar effects to the adaptive immune system. The

1,25(OH)2D3 has been shown to down-regulated T cell proliferation in cattle (Waters et

al. 2003), mice (Koizumi et al. 1985, Bhalla et al. 1984), and humans (Lemire et al.

1984). The 1,25(OH)2D3 reduced the production of the pro-inflammatory cytokines INF-γ

and IL-17A in all three species (Jeffery et al. 2009, Tang et al. 2009a, Nelson et al.

2011). Nelson et al. (2011) reported that the addition of 25(OH)D3 to cultures of

mononuclear cells harvested from cattle blood stimulated with a derivative of

Mycobacterium bovis decreased expression of IFN- γ as previously mentioned but also

decreased IL-17F gene expression (Nelson et al. 2011). There is little information

available on 25(OH)D and its effects on acquired immunity in cattle. However, further in

vivo information is available supporting the hypothesis that 1,25(OH)2D3 directly effects

the adaptive immune system of cattle. Reinhardt et al. (1999) reported an intramuscular

injection of 200 µg of 1,25(OH)2D3 at 0 and 7 days relative to E. coli J5 vaccinations

increased milk whey anti-J5 IgM titers as well as milk whey IgG antibodies to E. coli J5

29

vaccine compared to their counterparts receiving only the vaccine with no 1,25(OH)2D.

Milk whey IgA titers were also significantly increased in this same experiment

(Reinhardt, Stabel and Goff 1999). Although calcium concentrations were not measured

in this experiment, it is logical to assume from the 1,25(OH)2D3 injection that serum

calcium concentration increased. The increases in IgM, IgG, and IgA could have been

directly caused by the immunomodulatory effects of 1,25(OH)2D or as a result of the

assumed increase in calcium altering calcium mediated immunity.

In order for the innate immune system to react to an invading pathogen, innate

immune cells like monocytes, macrophages, neutrophils and others must be able to

recognize the pathogen to mount the appropriate response. This recognition occurs

through the binding of pathogen associated molecular patterns (PAMP) to pattern

recognition receptors (PRR) displayed on these innate immune cells (Akira and Hemmi

2003, Sadeghi et al. 2006, Beutler 2004, Sheldon et al. 2008). Toll-like receptors (TLR)

are a type of PRR commonly found on mammalian monocytes and macrophages

(Rainard and Riollet 2006). If a PAMP binds a TLR, a defense cascade is initiated and

eventually nuclear factor КB and transcription factor AP-1 will be activated, promoting

the transcription of host defense genes and proinflammatory cytokines (Netea et al.

2004, Asea et al. 2002, Akira and Takeda 2004). Phagocytic cells like neutrophils and

macrophages become an important player in host defense after the binding of TLRs by

PAMPs and are the major contributors to host defense in the udder, uterus, and

respiratory tract in bacterial infection situations (Hill, Shears and Hibbitt 1978, Sheldon

et al. 2009b, Bals and Hiemstra 2004).

30

Several research groups recently discovered that vitamin D signaling contributes

to the TLR-mediated activation host-defense mechanisms. Liu et. al (2006) showed in

human macrophages and Nelson et al (2010) showed in bovine macrophages that

stimulation by TLR ligands such as LPS or lipopeptides stimulates an increase in the

expression of the enzyme CYP27B1 and the VDR (Liu et al. 2006b, Wang et al. 2004a).

The increased expression of these two facilitates an increased amount of transcriptional

influence from 1,25(OH)2D. In the human macrophage, synthesis of 1,25(OH)2D

upregulates -defensin 4A (DEFB4A) and cathelicidin (LL-37 or CAMP) antimicrobial

peptide production. Accordingly, Liu et al. (2007, 2009) demonstrated that

antimycobacterial activity of human macrophages depended on vitamin D-mediated

upregulation of DEFB4A and CAMP gene expression.

Somewhat similar, synthesis of 1,25(OH)2D3 by bovine monocyte cultures

stimulates expression of iNOS and several -defensin (DEFB3, DEFB4, DEFB6,

DEFB7, and DEFB10) genes (Nelson et al. 2010b, Merriman et al. 2015). The authors

further showed that expression of the iNOS and -defensin genes was dependent on

the concentration of 25(OH)D3 added to the monocyte cultures (0 to 100 ng/mL of

25(OH)D; linear effect, P < 0.05), and activity of the 1-OHase enzymes. Nitric oxide

and the bovine -defensins antimicrobial peptides contribute to antimicrobial activity of

bovine innate immune cells and will be discussed further in the following paragraphs.

Function of -Defensins

Of the three families that form the antimicrobial peptide family, cathelicidins and

β-defensins are the only 2 families found in the bovine animal (Fjell et al. 2008, White,

31

Wimley and Selsted 1995, Kosciuczuk et al. 2014). Cathelicidins range from 12 to 80

amino acid residues but most are 23 to 37 amino acid residues and fold into an α-helical

structure (Gennaro and Zanetti 2000). Expression of this family of antimicrobial peptides

is induced by 1,25(OH)2D in many innate immune cells including macrophages (Wang

et al. 2004b). In cattle, 1,25(OH)2D had no effect on the expression of cathelicidins in

monocyte cultures (Nelson et al. 2010b).

Both α-defensins and β-defensins are stabilized with three disulfide bonds and

both are cationic with arginine as the primary cationic residue. α-defensins are made up

of 29 to 35 amino acids. β-defensins are slightly larger than α-defensins consisting of 38

to 42 amino acids. α-defensins and β-defensins are also different in that their cysteine

pairs are not the same. The α-defensins share disulfide bonds between cysteines 1-6,

2-4, and 3-5 whereas β-defensins have a 1-5, 2-4, and 3-6 arrangement relative to the

amino terminus.

All of the defensins work in a similar fashion when interacting with and killing

microorganisms (White et al. 1995, Patterson-Delafield et al. 1981, Lehrer et al. 1989).

This hypothesis has been demonstrated in a number of experiments using insect

defensins. Some authors have demonstrated a loss of cytoplasmic potassium ion,

membrane depolarization, a decrease in cytoplasmic ATP, and respiration inhibition

when observing the interaction between insect defensins and bacteria (White et al.

1995, Gálvez et al. 1991, Cociancich et al. 1993). More recent experiments reported a

large portion of gram positive bacteria are initially eliminated through the mechanism of

action mentioned previously but a substantial subpopulation of bacteria will survive the

initial membrane attack. Depending on the strain of bacteria, they will survive and

32

proliferate or succumb to additional antimicrobial activities of these proteins (Sahl et al.

2005). β-defensin expression has been shown to be directly correlated with infection

incidence in humans (Nomura et al. 2003, Ong et al. 2002). When specifically studying

the effects of 1,25(OH)2D3 on antimicrobial peptide gene expression, Wang et al (2004)

reported the presence of the vitamin D response element on the promotor of the

DEFB4A gene in some human cells and showed 1,25(OH)2D3 was able to induce β-

defensin mRNA in primary keratinocytes and lung adenocarcinoma cells (Wang et al.

2004b). Merriman et al. (2015) showed an increase in bovine β-defensin 3 (DEFB3),

DEFB4, DEFB6, DEFB7, and DEFB10 in cultured monocytes stimulated with LPS in the

presence of 1,25(OH)2D3 indicating that interaction between TLR4 and the vitamin D

pathway adds to β-defensin expression in monocytes (Merriman et al. 2015). However,

unlike the human DEFB4A promoter, they also concluded that the VDR does not

directly target β-defensins and their increased expression is a secondary response to

1,25(OH)2D3 in cattle by using a protein translation inhibitor, cycloheximide. The use of

this inhibitor blocked upregulation of these β-defensins (Merriman et al., 2015.)

The bovine -defensins (DEFB1 , DEFB3, DEFB4, DEFB5, DEFB9, DEFB10,

and DEFB13) lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide

(TAP) are expressed in the bovine mammary gland, specifically in the mammary lymph

node, epithelium, and parenchyma (Kosciuczuk et al. 2014, Roosen et al. 2004). A

screening of the uterus was found to express LAP, DEFB4, DEFB5, and novel bovine β-

defensins 19 (DEFB19), DEFB123, and DEFB124 encoding genes (Davies et al. 2008).

They do mention the presence of a number of cell types in the samples taken so it is not

totally clear if these proteins are expressed in endometrial cells. Both TAP and LAP are

33

produced by bovine respiratory epithelia (Ackermann et al., 2010.) Remembering that

1,25(OH)2D3 can increase expression of many bovine β-defensins in monocytes, the

presence of multiple β-defensins in tissues frequently infected within the cattle

population shows the potential of vitamin D to bolster the immune system and possibly

prevent common cattle diseases.

Function of Nitric Oxide

The iNOS catalyzes nitric oxide production from arginine and is an important

factor in the macrophage defense arsenal (Liu and Gross 1996, MacMicking, Xie and

Nathan 1997). Nitric oxide is a reactive nitrogen molecule, and when produced in

macrophages it acts as a bactericidal and signaling molecule through generation of

peroxynitrate molecules and nitrosylated proteins (Stuehr and Nathan 1989, Moncada,

Palmer and Higgs 1989, Hibbs et al. 1988). The role of nitric oxide in macrophage

antimicrobial activity has been shown in rodent models of bacterial infection (Vazquez-

Torres et al. 2000). Inbred mice that are unable to produce interferon-γ (INF- γ), a

macrophage-activating cytokine, had no defense against a virus. When treated with a

source of INF- γ and given an iNOS inhibitor, macrophages were activated but viral

replication persisted. Only when treated with INF- γ and not treated with an iNOS

inhibitor were the macrophages able to subdue and eliminate the virus (Karupiah et al.

1993). iNOS has been shown to play an important role in macrophage deterrence of

fungal and protozoan infections as well (Lane et al. 1994, Liew et al. 1991). Bovine

monocytes respond to 1,25(OH)2D3 and stimulate expression of iNOS (Nelson et al.

2010b, Merriman et al. 2015). Intramammary 1,25(OH)2D3 treatment increased

expression of iNOS in total somatic cells. In this same experiment, somatic cells were

34

sorted to isolate macrophages according to the presence of cluster of differentiation 14

(CD14+). These isolated macrophages showed a significant increase in iNOS

expression with the 1,25(OH)2D3 treatment (Merriman et al. 2015). This stimulated

production was maintained through the addition of the protein translation inhibitor

cycloheximide indicating the increased expression is a primary response to the active

vitamin D hormone and that the VDR directly targets iNOS genes.

Vitamin D and Disease

Many observational studies have been conducted looking at the association

between vitamin D status and disease. The most common association discussed

between vitamin D status and disease in humans is that between poor vitamin D status

and cardiovascular disease (Wang et al. 2008, Kilkkinen et al. 2009, Anderson et al.

2010). Others have found associations between low serum 25(OH)D and prevalence of

hypertension and both forms of diabetes mellitus (Hintzpeter et al. 2008). In humans,

the VDR is expressed by some antigen-presenting cells including dendritic cells and

macrophages and can be induced into expression by lymphocytes (Mathieu and Adorini

2002). This discovery has led many to believe if the receptor is abundantly expressed

on these particular immune cells it must play a role as an immunomodulatory effector.

Vitamin D effects T cell development and maturation resulting in fewer pro-inflammatory

Th17 cells and an increase in the presence of T-regulatory cells (Tang et al. 2009b).

Vitamin D had a direct effect on naïve CD4+ T cells to enhance the development of Th2

cells which have many functions, one of which is maximizing bactericidal activity of

phagocytic cells such as macrophages (Boonstra et al. 2001). Vitamin D has also been

35

shown to effect human monocytes by inhibiting production of inflammatory cytokines

such as IL-1, IL-6, IL-8, IL-12, and TNFα (Almerighi et al. 2009).

Cows experience an immunosuppressed state during the time of parturition,

which is classified as three weeks before calving and three weeks after calving (Loiselle

et al. 2009). During this period, the animal is vulnerable to microbial and viral attack.

Management practices have focused more energy into disease prevention in recent

years as it is almost always more economically advantageous to prevent disease

instead of treating disease. As a result, the incidence of common diseases such as

mastitis during the periparturient period have been decreasing over time (Bradley 2002).

Mastitis and metritis in the cow and respiratory disease in the calf are the most common

diseases associated with parturition (Fleischer et al. 2001). At any time, mastitis is the

most commonly treated disease in lactating dairy cattle (Mitchell et al. 1998). Treating

mastitis costs producers a substantial amount of money for treatment, discarded milk

loss, and overall losses in milk production through the current and future lactations (Bar

et al. 2008). Metritis is also detrimental to producers as cows who contract metritis have

more difficulty becoming pregnant in future inseminations and often do not resume

cyclicity as fast as animals that did not contract the disease (Huszenicza et al. 1999,

Könyves et al. 2009). Metritis and mastitis, like most other diseases, will also cause a

decrease in dry matter intake, which is directly related to milk production and proper

immune function (Könyves et al. 2009, Bareille et al. 2003).

During mastitis, the vitamin D pathway is upregulated. Escherichia coli,

Streptococcus agalactiae, Klebsiella species, Staphylococcus aureus, and

Staphylococcus species are the most common pathogens for clinical mastitis cases

36

(Lago et al. 2011, Schukken et al. 2011, Oliveira, Hulland and Ruegg 2013, Wilson,

Gonzalez and Das 1997). Mammary tissue as well as somatic cells and sorted

macrophages (CD14+) from milk of clinically infected quarters show massive increases

in gene expression of iNOS and CYP27B1 when compared to samples taken from

healthy contralateral quarters (Nelson et al. 2010a). VDR and RANTES gene

expression had similar increases in clinically infected mammary tissue and somatic cells

from milk but not in CD14+ sorted cells (Nelson et al. 2010a).

Scientists at the USDA National Animal Disease Center studied the effects of

intramammary treatment of cows with 25(OH)D on experimental mastitis (Lippolis et al.

2011). In this experiment, mastitis was induced in ten cows by an intramammary

infusion of 500 CFU of Streptococcus uberis strain 0140. Five of the animals received a

daily intramammary infusion of a solution containing 100 µg of 25(OH)D3 while the other

five received only fetal bovine serum. Animals receiving the 25(OH)D3 treatment had

lower bacterial counts in milk, lower body temperatures, and somatic cell counts

(Lippolis et al. 2011). The control cows also suffered from daily dry matter intake and a

shorter time to milk production decline (Lippolis et al. 2011). Merriman et al. (Merriman

2015) conducted an experiment using LPS combined with 25(OH)D3 to observe the

effects and compare immune response among treatments. Each lactating Holstein cow

was healthy and received one treatment per quarter. Treatments included a control, 5

µg of LPS, 100 µg of 25(OH)D3, or a combination of the 25(OH)D3 treatment and LPS.

Cows showed no difference between LPS treated and LPS + 25(OH)D3 treated

quarters. LPS caused significant increases in expression of CYP24A1, VDR, iNOS,

DEFB7, DEFB3, and DEFB10 (Merriman 2015). In conclusion of the study the author

37

found that intramammary 25(OH)D3 does not influence the immediate mammary

immune response to LPS.

Another experiment from Merriman et al. (2017) used intramammary infusions of

1,25(OH)2D3 to observe its effects on healthy mammary glands. Infusion of 10 µg of

1,25(OH)2D3 increased iNOS and DEFB7 gene expression in total somatic cells more

than two-fold compared to placebo-treated glands within eight hours after treatments

(Merriman et al. 2017b). In the same experiment the authors used cows with subclinical

mastitis and treated with intramammary 1,25(OH)2D3 at the same concentration as

previously mentioned and placebo. Milk somatic cells from 1,25(OH)2D3 treated glands

showed increased gene expression of CYP24A1, DEFB4, DEFB7, and iNOS when

compared to cells from placebo treated glands. The 1,25(OH)2D3 treatment also

resulted in higher serum 1,25(OH)2D concentrations (55 vs 33 pg/mL) compared to

placebo. The results from this experiment affirm the hypothesis that the vitamin D

pathway is upregulated during mastitis.

Metritis is a disease that occurs exclusively during the peripartum period with the

vast majority of cases occurring early postpartum (Sheldon et al. 2008). Escherichia coli

and Trueperella pyogenes are the most common bacteria isolated from the uteri of cows

with metritis, and T. pyogenes becomes the predominant pathogen in cases of

endometritis (Machado et al. 2012). Uterine infection in cattle results in a massive influx

of neutrophils and macrophages and the persistence of/presence of > 10% of the cells

as neutrophils is associated with endometritis and their presence is used to define

subclinical endometritis in the absence of clinical signs (Sheldon et al. 2009a, Zerbe et

al. 2003, Sheldon et al. 2008). Martinez (2014) reported that cows fed 25(OH)D3 had

38

reduced incidence of retained placenta which most likely led to the reduction in metritis

incidence. Postpartum hypocalcemia is associated with reduced neutrophil function

(Ducusin et al. 2003). An experiment conducted by Matrinez et al (2012) reported

attenuated neutrophil function in plasma of subclinical hypocalcemic cows during the

first 3 DIM. Cows that received 1,25(OH)2D3 injections within 6 h of calving had

enhanced neutrophil function compared to their placebo-treated cohorts (Vieira-Neto et

al. Under Review).

Respiratory disease is most prevalent in beef cattle during the transition period

from nursing through weaning (Griffin 1997). Pasteurella species and Haemophilus

somnus are usually the culprits behind bacterial bovine respiratory disease or shipping

disease (Glock 1998). Bovine respiratory syncytial virus (BRSV) is the major cause of

bovine respiratory disease in calves during the first year of life (Larsen 1999).

Mycobacterium bovis is another pathogen that is not as common as the previously

referenced bacteria but has the potential to cause major damage to herds and people

as the bacteria can cause tuberculosis in both humans and cattle (Davies 2006). Upper

respiratory infections and tuberculosis have been shown to be inversely correlated with

vitamin D status in humans in certain parts of the world (Martineau 2012, Ginde,

Mansbach and Camargo 2009a, Ginde, Mansbach and Camargo 2009b, Williams,

Williams and Anderson 2008). In the pre-antibiotic period, cod liver oil containing

vitamin D3 was successfully used to treat tuberculosis (Martineau et al. 2007, Williams

1849). Respiratory syncytial virus (RSV) is the most common pathogen for lower

respiratory tract infections in children worldwide (Stensballe, Devasundaram and

Simoes 2003).

39

Incidence of the RSV and other lower respiratory tract infections have been

associated with vitamin D deficiency (Stensballe et al. 2003, Wayse et al. 2004,

Karatekin et al. 2009). Also, incidence of viral infections in children often peaks during

the winter months when cutaneous vitamin D synthesis is at its lowest point (Stensballe

et al. 2003). Sacco et al. (2012) conducted an experiment using calves supplemented

1,700 IU/kg of feed of vitamin D compared to calves receiving 17,900 IU/kg of feed

(Sacco et al. 2012). Calves receiving the high dose of vitamin D had 25(OH)D serum

concentrations of 175 ng/mL whereas calves receiving the low dose had serum

concentrations below 40 ng/mL. All calves were unvaccinated and were subjected to an

RSV challenge. The high vitamin D calves had greater expression of IL-8 and IL-12p40

genes in lung tissue compared with low vitamin D calves and had numerically increased

gene expression of TNF-α, IL-1β, IL-6, and INF-γ (Sacco et al. 2012).

Dietary Sources of Vitamin D

Current data on serum 25(OH)D in cattle and its effects on the bovine immune

system are still poorly understood. Associations have been made in the human

population between vitamin D status and disease incidence and it appears to be

beneficial to keep serum 25(OH)D concentrations in cattle above 30 ng/mL. For

supplementary vitamin D, grass hays such as brome, bermuda, timothy, and clover

contain the greatest concentrations of vitamin D between 570 and 1440 U.S.P. units of

vitamin D per pound of grass on an air-dry basis (Wallis, Kennedy and Fishman 1958).

Next are legume hays such as alfalfa that contain between 200 and 690 units (Wallis et

al. 1958, Wallis 1944). Silages like corn silage containd 70 to 110 units (as-is basis) and

green forages are last with low total vitamin D content (Wallis et al. 1958, Bechtel et al.

40

1936). However, many forages have a vitamin D content of less than 600 IU/kg of dry

matter (DM). This puts housed animals at a fairly high risk for vitamin D deficiency as

they are not receiving the ultraviolet radiation to endogenously create vitamin D while

also eating feed that is low in vitamin D content (Ballet, Robert and Williams 2000).

Many producers will supplement vitamin D3 to the ration to make up for the poor vitamin

D content of total mixed rations (TMR) in order to keep serum 25-hydroxy vitamin D

concentrations adequate.

Vitamin D Requirements for Cattle

As mentioned previously, 25(OH)D concentration in serum is the best indicator of

the vitamin D status of the dairy cow (Horst et al. 1994b). Serum concentrations greater

than 20 ng/mL are considered to be required for normal calcium and phosphate balance

(NRC 2001). Some authors have stated 30 ng/mL should be considered as the lower

threshold for sufficient metabolism in order to maintain proper mineral metabolism as

well as offer enough substrate to benefit the immune system (Nelson et al. 2016b).

Dairy cows with serum 25(OH)D concentrations of less than 5 ng/mL are considered to

be vitamin D deficient (Horst, Goff and Reinhardt 1994a). The absolute minimum dose

of vitamin D3 in order to prevent rickets in dairy calves is 300 IU per day (Bechtel,

Hallman and Huffman 1935). They are also considered to be at risk for vitamin D toxicity

at concentrations ranging from 200 to 300 ng/mL (Horst et al. 1994b, NRC 2001).

The current edition of the dairy NRC recommends vitamin D3 supplementation of

21,000 IU per day for lactating Holsteins and 21,900 IU for dry Holsteins (NRC 2001).

There is little to no titration research done to back these recommendations though and

an optimal dose of vitamin D3 for dairy cattle has not been truly established. There are

41

large differences in recommendations ranging from 12,000 IU (Wallis 1946) to 21,900

(NRC 2001) as well as the differences between what is recommended and what

producers actually feed which is typically 30-50,000 IU (Nelson et al. 2016b, Weiss

1998).

The geographical latitude of a farms location and the season has a large effect

on the amount of endogenously produced vitamin D as the synthesis of vitamin D3 is

dependent on UV radiation (Norman 1998, Webb, Kline and Holick 1988, Stamp and

Round 1974). The majority of vitamin D3 production occurs in the summer months with

little to no vitamin D3 generation during the winter months depending on the latitude

(Norman 1998). Generally, as latitude increases, biosynthesis of vitamin D3 decreases

(Webb et al. 1988, Loomis 1967). With this in mind, appropriate dosage of vitamin D3 for

dairy cattle can vary greatly depending on the location of the farm. Also, the current

recommendation of supplemental vitamin D3 for dairy cows does take the naturally

occurring vitamin D2 of the plants into account.

An observational study was recently conducted to determine the typical vitamin D

status of dairy cows in the U.S. Over 700 samples were taken from cows at different

points of lactation receiving different doses of supplemental vitamin D. The authors

observed that cows fed supplemental vitamin D at a rate of 30,000 to 50,000 IU/d,

approximatley 1.5 to 2.5 times the NRC recommendation, had serum 25(OH)D

concentrations of 40 to 100 ng/mL with an average of 68 ng/mL, which more closely

matches the Rhône Poulenc Animal Nutrition recommendations for a lactating dairy cow

(Nelson et al. 2016b, Poulenc 1993). A number of experiments have been done using

25(OH)D3 as the supplemental vitamin D metabolite. In this case, the liver hydroxylation

42

step required for the conversion of vitamin D3 to 25(OH)D3 is skipped. Experiments

using this method of supplementation show rapid increase and sustained

concentrations of serum 25(OH)D (Wilkens et al. 2012, Weiss et al. 2015, Martinez

2014). These papers also used a negative dietary cation anion difference (DCAD) to

observe the combined effects of negative DCAD with 25(OH)D3 supplementation mainly

on calcium metabolism. The papers showed better calcium status in animals

supplemented with both negative DCAD and 25(OH)D3.

Vitamin D deficiency in cattle can lead to osteomalacia, hypocalcemia, and

hypophosphatemia as well as mobility limitations due to bent forelegs, stiff and swollen

joints, and thickening of the metacarpal and metatarsal bones. Offspring of vitamin D

deficient cows as well as calves that become vitamin D deficient after birth show signs

of rickets (Wallis 1938). Concentrations of 25(OH)D that facilitate optimal immune

function have not been determined but are hypothesized to be no lower than 30 ng/mL

(Hollis 2005, Nelson et al. 2012).

Along with the negative consequences of vitamin D deficiency, vitamin D toxicity

also has severe negative consequences. It is difficult to determine the exact amount at

which vitamin D toxicity occurs. In addition, the exact mechanism that leads to toxicity is

undefined. A number of hypotheses have been formulated to explain the mechanism of

vitamin D toxicity. All of the proposed mechanisms involve some form of vitamin D

reaching the vitamin D receptor and causing exaggerated gene expression (Jones

2008). The first theory is that vitamin D intake raises serum concentration of the active

hormone, 1,25(OH)2D, which increase cellular concentrations of 1,25(OH)2D resulting in

exacerbated gene expression (Jones 2008). This is a possible mechanism in the dairy

43

cow as elevated 1,25(OH)2D concentrations have been observed after feeding 25(OH)D

(Weiss et al. 2015) and vitamin D3 (Weiss et al. 2015, Hibbs and Pounden 1955) and

after intramuscular injections of large amounts of vitamin D3 (Littledike and Horst 1982).

Alternatively, elevated 25(OH)D concentrations exceed the DBP capacity which may

allow for free or unbound 25(OH)D to enter the cell and directly affect gene expression

(Jones 2008). Some evidence indicates that 25(OH)D can activate transcription, but this

evidence stems from highly artificial in vitro experimentation (Uchida, Ozono and Pike

1994).

The final theory of vitamin D toxicity is similar to the previous theory. Excessive

intake of vitamin D could raise serum vitamin D and 25(OH)D so much that the DBP

releases 1,25(OH)2D in order to mostly bind the 25(OH)D which has a much higher

affinity for the DBP (Jones 2008). This unbound or free 1,25(OH)2D can then enter the

cell and effect gene transcription. Because 1,25(OH)2D is a secosteroid, many

researchers assume that it mimics other steroids and that its free form is the most

important for cell activity (Jones 2008). There is some evidence of increased free

1,25(OH)2D and 25(OH)D in humans under the effects of vitamin D toxicity caused by

large intakes of vitamin D (Pettifor et al. 1995). Authors concluded that increased

concentrations of 25-hdyroxyvitamin D and other metabolites displaced 1,25(OH)2D

from the DBP creating free 1,25(OH)2D (Pettifor et al. 1995). Evidence for causes of

vitamin D toxicity in cattle is limited. Some experiments have been conducted where

supraphysiologic concentrations of serum vitamin D were induced by intramuscular

injection of upwards of 15 x 106 IU of vitamin D3 at time zero with a subsequent injection

of no more than 5 x 106 IU of the same product (Littledike and Horst 1982). This high

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serum vitamin D lead to extreme concentrations of 25(OH)D as well as the active form

of vitamin D. Vitamin D toxicity was high in nonlactating pregnant cows and very often

led to metastatic calcification and death. The increased concentrations of vitamin D3,

25(OH)D, and 1,25(OH)2D ultimately led to greater intestinal absorption of calcium as

well as greatly increased osteoclastic activity both contributing to supraphysiologic

concentrations of calcium in blood reaching over 12 mg/dL. However, no signs of

vitamin D toxicity were observed in nonlactating nonpregnant cows that received the

same treatment (Littledike and Horst 1982). The toxicity threshold of 200 to 300 ng/mL

of 25(OH)D is set because sustained concentrations this high could lead to

hypercalcemia although no data shows that keeping just 25(OH)D at these

concentrations will eventually lead to hypercalcemia and organ calcification soon after.

It is currently unknown if 25(OH)D concentrations of 200 to 300 ng/mL is sufficiently

high enough to saturate the DBP (Jones 2008).

Use and Absorption of 25(OH)D

Currently in the U.S., vitamin D3 is the primary recommended vitamin D feed

additive for cattle. Some products used to supplement vitamin D3 in dairy cows include

ROVIMIX® D3-500 and ROVIMIX® AD3 1000/200. Currently the use of 25(OH)D3

products in dairy cattle is not approved in the U.S. but in other countries (i.e., Australia)

the use of ROVIMIX® Hy.D® 1,25%, a commercial product of 25(OH)D3, is marketed

for use in dairy cows.

Feeding 25(OH)D3 is an alternative to vitamin D3 and is largely successful in the

meat poultry industry. Use of 25(OH)D over vitamin D3 increased weight gain and feed

efficiency and reduced incidence of leg abnormalities in broilers (Ward 2012, Parkinson

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and Cransberg 2001, Zhang et al. 1997). Supplementation of 25(OH)D3 increased egg

production, hatchability, and the number of hatched chicks per hen in broiler breeds

(Ward 2012). The 25(OH)D3 form absorbed better in healthy individuals (Sitrin and

Bengoa 1987), patients with bone diseases (Stamp, Haddad and Twigg 1977), and in

individuals suffering from fat malabsorption (Sitrin and Bengoa 1987, Davies, Mawer

and Krawitt 1980, Krawitt and Chastenay 1980).

The differences in absorption between vitamin D3 and 25(OH)D3 likely stem from

the molecular makeup of the molecules. Ingested vitamin D3 eventually forms micelles

and is primarily absorbed into the lymph (Nechama et al. 1977, Sitrin et al. 1982). The

25(OH)D3 form is a more polar compound and is absorbed more rapidly from the

proximal jejunum and into the portal vein (Nechama et al. 1977, Sitrin et al. 1982). The

25(OH)D3 is absorbed independently of fat absorption and a low percentage of

25(OH)D3 is carried in the lymph chylomicrons (Sitrin and Bengoa 1987). In rats, ligation

of the bile ducts greatly attenuated absorption of vitamin D3 but had almost no effect on

25(OH)D3 absorption (Nechama et al. 1977). Vitamin D binding proteins have been

identified within the intestine that have affinity for 25(OH)D at least 1,000 times that of

other vitamin D metabolites (Nechama et al. 1977). Use of 25(OH)D3 in broiler chickens

has been largely successful due to the unique properties of the molecule but also

because the chick pancreas and liver do not fully develop until 14 to 21 days of age

(Ward 2012). The absorption of 25(OH)D3 has been reported to be more efficient (83%)

than vitamin D3 (66%) in the chick (Bar et al. 1980). The same group reported greater

secretion of vitamin D3 (20%) than 25(OH)D3 (7%) (Bar et al. 1980). The pancreas and

liver of the young calf have been shown to take over seven days to be fully functional

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(Huber et al. 1961). The use of 25(OH)D3 supplementation for dairy cows and calves

shows large potential based on the success it has had in the poultry industry.

Summary

Vitamin D and its derivatives are important in calcium, magnesium, and

phosphorus mineral metabolisms. More recent information has shown vitamin D can

also play a critical role in innate immune functionality. A summary of many recent

studies involving vitamin D and its effects on the innate immune system would show a

boundless potential for providing benefits to the system to combat pathogens that are

frequently involved in causing many diseases in dairy cattle. Studies have shown that

1,25(OH)2D3 can affect innate immune responses in the mammary gland. A number of

shared genes between mammary tissue, endometrial tissue, and tracheal tissue offering

the potential for vitamin D to have systemic antimicrobial and antiviral effects. The mode

of action within the mammary gland is currently being determined but is likely to be

through the recognition of PAMPs by TLRs located on CD14+ cells. This recognition

stimulates expression of the vitamin D activating enzyme CYP27B1 which leads to the

increased local production of 1,25(OH)2D. This PAMP TLR interaction also leads to an

increase in the amount of VDRs within the macrophage. The active hormone has been

shown to indirectly increase expression of several β-defensins genes and directly

increase expression of the iNOS gene. As serum concentrations of 25(OH)D the best

gauge for an animal’s vitamin D status, it is important to keep concentrations within

adequate range, but below toxic levels, in order to provide enough substrate for

CYP27B1 to create active vitamin D. Toxic concentrations should also be considered

because they have shown to be detrimental to animal health (Littledike and Horst 1982).

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The increased expression of these genes within commonly infected cattle tissues may

aid the animal in defending itself from pathogens.

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Figure 2-1. General vitamin D pathway. The 7-dehydrocholesterol is present in the skin and is transformed

into vitamin D3 when subjected to ultraviolet light. Vitamin D3 can be introduced to the body through

the diet as well and is converted by the 25-hydroxylase enzyme within the liver to 25(OH)D. The

25(OH)D can be activated to form 1,25(OH)2D3 by the 1α-hydroxylase enzyme. The formation of

24, 25-dihydroxyvitamin D3 is also common especially in high concentrations of 25(OH)D. Modified

from Nelson 2010.

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CHAPTER 3 FEEDING SUPPLEMENTAL 25-HYDROXY VITAMIN D3 TO INCREASE MASTITIS

RESISTANCE IN LACTATING DAIRY COWS

Summary

Objectives were to determine the effects of feeding supplemental 25(OH)D3 on

concentrations of 25(OH)D and minerals in serum, lactation performance, and mastitis

resistance in dairy cows. Sixty multiparous, pregnant lactating Holstein cows with SCC

< 165,000/mL were randomly assigned to receive a daily dietary supplement containing

1 or 3 mg vitamin D3 (1mgD or 3mgD), or 1 or 3 mg 25(OH)D3 (1mg25D or 3mg25D) for

28 days (n = 15/treatment). Blood and milk were sampled at 0, 7, 14, and 21 d for

measurements of vitamin D metabolites, minerals, and energy metabolites in serum.

Samples of milk were collected and cells isolated for quantification of relative gene

expression of genes involved in vitamin D and antimicrobial responses. On d 21, cows

fed 1mgD and 3mg25D received an intramammary challenge with Streptococcus uberis.

The 1mg25D and 3mg25D cows had greater serum 25D concentrations at 7, 14 and 21

d, greater serum 24,25-hydroxyvitamin D at 21 d, and smaller concentrations of vitamin

D at 21 d compared with cows fed 1mgD and 3mgD (25D = 62 ± 7, 66 ± 8 ng/mL, 135 ±

15, and 232 ± 26 ng/mL; 24,25D = 4.9, 4.0, 11.8, and 30.6 ± 2.8 ng/mL; vitamin D = 7.6,

15.7, 1.9, and 3.1 ± 1.6 ng/mL for 1mgD, 3mgD, 1mg25D and 3mg25D, respectively, at

21d). The 3mg25D cows had greater concentrations of total calcium and phosphorus at

21 d compared with other treatments (Ca = 2.38, 2.4, 2.37, 2.48 ± 0.02 mM; P = 1.69,

1.87, 1.88 and 2.10 ± 0.08 mM for 1mgD, 3mgD, 1mg25D and 3mg25D, respectively).

Yields of milk and milk components, DMI, BW, and concentrations of NEFA, BHBA,

glucose, 1,25-dihydroxyvitamin D and magnesium did not differ among treatments.

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Gene expression of VDR, CYP27B1, CYP24A1, DEFB7, and iNOS in cells isolated from

milk did not differ among treatments in the first 21 d of the experiment. Cows fed

3mg25D had less severe mastitis at 60 and 72 h after challenge with S.

uberis compared with cows fed 1mgD. The 3mg25D cows also had slightly smaller (P =

0.06) rectal temperature compared with 1mgD cows during the challenge period (38.9

vs. 39.1 oC). Expression of previously mentioned genes in milk cells did not differ

between 1mgD and 3mg25D during the challenge period. Feeding 25(OH)D increased

serum 25(OH)D more effectively than supplemental vitamin D, resulting in increased

serum mineral concentrations and less severe mastitis in lactating dairy cows.

Introductory Remarks

Evidence has accumulated in recent years showing that vitamin D may increase

mastitis resistance of dairy cows. Merriman et al. (2015) showed multiple β-defensin

genes as well as the iNOS gene are upregulated by the vitamin D pathway in

monocytes from cattle (Merriman et al. 2015). Expression of multiple β-defensins was

increased with increasing concentrations of both 1,25(OH)D3 and 25(OH)D (Merriman et

al. 2015). Using the NRC as a guide, modern dairy cows receive adequate

supplemental vitamin D to maintain 25(OH)D concentrations at approximately 68 ng/mL

for lactation (Nelson et al. 2016b). However, concentrations averaging 68 ng/mL will put

the cow in a state to prevent the onset of rickets and some other bone diseases but may

not be optimizing function of the vitamin D mediated portion of the innate immune

system. There is little known about the consequences that arise from having serum

25(OH)D concentrations high enough to prevent bone disease but still relatively low.

Some cows have 25(OH)D concentrations under 30 ng/mL even with the NRC

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recommended vitamin D3 supplementation (Nelson et al. 2016b). Feeding 25(OH)D3

offers an alternative dietary source of vitamin D to achieve elevated serum 25(OH)D

concentrations above normal physiological concentrations in cows achieved from

feeding the NRC vitamin D3 recommendations. There is also little known about the

consequences that arise from increasing serum 25(OH)D to supra-physiological

concentrations from directly feeding 25(OH)D. Some data show vitamin D toxicity

occurring in unison with greatly elevated serum 25(OH) concentrations (Littledike and

Horst 1982). Increased serum 25(OH)D concentrations from the aforementioned

experiment were a result of extremely high concentrations of vitamin D3 from a vitamin

D3 injection and not from directly feeding 25(OH)D3. Cows from Littledike et al (1982)

experienced effects of vitamin D toxicity at 25(OH)D concentrations of approximately

200 ng/mL. More recent data show when supra-physiologic concentrations of 25(OH)D

are achieved through feeding, 25(OH)D concentrations will likely have to exceed 300

ng/mL before toxicity can occur (Jones 2008). Although there is an assumed risk of

vitamin D toxicity in the presence of supra-physiologic 25(OH)D concentrations, there

may also be some immune and mineral concentration benefits that result from these

high concentrations as well. The hypothesis is that feeding 25(OH)D will improve

vitamin D-mediated immunity and mastitis resistance of lactating dairy cows.

Materials and Methods

Cows and Housing

The experiment was conducted at the University of Florida Dairy Unit to

characterize the effects of two amounts of dietary vitamin D3 and 25-hydroxyvitamin D3

on vitamin D and mineral metabolism and resistance to mastitis. The experimental

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procedures and care and treatment of animals were approved by the University of

Florida Institutional Animal Care and Use Committee protocol number 201408677.

Sixty lactating Holstein cows were enrolled in the experiment between February

and May of 2016. Cows were selected for enrollment based on the following criteria:

pregnant, multiparous cows with no recent mastitis, somatic cell count 110 at

enrollment (Table 2-1). Cows were moved to the experimental free-stall barn after being

selected for the experiment in order to acclimate to the environment and individual

feeding gates (Calan Broadbent Feeding System, American Calan Inc., Northwood,

NH.) Selection and subsequent enrollment of animals for each enrollment block was

done every 2 to 3 weeks. Cows were enrolled in groups of 12 for a total of five

enrollment blocks. All cows within enrollment block were housed together after

enrollment in a free-stall barn with sand bedded stalls and capacity for thirty cows. No

more than two enrollment blocks (24 cows) were housed together at any point. Cows

were randomly assigned to a feeding gate on the day of enrollment.

Experimental Design and Treatments

The experiment was a randomized complete block design with a two by two

factorial arrangement of treatments. Within each enrollment block, cows were blocked

based on average milk yield from two weeks prior to enrollment (Table 2-1). Within each

block, cows were then randomly assigned to receive one of four treatments. Treatments

were arranged as a factorial with two sources of vitamin D, vitamin D3 or 25(OH)D3, and

two different amounts, 1 mg and 3 mg. Therefore, the four treatments consisted of 1 mg

vitamin D3 (1mgD, 15 cows) 1 mg 25-hydroxyvitamin D3 (1mg25D 15 cows) 3mg vitamin

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D3 (3mgD, 15 cows) and 3 mg 25(OH)D3 (3mg25D, 15 cows.) A top-dress supplement

for each treatment was prepared by mixing a vitamin D3 concentrate (DSM Nutritional

Products, LCC, Parsippany, NJ) or a 25(OH)D3 concentrate (DSM Nutritional Products)

with cornmeal in order to provide either 1 or 3 mg of each source in 100g of the mixture.

Treatments were fed to all cows for 28 days. Cows in the last two blocks of enrollment

(blocks 4 and 5, 24 cows) received the treatments for a total of 56 days.

Milk Yield, Body Weight, and Feed Intake

Cows were milked twice daily at 1000 h and 2200 h. Milk production was

recorded twice daily electronically using Afi Milk Meters (AfiFlo milk meters, S.A.E.

Afikim, Israel.) Milk samples were taken on day