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EFFECTS OF CALCITRIOL ON CALCIUM METABOLISM AND IMMUNE FUNCTION IN DAIRY COWS AND THE INTERRELATION BETWEEN GESTATION LENGTH AND
PERFORMANCE OF HOLSTEIN COWS AND THEIR OFFSPRINGS
By
ACHILLES VIEIRA NETO
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
2016
© 2016 Achilles Vieira Neto
To my parents and grandparents for their endless support and encouragement
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ACKNOWLEDGMENTS
I would like to recognize and express my profound gratitude to my advisor Dr.
José Eduardo Portela Santos for giving me the opportunity of pursing my Master of
Science degree under his mentorship. I am greatly thankful for his guidance,
encouragement, and excellent graduate training. Dr. Santos taught me the importance
of work ethics and commitment to high quality research, and he helped me with the
development of critical thinking, allowing me to grow as a scientist. I admire his passion
for science and he will always be a professional role model for me. I am honored to
have spent the last few years close to one of the greatest researchers on animal health,
reproduction and nutrition in dairy cows.
I would like to extend my gratitude to my supervisory committee members, Dr.
Klibs Galvão, Dr. Corwin Nelson and Dr. William Thatcher for the support during this
journey, for the ideas, discussion and, especially, for the advices that helped me to
improve my knowledge. I am really proud to having been able to spend time and have
scientific dialogues with one of the most knowledgeable reproductive physiologists, Dr.
William Thatcher. He has been a mentor and a role model for me; he is an example of
passionate scientist, one who has infinite curiosity and endless desire to understand
biological processes. More importantly, he is a mentor who provides full dedication to
graduate students.
I would like to extend my deep appreciation to the current graduate students
members of our research group, Camilo Lopera Higuita, Francisco Rebolo Lopes Jr.,
Letícia D. P. Sinedino, Marcos Zenobi, and Roney Zimpel, and extend my
acknowledgments to the former members of Dr. Santos’ laboratory, Dr. Eduardo S.
Ribeiro, Dr. Fábio S. Lima, Gabriel C. Gomes, Dr. Leandro F. Greco, Dr. Natalia
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Martinez-Patiño, and Dr. Rafael S. Bisinotto. Without their valuable help and exemplary
conduct as graduate students, I am sure that this work would have not been completed.
I am glad to have worked with you all, and each one of you has taught me something
that I will take for life.
I would like to thank all the interns, visiting students and professors, André Dias,
Barbara Piffero, Prof. Bolivar Nóbrega de Faria, Bruno Costa, Camille Richie,
Christopher Colston, Claudia Rodriguez, Diana Roldon, Diandra Leziér, Prof. Fernanda
Paiva, Isabella Morales, Izabella Lima, João Neto, Joilson Reis, Lucas Menezes, Prof.
Maria Lucia Gambarini, Murilo Rômulo, Paula Cristina, Priscilla Ferraz, Rafaela
Tavares, Raquel Mello, Prof. Ricarda Santos, Rodrigo Moreira , Rodrigo Gardinal,
Rodrigo Gennari, Sara Knollinger, and William Ortiz, who visited our laboratory and
interacted with our research group assisting with experiments, but also offering us their
knowledge and friendship.
I extend my sincere appreciation to Todd Pritchard, Erick Lockyer, Patricia Best,
and Travis Fulchur from the University of Florida Dairy Research Unit for assistance
during the conduct of one of my experiments. I would like to extend my gratitude to
Ronald St. John, Nilo Francisco and Hernan from Alliance Dairies for allowing me to use
their cows and facilities during one my pilot experiments. I also extend my gratitude to
the faculty members and staff of the Department of Animal Sciences, specially Sergei
Sennikov, Pam Krueger, and Renee Parks-James.
I would like to thank my girlfriend, Renata Ramos, for her love, support, and
friendship. Her enthusiasm and optimism made the process of writing the thesis easier
and more enjoyable, especially in those moments when she was awake until 4 AM
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helping me reviewing this thesis. In addition, she gave me strength and comfort during
the difficult moments of this journey.
Finally, I would like to acknowledge the support of my family in all these years,
starting during my veterinary education, my externship at the University of Florida, and
now the completion of my Master of Science degree. The distance that separated us in
the last years has not prevented them to continue nurturing our love. I specially thank
my parents, Nilde and Valter for keeping me on track and for stimulating my passion for
science. My admiration and respect go to my grandparents Aldoino, Renilde, and Dona
Nita. Last but not least, I extend my gratitude to my grandfather Achilles [in memory] for
showing me that small things in life are more important than we think they are.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES .......................................................................................................... 10
LIST OF FIGURES ........................................................................................................ 11
LIST OF ABBREVIATIONS ........................................................................................... 13
ABSTRACT ................................................................................................................... 16
CHAPTER
1 INTRODUCTION .................................................................................................... 18
2 LITERATURE REVIEW .......................................................................................... 21
Vitamin D Metabolites ............................................................................................. 22
Mechanism of Action of 1α,25(OH)2D3 .................................................................... 24
Role of 1α,25(OH)2D3 in Controlling Mineral Absorption in the Gastrointestinal Tract .................................................................................................................... 25
Role of 1α,25(OH)2D3 in Controlling Bone Mineral Resorption ................................ 28
Role of 1α,25(OH)2D3 in Controlling Mineral Absorption in the Kidney ................... 29
Endocrine Control of 1α,25(OH)2D3 Metabolism ..................................................... 31
Role of 1α,25(OH)2D3 on the Immune System ........................................................ 32
Recommendations for Dietary Vitamin D Metabolites for Dairy Cows ..................... 33
Importance of Hypocalcemia in Dairy Cows ........................................................... 34
Risk Factors for Hypocalcemia ............................................................................... 37
Use of Different 1α,25(OH)2D3 Intermediates to Control Hypocalcemia ................. 39
Oral Administration of Ca Pastes to Control Hypocalcemia .................................... 40
Negative Dietary Cation-Anion Difference to Control Hypocalcemia ...................... 41
Gestation Length .................................................................................................... 43
Endocrine Control of Parturition .............................................................................. 45
Association of Gestation Length and Postpartum Responses ................................ 47
3 USE OF 1α,25-DIHYDROXYVITAMIN D3 (CALCITRIOL) TO MAINTAIN POSTPARTUM BLOOD CALCIUM (CA) AND IMPROVE IMMUNE FUNCTION IN DAIRY COWS .................................................................................................... 53
Summary ................................................................................................................ 53
Background ............................................................................................................. 54
Materials and Methods............................................................................................ 57
Experiment 1 .................................................................................................... 58
Experiment 2 .................................................................................................... 59
Experimental design, cows, and housing ................................................... 59
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Diets, feeding, and analyses of dietary ingredients .................................... 59
Calcitriol formulation .................................................................................. 60
Measurements of yields of milk and milk components ............................... 61
Body weight and body condition ................................................................ 62
Blood minerals, metabolites and hormones ............................................... 63
Urinary excretion of minerals and creatinine .............................................. 64
Complete blood cell counts and neutrophil function ................................... 65
Statistical Analysis ............................................................................................ 66
Results .................................................................................................................... 68
Experiment 1 .................................................................................................... 68
Experiment 2 .................................................................................................... 69
Concentrations of calcitriol and calcidiol in plasma .................................... 69
Concentrations of minerals in whole blood and plasma and prevalence of subclinical hypocalcemia .................................................................... 70
Concentrations of PTH, serotonin, and bone resorption marker in plasma .................................................................................................... 71
Composition and yield of colostrum and milk in the second milking postpartum .............................................................................................. 72
Dry matter intake, milk yield, BW and BCS ................................................ 72
Concentrations of metabolites in plasma ................................................... 72
Urinary excretion of minerals ..................................................................... 73
Blood cell count and neutrophil function .................................................... 73
Discussion .............................................................................................................. 74
Final Remarks ......................................................................................................... 80
4 ASSOCIATION AMONG GESTATION LENGTH AND HEALTH, PRODUCTION AND REPRODUCTION IN HOLSTEIN COWS AND IMPLICATIONS TO THEIR OFFSPRINGS ........................................................................................................ 98
Summary ................................................................................................................ 98
Background ............................................................................................................. 99
Materials and Methods.......................................................................................... 102
Farms and Cows ............................................................................................ 102
Management and Feeding of Calves and Heifers .......................................... 105
Data Collection ............................................................................................... 105
Dam data ................................................................................................. 105
Heifer data ............................................................................................... 107
Gestation Length Category............................................................................. 108
Statistical Analyses ........................................................................................ 109
Dam data ................................................................................................. 109
Heifer data ............................................................................................... 109
Results .................................................................................................................. 110
Health Performance of Dams ......................................................................... 110
Production Performance of Dams .................................................................. 112
Reproductive Performance of Dams .............................................................. 112
Health Performance of Female Offsprings ..................................................... 113
Reproductive Performance of Female Offsprings ........................................... 114
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Discussion ............................................................................................................ 114
Final Remarks ....................................................................................................... 122
5 CONCLUSIONS ................................................................................................... 134
REFERENCE LIST...................................................................................................... 138
BIOGRAPHICAL SKETCH .......................................................................................... 156
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LIST OF TABLES
Table page 3-1 Ingredient composition and nutrient content of pre- and postpartum diets in
experiment 2 ....................................................................................................... 82
3-2 Effect of treatment with Calcitriol on yield and composition of colostrum and milk from the second milking postpartum from Holstein cows in experiment 2 ... 84
3-3 Effect of treatment with calcitriol on performance of Holstein cows in experiment 2 ....................................................................................................... 85
3-4 Effect of treatment with calcitriol on blood cell count of Holstein cows in experiment 2 ....................................................................................................... 86
4-1 Association between gestation length category and incidence of diseases, culling and mortality in Holstein cows ............................................................... 123
4-2 Cox’s regression model for time to diagnosis of disease up to 90 DIM in Holstein cows ................................................................................................... 124
4-3 Cox’s regression model for time to removal from the herd up to 300 DIM in Holstein cows ................................................................................................... 125
4-4 Association between gestation length category and milk production in Holstein cows ................................................................................................... 126
4-5 Reproductive performance in Holstein cows according to gestation length category ............................................................................................................ 127
4-6 Cox’s regression model for time to pregnancy up to 300 DIM in Holstein cows 128
4-7 Survival and reproduction of Holstein heifers born live according to gestation length ................................................................................................................ 129
4-8 Cox’s regression model for time to removal from the herd up to 300 days of age in Holstein heifers ...................................................................................... 130
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LIST OF FIGURES
Figure page 2-1 Regulation of transcellular epithelial calcium transport by 1α,25(OH)2D3 ........... 50
2-2 1α,25(OH)2D3 regulates osteoclastogenesis by reciprocal regulation of receptor activator of NF-κB ligand and osteoprotegerin ..................................... 51
2-3 Diagram with percentage of filtrate calcium and magnesium reabsorption through the nephron ........................................................................................... 52
3-1 Diagram representing analyzes of neutrophil function using flow cytometry ...... 87
3-2 Concentrations of total Ca, total Mg, and total P in plasma of cows after dosing 0, 200, or 300 µg of 1α,25-dihydroxyvitamin D3, and concentrations of calcitriol total Ca and total Mg in plasma of dairy cows in after dosing 300 μg of 1α,25-dihydroxyvitamin D3 on day 0 and another 50 μg on day 6 in experiment 1 ....................................................................................................... 89
3-3 Concentrations of calcitriol and calcidiol in plasma of dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ..... 91
3-4 Concentrations of ionized Ca in whole blood and total Ca, total P, and total Mg in plasma of dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ........................................................ 92
3-5 Daily prevalence of subclinical hypocalcemia in dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ..... 93
3-6 Concentrations of PTH, serotonin, and CTX-1 in plasma of dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ....................................................................................................... 94
3-7 Concentrations of BHBA, NEFA, glucose, and urea N in plasma of dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 .............................................................................................. 95
3-8 Daily excretion of Ca and Mg in urine of dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ....................... 96
3-9 Percentage of neutrophils positive for phagocytosis and oxidative burst percentage, and mean fluorescence intensity for phagocytosis, and for oxidative burst in neutrophils from dairy cows receiving an injection of placebo or 300 µg of 1α,25-dihydroxyvitamin D3 in experiment 2 ....................... 97
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4-1 Survival curves for time to diagnosis of disease up to 90 DIM and time to removal from the herd up to 300 DIM in Holstein cows according to gestation length category ................................................................................................. 131
4-2 Survival curves for days postpartum to pregnancy in Holstein cows up to 300 DIM according to gestation length category ..................................................... 132
4-3 Survival curves for age at removal from the herd up to 300 days of age in heifers according to gestation length category of their dams ............................ 133
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LIST OF ABBREVIATIONS
1α,25(OH)2D3 1α,25-dihydroxyvitamin D3
25(OH)D3 25-hydroxyvitamin D3
ADF Acid detergent fiber
ATPase Adenosine 5'-triphosphatase
BCS Body condition score
BHBA Beta-hydroxybutyric acid
BW Body weight
Ca Calcium
CaCl2 Calcium chloride
cAMP Cyclic adenosine monophosphate
CH Clinical hypocalcemia
CP Crude protein
CV Coefficient of variation
CYP24A1 24-hydroxylase
CYP27A1 25-hydroxylase
CYP27B1 1-α-hydroxylase
DCAD Dietary cation-anion difference
DHR 5-µM dihydrorhodamine 123
DIM Days in milk
DM Dry matter
DMI Dry matter intake
DNA Deoxyribonucleic acid
ECM Energy-corrected milk
EDTA Ethylenediaminetetraacetic acid
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FCM Fat-corrected milk
FGF23 Fibroblast growth factor 23
GL Gestation length
h Hour
iCa Ionized calcium
iMg Ionized magnesium
LSM Least squares means
mEq Miliequivalent
MFI Mean fluorescence intensity
Mg Magnesium
mM Milimolar
mRNA Messenger ribonucleic acid
miRNA Micro ribonucleic acid
Na Sodium
NDF Neutral detergent fiber
NEFA Non-esterified fatty acids
NEL Net energy for lactation
NKCC2 furosemide-sensitive Na+-K+-2Cl- co-transporter
OM Organic matter
P Phosphorus
P/AI Pregnancy per artificial insemination
P-value Probability value
PMCA1b Plasma membrane calcium ATPase
PO43- Phosphate
PTH Parathyroid hormone
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RANK Receptor activator of nuclear factor kappa-B
RANKL Receptor activator of nuclear factor kappa-B ligand
RNA Ribonucleic acid
RXR Retinoid-x receptor
SCH Subclinical hypocalcemia
SCS Somatic cell score
SD Standard deviation
SEM Standard error of the mean
tCa Total calcium
TRPM6 Transient receptor potential channel melastatin member 6
TRPM7 Transient receptor potential channel melastatin member 7
TRPV5 Transient receptor potential vanilloid channel member 5
TRPV6 Transient receptor potential vanilloid channel member 6
TRT Treatment
V-ATPase Vacuolar-type proton pump
VDR Vitamin D receptor
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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 CALCITRIOL ON CALCIUM METABOLISM AND IMMUNE FUNCTION
IN DAIRY COWS AND THE INTERRELATION BETWEEN GESTATION LENGTH AND PERFORMANCE OF HOLSTEIN COWS AND THEIR OFFSPRINGS
By
Achilles Vieira Neto
August 2016
Chair: José Eduardo Portela Santos Major: Animal Sciences
Objectives of the experiment presented in Chapter 3 were to determine the
effects of a slow-release injectable formulation of 1α,25-dihydroxyvitamin D3 (calcitriol)
on mineral metabolism and measures of immune function in recently calved Holstein
cows. Calcitriol increased blood concentration of ionized Ca, and plasma concentrations
of total Ca and total P, whereas reduced concentrations of total Mg. Urinary excretion of
Ca and Mg increased with calcitriol. Calcitriol did not affect production performance in
the first 36 DIM, but it improved measures of innate immune function. Administration of
injectable calcitriol at calving maintained blood Ca and P postpartum and improved
immune function. Objectives of the study presented in Chapter 4 were to evaluate
associations among gestation length (GL) with health, production, and reproduction of
Holstein cows and their offspring. Gestation length was categorized based on deviations
from the average value. Cows with average GL had reduced morbidity and rate of
removal from the herd, and improved performance and rate of pregnancy compared
with cows with short or long GL. Heifers from dams with average GL had reduced
postweaning mortality, reduced rate of removal from the herd, and improved
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reproductive performance. Gestation length influences dam and offspring survival and
performance.
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CHAPTER 1 INTRODUCTION
The transition period that encompasses the last 3 to 4 weeks of gestation and the
first 3 to 4 weeks postpartum is of major scientific interest because the majority of health
problems that affect dairy cattle occur in this stage of the life cycle of cows. They have
long-term effects that impair productive and reproductive performance in the new
lactation. With the onset of lactation dramatic changes occur in dairy cows in part
because of the homeorhetic adaptation to cope with the increased needs for nutrients to
support colostrum and milk synthesis. The needs of calcium (Ca) increase abruptly
because of the synthesis of colostrum and milk and some cows are unable to control Ca
homeostasis properly leading to the development of clinical hypocalcemia (CH) or
subclinical hypocalcemia (SCH). The incidence of CH, also known as milk fever, ranges
from 3 to 7% (DeGaris and Lean, 2008), whereas SCH is much more prevalent and
affects at least 25 of the primiparous and 50% of the multiparous cows (Reinhardt et al.,
2011). The high incidence of SCH and the subsequent increased risk of development of
other diseases such as metritis (Martinez et al., 2012) demonstrate the need for the
strategies to mitigate this problem and associated economic losses that come along.
Several experiments have been conducted to evaluate dietary and
pharmacological strategies to improve Ca homeostasis in dairy cows in the first days
postpartum. Some of these manipulations include the use of diets with very low
concentrations of Ca to induce a negative Ca balance or the manipulation of the mineral
composition of the ration to induce a compensated metabolic acidosis by feeding
acidogenic salts that cause a negative dietary cation-anion difference (DCAD) during
the last weeks of gestation. The use of calcitriol, also known as 1α,25-dihydroxyvitamin
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D3 [1α,25(OH)2D3], has been evaluated during late gestation as a pharmacological
method to improve Ca metabolism. When 1α,25(OH)2D3 was used prepartum, the
inability to predict calving resulted in cows receiving multiple treatments which can have
implications to the endogenous synthesis of 1α,25(OH)2D3 and result in inability to
control Ca homeostasis once the exogenous vitamin is cleared from the body. Because
1α,25(OH)2D3 induces an increase in blood Ca within hours of treatment, ideally cows
would be treated immediately before calving. There is lack of detailed information on the
use of 1α,25(OH)2D3 immediately before or after calving and the subsequent
implications to Ca and mineral metabolism and performance of dairy cows. It was
hypothesized that a slow-release injectable formulation of 1α,25(OH)2D3 would sustain
blood concentrations of ionized (iCa) and total Ca (tCa) and improve measures of
immune function in early lactation cows. The effects of 1α,25(OH)2D3 on bone
remodeling, renal reabsorption and intestinal absorption of Ca and phosphate are well
established. Thus providing exogenous 1α,25(OH)2D3 immediately after calving was
postulated to help multiparous dairy cows to supply the sudden needs for Ca by the
mammary gland at the onset of lactation. Therefore, the objectives of the experiment
presented in Chapter 3 was to determine the effects of a slow-release injectable
formulation of 1α,25(OH)2D3 on mineral metabolism and measures of immune function
in recently calved multiparous Holstein cows.
Gestation length in dairy cows, which is characterized by the period between
conception and calving, is of great importance for dairy producers because the days
pregnant or the anticipated date of calving is used for dry off, movement between
groups, health and nutritional decisions. Gestation length has implications for the
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duration of the dry period in parous cows and, to a large extent, defines the number of
days cows spend receiving the transition rations, which can have implications to
peripartum diseases like hypocalcemia as many of those diets include manipulations in
the mineral content by use of acidogenic salts. Some of the factors that influence
gestation length are well characterized in dairy cattle and they include gender of the
calf, season of calving, breed and age of the cows. However, limited information exists
regarding the importance of duration of gestation and associations with health,
productive and reproductive performance of the dams. Even more limited is the long-
term impact of gestation length on the offspring. Of interest are the impacts of gestation
length on growth, health, survival and reproduction of female offsprings in dairy farms.
Therefore, it was hypothesized that an abnormal gestation length, either shortened or
extended, is associated with impairments in health, productive and reproductive
performance in Holstein cows and their offsprings. The objectives of the study
presented in Chapter 4 were to investigate the associations between abnormal
gestation length and health, survival, production, and reproduction in Holstein cows and
survival and reproduction of their female offsprings.
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CHAPTER 2 LITERATURE REVIEW
The subject of calcium (Ca) metabolism during the transition period in dairy cows
is of major importance because of the potential health and economic impacts when Ca
homeostasis is altered. Hypocalcemia is a known disturbance of the mineral metabolism
in dairy cows and associated with development of other diseases during the early
postpartum period. Studies focusing in the use of dietary methods to improve Ca
metabolism have contributed to the prevention of clinical (CH) and subclinical
hypocalcemia (SCH); however, in spite of major efforts, at least 45% of the multiparous
cows suffer from SCH and prevention needs further research.
In the early 1900s, scientists found evidence of a substance, which later became
known as vitamin D, associated with growth regulation and bone Ca deposition, and
successfully prevented rickets (McCollum et al., 1922). Many years after this original
observation, studies demonstrated that exposure of the skin to sunlight is the main
source of vitamin D in mammals (Glerup et al., 2000), whereas the secondary source of
vitamin D is the diet.
Vitamin D represents a group of fat soluble secosteroids that are synthesized
under ultraviolet B radiation by the metabolism of cholesterol in animals, or ergosterol in
fungi and protozoa. The active form of vitamin D, 1α,25(OH)2D3 or calcitriol, is known to
be a Ca-regulating hormone, which plays an important role in mineral metabolism, and
was recently reported to also be involved with the innate immune function (Liu et al.,
2006; Wang et al., 2004; White, 2012).
The period between the conception and the parturition characterize the gestation
length (GL). Studies have demonstrated that manipulating the diet during the last weeks
22
of gestation can reduce the risk of CH at the onset of lactation. Also, GL allows for
proper timing of drying off cows, which has implications to weight gain and feeding of
dry cows; therefore, GL plays an important role in management of dairy cows. The
influence of various genetic and non-genetic factors on GL had been extensively
investigated. Such factors include breed and age of the cow, seasonality, gender of the
calf, and sire predicted transmitting ability for duration of gestation. Despite these
findings, the association between GL and health, productive and reproductive
performance of dairy cows, and their offsprings remain incompletely elucidated.
The objectives of this literature review are to provide a comprehensive
understanding of the 1α,25(OH)2D3 metabolism, its role in the regulation of mineral
homeostasis, dietary needs of 1α,25(OH)2D3 and its metabolites for dairy cows, use of
1α,25(OH)2D3 to prevent CH in dairy cows, Ca homeostasis during the periparturient
period of dairy cows, CH and SCH in dairy cows, possible treatments and prevention for
those Ca-related disorders, endocrine control of parturition, factors associated with
changes in GL, metabolic changes during the periparturient period, and association of
GL with postpartum parameters.
Vitamin D Metabolites
The synthesis of 1α,25(OH)2D3 starts in the skin of animals under exposure to
ultraviolet light B radiation, at 280 to 320 nm, which cleaves the B ring of 7-
dehydrocholesterol, a cholesterol precursor, between C9 and C10 to form a secosteroid
named previtamin D3 (Havinga, 1973). The isomerization of previtamin D3 into vitamin
D3 occurs in the skin through a temperature dependent reaction, which is enhanced by
raising the temperature (Hanewald et al., 1961). Vitamin D3 can also be obtained from
dietary sources of animal origin and it is absorbed in the small intestine. In fungi and
23
protozoa, ultraviolet light B radiation can form a similar compound, vitamin D2, by the
same photochemical reaction, with the only difference that, in this case, the substrate is
ergosterol instead of 7-dehydrocholesterol. In contrast with vitamin D2, vitamin D3 is
more efficiently converted into 1α,25(OH)2D (Trang et al., 1998); however, both forms
may become active through addition of two hydroxyl groups, with the first taking place in
the liver (Ponchon et al., 1969) and the second in the kidney (Fraser and Kodicek,
1970).
Vitamin D3 produced in the skin is released into the bloodstream where it binds to
the vitamin D binding protein and is transported to the liver. Multiple 25-hydroxylases
add a hydroxyl group to C25 of vitamin D3 to form 25(OH)D3 (Blun et al., 1968; Horsting
and DeLuca, 1969), a process that occurs in hepatic mitochondria and microsomes
(Bhattacharyya and DeLuca, 1974). The enzyme 25-hydroxylase present in hepatic
mitochondrion is a member of the cytochrome P-450 family, and is also known as
CYP27A1 because, in addition of the hydroxylation of C25 of the vitamin D3, it also adds
a hydroxyl group to C27 of cholesterol-derived intermediates (Araya et al., 2003). The
25(OH)D3, also known as calcidiol, released in the bloodstream binds to vitamin D
binding protein, and represents the major circulating form of vitamin D3 metabolites
present in the plasma of normal cows at concentrations varying between 20 to 50 ng/mL
(Horst et al., 1994). Eventually, 25(OH)D3 is transported into the mitochondria of the
kidney, in which hydroxylation of C1 occurs (Fraser and Kodicek, 1970), resulting in the
bioactive form 1α,25(OH)2D3. The enzyme that adds the hydroxyl group to C1 of
25(OH)D3 is also a member of the cytochrome P-450 family and is called 1-α-
hydroxylase, also known as CYP27B1. This enzyme was cloned, sequenced, and
24
characterized in rats (St-Arnaud et al., 1997), mice (Takeyama et al., 1997) and humans
(Monkawa et al., 1997). Moreover, the reaction controlled by 1-α-hydroxylase is the
limiting process for the regulation of concentrations of 1α,25(OH)2D3 in the bloodstream;
therefore, it is the target enzyme by regulatory hormones and growth factors.
Catabolism of the 1α,25(OH)2D3 is performed by the mitochondrial enzyme
CYP24A1, which is able to catalyze multiple hydroxylation reactions at carbons C24 and
C23 of the side chain of both 25(OH)D3 and 1α,25(OH)2D3 (Tanaka et al., 1977). It is
believed that the 24-hydroxylation is the first step of a target cell C24 oxidation pathway
where the major function is to convert 25(OH)D3 or 1α,25(OH)2D3 to calcitroic acid
(Jones et al., 1998). Furthermore, calcitroic acid was identified as the principal biliary
excretory form of 1α,25(OH)2D3 (Esvelt et al., 1979).
Mechanism of Action of 1α,25(OH)2D3
The 1α,25(OH)2D3 binds to the vitamin D receptor (VDR), also known as nuclear
receptor subfamily 1, group 1, member 1, which is a member of the nuclear receptor
family of transcription factors. Binding of 1α,25(OH)2D3 to the VDR leads to the
formation of a heterodimer with a retinoid-X receptor (RXR) isoform, and the complex
VDR-RXR heterodimer recognizes specific DNA sequences also known as vitamin D
responsive elements (Haussler et al., 2011). Furthermore, VDR-RXR recruits
coactivators that interact in a ligand-dependent way being essential for the formation of
the initial transcription complex with RNA polymerase II (Kato, 2000). In the absence of
1α,25(OH)2D3, VDR interacts with co-repressor enzymes, which stimulates histone
deacetylase leading to locally compact chromatin packaging, whereas when
1α,25(OH)2D3, binds to VDR, there is interruption of VDR association with co-repressors
and VDR associates with co-activators, which results in local chromatin relaxation
25
(Carlberg and Seuter, 2010). Seuter et al. (2013) shows that chromatin accessibility is
increased by 1α,25(OH)2D3 through regulation of histone deacetylase and histone
acetyltransferase, facilitating gene expression. In addition, Lisse et al. (2013) reported
that the regulatory effects of 1α,25(OH)2D3 on cell function is not only through the
transcriptional changes and extends to posttranscriptional events regulated by micro
RNAs or miRNAs. According to Wang et al. (2005), the human and murine genomes
have about 1,000 genes with potential vitamin D responsive elements. In addition, the
main target genes for 1α,25(OH)2D3 were reviewed by Haussler et al. (2013), and
grouped into major biological networks, bone metabolism, mineral homeostasis,
1α,25(OH)2D3 detoxification, cell cycle control, immune function, energy metabolism.
Target genes for bacteria control has also been reported in humans and cattle (Liu et
al., 2006; Merriman et al., 2015; Nelson et al., 2010; Wang et al., 2004).
Role of 1α,25(OH)2D3 in Controlling Mineral Absorption in the Gastrointestinal Tract
Calcium absorption in the intestine is exclusively related to the action of the
1α,25(OH)2D3, since in nephrectomized rats 25(OH)D3 was not able to increase
intestinal Ca absorption (Boyle et al., 1972). There are two mechanisms involved in
absorption of Ca by the intestinal epithelium, which are defined as the paracellular and
the transcellular mechanisms. The paracellular absorption is mainly regulated by Ca
concentration in the lumen of the intestine, however 1α,25(OH)2D3 exerts nongenomic
actions on the intestinal tract that enhances the paracellular Ca transport by altering
charge-selective properties of the tight junctions between enterocytes (Tudpor et al.,
2008). Thus, the nongenomic action occurs because of binding of 1α,25(OH)2D3 to the
26
membrane-associated, rapid-response steroid-binding protein, which also increases
phosphate uptake in the intestine (Nemere et al., 2004).
Even though intestinal Ca absorption takes place in the duodenum, jejunum and
in the ileum, 1α,25(OH)2D3 regulates intestinal Ca absorption mainly in the duodenum
(Pansu et al., 1983). The actions of 1α,25(OH)2D3 on the transcellular transport of Ca
are regulated through the classical VDR, and the binding of 1α,25(OH)2D3 to VDR leads
to genomic actions to enhance transcription of target genes. In the duodenum, Ca
enters the enterocyte brush border membrane through the Ca channel transient
receptor potential vanilloid channel member 6, TRPV6, which is highly regulated by
1α,25(OH)2D3 (Van Cromphaut et al., 2001). Once inside the cell, Ca binds with high
affinity to the Ca binding protein, calbindin-D9k, via EF-hands region (Bredderman et al.,
1974), and the complex is diffused into the cytosol. Calbindin-D9k was described to be
regulated by 1α,25(OH)2D3 with reduced levels of mRNA in the intestine of VDR ablated
mice (Li et al., 1998). In the basolateral membrane, Ca is extruded from the enterocyte
by the plasma membrane Ca ATPase, PMCA1b, via an active and energy-requiring
process (Figure 2-1). Favus et al. (1983) showed that trifluoroperizine, a Ca-activated
ATPase inhibitor, reduced transcellular Ca transport that was induced by treatment with
1α,25(OH)2D3 in duodenal segments.
Yamagishi et al. (2006) reported that expression of genes for VDR, calbindin-D9k,
and PMCA1 was presented in the duodenum of dairy cows; however, the existence of
genes related to Ca transporting in rumen of cattle has not been determined. In sheep,
Wilkens et al. (2009) reported very low expression of TRPV6 in the ruminal epithelia
suggesting that the mechanism involved in Ca absorption in the rumen is different from
27
the duodenum. In vitro studies have shown the presence of an active Ca absorption in
the rumen, and suggested the presence of a Ca2+/H+ exchange mechanism (Schröder
et al., 1999). It is believed that non-dissociated short chain fatty acids can pass through
the apical membrane of the rumen epithelia, and in the cytosol they may dissociate to
deliver H+, which can be used to exchange for Ca2+ or iCa. Furthermore, Schröder et al.
(2001) showed that vitamin D3 did not influence active Ca transport in sheep rumen. As
the cow becomes older, there is a reduction in VDR in the intestinal tract (Horst et al.,
1990), which may lead to a decrease in the ability of 1α,25(OH)2D3 to regulate Ca
absorption. In contrast, according to Hansard et al. (1954), Ca absorption in beef steers
from 1 to 6 years of age did not differ, suggesting that the reduction on VDR may not be
of physiological relevance for a beef steer that has low requirements for Ca,
approximately 10 g of absorbable Ca per day, an amount that is far less than that
needed by the lactating dairy cow that requires 50 to 80 g of absorbable Ca according
when producing 30 to 50 kg of milk per day (NRC, 2001). Because older dairy cows are
more susceptible to disruptions of Ca homeostasis, it is possible that a reduction in VDR
in the small intestine that occurs with age might play a role in the risk for CH and SCH
(Reinhardt et al., 2011).
In cattle, ionized magnesium (iMg) absorption occurs through the ruminal
epithelia and is driven by potential difference of the apical membrane. Some factors that
influence ruminal iMg absorption includes ruminal pH, polarization of the apical
membrane of the ruminal papillae, and concentrations of ionized potassium (K+) in the
rumen (Leonhard-Marek and Martens, 1996). Transient receptor potential melastatin
subtype 7, TRPM7, is present in the apical membrane and is thought to mediate iMg
28
transport (Schweigel et al., 2008). In addition, there is another mechanism involved in
Mg transport in the rumen, which consist of a co-transport with an anion, also known as
potential difference independent, although the exact mechanism remains unknown. The
extrusion of Mg is mediated by Na/Mg exachanger, which is regulated by cellular Mg
status (Schweigel et al., 2008).
In addition to the nongenomic actions of 1α,25(OH)2D3 on phosphorus (P)
absorption, 1α,25(OH)2D3 stimulates duodenal uptake of P through VDR by increasing
the expression of the sodium (Na)-P cotransporter channel in the apical membrane
(Yagci et al., 1992). The mechanism involved in P transport through the basolateral
membrane of enterocytes it is also not well established.
Role of 1α,25(OH)2D3 in Controlling Bone Mineral Resorption
The 1α,25(OH)2D3 plays an important role in regulating bone remodeling through
its indirect actions on osteoclast activity. Osteoblasts express receptor activator of
nuclear factor kappa-B ligand (RANKL) in the cell membrane, whereas osteoclasts have
the RANK present in their membranes. Binding of the RANKL from the osteoblasts into
the RANK molecule on immature osteoclasts induces maturation and differentiation of
osteoclasts increasing their activity and leading to bone resorption (Figure 2-2).
Kitazawa et al. (2003) showed that 1α,25(OH)2D3 augments osteoclastogenesis by
increasing expression of RANKL gene in osteoblasts through vitamin D responsive
elements. Furthermore, osteoprotegerin is a protein produced by osteoblasts, which can
bind to the RANKL and prevents the formation of the RANK-RANKL complex and,
therefore, prevents osteoclast differentiation and bone resorption (Udagawa et al.,
2000). Kondo et al. (2004) reported that 1α,25(OH)2D3 downregulates osteoprotegerin
29
expression by accelerating the degradation of osteoprotegerin mRNA and by
transrepressing the osteoprotegerin gene.
Mature osteoclasts produce a large amount of protons by carbonic anhydrase II,
which catalyzes the hydration of carbon dioxide to form bicarbonate and protons. A
vacuolar-type proton pump, V-ATPase, pumps protons into the resorption lacuna
decreasing the pH (Blair et al., 1989). Shapiro et al. (1989) reported that 1α,25(OH)2D3
induces de novo production of carbonic anhydrase II at the protein and mRNA levels. In
addition, secretion of lysosomal enzymes such as cathepsin K acts on digestion of
collagen in the organic matrix, and the acidic media converts Ca salts into soluble
forms. Hydroxyapatite is then dissolved releasing iCa and PO43-, which are absorbed by
the osteoclast and released in to the bloodstream. As a result of matrix digestion,
peptide fragments are generated by collagen degradation, which are used as bone
resorption markers (Calvo et al., 1996). Thus, C-terminal telopeptide of type I collagen,
also known as B-CrossLaps, is an important bone resorption marker used to evaluate
born turnover (Rosen et al., 2000).
Role of 1α,25(OH)2D3 in Controlling Mineral Absorption in the Kidney
Calcium in the ionized form comprises about 48 to 52% of the tCa concentration
present in blood plasma, with the remaining portion bound to plasma proteins, mainly
albumin, or in the form of complexed salts such as bicarbonate, phosphate, and lactate.
Ionized Ca and complexed fractions are filtered in the glomeruli. In the case of Mg,
almost 70% is presented as iMg, and the rest as complexed with filterable anions in the
plasma (De Rouffignac and Quamme, 1994), which are present in the initial glomerular
filtrate. The kidney excretes only 1 to 2 % of filtered iCa, and 3 to 5% of filtered iMg
(Figure 2-3). Calcium reabsortion from the filtrate occurs passively in the proximal
30
tubule and the thick ascending limb of Henle Loop, whereas it is actively reabsorbed in
the distal convoluted and connecting tubules (Seldin, 1999; Dimke et al., 2010).
Reabsorption of Na and water from the lumen of the proximal convoluted tubule
increases iCa concentration in the filtrate and enhances passive reabsorption of iCa
(Seldin, 1999). It has been reported that the tubule fluid/filterable Ca ratio ranges from
1.0 to 1.2 in the proximal convoluted tubule (Suki, 1979). For iMg, reabsorption in the
proximal convoluted tubule requires a tubule fluid/filterable Mg ratio of 1.9 (Le Grimellec,
1975); thus water removal is required for iMg reabsorption, and only 10 to 20% is
reabsorbed in this part of the nephron (Le Grimellec et al., 1973). In the thick ascending
limb of the Loop of Henle, reabsorption of iCa and iMg from the lumen occurs passively
because of the generation of a lumen-positive transepithelial voltage (Dimke et al.,
2010). This electric gradient happens by the uptake of Na+, K+ and two chloride (Cl-)
ions by the NKCC2 (furosemide-sensitive Na+-K+-2Cl- co-transporter) with subsequent
secretion of K+ (Greger and Velazquez, 1987), and by the reabsorption of Na+ that
reduces the intraluminal Na+ concentration, which lead to backflux of Na+ from the
interstitium into the tubular lumen (Greger, 1981). Parathyroid hormone (PTH) increases
cAMP and enhances Na+/Cl- transport in the thick ascending limb of Loop of Henle
resulting in increased reabsortion of iCa and iMg (Di Stefano et al., 1990). Furthermore,
Ca sensing receptors expressed in the basolateral membrane of the thick ascending
limb of the Henle Loop can inhibit vasopressin-induced cAMP by as much as 90%,
decreasing iCa and iMg reabsorption (Desfleurs et al., 1998). The thick ascending limb
of Henle Loop is the main location for iMg reabsorption with 64% of filtered iMg
reabsorbed, whereas only 20 to 25% of the filtered iCa is reabsorbed. The reason for
31
this discrepancy is not entirely clear, but may be related to the delivery of divalent
cations to the bend of Henle Loop or permeability differences for those cations within
the thick ascending limb of Loop of Henle (Dimke et al., 2010).
Final excretion of iCa and iMg into the urine is determined at the distal portion of
the nephron, and is regulated through the active reabsorption of iCa and iMg. Ionized
Ca reabsortion in the distal convoluted tubules occurs through three mechanisms; entry
of iCa across the apical membrane by the transient receptor potential vanilloid channel
member 5, TRPV5; cytosolic diffusion of iCa bound to calbindin-D28K; and active
extrusion of iCa across the basolateral membrane by the Na+/Ca2+ exchanger and the
plasma Ca ATPase, similarly to the epithelial absorption that occurs in the intestine
(Figure 2-1). In addition, vitamin D-deficient rats showed that 1α,25(OH)2D3 increased
expression of TRPV5 mRNA and protein in epithelial cells of the distal convoluted
tubule and connecting tubule (Hoenderop et al., 2001). Also, 1α,25(OH)2D3 enhances
calbindin-D28K expression and accelerates PTH-dependent Ca transport (Friedman et
al., 1993). Active reabsortion of iMg in the distal convoluted tubules occurs similarly to
the mechanism in the intestine through TRPM6 and TRPM7 as previously mentioned.
Endocrine Control of 1α,25(OH)2D3 Metabolism
The main regulatory factors of the 1α,25(OH)2D3 endocrine function consist in
regulation of the renal enzyme 1-α-hydroxylase. The most important determinant of 1-α-
hydroxylase activity is its own product, the 1α,25(OH)2D3. High concentrations of
1α,25(OH)2D3 in the blood causes a downregulation of 1-α-hydroxylase and a reduction
in the syntheses of 1α,25(OH)2D3. Extra-renal activity of the 1-α-hydroxylase has been
reviewed by Norman (2008) and presence of mRNA, protein, and enzymatic activity
were detected in the colon, dendritic cells, human brain, mammary gland, pancreas,
32
parathyroid glands, placenta, prostate and in the skin, and keratinocytes; however, the
contribution of extra-renal 1-α-hydroxylase in controlling 1α,25(OH)2D3 concentration in
the bloodstream remains unclear.
Low concentrations of iCa in the blood results in the production and secretion of
PTH, which acts on the kidney stimulating production of 1-α-hydroxylase. As a negative
feedback loop, the parathyroid gland expresses VDR, and 1α,25(OH)2D3 suppresses
PTH synthesis by downregulation of PTH gene expression (DeMay et al., 1992). In
addition, Ca represses PTH, and both, PTH and 1α,25(OH)2D3 establish a protective
system against hypercalcemia in mammals because they are tightly regulated by each
other. In the bone, 1α,25(OH)2D3 stimulates the expression of fibroblast growth factor
(FGF) 23 in osteocytes (Kolek et al., 2005). In a negative feedback loop, FGF23
represses 1-α-hydroxylase reducing 1α,25(OH)2D3. Moreover, P stimulates FGF23
production, and both, 1α,25(OH)2D3 and FGF23 establish a protection system against
hyperphosphatemia.
Regulation of CYP24A1 for the catabolism of 1α,25(OH)2D3 also plays a role
regulating 1α,25(OH)2D3 endocrine function. According to Ohyama et al. (1994),
1α,25(OH)2D3 activates transcription of the CYP24A1 gene, which is the most potentially
regulated gene by 1α,25(OH)2D3 as it has several vitamin D responsive elements and is
induced in most cells that have the VDR. In addition, FGF23 stimulates CYP24A1,
reducing 1α,25(OH)2D3 concentrations in the blood.
Role of 1α,25(OH)2D3 on the Immune System
Parallel to the functions on mineral metabolism, 1α,25(OH)2D3 plays an important
role in regulating the immune system. It has been shown that in dairy cows
1α,25(OH)2D3 modulates the innate immune response in bovine monocytes, and
33
monocytes are able to produce 1α,25(OH)2D3 in response to toll-like receptor signaling
(Nelson et al., 2010). Toll-like receptors are protein receptors that recognize molecules
derived from microbes such as bacterial lipoprotein, peptidoglycans, glycolipids,
lipoglycan, and lipopolysaccharides. The ability of monocytes to convert 25(OH)D3 into
1α,25(OH)2D3 through toll-like receptors allows an increase of 1α,25(OH)2D3
concentrations in the infection site without raising its blood concentration and altering
mineral metabolism. Furthermore, 1α,25(OH)2D3 increases gene expression of inducible
nitric oxide synthase and regulation upon activation, normal T-cell expressed and
secreted, RANTES, in activated monocytes (Nelson et al., 2010). Induction of inducible
nitric oxide synthase increases production of nitric oxide, which is known to be a
fundamental component of the antimicrobial response (Bogdan, 2001), whereas the
protein RANTES is a chemo-attractant for T-helper cells and monocytes to the site of
inflammation (Schall, 1991). In addition, 1α,25(OH)2D3, has been shown to initiate a
host defense response through β-defensins in cattle, providing further evidence of its
contribution to the innate immune system (Merriman et al., 2015). Studies using an in
vivo model, the authors showed that intramammary infusion of 25(OH)D3 into the gland
infected with Streptoccus uberis was able to reduce bacterial counts in milk,
consequently reducing symptomatic effects of mastitis (Lippolis et al., 2011).
Recommendations for Dietary Vitamin D Metabolites for Dairy Cows
Measurements of serum concentration of 25(OH)D3 have been used as an
indicator of vitamin D3 status in order to determine the requirements of vitamin D3 for
dairy cows. According to Horst et al. (1994), plasma concentrations of 25(OH)D3 below
5 ng/mL are indicative of vitamin D3 deficiency and concentrations greater than 200
ng/mL would indicate potential for vitamin D3 toxicity. It is accepted that cows with
34
adequate vitamin D3 status have plasma concentrations of 25(OH)D3 between 20 and
50 ng/mL (Horst et al., 1994). Presumably, cows having an increased concentration of
25(OH)D3 during the transition period would have more substrate available for formation
of 1α,25(OH)2D3, therefore, maintaining Ca homeostasis. Wilkens et al. (2012)
supplemented cows with 25(OH)D3 orally, and the treated group had 25(OH)D3
concentrations in plasma greater than 100 ng/mL, whereas the control group had
concentrations between 20 and 50 ng/mL. Despite these findings, concentrations of
1α,25(OH)2D3 in plasma did not differ between treatments and, therefore, treatment did
not benefit Ca homeostasis.
According to the 7th edition of Nutrient Requirement of Dairy Cattle published in
2001 (NRC, 2001), 21,000 IU of vitamin D3 is required per day for lactating Holstein
cows and late gestation dry cows (NRC, 2001). Although conclusive data are not
available to quantitatively adjust the current vitamin D3 requirements in dairy cattle, the
housing system used in most dairy herds should be considered for dietary vitamin D3
recommendations. Hymoller et al. (2009) reported that dairy cows housed without
exposure to sunlight and fed amounts of vitamin D3 according to the 7th edition of
Nutrient Requirement of Dairy Cattle (NRC, 2001) had significantly less plasma
concentration of 25(OH)D3 than cows fed no supplemental vitamin D3 but housed
outside exposed to sunlight during summer. There is still a lack of information to proper
establish the requirements of vitamin D3 for dairy cows during different physiological
states and, therefore, further studies are needed.
Importance of Hypocalcemia in Dairy Cows
Adult dairy cows in normocalcemic status maintain tCa concentration between
8.5 and 10.0 mg/dL, 2.25 to 2.5 mM. Ionized Ca, the biologically active form, represents
35
about 48 to 52% of the tCa, whereas 50% of the tCa is bound to proteins such as
albumin, and the remaining tCa is bound to soluble anions such as bicarbonate, citrate,
phosphate, and lactate. For instance, in a 600 kg dairy cow there are 3.0 to 3.5 g of Ca
in the plasma pool, 8 to 9 g of Ca in extracellular fluids, and between 7.8 and 8.5 kg of
Ca within the skeleton. Furthermore, less than 1 g of Ca is stored in the cytosol. In high-
producing lactating dairy cows, 30 to 60 g of Ca are withdrawn from those pools every
day (e.g., colostrum contains 1.7 to 2.3 g of Ca per kg; milk contains 1.1 g of Ca per kg).
At the onset of lactation, there is a sudden increase in Ca requirements primarily by the
mammary gland to support milk production; however, some cows are not able to
successfully supply this need and consequently develop hypocalcemia. Reduction of
urinary excretion of Ca, increased Ca removal from bone and intestinal absorption of Ca
are mechanisms used to prevent a reduction in concentrations of tCa in blood.
Clinical hypocalcemia is defined as blood tCa concentration below 6.0 mg/dL
with presence of clinical signs. According to Smith (2002), CH can be discernible in
three stages, with stage 1 consisting of cows presenting signs of hypersensitivity and
excitability. Mild ataxia, with fine tremors of the flanks and triceps can also be observed.
During stage 2, cows are unable to stand but can maintain sternal recumbency, smooth
muscle paralysis can lead to gastrointestinal stasis that is manifested as bloat. Cows
often tuck their heads into their flanks during this stage. Stage 3 is characterized by
cows losing consciousness progressively to the point of coma, subnormal body
temperature and cold extremities or inability of thermoregulation, as well as complete
muscle flaccidity, heart rate can approach 120 beats per minute, and peripheral pulses
may be undetectable. The prevalence of CH is generally low, but it increases as cows
36
age. Reinhardt et al. (2011) found incidences of 1% for first lactation, 4% for second
lactation and between 6 and 13% for third and greater lactation.
Subclinical hypocalcemia is defined as tCa concentration in plasma below 8.59
mg/dL without presentation of clinical signs (Martinez et al., 2012). The importance of
SCH is usually underestimated as no obvious clinical signs are observed; however, this
condition is highly prevalent during the first few days postpartum. According to
Reinhardt et al. (2011), primiparous cows had a prevalence of SCH of 25%, whereas
multiparous cows had prevalence greater than 41% in the first 2 DIM. The use of diets
with low concentration of Ca during the dry period and a diet rich in strong anions are
effective in preventing CH; however, prevalence of SCH still remains high and needs
further research.
Cows presenting either CH or SCH have impaired longevity and productivity
(Murray et al., 2008), which result in important economic losses to dairy producers.
Furthermore, cows with CH or SCH are more susceptible to develop other periparturient
disorders such as dystocia and ketosis (Curtis et al, 1983), displaced abomasum
(Massey et al., 1993), uterine prolapse (Risco et al., 1984), and retained placenta
(Melendez et al., 2004). In addition, cows presenting SCH are more susceptible to
develop uterine disease, as the condition is positively associated with a decrease in
blood Ca in the first 3 DIM (Martinez et al., 2012). Moreover, normocalcemic cows have
greater rate of pregnancy compared with cows with SCH (Martinez et al., 2012).
Martinez et al. (2014) induced SCH in dry cows and showed that cytosolic iCa in
neutrophils was less, which compromised their ability to phagocytize and kill bacteria.
These findings help explain how SCH contributes to the development of diseases during
37
the postpartum period in dairy cows, and additional studies are needed for successful
preventive herd health and nutritional programs to maintain Ca homeostasis during the
transitional period in dairy cows.
Risk Factors for Hypocalcemia
An important factor that impairs Ca homeostasis is metabolic alkalosis, which
predisposes cows to develop CH and SCH (Craige and Stoll, 1947). Phillippo et al.
(1994) suggested that metabolic alkalosis decreases the tissue responsiveness to PTH
resulting in reduced production of 1α,25(OH)2D3 and, therefore, impairing Ca
homeostasis. In addition, metabolic alkalosis during the periparturient period is known to
be associated with diets containing more equivalents of strong cations than strong
anions, which increases retention of HCO3- (Stewart, 1983). Under alkalosis, PTH
secretion is reduced and PTH activity is important for maintaining Ca balance during
colostrogenesis and the onset of lactation.
Magnesium plays an important role in the ability of PTH to act on its receptor.
Parathyroid hormone response on target tissues is dependent on the activation of the
enzymes adenylate cyclase (Nissenson et al., 1985) and phospholipase C (Dunlay and
Hruska, 1990). Both, adenylate cyclase and phospholipase C have an iMg binding site,
which requires iMg binding for full activity (Rude, 1998). Van de Braak et al. (1987)
suggested that blood Mg concentration below 0.65 mM increases the risk of
development of CH and SCH. In fact, Lean et al. (2006) demonstrated that diets low in
Mg predispose cows to CH; therefore, it is essential to supply sufficient Mg to fulfill the
needs of periparturient cows.
Cows fed low dietary P during late gestation had increased concentrations of tCa
in plasma after calving (Barton et al., 1987). Apparently, diets with high P may increase
38
phosphate concentration in blood, which stimulates the secretion of FGF23 that inhibits
1-α-hydroxylase in the kidney. The end result is a potential reduction in synthesis of
1α,25(OH)2D3 that may impair Ca homeostasis. Although the mechanism of FGF23
inhibition of 1-α-hydroxylase is well established, further research on dietary P during late
gestation and subsequent endocrine regulation of 1α,25(OH)2D3-mediated Ca control is
warranted.
According to Boda and Cole (1954) and Goings et al. (1974), feeding diets with
low Ca reduced the risk of CH; however, the association between concentration of
dietary Ca in prepartum diets and incidence of CH is controversial, and often times
confounded with intake of strong cations. According to Goff (2000), Ca concentration in
diets during the prepartum period has little influence on the incidence of CH when fed at
levels above the daily requirement of the cow. A typical late gestation dairy cow
weighing 750 kg requires approximately 22 g of absorbable Ca (NRC, 2001), which is
easily met by most diets. Therefore, feeding diets that supply Ca beyond the needs of
the late gestation cow is very common. Goff and Horst (1997) reported that prepartum
diets containing high concentration of strong cations, such as K and Na, independently
of Ca concentration in the diet, increased the risk of CH. Diets composited with alfalfa,
are rich in Ca, however, they are rich in K as well, thus the increase risk for CH is
because of the effects of K on blood pH and its consequences, and not because of Ca
concentration. In contrast, McNeill et al. (2002) concluded that high Ca diets are an
important risk factor for CH. Oetzel (1991) found that dietary Ca concentration of 1.16%
had the highest risk for development of CH. In addition, literature reviews conducted by
Lean et al. (2003) suggested limiting the prepartum intake of Ca to 60 g per day.
39
The age of cows is an important predisposing factor for CH. As cows age, the
risk for CH and SCH increases. Reinhardt et al. (2011) showed that the prevalence of
CH was 1% for primiparous, and 4, 6, 10, 8, and 13% for second, third, fourth, fifth and
sixth lactation. The increased risk for older cows is probably associated with a reduction
in the ability to quickly remodel bone either because less osteoclastic activity or
because the decreased number of 1α,25(OH)2D3 receptors in the small intestine of older
cows (Horst et al., 1990).
Use of Different 1α,25(OH)2D3 Intermediates to Control Hypocalcemia
The use of 1α,25(OH)2D3 metabolites attempting to prevent the development of
hypocalcemia has been extensively investigated starting in the 1960s, with emphasis
increased after the 1970s. Several routes of administration have being tested, including
oral, intravenous, and intramuscular. In addition, different metabolites such as
25(OH)D3, 1α,25(OH)2D3 and vitamin D analogues have been investigated.
Hove and Kristiansen (1982) administered 500 µg of 1α,25(OH)2D3 orally to
cows predisposed to develop CH as they were fed a diet during prepartum with high
concentrations of Ca. The authors concluded that when 1α,25(OH)2D3 was
administered 1 to 3 days before parturition, then it had a preventative effect. In contrast,
administration 4 to 5 days before calving or in the day of calving was not effective in
reducing milk fever. Gast et al. (1979) administered 400 μg 1α,25(OH)2D3
intramuscularly 5 days before calving to cows predisposed to develop CH, followed by
reinjection every 5 days until calving. Treatment was successful to prevent CH resulting
in greater serum concentration of tCa and P. On the other hand, Goff et al. (1987),
working with cows fed high dietary Ca rations, administrated an analogue of
1α,25(OH)2D3 intramuscularly 5 days before the expected calving date with reinjection
40
every 7 days until calving. Treatment was effective to prevent CH; however, cows that
received the treatment 7 days or within 24 hours before calving still developed CH.
According to Jorgensen et al. (1978), intramuscular injection of 4 or 8 mg of
25(OH)D3 administrated 3 days before the expected day of calving and repeated at 7-d
intervals if needed, effectively reduced the incidence of CH. In contrast, a study using
123 New Zealand dairy cows in a pasture-based system demonstrated that
administration of 4 mg of 25(OH)D3 intramuscularly between 3 and 10 days before
calving failed to prevent CH (Allsop and Pauli, 1985).
Oral Administration of Ca Pastes to Control Hypocalcemia
The oral administration of large amounts of Ca salts aiming to increase
concentration of tCa in blood, through stimulus of the ruminal paracellular absorption
mechanism, has been investigated during the transition period in dairy cows. The oral
administration of Ca salts such as CaCl2 increases concentrations of tCa in plasma
likely because of the high solubility of the salt that results in large quantities of iCa in the
lumen of the digestive tract associated with the acidogenic effects of the salt because of
the faster and more extensive absorption of Cl- ions (Goff and Horst, 1994). Acidification
of the blood increases PTH receptor responsiveness to the PTH, which favors Ca
absorption in the gut and bone remodeling (Goff et al., 2014). Moreover, Oetzel (1993)
showed a reduction in the incidence of CH and displaced abomasum followed by the
administration of a commercial CaCl2 paste containing 54 g of Ca at 12 h before
calving, at calving, and at 12 and 24 h after calving.
Calcium salts, such as CaCl2, have several disadvantages. Aqueous solution of
CaCl2 and some gel products are very caustic and can cause ulceration of the mouth
and digestive mucosa in cows (Wermuth, 1990). The reduction in blood pH is beneficial
41
because increases the responsiveness of the PTH receptor in bone and renal tissues,
consequently, increasing 1α,25(OH)2D3 production and Ca absorption (Goff et al.,
1991). In contrast, an excessive decrease in blood pH can cause metabolic acidosis
(Goff and Horst, 1994), consequently compromising dry matter intake during a period of
a negative energy balance, aggravating energy metabolism. Calcium propionate, an
alternative Ca salt, may be safer than CaCl2 because it does not have the same effect
on blood pH, in addition it is a source of propionate for gluconeogenesis during a period
of negative energy balance. Calcium chloride has 36% of soluble Ca, whereas Ca
propionate has only 21.5%. At a given amount of salts administered, Ca propionate
might have a less sustained impact on blood concentration of tCa (Goff and Horst,
1993).
Negative Dietary Cation-Anion Difference to Control Hypocalcemia
Dietary cation-anion difference (DCAD) is the difference between the number of
equivalents of strong cations relative to strong anions present in the diet. A negative
DCAD consist of a diet with a greater amount of equivalents of strong anions relative to
strong cations which is typically calculated by the formula proposed by Ender et al.
(1962) as DCAD = [mEq of K+ + mEq of Na+] – [mEq of Cl- + mEq of SO42-]. This
formula assumes that the bioavailability of the four strong anions is equal and no other
ion influences acid-base balance, which has been questioned by others, either because
of the lower bioavailability of sulfur (S) or the potential contributions of other strong ions
in the diet (Horst et al., 1997; Goff, 2000; Tucker et al., 1991). Nevertheless, Lean et al.
(2006) demonstrated in a meta-analysis that the formula initially proposed with only K,
Na, Cl and S2- was the most accurate for predicting the risk of CH in dairy cows.
42
There are several salts in which the anion component is more absorbable than
the cation, making them acidogenic salts. Among them are CaCl2, CaSO4, MgCl2,
MgSO4, NH4Cl, and (NH4)2SO4. In addition, feeding acids of strong anions such as HCl
and H2SO4 can decrease the DCAD (DeGaris and Lean, 2008). When acidogenic salts
are fed, the absorption of strong anions is greater than the absorption of strong cations,
thereby increasing the absorption of negatively charged ions, which induces secretion of
bicarbonate into the digestive lumen concurrent with retention of H+. This process of
loss of HCO3- and retention of H+ results in a reduction in the base excess and a
concurrent reduction in blood pH. Cows fed diets with a negative DCAD have been
shown to develop a compensated metabolic acidosis with reduced partial pressure of
CO2 and increased excretion of H+ through the urine (Hu and Murphy, 2004). If
uncompensated, then the metabolic acidosis might have detrimental effects on the cow.
The rationality of the negative DCAD consists that under a metabolic acidosis,
the affinity of the PTH receptors to PTH is enhanced in different tissues (Goff et al.
2014). Furthermore, induction of metabolic acidosis with acidogenic salts increases
secretion of PTH (Lopez et al., 2002), and the expression of PTH receptor in the kidney
of cattle (Rodriguez et al., 2016), therefore, potentially stimulates 1α,25(OH)2D3
production. Altogether, cows are able to improve Ca homeostasis and reduce the
incidence of CH and SCH, although SCH remains relatively prevalent in postpartum
cows in spite of dietary manipulations. It is clear that only manipulating the prepartum
DCAD is not sufficient to assure proper Ca homeostasis in early lactation and
opportunities exist for further improvements either through additional dietary or
pharmacological means.
43
Gestation Length
The period between conception and parturition is defined as GL. There is lack of
information determining what should be a normal GL, however, many authors have
shown that the average GL of dairy cows vary between 277 and 285 days with some
variation attributed to breed of the cows (Brakel et al., 1952; Norman et al., 2009).
Jersey cows had a mean GL of 278 d, which was shorter than the 280 days for
Holsteins, and 282 days for Guernseys (Silva et al., 1992).
From a management standpoint, the importance of accurately predicting GL is to
use the expected calving date to determine the proper dry date that defines the length
of the dry period for each cow. The recommendation for dry period length has been
between 6 and 8 weeks (Dix Arnold and Becker, 1936). Pezeshki et al. (2007) showed
that primiparous cows had increased milk production with 56 days of dry period
compared with primiparous cows dried for 35 and 42 d; however, prepartum multiparous
cows had similar milk production independently of being dried for 35, 42 or 56 d.
Therefore, the length of the dry period for maximal milk production varies according to
parity. According to Kuhn et al. (2005), cows with proper dry period length maximize
milk production in the next lactation. In addition, others have investigated shortening the
length or completely omitting the dry period, and the latter results in a clear reduction in
milk production in the next lactation (Van Knegsel et al., 2014), whereas reducing the
dry period length to 30 days in multiparous cows results in no negative impact on
subsequent lactation compared with the more traditional 60 d. Because cows with
shortened GL might also have a shortened dry period, this may reduce milk production
in the subsequent lactation. Furthermore, determining the proper length of gestation
allows for adequate time of exposure to the transition diet prepartum. Most farms feed
44
diets containing a negative DCAD during the last 3 weeks of gestation with the aim of
improving Ca homeostasis at the onset of lactation (Moore et al., 2000). Cows with
shortened GL would also have reduced exposure to the prepartum diet, which might
affect mineral metabolism in early lactation.
Several factors have been shown to influence the duration of gestation in dairy
cows. Age of the cow has been identified as an important risk factor, and multiparous
cows had longer GL compared with primiparous cows (Nogalski and Piwczyński, 2012;
Przysucha and Grodzki, 2009). In addition, older nulliparous had longer GL when
compared with nulliparous (McClintock et al., 2003). Moreover, gender of the calf and
the number of calves also have been demonstrated to influence GL. Male calves had 1
day longer GL than female calves, and twin gestation resulted in 8 days shorter
gestation than singleton pregnancy (Foote, 1981). In agreement, Cady and Van Vleck
(1978) found 5 days shorter gestation when twins were being carried compared with
singletons. Seasonality has been shown to influence GL, and cows calving during the
warm season had 2.8 days shorter GL than cows calving during the cool season of the
year (DuBois and Williams, 1979). This effect of season seems to be mediated by heat
stress inducing hyperthermia. When cows in the summer months received shade plus
evaporative cooling prepartum, body temperature decreased by 0.4 oC in the afternoons
and GL was 3 to 4 days longer than cows receiving only shade (Tao and Dahl, 2013).
Furthermore, Nogalski and Piwczyński (2012) found a linear relationship between GL
and calf body weight, with calves from longer GL having greater body weight than
calves from cows with shorter GL. Holm et al. (1996) reported that lambs born from in
vitro produced embryos had increased GL and birth weight compared with lambs born
45
from in vivo produced embryos. In cattle, in vitro produced embryos had increased GL
and birth weight compared with calves originated from artificial insemination (Lazzari et
al., 2002; Van Wagtendonk-de Leeuw et al., 1997). In contrast, in humans, newborns
originated from transfer of fresh embryos had shorter GL than newborns naturally
conceived, whereas newborns from transfer of frozen embryos did not have a difference
in GL compared with those naturally conceived (Pingboard et al., 2014).
Endocrine Control of Parturition
Parturition represents the physiological process of bringing forth the offspring.
The three stages of this process are; initiation of myometrial contractions and removal
of progesterone block, expulsion of the fetus, and, expulsion of the fetal membranes.
Liggins and Thorburn (1994) demonstrated in sheep, that parturition is triggered by the
fetus through increased activity of the hypothalamic-pituitary-adrenal axis. Hypoxemia is
a potent stimulus to increased hypothalamic-pituitary-adrenal axis activity in the fetus
(Matthews and Challis, 1996), and results in parturition. Moreover, hypoglycemia may
result in parturition because of accelerated maturation of the hypothalamic-pituitary-
adrenal axis endocrine pathway (McMillen et al., 1995). Adrenocorticotropic hormone is
progressively increased in fetal plasma during late gestation (Norman et al., 1985). In
addition, fetal cortisol increases in an exponential fashion during the last 10 days of
gestation, presenting the highest concentration immediately before the onset of
parturition (MacIsaac et al., 1985).
Cortisol plays an important role in the final maturation of some fetal tissues such
as the lungs and the kidneys (Liggins, 1994). In the lungs, glucocorticoids induce
structural changes, such as thinning of the alveolar wall and increase potential lung gas
volume (Polglase et al., 2007). In addition, glucocorticoid stimulates the synthesis of
46
surfactant (Liggins, 1969). Components of the surfactant include lipids and proteins,
which prevent alveolar collapse and permit normal respiration (Goerke, 1998).
Stonestreet et al. (1983) showed that glucocorticoid accelerates renal function, as it
increased glomerular filtration rates in fetal lambs exposed to glucocorticoid. In addition,
fetal exposure to glucocorticoid accelerated postnatal development of tubular
reabsorptive capabilities for Na, K, other osmotic particles, water, and urea (Slotkin et
al., 1992). Fetal cortisol alters steroidogenesis on the placenta, reducing progesterone
synthesis and increasing estrogen concentration (Challis and Patrick, 1981; Jenkin and
Thorburn, 1985). Furthermore, fetal corticoids stimulate the placenta to synthetize
prostaglandin F2α, which causes luteolysis of the corpus luteum and consequently
decreases progesterone concentration. The shift in progesterone and estradiol
concentrations activates the myometrium, resulting in increased muscle contraction and
secretion of mucus particularly from the cervix. Oxytocin appears to have an important
role in stimulating uterine contractions (Zhuge et al., 1995). In addition, increased
numbers of oxytocin receptors were found in the placenta during labor (El Alj et al.,
1990).
Prostaglandin F2α stimulates the synthesis of relaxin by the placenta (Sherwood
et al., 1976), loosening up the supportive tissues at the birth canal facilitating the fetus
progress. Progression of uterine contractions forces the placement of fetal feet and
head into the birth canal, therefore increasing the pressure on the fetal membranes with
eventually rupture. The fluid from fetal membrane rupture serves to lubricate the birth
canal. The fetus in the birth canal undergoes hypoxia and this leads to movement of the
fetus, which helps it to pass through the birth canal. The duration of parturition is
47
variable between species; in the cow, myometrium contractions vary between 2 to 6
hours, fetal expulsion varies between 30 to 60 min, and fetal membrane expulsion
varies between 2 to 12 h for the third stage (Senger, 2003).
Infection and abnormal inflammatory processes in the utero-placenta can induce
parturition. Microorganisms are recognized by toll-like receptors leading to a
proinflammatory response (Goldenberg et al., 2000). This response is characterized by
the release of cytokines including tumor necrosis factor α, interleukin-1α, and
interleukin-6, interleukin-8 (Arntzen et al., 1998), metalloproteinases including
collagenase, and gelatinase (Vadillo-Ortega and Estrada-Gutiérrez, 2005), and products
of lipoxygenase and cyclooxygenase pathway (Romero et al., 1987). Parturition may be
initiated by uterine contractions caused by an increased in prostaglandin synthesis,
which is stimulated by cytokines (Christiaens et al., 2008), eventually with rupture of the
fetal membranes. Moreover, metalloproteinases may weaken the fetal membranes,
facilitating rupture, and they can also alter the structure of the collagen present in the
cervix, which leads to cervical ripening (Stygar et al., 2002). These effects of abnormal
inflammatory response may induce parturition, which is usually associated with
premature birth.
Association of Gestation Length and Postpartum Responses
Premature birth is an important cause of increased mortality in newborns in
humans (Kramer et al., 2000), and represented 17.9% of causes of infant death in 2013
(Osterman et al., 2015). Reduction in GL has also been associated with intraventricular
hemorrhage because of increased fragility of the vasculature in the brain of premature
newborns (Ballabh, 2010). Moreover, lung immaturity, a consequence of shortened GL,
predisposes premature newborns to chronic lung disease (Baraldi and Filippone, 2007).
48
Hofman et al. (2004) reported that children between 4 and 10 years of age, who were
born prematurely, had reduced insulin sensitivity, which was suggested to increase the
risk for diabetes mellitus and other diseases associated with insulin resistance. In dairy
cows, shortened and prolonged GL have been reported to increase number of stillbirths
(Nogalski and Piwczyński, 2012). In contrast, Meyer et al. (2000) concluded that
shortened, but no extended GL, was associated with increased risk of stillbirth. In
addition, Bell and Roberts (2007) concluded that shortened GL increased the incidence
of uterine infection, metritis and/or endometritis. According to Huzzey et al. (2007), the
odds of having metritis increased 1.22 for every day that GL decreased. In addition,
Markusfeld (1984) concluded that cows with shortened GL were at great risk for
retained fetal membranes, and heifers with prolonged GL had greater risk of having
metritis. In agreement, Muller and Owens (1973) demonstrated that shortened GL was
associated with increased incidence of retained fetal membranes. DuBois and Williams
(1980) reported that cows having retained fetal membranes and/or metritis had 5.3 days
shorter GL than cows without those diseases. Norman et al. (2011) concluded that
intermediate GL was optimal for calving ease, incidence of stillbirth, survival, and
productive life. Moreover, shortened and prolonged GL were associated with decreased
survival of the offspring (Jenkins et al., 2016).
In general, abnormal GL either shortened or prolonged have been associated
with decreased productive performance. Jenkins et al. (2016) concluded that shortened
and prolonged GL were associated with decreased yields of milk, protein, and fat. In
agreement with that, Norman et al. (2011) found that cows with intermediate GL tended
to have the highest milk production. Furthermore, Norman et al (2009) reported a
49
decrease in productive life when sires with predicted transmitting ability for longer GL
were used.
50
Figure 2-1. Regulation of transcellular epithelial calcium transport by 1α,25(OH)2D3. Epithelial calcium transport is stimulated by 1α,25(OH)2D3 by induction of the apical calcium channel (TRPV6 in the intestine or TRPV5 in the kindey) that enhances calcium entry, the cytosolic calcium binding protein (CaBP; calbindin) that facilitates calcium movement across the cell, and the basolateral plasma membrane calcium ATPase (PMCA1) that pumps calcium from the cell. Modified from Dusso et al. (2000).
51
Figure 2-2. 1α,25(OH)2D3 regulates osteoclastogenesis by reciprocal regulation of receptor activator of NF-κB (RANK) ligand (RANKL) and osteoprotegerin (OPG). 1α,25(OH)2D3-VDR increases the expression of RANKL on the surfaces of the osteoblast. RANKL interaction with its receptor, RANK, promotes maturation of osteoclast progenitor cells to mature osteoclasts, the bone-resorbing cells. 1α,25(OH)2D3-VDR also represses the expression of OPG, a decoy receptor that binds RANKL and prevents RANK-mediated osteoclastogenesis. Modified from Dusso et al. (2005).
52
Figure 2-3. Diagram with percentage of filtrate calcium and magnesium reabsorption through the nephron. Modified from Quamme (1997).
53
CHAPTER 3 USE OF 1α,25-DIHYDROXYVITAMIN D3 (CALCITRIOL) TO MAINTAIN
POSTPARTUM BLOOD CALCIUM AND IMPROVE IMMUNE FUNCTION IN DAIRY COWS
Summary
Objectives were to determine effects of an injectable formulation of 1α,25-
dihydroxyvitamin D3 on mineral metabolism and measures of immune function in
Holstein cows. In experiment 1, cows received different amounts of 1α,25-
dihydroxyvitamin D3 to determine the dose needed to increase plasma concentrations of
Ca. Based on those results, experiment 2 was designed and 300 μg was deemed
sufficient. In experiment 2, multiparous cows were assigned randomly to receive only
vehicle (control, n = 25) or 300 μg of 1α,25-dihydroxyvitamin D3 (Calcitriol, n = 25)
subcutaneously within 6 h of calving. Blood was sampled before treatment and 12 h
later, and then until 15 DIM and analyzed for concentrations of ionized Ca (iCa), total
Ca (tCa), Mg (tMg), and P (tP), metabolites and hormones. Urine was sampled in the
first 7 DIM and analyzed for concentrations of Ca, Mg and creatinine. Neutrophil
function was evaluated in the first week postpartum. Intakes of DM and production
performance were evaluated for the first 36 DIM. Calcitriol administration increased (P <
0.01) concentrations of calcitriol within 12 h of application from 51 to 427 pg/mL, which
returned to baseline within 5 d. Blood concentrations of iCa and plasma tCa increased
24 h after treatment with Calcitriol. Concentrations of iCa (control = 1.08 vs. Calcitriol =
1.20 mM), tCa (control = 2.23 vs. Calcitriol = 2.33 mM), and tP (control = 1.47 vs.
Calcitriol = 1.81 mM) remained elevated in cows treated with Calcitriol until 3, 5 and 7
DIM, respectively, compared with control cows, whereas concentration of tMg (control =
0.76 vs. Calcitriol = 0.67 mM) was less in Calcitriol than control cows until 3 DIM.
54
Concentration of PTH decreased in Calcitriol compared with control cows (control = 441
vs. Calcitriol = 336 pg/mL). Calcitriol treatment tended to increase plasma concentration
of BHBA (control = 0.82 vs. Calcitriol = 0.97 mM), but concentrations of glucose, NEFA,
serotonin and CTX-1 in plasma did not differ between treatments. Cows treated with
Calcitriol excreted more urinary Ca (control = 0.5 vs. Calcitriol = 2.1 g/d) and Mg (control
= 4.5 vs. Calcitriol = 5.0 g/d) in the first 7 and 2 DIM, respectively, than control cows.
Calcitriol improved neutrophil function compared with control cows. The percentage of
neutrophils in blood with oxidative burst activity (control = 31.9 vs. Calcitriol = 40.6%),
mean fluorescence intensity (MFI) for oxidative burst (control = 90,900 vs. Calcitriol =
99,746), and MFI for phagocytosis (control = 23,887 vs. Calcitriol = 28,080) were all
greater for Calcitriol than control cows. Intake of DM and yields of milk and milk
components did not differ between treatments. Administration of 300 μg of calcitriol at
calving was safe and effective in increasing blood concentration of iCa, and plasma
concentrations of calcitriol, tCa, and tP for the first few days after treatment, and
improved measures of innate immune function in early lactation Holstein cows.
Background
A large proportion of dairy cows undergo a period of disruption in Ca
homeostasis with the onset of colostrogenesis and lactation. The large demands for Ca
for colostrum and milk synthesis induce a sudden drop in blood concentrations of
ionized (iCa) and total Ca (tCa), resulting in some cows developing clinical
hypocalcemia, also known as milk fever (DeGaris and Lean, 2008). Normal
concentration of tCa in blood usually ranges between 2.2 and 2.7 mM, but the onset of
lactation results in sequestration of Ca in the mammary gland followed by loss with
colostrum secretion, which can represent 7 to 10 fold the estimated amount of tCa
55
present in blood of a cow (Horst et al., 2005), thereby resulting in reductions in blood
tCa to values lesser than 2.2 mM. The inability of the cow to reestablish concentrations
of iCa and tCa in blood, either because of inadequate intestinal absorption, bone
resorption and/or urinary reabsortion is responsible for the development hypocalcemia
in the first days of lactation. Reinhardt et al. (2011) used the cut-off values of serum
concentrations of tCa between 1.4 and 2.0 mM as indicating cows with subclinical
hypocalcemia and concentrations below 1.4 in mM as indicating clinical hypocalcemia.
Using those cut-off values, the authors reported prevalences of 1% clinical
hypocalcemia and 25% subclinical hypocalcemia for primiparous cows, and 7% clinical
hypocalcemia and 47% subclinical hypocalcemia for multiparous cows in the first 48 h
of calving. Cows that develop subclinical and clinical hypocalcemia have impaired
subsequent health and reproduction. For instance, cows with either clinical or
subclinical hypocalcemia were more susceptible to develop other periparturient
disorders such as dystocia and ketosis (Curtis et al, 1983), displaced abomasum
(Massey et al., 1993), uterine prolapse (Risco et al., 1984), retained placenta (Melendez
et al., 2004), and metritis (Martinez et al., 2012). Thus, susceptibility to develop other
periparturient diseases, particularly those affecting the reproductive tract increases in
dairy cows unable to maintain blood concentrations of Ca in early lactation. In fact, it’s
been suggested that both clinical hypocalcemia (Kimura et al., 2006) and subclinical
hypocalcemia (Martinez et al., 2014) depress immune function that predispose cows to
diseases.
Dietary and therapeutic strategies are available to minimize the risk of
hypocalcemia in dairy cattle (DeGaris and Lean, 2008). One of the most common
56
methods to prevent hypocalcemia is the manipulation of the prepartum dietary mineral
content by altering the dietary cation-anion difference (DCAD) of the ration. Diets with
negative DCAD induce a compensated metabolic acidosis that promotes increases in
the concentrations of iCa and tCa in blood of cows immediately after calving (DeGaris
and Lean, 2008). Nevertheless, in spite of preventative measures such as diets with
negative DCAD, at least 47% of the multiparous cows experience subclinical
hypocalcemia in the first 48 h after calving (Reinhardt et al., 2011). As cows develop
subclinical hypocalcemia and the concentration of blood iCa drops, the parathyroid
gland rapidly increases secretion of parathyroid hormone (PTH), which activates renal
re-absorption of urinary Ca, osteoclastic bone resorption, and increases renal
production of the active form of vitamin D3, 1α,25-dihydroxyvitamin D3. The 1α,25-
dihydroxyvitamin D3 enhances intestinal Ca absorption (Pansu et al., 1983), bone
resorption (Kitazawa et al., 2003) and urinary Ca and P reabsorption (Hoenderop et al.,
2001). Several authors investigated use of vitamin D3 metabolites to prevent
development of clinical hypocalcemia in dairy cows (Jorgensen et al., 1978; Gast et al.,
1979; Allsop and Pauli, 1985). Goff et al. (1988) administered an analogue to 1α,25-
dihydroxyvitamin D3 intramuscularly to prepartum cows at greater risk of developing
clinical hypocalcemia starting 7 days before the expected day of calving and repeated
each 7 days until calving. Incidence of clinical hypocalcemia decreased from 85% in
untreated controls to 43 and 29% in cows receiving 100 and 150 μg of 24-F-1,25-
dihydroxyvitamin D3; however, the repeated treatments prepartum seems to impair the
endogenous vitamin D3 synthesis postpartum in treated cows that eventually develop
CH. Hove and Kristiansen (1982) administered 500 μg of 1α,25-dihydroxyvitamin D3
57
orally to 15 cows predisposed to develop clinical hypocalcemia and then grouped them
according to the day the treatment was administered relative to calving as those
receiving within 24 h of calving, 1 to 3 days before calving, or 4 to 5 days before calving.
A group of 8 cows remained as untreated controls. Administration of 1α,25-
dihydroxyvitamin D3 1 to 3 days before calving was able to increase blood Ca and P
compared with all other groups. An issue with such strategies prepartum is to predict
when the cow will calve such that administration of the active vitamin D3 occurs at the
proper time. Another option is to use immediately after calving in an attempt to prevent
the decline in blood Ca that occurs postpartum.
It was hypothesized that a slow-release injectable formulation of 1α,25-
dihydroxyvitamin D3 administered in the first hours after parturition would sustain blood
concentrations of iCa and tCa and improve measures of immune function in early
lactation cows. Therefore, the objectives of the present study were to determine the
effect of an injectable formulation of 1α,25-dihydroxyvitamin D3 on Ca homeostasis,
measures of immune function, energy metabolism, and productive performance of early
lactation dairy cows.
Materials and Methods
Two experiments were conducted at the University of Florida Dairy Unit to
characterize the responses to administration of a single dose of 1α,25-dihydroxyvitamin
D3 in early lactation multiparous Holstein cows. The experiments were approved by the
University of Florida Institutional Animal Care and Use Committee protocol number
201408679.
58
Experiment 1
Experiment 1 was designed to determine the appropriate dose of 1α,25-
dihydroxyvitamin D3 to increase plasma concentrations of Ca in a sustained manner
without subsequent hypocalcemia. Twenty-four multiparous Holstein cows were
randomly assigned to receive 1 of 3 treatments within 6 h of calving. Treatments
consisted of administration of a subcutaneous injection in the ischioanal fossa of vehicle
only (0 μg, 8 cows), or a solution containing 200 μg of 1α,25-dihydroxyvitamin D3 (9
cows), or 300 μg of 1α,25-dihydroxyvitamin D3 (7 cows). Details of the injectable
preparations are presented in Experiment 2. Blood was sampled by puncture of the
coccygeal vessels into lithium-heparinized evacuated tubes (Vacutainer, Becton
Dickinson, Franklin Lakes, NJ) immediately before administration of treatments, and
daily until 15 DIM. Tubes were placed in ice and transported to the laboratory within 30
min and centrifuged for 15 min at 2,500 x g for plasma separation. Plasma samples
were transferred into microtubes and stored frozen at -20°C until analyses. Plasma
samples were analyzed for concentrations of tCa, total P (tP), and total Mg (tMg) as
described in experiment 2.
In order to assess if the dose of 1α,25-dihydroxyvitamin D3 would affect
concentrations of 1α,25-dihydroxyvitamin D3 without the interference of early lactation
hypocalcemia, another four multiparous Holstein in late lactation received a
subcutaneous injection in the ischioanal fossa of 300 μg 1α,25-dihydroxyvitamin D3 on
day 0 followed by another dose of 50 μg on day 6. Blood was sampled by puncture of
the coccygeal vessels into lithium-heparinized evacuated tubes (Vacutainer, Becton
Dickinson) immediately before administration of treatment, and at 4, 8, 12, and 24 h,
daily until day 15, and at 12 h after the second administration on day 6. Tubes were
59
placed in ice and transported to the laboratory within 30 min and centrifuged for 15 min
at 2,500 x g for plasma separation. Plasma samples were transferred into microtubes
and stored frozen at -20°C until analyses. Plasma samples were analyzed for
concentrations of tCa, tMg, and 1α,25-dihydroxyvitamin D3 as described in Experiment
2.
Experiment 2
Experimental design, cows, and housing
The experiment was a randomized complete block design. Holstein cows were
blocked by parity as lactation 2 or lactation greater than 2 and sequence of calving and,
within each block, assigned randomly to 1 of 2 treatments within 6 h of calving. All
treatments were performed before colostrum milking. The dose chosen of 1α,25-
dihydroxyvitamin D3 was based on results from experiment 1. Treatments were
administration of a subcutaneous injection in the ischioanal fossa of vehicle only
(control, 25 cows) or a solution containing 300 μg of 1α,25-dihydroxyvitamin D3
(Calcitriol, 25 cows).
Prepartum cows were housed in a free-stall barn in the last 4 weeks of gestation.
Cows were observed 4 times daily, at 6-h intervals for signs of parturition to assure that
experiment enrollment occurred within 6 h of calving. Upon calving and experiment
enrollment, postpartum cows were housed in a free-stall barn equipped with individual
feeding gates (American Calan Inc., Northwood, NH) that were assigned randomly to
each cow. Individual cow DMI was measured for the first 36 DIM.
Diets, feeding, and analyses of dietary ingredients
Prepartum cows were fed once daily and postpartum cows twice daily. Diets
were fed as TMR for ad libitum intake. In the postpartum period, amounts of diets
60
offered and refused were measured daily for individual cows to calculate DMI.
Prepartum cows received the same diet formulated to have a negative calculated DCAD
to minimize the risk of clinical and subclinical hypocalcemia. Postpartum cows received
the same TMR to meet or exceed the nutrient requirements for a lactating Holstein cow
weighing 650 kg of BW and producing 40 kg of 3.5% FCM/d when intake averaged 23
kg of DM in the first 36 DIM (NRC, 2001).
Individual forages, brewer’s grains and concentrate mixtures were sampled
weekly and dried in air-forced oven at 55C for 48 h and moisture loss recorded.
Individual dried samples were ground to pass through a 1.0-mm mesh screen. Samples
were composited monthly and analyzed for OM (512 °C for 8 h), NDF using a heat
stable α-amylase and ADF, N using an automated quantitative combustion digestion
method, starch using an enzymatic digestion method and subsequent quantification of
glucose, fat by acid hydrolysis, and minerals using inductively coupled plasma mass
spectrometry at a commercial laboratory (Dairyland Laboratories Inc., Arcadia, WI).
Table 3-1 depicts the ingredient composition and nutrient content of TMR fed in the pre-
and postpartum periods.
Calcitriol formulation
A stock solution of calcitriol was prepared by dissolving 10 mg of crystalline
powder 1α,25-dihydroxyvitamin D3 (Cayman Chemical, Ann Arbor, MI) with ethanol
99.5% (1 mL; Sigma-Aldrich, St. Louis, MO). Further dilution of 1 to 10 with ethanol was
done on the day of use. The concentration of 1α,25-dihydroxyvitamin D3 in the ethanol
solution was measured using an absorbance reader (BioTek Synergy HT, BioTek
Instruments, Winooski, VT) to assure that 1α,25-dihydroxyvitamin D3 was properly
61
diluted. To prepare a slow-release formulation, glyceryl trioctanoate (Sigma-Aldrich, St.
Louis, MO) was used as a source of medium chain triglycerides and 2,6-di-tert-butyl-4-
methylphonel (Sigma-Aldrich, St. Louis, MO) as an antioxidant to achieve a 95%
oleaginous solution with 1α,25-dihydroxyvitamin D3 at the concentration of 50 μg/mL.
The final solution was mixed continuously using a vortex for at least 20 min. All the
procedures were done aseptically under a laminar flow hood using autoclaved
laboratory materials. A control solution without the 1α,25-dihydroxyvitamin D3 crystalline
powder was prepared following the same steps. Solutions were stored at 8°C and used
within 14 d. Before administration to cows, the solutions were brought up to ambient
temperature and mixed thoroughly for 1 min. Calcitriol treated cows received 6 mL of
the 95% oleaginous solution with 1α,25-dihydroxyvitamin D3 at the concentration of 50
μg/mL, and control treated cows received 6 mL of the control solution without the 1α,25-
dihydroxyvitamin D3 crystalline powder.
Measurements of yields of milk and milk components
Cows were milked twice daily at 1000 and 2200 h, and the milk production was
recorded automatically using milk meters (AfiFlo milk meters, S.A.E. Afikim, Israel). The
yield and composition of colostrum and milk from second milking were analyzed
separately from other milk measurements.
Milk samples were collected on every milking during the first 5 DIM, after which
milk was sampled in two consecutive milkings twice a week until 36 DIM. Samples were
submitted to the Southeast DHI laboratory in Belleview, FL, and analyzed for
concentrations of fat, true protein, lactose and somatic cells. The yields of 3.5% FCM
and ECM were calculated as follows: FCM = [(0.4324 x kg milk) + (16.218 x kg fat)];
62
ECM = [(0.3246 x kg milk) + (12.86 x kg fat) + (7.04 x kg of protein)]. The SCS was
calculated according to the following formula: SCS = Log10 (SCC/12.5)/Log10 2.
The Ca and Mg contents of colostrum and milk from the second milking
postpartum were analyzed. Frozen sampled of colostrum and milk were thawed,
thoroughly homogenized, and an aliquot of 2.5 mL was pipetted into 50 mL tubes.
Twenty-five milliliters of trichloroacetic acid 24% and 22.5 mL of deionized water were
added to each tube. Samples were agitated and homogenized every 5-min for 30 min.
The solution was filtrated and a 5 mL aliquot of the filtrate was transferred to another 50
mL tube. One milliliter of lanthanum chloride 5% and 44 mL of deionized water were
added to each tube to result in a 50 mL solution. Samples were then analyzed by
atomic absorption using a spectrophotometer (AAnalyst 200, Perkin-Elmer Inc.,
Waltham, MA) equipped with Ca and Mg specific hollow cathode lamps. The amounts of
Ca and Mg secreted in the first day postpartum was calculated based on yields of
colostrum and milk from second milking and the respective concentrations of Ca and
Mg.
Body weight and body condition
Cows were weighed twice a day after each milking as they left the parlor using a
walk-through scale (AfiWeigh, S.A.E. Afikim). The BW were averaged for each day for
the first 36 DIM for statistical analysis. Twice a week, cows had their body condition
scored by the same evaluator using a 5-point scale (1 = emaciated to 5 = obese)
divided into 0.25 points according to Ferguson et al. (1994) as depicted in the Elanco
BCS chart (Elanco Animal Health, 2009).
63
Blood minerals, metabolites and hormones
Blood was sampled by puncture of the coccygeal vessels into lithium-heparinized
evacuated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) immediately before
administration of treatments, 12 h after, and again at 1, 2, 3, 5, 7, 9, 12, and 15 DIM.
Tubes were placed in ice and transported to the laboratory within 30 min and
centrifuged for 15 min at 2,500 x g for plasma separation. Plasma samples were
transferred into microtubes and stored frozen at -20°C until analyses. Blood was also
sampled immediately before administration of treatments, 12 h later, and at 1, 2, 3 and
5 DIM and analyzed for concentrations of iCa, Na+, K+, and glucose using a handheld
biochemical analyzer (i-Stat, Abbott Laboratories, Princeton, NJ).
Concentrations of calcitriol in plasma were analyzed by ELISA (1α,25(OH)2
Vitamin D ELISA, IBL-America, Minneapolis, MN) according to the manufacturer's
instructions. The intra- and interassay CV were 8.0 and 11.6%, respectively.
Concentrations of calcidiol in plasma were analyzed by ELISA (25(OH) Vitamin D
ELISA, DRG Instruments GmbH, Germany) according to the manufacturer's
instructions. The intra- and interassay CV were 6.1 and 11.2%, respectively.
Concentrations of tCa and tMg in plasma were analyzed by atomic absorption
(AAnalyst 200, Perkin-Elmer Inc., Waltham, MA) as previously described (Martinez et al.
2012). Intra and interassay CV were, respectively, 2.1 and 5.9% for tCa, and 4.2 and
4.7% for Mg. Concentrations of tP were quantified in plasma using the molybdenum
blue method (Quinlan and DeSesa, 1955). The intra and interassay CV were 3.3 and
4.1%. Subclinical hypocalcemia was defined based on concentrations of tCa in plasma
below 2.125 mM (Martinez et al., 2016).
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Concentrations of NEFA (NEFA-C Kit; Wako Diagnostics Inc., Mountain View,
CA) and BHBA (Wako Autokit 3-HBt; Wako Diagnostics Inc., Mountain View, CA) in
plasma were analyzed by colorimetric methods. The intra- and interassay CV were,
respectively, 2.5 and 3.4% for NEFA and 4.3 and 7.0% for BHBA. Concentrations of
urea N and glucose in plasma were determined by colorimetric continuous flow analysis
(Autoanalyzer II; SEAL Analytical, Segensworth, Fareham Hampshire, UK) using a
modification of the method described by Gochman and Schmitz (1972).
Concentrations of serotonin in plasma were analyzed by ELISA (Serotonin EIA
kit, Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions.
The intra- and interassay CV were 10.2 and 21.2%, respectively. Intact PTH was
determined using an ELISA assay (Bovine Intact PTH ELISA Kit, Immutopics Inc., San
Clemente, CA) according to the manufacturer`s instructions. The intra- and interassay
CV were 7.1 and 15.7%, respectively. The bone resorption marker C-telopeptide of type
I collagen (CTX-1) was measured in plasma by ELISA (Bovine CTX-1 ELISA Kit,
NeoScientific, London, UK), and the intra- and interassay CV were 6.2 and 14.1%,
respectively.
Urinary excretion of minerals and creatinine
Urine samples were collected at 1, 2, 3, 5 and 7 DIM. The perineal area was
massaged until a clean and copious stream of urine was obtained. Samples were frozen
at -20°C for later analyses of concentrations of creatinine, Ca, and Mg. Samples were
analyzed for creatinine using a commercial colorimetric method (Creatinine Urinary
Detection Kit, Arbor Assays, Ann Arbor, MI). Samples were diluted 1 to 400 with 0.5%
lanthanum chloride and concentrations of Ca and Mg were analyzed by atomic
absorption using a spectrophotometer equipped with Ca and Mg specific hollow cathode
65
lamps (AAnalyst 200, Perkin-Elmer Inc., Waltham, MA). The intra- and interassay CV
for creatinine were 2.2 and 8.6%, respectively. Urinary Ca and Mg assays were
performed as a single run and the intraassay CV were 5.4 and 1.3%, respectively.
Creatinine was used as a marker to estimate daily urinary volume based on the
constant excretion of 29 mg of creatinine per kg of BW per day (Valadares et al., 1999).
The estimate of daily urinary volume (kg) is: BW kg x 29/urinary concentrations of
creatinine mg/L (e.g. 680 kg x 29/450 mg/L urine = 43.82 L of urine/day). Daily urinary
excretions of Ca and Mg were calculated based on the urinary volume and the
concentrations of those minerals in the urine samples.
Complete blood cell counts and neutrophil function
Blood was sampled by puncture of the coccygeal blood vessels into evacuated
tubes (Vacutainer, Becton Dickinson) containing either lithium heparin or K2 EDTA at 2,
4, and 6 DIM. Samples were kept at room temperature, protected from light, and
transported to the laboratory within 2 h of collection. Samples collected with K2 EDTA
were used for analysis of differential blood cell count using an automated hematology
analyzer (ProCyte Dx Hematology Analyzer; Idexx Laboratories, Westbrook, ME).
Samples collected with heparin were used for flow cytometric assays to quantify
neutrophil phagocytosis and oxidative burst.
Function of neutrophils in vitro was assayed by dual color flow cytometry
according to Martinez et al. (2012). Escherichia coli isolated from milk from a cow with
mastitis was labeled with propidium iodide and used to quantify neutrophil phagocytosis
and intracellular killing. Briefly, blood was pipetted into four tubes and 5-µM
dihydrorhodamine 123 (DHR) was added to all the tubes. Tube 1 was used as a
negative control. Phorbol 12-miristate 13-acetate was added to tube 2 to induce
66
oxidative burst activity in neutrophils as a positive control. In tubes 3 and 4, propidium
iodide labeled E. coli were added to the tubes to achieve a bacteria to neutrophil ratio of
40 to 1. The samples were analyzed using an Accuri C6 (BD Biosciences, San Jose,
CA) digital analyzer flow cytometer. Forward and side scatter gating of granulocytes
resulted in more than 90% neutrophils selected in the granulocyte cell population
evaluated. Data acquisitions from at least 10,000 cells per sample were analyzed using
Flowjo software (version 10.0.8, Tree Star Inc., Ashland, OR). Gating of neutrophils to
establish phagocytosis and oxidative burst activity was based on the negative control
and positive control as shown on Figure 3-1. Fresh blood from a single reference cow
was used in every assay throughout the experiment and results of neutrophil function
were expressed relative to the reference cow. The parameters analyzed were
percentage of neutrophils that phagocytized bacteria and the percentage of neutrophils
with phagocytosis-induced oxidative burst. Furthermore, histogram analysis for mean
fluorescence intensity (MFI) of oxidized DHR and propidium iodide-labeled bacteria
were used to estimate the neutrophil mean oxidative burst intensity as an indication of
the intensity of reactive oxygen species produced per neutrophil, and neutrophil mean
phagocytic activity as an indication of the number of bacteria phagocytized per
neutrophil, respectively.
Statistical Analysis
Continuous data were evaluated for distribution of the residuals and homogeneity
of variance before statistical analyses. Data with violation of the assumptions of
normality were subjected to Box-Cox transformation using the TRANSREG procedure
of SAS before statistical analysis. When data was transformed, then the LSM were back
transformed and the SEM calculated (Jørgensen and Pedersen, 1998).
67
In experiment 1, the effects of 0, 200 or 300 μg of 1α,25-dihydroxyvitamin D3 on
plasma concentrations of tCa, Mg, and P were analyzed using the MIXED procedure of
SAS ver. 9.4 (SAS/STAT, SAS Institute Inc., Cary, NC). The statistical models included
the fixed effects of dose (0 vs. 200 vs. 300 μg), day after treatment, and the interaction
between dose and day after treatment, and the random effect of cow nested within
dose. Measurements taken immediately before dosing were used as covariates in the
statistical models. When a single dose was used, then means and SD were calculated
using the MEANS procedure of SAS (SAS/STAT, SAS Institute Inc.).
In experiment 2, continuous data were analyzed using the MIXED procedure of
SAS version 9.4 (SAS/STAT, SAS Institute Inc., Cary, NC). The statistical models
included the fixed effects of treatment (control vs. Calcitriol), day postpartum, and the
interaction between treatment and day postpartum, and the random effects of block and
of cows nested within treatment and block. Measurements taken on day 0 before
treatment administration were used as covariates for adjustment during analyses. The
covariance structure with the smallest Akaike’s information criterion was chosen, and
most analyses used the first-order autoregressive structure for equally spaced
measurements or spatial power for unequally spaced measurements. The Kenward-
Roger method was used to obtain the approximate degrees of freedom. When the F-
test for an interaction between treatment and day postpartum was significant, means
were then portioned using the SLICE command in SAS.
The incidence and daily prevalence of subclinical hypocalcemia were analyzed
by logistic regression using the GLIMMIX procedure of SAS fitting a binary distribution.
For incidence of SCH, the model included the fixed effect of treatment and the random
68
effect of block. For the daily prevalence of SCH, the model included the fixed effects of
treatment, day, interaction between treatment and day, and the random effects of block
and cow nested within treatment and block.
Treatment differences with P ≤ 0.05 were considered significant, whereas
tendencies for differences were reported if 0.05 < P ≤ 0.10.
Results
Experiment 1
Treatment with 1α,25-dihydroxyvitamin D3 administered as a single dose of either
200 or 300 μg increased (P < 0.05) the concentrations of plasma tCa within 24 h of
treatment and they remained elevated for the first 3 after treatment compared with cows
receiving 0 μg of 1α,25-dihydroxyvitamin D3 (Figure 3-2A). Similar to concentrations of
tCa, treatment with 200 or 300 μg of 1α,25-dihydroxyvitamin D3 increased (P < 0.05) the
concentrations of tP in plasma in the first 3 days after treatment compared with cows
receiving 0 μg 1α,25-dihydroxyvitamin D3 (Figure 3-2C). Concentrations of tCa and tP in
plasma did not differ between treatments beyond the third day after treatment with
1α,25-dihydroxyvitamin D3. Treatment with 200 μg or 300 μg of 1α,25-dihydroxyvitamin
D3 reduced (P < 0.05) the concentrations of tMg in plasma in the first 4 days after
treatment compared with cows receiving 0 μg 1α,25-dihydroxyvitamin D3 (Figure 3-2B),
with no differences between treatments after 5 DIM.
Administration of 300 μg 1α,25-dihydroxyvitamin D3 to late lactation dairy cows
increased plasma 1α,25-dihydroxyvitamin D3 from 22.5 ± 6.5 to 488.9 ± 31.4 pg/mL in
24 h (Figure 3-2D). Furthermore, the second administration of 50 μg 1α,25-
dihydroxyvitamin D3 on day 6 increased plasma 1α,25-dihydroxyvitamin D3 from 10.7 ±
1.9 pg/mL immediately before administration to 50.8 ± 7.4 in 12 h. Concentration of tCa
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in plasma increased from 2.52 ± 0.14 to 3.02 ± 0.24 mM during the first 48 h after
treatment with 300 μg 1α,25-dihydroxyvitamin D3 (Figure 3-2E), and increased from
2.92 ± 0.24 to 3.08 ± 0.15 mM during the first 24 h after the second treatment with 50 μg
1α,25-dihydroxyvitamin D3 on day 6. From day 7 to day 15 after first treatment,
concentration of tCa in plasma decreased from 3.08 ± 0.15 to 2.41 ± 0.13 mM, and
remained in the normocalcemic range. Concentrations of tMg in plasma decreased from
0.97 ± 0.12 to 0.81 ± 0.06 mM during the first 24 h after treatment with 300 μg 1α,25-
dihydroxyvitamin D3 (Figure 3-2F). The second treatment with 50 μg 1α,25-
dihydroxyvitamin D3 6 days later had minor impacts on plasma tMg concentrations.
Collectively, these results showed a sustained increased in plasma tCa in dairy
cows for at least 3 days after administration of 300 μg of 1α,25-dihydroxyvitamin D3 with
no apparent signs of subsequent hypocalcemia after the exogenous 1α,25-
dihydroxyvitamin D3 is catabolized to intermediate metabolites by hydroxylation by
CYP24 or hydroxylation of C-23 to lesser active metabolites (Harant et al., 2000). It was
judged that the second dose of 50 μg 1α,25-dihydroxyvitamin D3 would not be
necessary because tCa concentrations remained in the normocalcemic range.
Therefore, the 300 μg dose of 1α,25-dihydroxyvitamin D3 as a single treatment was
chosen for experiment 2.
Experiment 2
Concentrations of calcitriol and calcidiol in plasma
Treatment with Calcitriol increased (P < 0.01) the concentrations of calcitriol in
plasma compared with control cows starting 12 h after treatment and differences were
observed until 3 DIM, after which the concentrations did not differ between treatments
(Figure 3-3A). Concentrations of calcidiol in plasma did not differ between treatments,
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and averaged 55.4 ± 2.5 ng/mL for control and 56.1 ± 2.5 ng/mL for Calcitriol in the first
3 DIM (Figure 3-3B).
Concentrations of minerals in whole blood and plasma and prevalence of subclinical hypocalcemia
Treatment with Calcitriol within 6 h of calving increased (P < 0.01) the
concentrations of iCa in whole blood compared with control cows starting 24 h after
treatment and differences were observed until 3 DIM, after which the concentrations did
not differ between treatments (Figure 3-4A). Concentrations of iCa in whole blood
averaged 1.08 ± 0.03 and 1.20 ± 0.03 mM for control and Calcitriol in the first 5 DIM.
Similar to iCa, Calcitriol increased (P < 0.01) the concentrations of tCa in plasma
compared with control cows 24 h after treatment, and this increment lasted until 5 DIM
(Figure 3-4B). In the first 5 DIM, concentrations of tCa averaged 2.12 ± 0.04 mM for
control and 2.44 ± 0.04 mM for Calcitriol-treated cows; however, treatment with
Calcitriol resulted in smaller (P < 0.05) concentrations of tCa in plasma between 9 and
15 DIM compared with control cows, although cows in both treatments had mean tCa
concentrations in plasma within the normocalcemic range during this period.
Calcitriol reduced (P < 0.05) the prevalence of subclinical hypocalcemia in the
first 3 DIM compared with control (Figure 3-5), and the daily prevalence rate in the first 3
DIM averaged 56.6 and 6.8% for control and Calcitriol, respectively. The daily
prevalence of subclinical hypocalcemia did not differ between treatments on days 5, 7
and 15 postpartum, but it was greater for Calcitriol than control on days 9 and 12
postpartum. At 9 and 12 DIM, the daily prevalence rate averaged 1.0 and 14.4% for
control and Calcitriol, respectively.
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Treatment with Calcitriol increased (P < 0.01) the concentrations of tP in plasma
compared with control cows starting 24 h after treatment and the difference lasted until
7 DIM (Figure 3-4C), after which they normalized and no longer differed between
treatments. In the first 7 DIM, concentrations of tP in plasma averaged 1.47 ± 0.07 mM
for control and 2.13 ± 0.07 mM for Calcitriol-treated cows. Concentrations of tMg
decreased in both treatments after calving, but Calcitriol induced a more accentuated
decline (P < 0.01) in concentrations of tMg in plasma compared with control from 24 h
after treatment until 3 DIM (Figure 3-4D). In the first 3 DIM, concentrations of tMg in
plasma averaged 0.75 ± 0.02 mM for control and 0.65 ± 0.02 mM for Calcitriol-treated
cows. Concentrations of tMg in plasma increased after 5 DIM and they did not differ
between treatments after 3 DIM.
Concentrations of PTH, serotonin, and bone resorption marker in plasma
Treatment with Calcitriol reduced (P = 0.04) the concentrations of PTH in plasma
compared with control cows starting 24 h after treatment and differences were observed
until 3 DIM, after which the concentrations did not differ between treatments (Figure 3-
6A). Concentrations of PTH in plasma averaged 475 ± 48 pg/mL for control and 325 ±
32 pg/mL for Calcitriol in the first 3 DIM. Treatment with Calcitriol tended (P = 0.10) to
increase concentrations of serotonin in plasma compared with control cows during the
first 7 DIM, and averaged 1,350 ± 162 and 1,694 ± 203 ng/mL for control and Calcitriol,
respectively (Figure 3-6B). Furthermore, concentrations of the bone resorption marker
CTX-1 increased (P < 0.05) in the first 7 DIM in both treatments, from a mean of 1.07
ng/mL at calving to a mean of 1.64 ± 0.09 and 1.68 ± 0.09 ng/mL for control and
Calcitriol in the first 7 DIM (Figure 3-6C). Nevertheless, treatment did not affect
concentrations of CTX-1 in plasma of dairy cows in the first week postpartum.
72
Composition and yield of colostrum and milk in the second milking postpartum
Treatment with Calcitriol did not affect yields of colostrum or milk during the
second milking postpartum, as well as their nutrient composition (Table 3-2). Cows
produced an average 7.0 kg of colostrum and 6.7 kg of milk in the second postpartum
milking. The similar contents of fat, protein and lactose resulted in no difference in NE
secreted as colostrum and milk in the first day postpartum. The concentrations of Ca
and Mg in colostrum and milk from the second postpartum milking did not differ between
treatments. Cows lost an average of 13.9 g of Ca and 2.9 g of Mg in colostrum and 10.0
and 1.9 g of Ca and Mg in milk in the second milking, respectively. Collectively, cows
lost 23.9 g of Ca and 4.8 g of Mg in the first day postpartum.
Dry matter intake, milk yield, BW and BCS
Dry matter intake in the first 7 weeks postpartum did not differ between
treatments (Table 3-3). Treatment with Calcitriol did not affect yields of milk, milk
components, concentrations of fat, true protein and lactose in milk, or yields of 3.5%
FCM or ECM. Lack of changes in intake and production resulted in no differences in the
caloric content of milk, energy output as milk, mean or change in BW and BCS, and
energy balance. The SCS was not influenced by treatment.
Concentrations of metabolites in plasma
Treatment with Calcitriol tended (P = 0.08) to increase the concentrations of
BHBA in plasma compared with control (Figure 3-7A). Concentrations of BHBA in
plasma averaged 0.82 ± 0.05 and 0.97 ± 0.06 mM for control and Calcitriol during the
first 15 DIM. Calcitriol did not affect the concentrations of NEFA in early lactation
compared with control (Figure 3-7B), and concentrations of NEFA in plasma averaged
561 ± 40 and 616 ± 44 mM for control and Calcitriol, respectively, during the first 15
73
DIM. Similar to NEFA, Calcitriol did not affect the concentrations of glucose in plasma in
the first 2 weeks postpartum (Figure 3-7C), and they averaged 54.1 ± 1.1 and 56.1 ± 1.1
mM for control and Calcitriol, respectively. Moreover, concentrations of urea nitrogen in
plasma did not differ between treatments, and averaged 9.3 ± 0.3 and 9.3 ± 0.3 mg/dL
for control and Calcitriol, respectively, in the first 15 DIM (Figure 3-7D).
Urinary excretion of minerals
Treatment with Calcitriol increased (P = 0.01) the excretion of Ca in urine in the
first 7 DIM compared with control cows, and the daily loss averaged 0.52 ± 0.26 and
2.07 ± 0.26 g/d for control and Calcitriol, respectively, during the first 7 DIM (Figure 3-
8A). Similarly, treatment with Calcitriol increased (P = 0.02) the daily urinary loss of Mg,
but this effect was observed only in the first 2 DIM (Figure 3-8B). Daily urinary loss of
Mg averaged 4.49 ± 0.42 and 5.03 ± 0.42 g/d for control and Calcitriol, respectively,
during the first 7 DIM.
Blood cell count and neutrophil function
Calcitriol increased (P = 0.04) the hematocrit compared with control during the
first 6 DIM, but concentration of hemoglobin did not differ between treatments (Table 3-
4). The number of erythrocytes, reticulocytes, platelets, total leukocytes, or distinct
types of leukocytes such as neutrophils, lymphocytes, and monocytes per microliter of
blood did not differ between treatments. Calcitriol tended (P = 0.07) to increase the
concentration of eosinophils plus basophils in blood compared with control cows. The
blood values for erythrocytes, leukocytes and platelets were within the normal range
expected for lactating dairy cows.
Treatments did not affect the percentage of neutrophils in blood with phagocytic
activity during the first 6 DIM (control = 53.9 ± 4.4 vs. Calcitriol = 60.9 ± 4.4; Figure 3-
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9A); however, Calcitriol increased (P = 0.03) the MFI for number of bacteria
phagocytized per neutrophil compared with control cows (control = 23,887 ± 1,324 vs.
Calcitriol = 28,080 ± 1,310; Figure 3-9C), and this response was more evident at 2 DIM.
Furthermore, Calcitriol increased (P = 0.03) percentage of neutrophils in blood with
oxidative burst activity (control = 31.9 ± 2.7 vs. Calcitriol = 40.6 ± 2.7; Figure 3-9B), and
this response was observed in the first 4 DIM. Calcitriol increased (P < 0.05) and tended
(P = 0.09) to increase the MFI for production of reactive oxygen species per neutrophil
in blood compared with control cows on days 2 and 4 postpartum, respectively (Figure
3-9D).
Discussion
Administration of 300 μg of 1α,25-dihydroxyvitamin D3 within 6 h of parturition
was efficacious and safe to increase concentrations of Ca in plasma of dairy cows in a
sustainable manner, which reduced the daily prevalence rate of subclinical
hypocalcemia in the first 3 DIM. Administration 300 μg of 1α,25-dihydroxyvitamin D3
increased concentrations of 1α,25-dihydroxyvitamin D3 in plasma similar to those
observed in cows with clinical hypocalcemia (Horst et al., 1978), and concentrations
remained elevated for 3 d, after which they returned to values below 50 pg/mL. This
elevated concentration of 1α,25-dihydroxyvitamin D3 in Calcitriol cows resulted in
increases in blood iCa, and plasma tCa and tP in the first 3 to 7 days postpartum.
Vitamin D3 is typically fed to dairy cows in the inactive form cholecalciferol with
the goal of promoting Ca absorption in the intestine to prevent hypocalcemia. The
dietary form has to be activated by subsequent hydroxylations in the liver and kidney in
order to produce the active 1α,25-dihydroxyvitamin D3; however, regulatory
mechanisms exist to maintain production of 1α,25-dihydroxyvitamin D3 in strict balance
75
with oscillatory Ca in order to avoid calcification of soft tissues. When blood
concentrations of iCa are low, then the stimulation of production and release of PTH
favor the synthesis of additional 1α,25-dihydroxyvitamin D3 (Horst et al., 1994). On the
other hand, the use of 1α,25-dihydroxyvitamin D3 and its precursors to prevent clinical
hypocalcemia have been studied previously (Goff et al., 1986; Goff et al., 1988; Hove
and Kristiansen, 1982). Goff et al. (1988) administered an analogue to 1α,25-
dihydroxyvitamin D3 intramuscularly to prepartum cows starting 7 days before the
expected day of calving and repeated each 7 days until calving. Incidence of clinical
hypocalcemia decreased from 85% in untreated controls to 43 and 29% in cows
receiving 100 and 150 μg of 24-F-1,25-dihydroxyvitamin D3; however, the repeated
treatments prepartum reduced concentrations of 1α,25-dihydroxyvitamin D3 in cows that
subsequently developed milk fever suggesting depressed endogenous production in
response to low blood iCa.
One of the issues with administration of 1α,25-dihydroxyvitamin D3 prepartum is
the selected dose and the frequency of administration. The inability to predict the day of
calving results in some cows receiving multiple treatments, which can downregulate
endogenous synthesis and result in hypocalcemia after 1 or 2 DIM. Also, if excessive
doses are administered, then toxicity can occur with signs such as anorexia and
metastatic calcification (Littledike et al., 1982). Increased concentrations of 1α,25-
dihydroxyvitamin D3 activates transcription of the CYP24A1 gene (Ohyama et al., 1994),
which hydroxylates the side chain to form less bioactive metabolites of vitamin D3.
Furthermore, increased concentrations of 1α,25-dihydroxyvitamin D3 downregulates 1-
α-hydroxylase (Henry, 1979), which suppresses endogenous synthesis of 1α,25-
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dihydroxyvitamin D3. Therefore, excessive doses or repeated administrations of 1α,25-
dihydroxyvitamin D3 possess a risk of toxicity or suppressed periods of endogenous
synthesis of the active vitamin D3. Because clinical hypocalcemia can be effectively
prevented by manipulating the prepartum DCAD with use of acidogenic salts, our
strategy was to use 1α,25-dihydroxyvitamin D3 immediately after calving to reduce the
risk of subclinical hypocalcemia and improve innate immune function (Martinez et al.,
2012; Martinez et al., 2014).
Calcitriol increased concentrations of iCa, tCa, and tP in dairy cows within 24
hours of treatment. The quick response likely occurred through the non-genomic actions
of 1α,25-dihydroxyvitamin D3 by binding to the membrane associated rapid response
steroid-binding receptor within the plasma membrane inducing increased absorption of
intestinal Ca (Larsson and Nemere, 2003; Tudpor et al., 2008) and P (Nemere et al.,
2004). In addition, 1α,25-dihydroxyvitamin D3 increases gene expression of vitamin D
responsive elements, which favors intestinal uptake of Ca and P (Li et al., 1998), bone
resorption (Kondo et al., 2004), and urinary reabsorption of Ca and P (Hoenderop et al.,
2001). Consequently, plasma tCa and tP concentration remained elevated until 5 to 7
DIM in Calcitriol compared with control cows. Although 1α,25-dihydroxyvitamin D3 is
expected to increase bone resorption (Kondo et al., 2004), treatment with Calcitriol was
unable to increase the concentrations of CTX-1 in plasma compared with those in
control cows. Concentrations of CTX-1 increased almost 60% after calving, indicating
active bone metabolism in both treatments in order to maintain Ca homeostasis.
Previous experiments using hydroxyproline as a bone resorption marker showed that
1α,25-dihydroxyvitamin D3 and its analogues did not increased plasma and urinary
77
excretion of hydroxyproline in nonlactating cows (Goff et al., 1986). Furthermore, cows
treated with Calcitriol had reduced PTH, and PTH has been shown to stimulate bone
remodeling (Hustmyer et al., 1995). The lack of differences in CTX-1 between
treatments suggests that the increases in concentrations of iCa and tCa and tP
observed with Calcitriol were likely the result of increased intestinal absorption, and not
bone resorption. It is possible that the absolute amounts of Ca and P reabsorbed by the
kidneys also increased in Calcitriol cows, but the transient hypercalcemia induced by
treatment with 1α,25-dihydroxyvitamin D3 doubled the amount of Ca lost in urine in the
first 7 days postpartum.
Calcium intake did not differ based on the fact that cows receive the same diets
pre- and postpartum and that DMI did not differ between treatments. Also, the loss of
Ca as colostrum and milk in the first day postpartum did not differ between treatments.
Because milk yield did not differ for the first 36 DIM, it is likely that loss of Ca and other
minerals as milk were the same between treatments in early lactation. Therefore, the
changes in blood Ca observed in Calcitriol cows were likely the result of intestinal
absorption.
Cows treated with Calcitriol had reduced concentrations of PTH compared with
control cows. Calcitriol increased concentrations of 1α,25-dihydroxyvitamin D3, which is
known to suppress PTH synthesis by downregulation of PTH gene expression (DeMay
et al., 1992). Also, increases in plasma concentrations of iCa stimulate Ca-sensing
receptors in the parathyroid gland that suppresses PTH synthesis and secretion (Chen
and Goodman, 2004). These responses likely explain the reduced concentrations of
PTH in Calcitriol-treated cows. Activation of Ca sensing receptors induced by increases
78
in iCa in the thick ascending limb of the Henle loop and convoluted tubules inhibits the
Na+/K+/2Cl- cotransporter system, which is important for paracellular reabsorption of Ca
and Mg (Dai et al., 2001). The changes in the Na+/K+/2Cl- cotransporter system induced
by increases in iCa causes a reduction in the lumen-positive voltage, which reduces the
paracellular mineral reabsorption in the kidney (Hebert et al., 1997). In fact, Calcitriol
increased urinary excretion of Mg. Thus, the increase in iCa combined with the reduced
concentrations of PTH in cows in Calcitriol resulted in increased urinary losses of Mg,
which likely explain the reduced concentrations of tMg in plasma compared with control
cows in the first 3 DIM.
Calcitriol tended to increase serotonin concentrations and serotonin plays a role
in bone remodeling and it is required for osteoclastogenesis (Chabbi-Achengli et al.,
2002). Laporta et al. (2013) reported that concentrations of serotonin in blood were
positively associated with serum concentrations of iCa, and cows with clinical
hypocalcemia had smaller concentrations of serotonin compared with cows without
hypocalcemia.
Subclinical hypocalcemia reduces DMI and compromises energy metabolism in
dairy cows (Martinez et al., 2014). It was initially expected that administration of 1α,25-
dihydroxyvitamin D3 would increase blood concentrations of iCa and eliminate
subclinical hypocalcemia, which would benefit appetite in dairy cows. Nevertheless,
results from the current experiment clearly demonstrated that DMI and productive
performance were unaffected by treatment of cows with Calcitriol. In fact, the cows in
Calcitriol had similar yields of milk and milk components, which resulted in no
differences in energy balance or concentrations of NEFA, glucose, or urea N in plasma.
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Martinez et al. (2014) showed that subclinical hypocalcemia increased concentrations of
NEFA in plasma, which was thought to be mediated by a depression in the release of
insulin, in spite of increased glucose concentrations. In spite of the benefits of Calcitriol
in reducing subclinical hypocalcemia in the first 3 DIM, it seems that it was not sufficient
to attenuate the lipolytic signals and depress the concentrations of NEFA in plasma. In
fact, cows treated with Calcitriol had slightly greater concentrations of BHBA in plasma
than control cows. Collectively, these results indicate that treatment with Calcitriol
immediately after calving does not benefit lactation performance or energy metabolism
in dairy cows in the first month postpartum.
The increased demand for Ca during the peripartum period has been shown to
decrease intracellular iCa stores in peripheral blood leukocytes (Kimura et al., 2006),
and cows with clinical hypocalcemia have compromised leukocyte function (Ducusin et
al., 2003; Kimura et al., 2006). Similarly, induction of subclinical hypocalcemia in dry
cows reduced the intracellular replenishment of iCa in neutrophils and compromised
function for at least 72 h after recovery from hypocalcemia (Martinez et al., 2014). Cows
that develop subclinical hypocalcemia have reduced neutrophil function based on less
phagocytosis and oxidative burst needed to kill pathogens (Martinez et al., 2012;
Martinez et al., 2014). Results from the current study showed that treatment with
Calcitriol blunted subclinical hypocalcemia in the first 3 DIM by increasing
concentrations of iCa and tCa in blood of dairy cows, which improved neutrophils
phagocytic and killing activities. Ionized Ca plays a key role in the activation of
neutrophils and receptor-mediated transient increases in cytosolic iCa are required for
the activation of phagocytes to clear invading pathogens. Increments in cytosolic iCa
80
induce activation of membrane-associated superoxide anion, NADPH oxidase,
degranulation, and activation of phospholipases needed during the inflammatory
response that contribute to the killing of phagocytized pathogens (Bréchard and
Tschirhart, 2008). It is suggested that the increments in iCa in the first few days
postpartum in Calcitriol cows likely favored innate immune response and improved
neutrophil function in early postpartum. In bovine monocytes, 1α,25-dihydroxyvitamin D3
increased expression of inducible nitric oxide synthase and regulates upon activation,
normal T-cell expressed and secreted, RANTES, improving innate immune system as
nitric oxide contributes to intracellular killing of bacteria, and RANTES, regulate
migration of T-helper cells (Nelson et al., 2010). Because Calcitriol increased
concentrations of 1α,25-dihydroxyvitamin D3 in plasma of dairy cows, one cannot
discount the possibility of a direct effect of vitamin D metabolites on immune cells
(Nelson et al., 2012).
Final Remarks
Administration of a single dose 300 μg of 1α,25-dihydroxyvitamin D3 increased
the concentrations of 1α,25-dihydroxyvitamin D3 in plasma of dairy cows for
approximately 3 d. Calcitriol was an efficacious and safe approach to increase
concentrations of iCa and tCa in blood of early postpartum dairy cows, which reduced
the prevalence of subclinical hypocalcemia and improved measures of innate immune
function. The dose selected did not seem to result in major depression in blood Ca after
the exogenous 1α,25-dihydroxyvitamin D3 cleared from blood, although concentrations
of tCa were slightly less for Calcitriol than control cows between 12 and 15 DIM.
Calcitriol increased urinary excretion of Ca and Mg, which reduced tMg concentration in
plasma. Neutrophil phagocytosis and oxidative burst were improved by treatment with
81
Calcitriol. Measures of production, such as intake of DM, milk production, and milk
composition, and concentrations of analytes in plasma associated with energy
metabolism did not differ between treatments, although concentration of BHBA tended
to increase in Calcitriol cows compared with controls. Collectively, results from the
current experiment indicate that treatment of dairy cows with a single injection of 300 μg
of 1α,25-dihydroxyvitamin D3 is an alternative to minimize subclinical hypocalcemia and
enhance innate immune function. Further experiments are needed to clarify the effects
of Calcitriol treatment on postpartum diseases and the carry over impacts on productive
and reproductive performance.
82
Table 3-1. Ingredient composition and nutrient content of pre- and postpartum diets in experiment 2
Diet1
Ingredients, % DM Prepartum Postpartum
Corn silage 50.0 21.6 Bermuda grass hay 17.0 --- Alfalfa hay --- 23.9 Brewer’s grains, wet 20.0 12.0 Corn, finely ground --- 19.7 Soybean hulls 3.4 --- Citrus pulp, dry --- 5.9 Whole cottonseed --- 4.8 Soybean meal, solvent extracted --- 5.7 Saturated free fatty acids2 --- 1.1 Prepartum mineral and vitamin3 4.0 --- Acidogenic salt product4 5.0 --- Mineral-vitamin protein mix5 --- 4.8 Mycotoxin binder6 0.6 0.5 Nutrient content, DM basis Net energy of lactation,7 Mcal/kg 1.53 1.67 Organic matter, % 93.0 ± 0.3 90.9 ± 0.5 Crude protein, % 13.6 ± 0.3 18.5 ± 0.8 Neutral detergent fiber, % 44.1 ± 2.3 29.8 ± 1.1 Forage neutral detergent fiber, % 30.3 ± 2.3 17.6 ± 0.8 Acid detergent fiber, % 26.5 ± 2.0 19.3 ± 0.5 Nonfibrous carbohydrates,8 % 34.1 ± 1.9 39.9 ± 0.5 Starch, % 20.0 ± 3.0 23.8 ± 0.8 Ether extract, % 4.5 ± 0.1 5.3 ± 0.3
83
Table 3-1. Continued.
Diet1
Ingredients, % DM Prepartum Postpartum
Ca, % 0.76 ± 0.02 0.87 ± 0.05 P, % 0.32 ± 0.01 0.35 ± 0.01 Mg, % 0.59 ± 0.03 0.45 ± 0.03 K, % 0.95 ± 0.11 1.62 ± 0.07 S, % 0.32 ± 0.02 0.23 ± 0.02 Na, % 0.05 ± 0.02 0.54 ± 0.01 Cl, % 0.68 ± 0.04 0.59 ± 0.06 DCAD,9 mEq/kg -123 ± 32 364 ± 41
1 Prepartum diet was fed from 255 days of gestation to calving and postpartum diet from calving to 42 DIM. 2 Energy Booster Mag; Milk Specialties, Eden Prairie, MN. 3 The prepartum mineral vitamin supplement contained (DM basis) 64.1% corn gluten feed, 8.2% calcium carbonate, 15.7% magnesium sulfate heptahydrate, 6.0% magnesium oxide, 2.3% sodium chloride, 0.42% Sel-Plex 2000 (Alltech Biotechnology, Nicholasville, KY), 0.27% Intellibond Vital 4 (Micronutrients, Indianapolis, IN), 0.002% ethylenediamine dihydriodide, 0.66% of a premix containing vitamins A, D and E, 0.37% Rumensin 90 (Elanco Animal Health, Greenfield, IN), and 2.0% ClariFly Larvicide (Central Life Sciences, Schaumburg, IL). Each kg contained 13.6% CP, 3.7% Ca, 0.7% P, 5.5% Mg, 0.9% K, 1.1% Na, 1.6% Cl, 2.6% S, 788 mg Zn, 180 mg Cu, 581 mg Mn, 9 mg Se, 4.4 mg Co, 16 mg I, 104,000 IU vitamin A, 30,000 IU vitamin D, 1,500 IU vitamin E, and 800 mg of monensin. 4 SoyChlor, West Central Soy, Landus Cooperative, Ames, IA. 5 The mineral-vitamin-protein premix contained (DM basis) 19.4% LysAAmet blood meal (Perdue Agribusiness, Salisbury, MD), 26.8% sodium sesquicarbonate, 14.4% DCAD Plus (Arm and Hammer Animal Nutrition, Trenton, NJ), 5.7% potassium chloride, 13.2% calcium carbonate, 4.0% dicalcium phosphate, 7.7% magnesium oxide, 6.6% sodium chloride, 0.22% Intellibond Vital 4 (Micronutrients, Indianapolis, IN), 0.39% Sel-Plex 2000 (Alltech Biotechnology, Nicholasville, KY), 0.0015% ethylenediamine dihydriodide, 0.32% of a premix containing vitamins A, D and E, 0.11% biotin 2%, 0.22% Rumensin 90 (Elanco Animal Health, Greenfield, IN), and 1.0% ClariFly Larvicide (Central Life Sciences, Schaumburg, IL). Each kg contains 17.2% CP, 6.2% Ca, 0.9% P, 4.5% Mg, 10.4% K, 11.5% Na, 7.2% Cl, 0.2% S, 605 mg Zn, 143 mg Cu, 490 mg Mn, 8 mg Se, 4.4 mg Co, 12 mg I, 160,000 IU vitamin A, 28,000 IU vitamin D, 1,500 IU vitamin E, and 460 mg of monensin. 6 NovaSilPlus, BASF, Florham Park, New Jersey. 7 Calculated using the NRC (2001) according to the chemical composition of the dietary ingredients and adjusted for 11 and 20 kg of DM intake for the pre- and postpartum periods, respectively. 8 Calculated as follows: NFC = DM – (ash + CP + ether extract + NDF – NDF insoluble CP). 9 Calculated as follows: DCAD = [(mEq of K) + (mEq Na)] – [(mEq of Cl) + (mEq of S)].
84
Table 3-2. Effect of treatment with Calcitriol on yield and composition of colostrum and milk from the second milking postpartum from Holstein cows in experiment 2
Treatment1
Item Control Calcitriol SEM P-value
Colostrum yield, kg 7.3 6.6 1.2 0.63 Content
Fat, % 4.37 4.21 0.33 0.72 True protein, % 10.73 10.04 0.45 0.28 Lactose, % 3.53 3.67 0.07 0.17 Net energy, Mcal/kg 1.14 1.10 0.04 0.43 Somatic cell score 6.11 6.09 0.28 0.97 Ca, g/L 1.91 1.99 0.09 0.51 Mg, g/L 0.40 0.42 0.01 0.37
Yield Fat, kg 0.39 0.31 0.07 0.47 True protein, kg 0.83 0.66 0.12 0.23 Lactose, kg 0.26 0.27 0.05 0.93 Ca, g 14.6 13.2 2.5 0.65 Mg, g 3.0 2.8 0.5 0.78
Milk yield from 2nd milking, kg 6.2 5.3 0.6 0.22 Content
Fat, % 3.85 3.78 0.31 0.86 True protein, % 9.00 8.60 0.44 0.51 Lactose, % 3.75 3.65 0.10 0.49 Net energy, Mcal/kg 1.01 0.98 0.04 0.54 Somatic cell score 5.51 5.46 0.32 0.88 Ca, g/L 1.77 1.76 0.06 0.81 Mg, g/L 0.32 0.31 0.01 0.72
Yield Fat, kg 0.26 0.22 0.03 0.49 True protein, kg 0.56 0.46 0.06 0.22 Lactose, kg 0.24 0.20 0.02 0.24 Ca, g 10.7 9.2 1.1 0.27 Mg, g 2.0 1.7 0.2 0.32
1 Cows received either an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) within 6 h after calving.
85
Table 3-3. Effect of treatment with calcitriol on performance of Holstein cows in experiment 2
Treatment1 P-value2
Item Control Calcitriol SEM TRT Day TRT x day
DM intake, kg/d 19.9 19.4 0.6 0.50 < 0.01 0.71 Milk, kg/d 34.0 34.1 1.0 0.94 < 0.01 0.08 3.5% FCM, kg/d 38.2 38.1 1.2 0.88 < 0.01 0.19 ECM, kg/d 37.0 36.6 1.0 0.55 < 0.01 0.21 3.5% FCM/DMI 2.07 2.15 0.08 0.40 < 0.01 0.73 Milk fat
% 4.35 4.27 1.0 0.55 < 0.01 0.21 Kg/d 1.44 1.44 0.11 0.62 < 0.01 0.59
Milk true protein % 3.11 3.05 0.04 0.33 < 0.01 0.99 Kg/d 1.04 1.02 0.03 0.53 < 0.01 0.10
Lactose % 4.66 4.63 0.03 0.35 < 0.01 0.53 Kg/d 1.60 1.59 0.05 0.87 < 0.01 0.12
Somatic cell score 3.62 3.53 0.44 0.89 < 0.01 0.74 Milk net energy
Mcal/kg 0.76 0.75 0.01 0.41 < 0.01 0.52 Mcal/d 25.6 25.3 0.8 0.70 < 0.01 0.12
Body weight Kg 632.5 648.4 12.8 0.39 < 0.01 0.91 Change, kg/d -0.90 -0.91 0.16 0.98 < 0.01 0.55
Body condition, 1 to 5 Mean 3.09 3.13 0.05 0.52 < 0.01 0.57 Change per week -0.07 -0.06 -0.01 0.35 < 0.01 0.83
Energy balance, Mcal/d -3.4 -4.4 0.9 0.41 < 0.01 0.85 1 Cows received either an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) within 6 h after calving. 2 TRT = effect of treatment; TRT x day = interaction between TRT and day.
86
Table 3-4. Effect of treatment with calcitriol on blood cell count of Holstein cows in experiment 2
Treatment1 P-value2
Item Control Calcitriol SEM TRT Day TRT x day
Hematocrit, % 29.7 31.3 0.5 0.04 < 0.01 0.72 Hemoglobin, g/dL 4.6 4.8 0.3 0.41 0.39 0.68 Erythrocytes, x 106/μL 5.9 6.0 0.1 0.53 0.32 0.89 Reticulocytes
x 1,000/μL 6.9 8.2 1.0 0.40 0.48 0.75 % 0.13 0.16 0.03 0.37 0.56 0.81
Platelets, x 1,000/μL 224 218 18 0.81 0.22 0.62 Leukocytes, x 1,000/μL 15.3 15.1 1.4 0.94 < 0.01 0.58 Neutrophils
x 1,000/μL 4.4 4.3 0.3 0.96 < 0.01 0.16 % 30.1 28.2 2.5 0.60 < 0.01 0.45
Lymphocytes x 1,000 /μL 8.2 8.3 1.0 0.95 0.93 0.52 % 56.0 57.3 2.8 0.74 < 0.01 0.34
Monocytes x 1,000/μL 1.7 1.7 0.2 0.89 < 0.01 0.97 % 12.3 12.1 0.8 0.88 < 0.01 0.92
Eosinophils and basophils
x 1,000/μL 0.14 0.22 0.03 0.07 < 0.01 0.95 % 1.0 1.3 0.2 0.35 < 0.01 0.33
1 Cows received either an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) within 6 h after calving. 2 TRT = effect of treatment; TRT x day = interaction between TRT and day.
87
Figure 3-1. Diagram representing analyzes of neutrophil function using flow cytometry. (A) Total cell population was selected to remove dead cells and cellular debris. (B) Neutrophil cells were gated based on granulometry and cell size. (C) Neutrophils were gated in order to achieve at least 90% of events negative for DHR and PI on tube 1, and to achieve at least 90% of events positive for DHR and negative for PI on tube 2 (D). (E-F) Represents the tube 3 and 4 were E. coli was added into the assay. The average value between tube 3 and 4 were used for analysis.
88
89
Figure 3-2. Concentrations of total Ca (A), total Mg (B), and total P (C) in plasma of cows after dosing 0, 200, or 300 µg of 1α,25-dihydroxyvitamin D3, and concentrations of calcitriol (D) total Ca (E) and total Mg (F) in plasma of dairy cows in after dosing 300 μg of 1α,25-dihydroxyvitamin D3 on day 0 and another 50 μg on day 6 in experiment 1. In panels A to C, values represent the LSM and SEM. In panels D to E, values represent the means and SD. In panel A, effects of dose (P = 0.65), day (P < 0.001), and interaction between dose and day (P = 0.02). In panel B, effects of dose (P < 0.01), day (P < 0.001), and interaction between dose and day (P = 0.78). In panel C, effects of dose (P = 0.82), day (P < 0.001), and interaction between dose and day (P = 0.19). Within a day, * denotes difference between treatments (P < 0.05).
90
91
Figure 3-3. Concentrations of calcitriol (A) and calcidiol (B) in plasma of dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P < 0.001), day (P < 0.001), and interaction between treatment and day (P < 0.001). Panel B, effects of treatment (P = 0.84). Within a day, * denotes difference between treatments (P < 0.05).
92
Figure 3-4. Concentrations of ionized Ca (iCa) in whole blood (A) and total Ca (B), total P (C), and total Mg (D) in plasma of dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P < 0.001), day (P < 0.001), and interaction between treatment and day (P = 0.06). Panel B, effects of treatment (P < 0.01), day (P < 0.001), and interaction between treatment and day (P < 0.001). Panel C, effects of treatment (P < 0.001), day (P < 0.001), and interaction between treatment and day (P < 0.001). Panel D, effects of treatment (P = 0.07), day (P < 0.001), and interaction between treatment and day (P < 0.001). Within a day, * denotes difference between treatments (P < 0.05).
0.9
1.0
1.1
1.2
1.3
1.4
0 0.5 1 2 3 5
Ion
ized
Ca,
mM
Day postpartum
Control
Calcitriol
*
*
*A
1.7
1.9
2.1
2.3
2.5
2.7
0 1 2 3 5 7 9 12 15
To
tal
Ca,
mM
Day postpartum
*
**
*
* *
B
*
1.0
1.4
1.8
2.2
2.6
0 1 2 3 5 7 9 12 15
To
tal
P, m
M
Day postpartum
*
* *
*
*
C
0.5
0.6
0.7
0.8
0.9
1.0
0 1 2 3 5 7 9 12 15
To
tal
Mg
, m
M
Day postpartum
*
*
*
D
93
Figure 3-5. Daily prevalence of subclinical hypocalcemia in dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Effects of treatment (P = 0.28), day (P < 0.01), and interaction between treatment and day (P < 0.01). Within a day, * denotes difference between treatments (P < 0.05).
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 1 2 3 5 7 9 12 15
Pre
val
ence
of
SC
H, %
Day postpartum
Control
Calcitriol*
*
*
*
*
94
Figure 3-6. Concentrations of PTH (A), serotonin (B), and CTX-1 (C) in plasma of dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P = 0.04), day (P < 0.10), and interaction between treatment and day (P = 0.17). Panel B, effects of treatment (P = 0.10), day (P = 0.33), and interaction between treatment and day (P = 0.82). Panel C, effects of treatment (P = 0.75), day (P = 0.61), and interaction between treatment and day (P = 0.61). Within a day, * denotes difference between treatments (P < 0.05).
200
300
400
500
600
0 1 3 5 7
PT
H,
pg
/mL
Day postpartum
Control
Calcitriol
*
*
A
500
1,000
1,500
2,000
2,500
0 1 3 5 7S
ero
ton
in, n
g/m
L
Day postpartum
B
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 1 3 5 7
CT
X-1
, n
g/m
L
Day postpartum
C
95
Figure 3-7. Concentrations of BHBA (A), NEFA (B), glucose (C), and urea N (D) in plasma of dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P = 0.08), day (P < 0.001), and interaction between treatment and day (P = 0.99). Panel B, effects of treatment (P = 0.34), day (P < 0.001), and interaction between treatment and day (P = 0.61). Panel C, effects of treatment (P = 0.29), day (P < 0.001), and interaction between treatment and day (P = 0.44). Panel D, effects of treatment (P = 0.98), day (P = 0.39), and interaction between treatment and day (P = 0.41). Within a day, * denotes difference between treatments (P < 0.05).
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 1 2 3 5 7 9 12 15
BH
BA
, m
M
Day postpartum
Control
Calcitriol
*A
350
450
550
650
750
850
0 1 2 3 5 7 9 12 15
NE
FA
, μM
Day postpartum
*
B
40
50
60
70
80
90
100
0 1 2 3 5 7 9 12 15
Glu
cose
, m
g/d
L
Day postpartum
C
*
7
8
9
10
11
0 1 2 3 5 7 9 12 15
Ure
a N
, m
g/d
L
Day postpartum
D
96
Figure 3-8. Daily excretion of Ca (A) and Mg (B) in urine of dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P = 0.01), day (P = 0.06), and interaction between treatment and day (P = 0.01). Panel B, effects of treatment (P = 0.02), day (P < 0.001), and interaction between treatment and day (P < 0.001). Within a day, * denotes difference between treatments (P < 0.05), whereas t denotes a tendency to differ (0.05 < P < 0.10).
0.0
1.0
2.0
3.0
4.0
5.0
1 2 3 5 7
Uri
nar
y e
xcr
etio
n o
f C
a, g
/d
Day postpartum
Control
Calcitriol
*
t
**
*
A
2.0
3.0
4.0
5.0
6.0
7.0
1 2 3 5 7
Uri
nar
y e
xcr
etio
n o
f M
g,
g/d
Day postpartum
**B
97
Figure 3-9. Percentage of neutrophils positive for phagocytosis (A) and oxidative burst percentage (B), and mean fluorescence intensity (MFI) for phagocytosis (C), and for oxidative burst (D) in neutrophils from dairy cows receiving an injection of placebo (control) or 300 µg of 1α,25-dihydroxyvitamin D3 (Calcitriol) in experiment 2. Panel A, effects of treatment (P = 0.17), day (P = 0.17), and interaction between treatment and day (P = 0.60). Panel B, effects of treatment (P = 0.03), day (P = 0.06), and interaction between treatment and day (P = 0.24). Panel C, effects of treatment (P = 0.03), day (P = 0.01), and interaction between treatment and day (P = 0.41). Panel D, effects of treatment (P = 0.09), day (P = 0.02), and interaction between treatment and day (P = 0.21). Within a day, * denotes difference between treatments (P < 0.05), whereas t denotes a tendency to differ (0.05 < P < 0.10).
98
CHAPTER 4 ASSOCIATION AMONG GESTATION LENGTH AND HEALTH, PRODUCTION AND
REPRODUCTION IN HOLSTEIN COWS AND IMPLICATIONS TO THEIR OFFSPRINGS
Summary
Objectives were to evaluate the association among gestation length (GL) and
health, productive and reproductive performance of Holstein cows, and survival and
reproductive performance of their offsprings. Cows with GL shorter or longer than 3 SD
from the population mean were eliminated from the data set resulting in 149 cows
excluded from the original 8,244 cows evaluated. A total of 8,095 Holstein cows and
3,623 female offsprings born alive on two farms using only AI were evaluated. Gestation
length averaged 276 ± 6 days in the 8,095 dams, and it was categorized as shortened
(SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d),
average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), and
extended (EGL; at least 1 SD above the population mean; mean = 285 d, range 283 to
296 d). Responses evaluated in dams included incidences of dystocia, stillbirth, retained
fetal membranes (RFM), metritis, mastitis, and other diseases within 90 DIM. Pregnancy
per AI (P/AI) at first AI and by 300 DIM, and time to pregnancy were evaluated. Milk
yield and removal from the herd by culling or death were recorded for the first 300 DIM.
Responses evaluated in female offsprings included pre- and postweaning mortality,
removal from the herd, and reproductive performance. Gestation length affected the
incidences of stillbirth, RFM, metritis, lameness, but not dystocia and mastitis in dams.
Morbidity and rate of morbidity within 90 DIM were greater for SGL and for SGL and
EGL, respectively. Rate of removal of dams from the herd was faster (adjusted hazard
ratio [AHR] = 1.38; 95% CI = 1.17 to 1.62) for SGL than AGL. Milk production was
99
greatest in AGL cows, but responses depended on parity. For primiparous cows, milk
production was less in SGL and EGL (AGL = 35.4, SGL = 34.6, EGL = 33.0 ± 0.4 kg/d),
whereas for multiparous cows, production was reduced in SGL, and increased in EGL
(AGL = 41.6, SGL = 38.6, EGL = 42.4 ± 0.3 kg/d). Pregnancy at first AI did not differ
among groups. Pregnancy rate was lesser (AHR = 0.91; 95% CI = 0.84 to 0.98) for EGL
than AGL, but it did not differ between AGL and SGL. Heifers from dams with GL that
deviated from AGL had increased mortality postweaning (AGL = 3.2 vs. SGL = 6.3 vs.
EGL = 5.1%). The rate of removal from the herd was greater for SGL (AHR = 1.84; 95%
CI = 1.31 to 2.60) and EGL (AHR = 1.97; 95% CI = 1.43 to 2.70) than AGL heifers.
Pregnancy at first AI and by 500 days of age of heifers did not differ between AGL and
the abnormal GL categories. Cows with GL within 1 SD of the population mean, ranging
from 270 to 282 d, had improved health, production, and reproduction. Heifers from
cows with GL within 1 SD of the population mean had improved health and
reproduction. Gestation length affects performance of both the dam and her offspring.
Background
Gestation length (GL) is the period between conception and parturition, and
defining the expected GL is of importance from a management standpoint as the
anticipated dates are used for dry off, movement between groups, health and nutritional
decisions. In some countries, GL is genetically manipulated in breeding programs to
favor reducing variability in calving patterns that facilitates seasonal production systems
such as those used in New Zealand. Data from the Livestock Improvement Corporation
in New Zealand show that some sires can reduce GL by as much as 20 d, which favors
rebreeding of cows in seasonally breeding production systems (LIC, 2016). In the US
and many other countries, GL is not part of the breeding program, but it is considered
100
for management decisions. In Holstein heifers and cows, GL averaged (± SD) 277.8 ±
5.5 and 279.4 ± 5.7, respectively (Norman et al., 2009).
Several factors have been identified to influence GL such as genetics, gender of
the calf, singleton or twin pregnancy, age of dam, and season of the year. Dairy cows
carrying male calves had a GL 1.1 days longer than cows carrying female calves (Silva
et al., 1992). Gestation with twins was shorter when compared with gestation with
singletons (Echternkamp and Gregory, 1999). Days of gestation increased linearly as
the lactation number increased (McClintock et al., 2003). Cows calving during the warm
season had 2.8 days shorter GL than cows calving during the cool season (DuBois and
Williams, 1979). Genetics influence GL and some sires have predicted transmitting
ability (PTA) to either shorten or extend GL (Norman et al., 2009). Nogalski and
Piwczyński (2012) found a linear relationship between GL and calf BW; longer the GL,
greater BW of the calf. Also, lack of proper alleviation of heat stress caused
hyperthermia in pregnant cows during the last 6 weeks of gestation, which reduced GL
by 3 to 4 days compared with cows provided evaporative cooling (Tao and Dahl, 2013).
This likely explains the link between season of the year and length of gestation (Norman
et al., 2009).
Several studies found large genetic variation in GL, and heritability has been
estimated for the possibility of selection through genetics (DeFries et al., 1959; Jamrozik
et al., 2005; Olson et al., 2009). Service sire heritability for GL is greater than the cow
sire (Hansen et al., 2004), 33 to 36% for service sire and 7 to 12% for cow sire (Norman
et al., 2009). Therefore, the use of semen with different PTA for GL may be an option to
alter GL (LIC, 2016). According to Norman et al. (2009), services sires can be used to
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either increase or decrease GL of cows; however, there is not enough information to
suggest that either shortening or extending GL would provide advantages to the dam
and the offspring in the Holstein breed under intensive production systems.
Implementation of selection for GL has to consider possible effects on calving traits,
survival of the calf and performance of the dam.
Norman et al. (2011) showed that intermediate GL, between 274 to 281 days
optimized lifetime productivity, calving ease, stillbirth rates, and the interval from calving
to first service in dams. Additionally, Jenkins et al. (2016) found that cows within the 5%
shortest GL of the study population and within the 5% longest GL of the study
population had reduced yields of milk, fat and protein, and reduced survival of
offsprings. Cows considered having abnormal GL, either shorter than 275 days or
longer than 281 d, had impaired health performance with increased incidence of
retained fetal membranes (RFM; Nogalski and Piwczyński, 2012). Furthermore, longer
gestations were associated with increased incidence of metritis (Markusfeld, 1984).
Although several factors have been identified to influence GL in dairy cows, a
comprehensive evaluation of the effect of GL on the dam’s performance has not been
done. Furthermore, it is possible that the GL of the dam can affect the performance of
the offspring, and this has not been evaluated. Better understanding of the potential
implications of GL with health, reproduction and productive performance of dairy cows,
and the long-term impacts on health, survival, and reproduction of the offsprings might
provide information that can be used to manage cows and calves selectively. Because
GL can be manipulated genetically, the identification of an optimum GL might become
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target for future use of sires that favor that particular duration of gestation to improve
dam’s productivity and have long-term benefits to the offspring.
We hypothesized that an abnormal GL, either shortened or extended, is
associated with impairments in health, productive and reproductive performance in
Holstein cows and their offspring. Objectives of the present study were to evaluate the
association between abnormal GL and health, productive and reproductive performance
in dairy cows, and survival and reproductive performance of their female offspring.
Materials and Methods
Farms and Cows
This study followed a retrospective observational design. Data from 8,244
Holstein cows (3,335 primiparous and 4,909 multiparous) from two commercial dairies
(Farm 1, n = 1,886; Farm 2, n = 6,358) located in Central California that calved between
January and December of 2013 were collected for the study. A cow was included only
once in the study and completed the study either at 300 DIM or when she was sold or
died.
Farm 1 was a dry lot dairy that milked approximately 1,550 cows four times daily
in the first 3 to 4 weeks postpartum and then twice daily thereafter. Production was
measured once a month, in two consecutive milkings, by the local DHIA laboratory in
Hanford, CA. The rolling herd average for 2013 was 12,050 kg of 3.5% FCM.
Primiparous and multiparous cows were grouped separately throughout the entire dry
and lactating periods. Dry lots had shades in the central area of the corrals and over the
feed bunk. Approximately 6 m2 of shaded area was available for lying. Shades were
located north-south and the dried manure was added twice weekly as bedding material
under the shades. The bedding under the shaded area was raked once daily. Soaker
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lines with nozzles were placed above the stanchions in the feed lane, and the system
was activated once ambient temperature reached 22oC and nozzles sprayed water for
approximately 1 min each 6 min. Lactating cows were fed the same TMR throughout the
lactation formulated to meet or exceed the nutrient requirements of a 680-kg cow
consuming 27 kg of DM and producing 45 kg of milk with 3.70% fat and 3.30% true
protein (NRC, 2001). Cows were fed twice daily and amounts offered were calculated
daily for an estimated 2 to 3% orts. Cows were dried once weekly, at 232 ± 3 days of
gestation unless production was perceived to be very low by the herd manager and
dried earlier. Dry cows were moved into a dry cow pen and fed a ration formulated for
limited weight gain when the average DMI was 13 kg/d. Prepartum nulliparous and
parous cows were moved into dry lots at 255 ± 3 days of gestation and fed similar diets
once daily, except that multiparous received acidogenic salts to minimize the risk of
hypocalcemia postpartum. Cows calved either in the dry lots or in group ± pens
adjacent to the prepartum pens.
Farm 2 milked approximately 5,400 cows thrice daily and the rolling herd average
for 2013 was 13,100 kg of 3.5% FCM. Milk yield was recoded daily for individual cows
using electronic milk meters (Perfection 3000, Boumatic, Madison, WI), and daily values
were averaged into monthly means for statistical analysis. Cows were housed in sand-
bedded free stalls in groups of 280 to 370 cows each. Dry cows were housed in free-
stalls, but cows and heifers in the last 3 weeks of gestation were moved to dry lots and
they calved either in the dry lots or in group maternity pens. Primiparous and
multiparous cows were grouped separately throughout the entire dry and lactating
periods. Barns were equipped with fans in the central area above the stalls. Soaker
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lines with nozzles were located above the stanchions in the feed lane. The system was
activated once ambient temperature reached 22oC, and nozzles sprayed water for
approximately 1 min each 6 min. All rations were fed as TMR. Lactating cows were fed
an early lactation diet for the first 3 to 4 weeks postpartum, then moved into a diet
labeled high cow ration fed to cows between 4 weeks and approximately 200 to 250
DIM, and then a diet labeled low cow ration fed to cows after 250 DIM. Change of diets
from the high cow ration groups to the low cow ration groups was based on milk yield of
the cow and space needs in the barns at the discretion of the herd manager. Dietary
ingredients were the same in all rations, but proportions of forage, concentrates and
protein supplements were altered to accommodate the nutrient needs of each group of
cows according to production level and group DMI. Diets were formulated for expected
DMI of 20, 28, and 25 kg/d for the early lactation, high group, and low group rations.
Cows were fed once daily and amounts offered were calculated daily for an estimated 2
to 3% orts. Cows were dried once weekly, at 232 ± 3 days of gestation unless
production was perceived to be very low by the herd manager and dried earlier. Dry
cows were moved into a dry cow pen and fed a ration formulated for limited weight gain
when the average DMI was 13 kg/d. Prepartum nulliparous and parous cows were
moved into dry lots at 255 ± 3 days of gestation and fed similar diets once daily, except
that multiparous received acidogenic salts to minimize the risk of hypocalcemia
postpartum. In both farms cows received treatments with bovine somatotropin (Posilac,
sometribove zinc suspension for injection, Elanco Animal Health, Greenfield, IN) every
14 days starting at 70 ± 3 DIM and treatments stopped 14 days before the expected dry
off date.
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Management and Feeding of Calves and Heifers
In both farms, newborn female calves were identified and received 4 L of
colostrum by esophageal tube feeding within 4 h of birth and another 2 L of colostrum in
the next feeding. Female calves in both farms were transported twice daily to the same
calf raising facility in Farm 2 where they remained until 63 days of age. Calves were
housed in individual calf hutches and had access to water and starter grain from birth
until they left the hutches at 63 days of age.
Calves were fed pasteurized hospital milk for the first 3 to 4 weeks of life,
depending on availability, after which they were fed milk replacer at 13% dry matter.
Calves were fed three times daily with 2 L of milk per feeding for the first 4 weeks of life,
followed by twice daily until the end of week 7, then once daily on the eighth week,
when they were weaned from milk. Calves remained in the individual hutches until
approximately 63 days of age, when they returned to the respective farm of origin and
were housed in group pens according to age. In both farms, heifers were fed two diets,
one before reaching the breeding group and another during breeding and pregnancy.
Diets were formulated according to NRC (2001) to achieve daily weight gain of 850 to
950 g/d.
Data Collection
Dam data
Data regarding health, production and reproduction were collected from the dairy
management software Dairy Comp 305 (Valley Agricultural Software, Tulare, CA).
Calving information such as dystocia, stillbirth, twin, gender of the calf, and season of
calving were recorded. Hot season was defined as the period between June 1 and
October 15, whereas the rest of the year was classified as cool season. This
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classification was based on elevated ambient temperature observed in the location of
the two farms in 2013 based on the local weather station in Hanford, CA. Cows were
classified as having RFM if the placenta was not expelled within 24 h of calving. Cows
were evaluated daily for diagnosis of diseases for the first 3 to 4 weeks postpartum.
Diseases were recorded for the first 90 DIM and included metritis (watery, reddish or
brownish vaginal discharge with foul smell), pneumonia (fever, increased respiratory
frequency and sounds at auscultation), lameness based on routine therapeutic hoof
trimming, displaced abomasum (auscultation followed by confirmation during corrective
surgery), and mastitis (abnormal milk and/or inflammation of the gland). Removal from
the herd, either by culling or death was collected up to 300 DIM. Milk yield was recorded
once a month during two consecutive milkings by the local DHIA laboratory in Farm 1
and daily in Farm 2.
Both farms followed the same reproductive management protocols. Cows had
the estrous cycle synchronized with 2 injections of 25 mg of PGF2α (Lutalyse Sterile
Solution, 5 mg/mL dinoprost as tromethamine salt, Zoetis, Florham Park, NJ)
administered 14 days apart at 37 and 51 ± 3 DIM. Cows were observed daily, in the
morning for signs of estrus based on removal of tail chalk and the voluntary waiting
period was 51 DIM. Cows not inseminated by 62 ± 3 DIM were enrolled in the Ovsynch-
56 timed AI protocol (Brusveen et al., 2008) such that every cow in both farms received
the first postpartum AI by 75 DIM. Cows that returned to estrus after any AI were
inseminated in the same morning and considered to be nonpregnant to the previous
breeding. Pregnancy was diagnosed by ultrasound at 32 ± 3 days after AI and pregnant
cows were reconfirmed 4 weeks later, at 60 days of gestation. Visualization of an
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amniotic vesicle with an embryo was the criterion used to determine pregnancy.
Nonpregnant cows were re-enrolled in the Ovsynch-56 protocol for reinsemination.
Pregnancy loss between 32 and 60 days of gestation was evaluated for the first
postpartum AI. For the purpose of this study, a cow was considered pregnant when a
positive diagnosis was determined on day 60 after insemination. Reproductive data
were collected until a cow was confirmed pregnant, sold, died, or up to 300 DIM,
whichever occurred first. Nonpregnant cows by 300 DIM were censored for analyses of
time to pregnancy.
Heifer data
Live born females moved to the individual hutches were followed for the first 300
days of life for survival. Individual calf disease incidence was not recorded properly, so
information was not collected for analyses. Survival during the preweaning and the first
300 days of life was analyzed.
Heifers in both farms were moved to the breeding group based on age and hip
height. Starting at 12 months of age, heifers with hip height greater than 130 cm were
moved to the breeding groups, otherwise all heifers were moved at 13 months of age.
Weekly cohorts of heifers were moved to dry lots equipped with stanchions with self-
lock ups for daily activities. Heifers had tailheads painted daily with chalk (All Weather
Paintstik, LA-CO Industries Inc., Elk Grove Village, IL), and removal of the chalk was
interpreted as an indication of estrus. Heifers were evaluated daily, in the morning
immediately after feeding, and any heifer showing signs of estrus such as accepting
mounts or with rubbed tailheads were inseminated on the same morning. Heifers that
did not show signs of estrus within 14 days of moving to the breeding pen had ovaries
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scanned by transrectal ultrasonography, and those with visible corpus luteum received
a dose of 25 mg of PGF2α that was repeated every 14-d until insemination.
Pregnancy was evaluated once every two weeks when heifers were 35 to 48
days after AI. Pregnancy diagnosis was performed by transrectal ultrasonography as
described previously. Heifers that returned to estrus after any AI and before a
pregnancy diagnosis were considered nonpregnant. Nonpregnant heifers on the day of
pregnancy diagnosis received a dose of PGF2α to synchronize the return to estrus.
Pregnancy per AI was calculated by dividing the number of heifers diagnosed pregnant
by the number of heifers that received AI. Reproductive performance was evaluated
until 500 days of age. For interval to pregnancy, nonpregnant heifers were censored
either when last observed in the farm because of being sold or dead, or at 500 days of
age.
Gestation Length Category
The GL was determined as the interval between the day or AI that resulted in
pregnancy and the day of calving. Descriptive statistics were used to characterize the
distribution of GL resulting in a mean and SD of 276 ± 7 days in the original population
of 8,244 cows. Cows with GL shorter or longer than 3 SD from the mean were removed
and considered outliers. This process resulted in 149 of the initial 8,244 cows enrolled
removed from the study. Therefore, 8,095 cows remained for data analyses and the
range of GL was 256 to 296.
Subsequent descriptive statistics were performed for GL from the 8,095 cows
remaining in the study, which resulted in a mean and SD of 276 ± 6 d. Three categories
of GL were created, those with average, shortened, or extended GL. Cows with GL
within ± 1 SD of the study population mean were classified as average GL (AGL, n =
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6,181, mean = 276, range 270 to 282 d). Cows with GL shorter than 1 SD from the
study population mean were classified as shortened GL (SGL, n = 762, mean = 266,
range 256 to 269 d). Cows with GL longer than 1 SD from the study population mean
were classified as extended GL (EGL, n = 1,152, mean = 285, range 283 to 296 d).
Therefore, AGL, SGL, and EGL represented 76.4, 9.4, and 14.2% of the study
population, respectively.
Statistical Analyses
Dam data
Continuous data such as milk yield or DIM at first AI were analyzed by ANOVA
using the MIXED procedure of SAS version 9.4 (SAS/STAT, SAS Institute Inc., Cary,
NC). Binary data such as incidence of diseases, morbidity, pregnancy per AI, and
pregnancy loss were analyzed by logistic regression using the GLIMMIX procedure of
SAS. Time to an event such as days postpartum to leaving the herd or pregnancy was
analyzed by the Cox’s proportional hazard regression model with the PHREG procedure
of SAS. All models included the fixed effects of gestation length category (AGL vs. SGL
vs. EGL), parity (primiparous vs. multiparous), season of calving (cool vs. hot), farm (1
vs. 2), gender of calf (male, female, twin). Nonsignificant (P > 0.10) interactions were
dropped from the final models. The random effect of maternal grandsire was included in
the model for continuous and binary data analyses to adjust for differences in genetic
merit. Pairwise comparisons among groups were performed using Tukey’s test.
Heifer data
Continuous data such as age at first AI were analyzed by ANOVA using the
MIXED procedure of SAS. Binary data such as incidence of death or pregnancy per AI
were analyzed by logistic regression using the GLIMMIX procedure of SAS. Time to an
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event such as days to leaving the herd or pregnancy was analyzed by the Cox’s
proportional hazard regression model with the PHREG procedure of SAS. All models
included the fixed effects of gestation length category (AGL vs. SGL vs. EGL), season
of calving (cool vs. hot), farm (1 vs. 2), and interactions between GL category and
season, and GL category and farm. Nonsignificant (P > 0.10) interactions were dropped
from the final models. The random effect of maternal sire was included in the model for
continuous and binary data analyses to adjust for differences in sire genetic merit.
Pairwise comparisons among groups were performed using Tukey’s test.
Significance was declared when P ≤ 0.05 and a tendency to differ when 0.05 < P
≤ 0.10.
Results
Factors associated with GL were parity of the dam, gender of the calf, singleton
or twin gestation, and season of calving. Primiparous cows had shorter (P < 0.01) GL
than multiparous cows (primiparous = 274.1 ± 0.2 vs. multiparous = 276.8 ± 0.2 d).
Cows carrying female calves had shorter (P < 0.01) GL than cows carrying male calves,
respectively 275.4 ± 0.1 and 276.7 ± 0.1 d. Moreover, in the case of cows carrying
twins, those carrying female-female (FF) or female-male (FM) had shorter (P < 0.05) GL
that cows carrying male-male (MM; FF = 274.3 ± 0.7, FM = 273.6 ± 0.5, and MM =
277.3 ± 0.6 d). Cows calving during the hot season had shorter (P < 0.01) GL than cows
calving during the cool season (hot = 274.7 ± 0.2 vs. cool = 276.2 ± 0.2 d).
Health Performance of Dams
Incidence of disease during the early postpartum was affected by the different GL
categories. The incidence of dystocia was greater (P = 0.04) in cows having SGL than
those with AGL, but no difference was observed when AGL and EGL was compared
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(Table 4-1). The incidence of stillbirth was greater (P < 0.01) in cows having SGL than
those with AGL or EGL; however, no difference was found when AGL and EGL was
compared. The incidence of RFM was 5-fold greater (P < 0.01) in SGL compared with
AGL or EGL, and EGL had a tendency (P = 0.08) to have greater incidence of RFM
compared with AGL. Likewise, the incidence of metritis almost doubled (P < 0.01) in
SGL compared with AGL or EGL, but no difference was observed between AGL and
EGL. Incidence of mastitis was not associated with GL, but cows with EGL had a
tendency (P = 0.08) for increased incidence of lameness compared with SGL. Because
of the differences in incidence of individual diseases, morbidity by 90 DIM was greatest
(P < 0.01) for SGL, followed by EGL and then AGL. Figure 4-1A depicts the survival
curves for days postpartum at the first diagnosis of disease up to 90 DIM according to
GL category. It is clear that most cows were first diagnosed with disease in the first 2 to
3 weeks postpartum. Cows with SGL or EGL had reduced (P < 0.05) days to diagnosis
of the first disease compared with cows with AGL (Table 4-2). The rate of morbidity was
56% faster (P < 0.01) for SGL and 11% faster (P < 0.05) for EGL compared with AGL.
In addition, the rate of morbidity was greater (P < 0.01) for primiparous than multiparous
cows, for cows calving during the hot than the cool season, and for cows calving twins
than those calving singleton either male or female.
Culling in the first 300 DIM increased (P < 0.01) in SGL compared with AGL or
EGL (Table 4-1), whereas death by 300 DIM increased (P < 0.01) in EGL compared
with AGL, but no difference was found between SGL and EGL. Figure 4-1B depicts the
survival curves for time to removal from herd either by culling or death by 300 DIM
according to GL category. Cows with SGL were removed from the herd at a faster (P <
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0.01) rate than AGL (Table 4-3). There was no difference between EGL and AGL. In
addition, multiparous cows were removed from the herd at a rate almost 2.6-fold faster
(P < 0.01) than primiparous cows; cows calving in the cool season were removed at a
faster (P < 0.01) rate than those calving in the hot season; and those calving twins were
removed at a faster (P < 0.01) rate than those calving singleton male or female calf.
Production Performance of Dams
Cows with SGL or EGL produced less (P < 0.01) milk compared with cows with
AGL (Table 4-4). Nevertheless, important interactions between GL category and parity
and GL category and farm were observed. Within primiparous cows, those with EGL
produced 2.4 fewer kg of milk (P < 0.01) than primiparous with AGL, whereas those with
SGL produced 0.8 fewer kg of milk (P < 0.01) than primiparous with AGL. On the other
hand, within multiparous cows, production of milk was less (P < 0.01) in cows with SGL
than those with AGL, but cows with EGL produced 0.8 more kg of milk (P < 0.01) than
multiparous with AGL. In Farm 1, SGL produced less (P < 0.01) milk than AGL, but no
difference was observed between AGL and EGL, whereas in Farm 2, both SGL and
EGL produced less (P < 0.01) milk than cows with AGL. Cows calving in the hot season
produced 0.5 kg/d more milk than those calving in the cool season. Gender of calf did
not affect daily milk yield in the first 300 DIM.
Reproductive Performance of Dams
The proportion of cows receiving at least one insemination was less (P < 0.01) in
cows with SGL compared with cows with AGL, whereas there was no difference
between AGL and EGL (Table 4-5). Despite the difference in percentage of cows
inseminated, the DIM at first AI did not differ with GL category. Pregnancy at first AI was
not affected by GL category either on day 32 or 60 after insemination. Gestation length
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was not associated with pregnancy loss in the first 60 days of gestation following the
first AI. Nevertheless, the rate of pregnancy was less (P = 0.01) for EGL than AGL,
which resulted in an extension of the medial days to pregnancy by 11 (Table 4-6; Figure
4-2). No differences were observed for rate of pregnancy between AGL and SGL,
although a larger (P < 0.01) proportion of AGL cows became pregnant by 300 DIM than
SGL (Table 4-5). Primiparous cows became pregnant faster (P < 0.01) than multiparous
cows, which resulted in 10 fewer median days to pregnancy (Table 4-6). Cows calving
in the hot season became pregnant 4 days earlier (P < 0.01) than cows calving in the
cool season, and those calving female calves tended (P = 0.08) to become pregnant
faster than cows calving twins.
Health Performance of Female Offsprings
A total of 3,623 heifers were born alive and moved to individual hutches, 2,880
from dams with AGL, 337 born from dams with SGL, and 406 born from dams with EGL.
Preweaning mortality was not associated with GL, however heifers born from
dams with SGL or EGL had greater (P < 0.01) postweaning mortality compared with
heifers born from cows with AGL (Table 4-7). As a result, a larger (P < 0.01) proportion
of SGL and EGL heifers died by 300 days of age compared with heifers born from cows
with AGL. Culling by 300 days of age tended (P = 0.06) to be greater for EGL compared
with AGL heifers. The differences in mortality and culling resulted in greater (P < 0.01)
proportions of EGL heifers leaving the herd by 300 days of age than AGL heifers (Table
4-7). In fact, the daily rate of removal of heifers from the herd was 84 and 97% greater
(P < 0.01) for SGL and EGL, respectively, than AGL (Table 4-8; Figure 4-3). Removal
from the herd was also affected by season of birth and heifers born during the hot
season left the herd at a faster rate than those born in the cool season.
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Reproductive Performance of Female Offsprings
The proportion of heifers receiving at least one insemination was greater (P <
0.01) for AGL compared with SGL or EGL (Table 4-7). Interestingly, heifers born from
dams with EGL were 6 and 7 days younger at first AI (P < 0.01) than heifers born from
dams with AGL and SGL, respectively. Pregnancy diagnosed on day 60 after the first AI
tended to be associated (P = 0.09) with GL category, and it was greater (P < 0.05) for
heifers born from dams with SGL than EGL. No statistical difference was observed for
pregnancy at first AI between AGL and SGL heifers. The advantages observed at first
AI remained by 500 days of age. Proportions of heifers that became pregnant by 500
days of age did not differ among gestation length categories (Table 4-7).
Discussion
Parturition is triggered by the increase in fetal glucocorticoid concentrations
because of maturation and activation of the fetal hypothalamic-pituitary-adrenal (HPA)
axis (Matthews and Challis, 1996). Cows carrying male singleton had longer GL than
cows carrying females. In addition, twin gestation of two males was longer than twins
with at least one female calf. It seems that, final development of the HPA axis may take
longer in males, thereby resulting in longer GL. Hypoxemia is a potent stimulus to
increased HPA axis activity in the fetus (Matthews and Challis, 1996), and results in
parturition. Primiparous cows are smaller than multiparous cows with reduced space for
the fetus in the abdominal cavity at the end of gestation, which might create a potential
stress to the fetus and alter blood flow and oxygen availability, thereby stimulating the
HPA axis activity. This might explain the shorter GL typically observed in primiparous
compared with multiparous cows. Cows calving during the hot season had 1.5 days
shorter GL than cows calving during the cool season. It is well described that heat
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stress can induce earlier parturition, and providing evaporative cooling to late gestation
cows under heat stress reverses this effect and extends gestation another 3 to 4 days
compared with cows not receiving evaporative cooling (Tao and Dahl, 2013). It is
possible that heat stress promotes HPA axis maturation and cortisol release by the
fetus leading to parturition sooner than cows calving during the cool season, which
explains the link between season of the year and the length of gestation observed in the
current manuscript and others (Norman et al., 2009).
Holstein cows with GL within 1 SD of the mean for the study population, with a
range of 270 to 282 days had reduced incidence of dystocia and stillbirths and the
lowest incidences of RFM, metritis, and lameness, which resulted in reduced morbidity
compared with cows with GL that deviated, shorter than 270 or longer than 282 d. The
rates of morbidity and removal from the herd were lowest for cows with AGL compared
with those with SGL or EGL. The benefits observed for cow health and survival when
GL was within 1 SD from the mean were extended to production and reproduction.
Furthermore, female offsprings born from dams with GL between 270 and 282 days had
improved postweaning survival, and proportion of heifers inseminated by 500 days of
age compared with heifers from dams with SGL or EGL.
Nogalski and Piwczyński (2012) and Hansen et al. (2004) established an
association between shortened and extended GL with increased incidence of stillbirths,
and cows with GL between 275 and 280 days had the greatest proportion of unassisted
calving and reduced incidence of stillbirth calves compared with cows with GL shorter
than 275 and longer than 280 d. Norman et al. (2011) reported increased incidence of
stillbirths in cows with GL shorter than 274 d, beyond which the incidence of stillbirths
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were no longer affected, even when cows calved after 281 days of gestation.
Markusfeld (1984) concluded that shortened GL increased the risk of RFM, whereas
extended GL increased that of metritis. Results from the current study are mostly in
agreement with those from others and reinforced the concept that Holstein cows with
SGL, in this case, with gestation shorter than 270 d, had increased incidence of
dystocia, stillbirths, RFM, and metritis. In contrast, cows with EGL did not have
increased incidence of dystocia, stillbirths or metritis, but had a tendency to have more
RFM. Stillbirths and RFM are important risk factors for metritis and it was expected that
cows with SGL, which had a 2.5-fold increase in stillbirths and 5-fold increase in RFM
would have increased incidence of uterine diseases, a 2-fold increase.
The control of timing of calving is complex and involves fetal-maternal
interactions (Matthews et al., 1995), but it is clear that timely onset of calving is critical
for perinatal outcomes in cattle. It is unknown what exactly made cows with SGL calve
earlier, but it has been shown that fetal stress such as hypoxia and hypoglycemia may
result in premature parturition because of accelerated maturation of the hypothalamic-
pituitary-adrenal axis endocrine pathway (McMillen et al., 1995). Upon parturition, the
dam’s surveilling immune system immediately builds an immune response against the
remnants of fetal tissues and, in dairy cattle, RFM is thought to arise from failure of this
mechanism of rejection of fetal tissues by the dam’s immune system. As gestation
progresses, major histocompatibility complex class I antigens are expressed on the
placenta, which is thought to facilitate recognition and rejection of fetal placental tissues
by the maternal immune system (Davies et al., 2004). Early parturition usually results in
immature development of the lungs in the offspring, which can result in inadequate
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breathing, acidosis and hypoxia, which likely explain the increased incidence of
stillbirths. The lesser viable calves from dams with SGL that results in increased risk of
RFM suggest that the maternal immune system is less capable of rejecting the fetal
components of the placenta. Detachment of the placenta requires collagenolysis and
proteolysis, which are carried out by maternal immune cells (Gross et al., 1985). Likely,
cows with SGL lack proper signals to activate the innate immune system required to
eliminate the placenta, and the combination of lesser immune response and presence
of remnant dead tissues likely explain the increased incidence of metritis.
It was clear that deviations from the AGL resulted in marked increases in the rate
of morbidity, and most diseases occurred in the first 3 weeks of lactation. In fact, the
high incidence of RFM and metritis in SGL cows made almost half of them to be
diagnosed with clinical disease by 8 DIM. In dairy cattle, approximately 45% of the
postpartum cows are diagnosed with at least one clinical disease in the first 60 days of
lactation, and 75% of the first diagnoses of diseases in a lactation occur in the first 3
weeks postpartum (Ribeiro et al., 2016). Given the increased morbidity in cows with
SGL, it is not surprising that the rate of removal from the herd in the first 300 DIM was
markedly increased. It is interesting to note that mortality was increased statistically only
in cows with EGL, but not in cows with SGL, although SGL had greater morbidity than
cows with AGL. Cows with EGL usually deliver larger and heavier calves, and dystocia
is a very important risk factor for mortality in dairy cows. Perhaps cows with EGL had
more calving trauma from larger calves that resulted in increased mortality, although
dystocia was not increased in cows with EGL in comparison with AGL.
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Norman et al. (2011) concluded that milk production of multiparous cows
increased as GL increased. The same authors also demonstrated that milk fat and true
protein were linearly and positively associated with GL, i.e., as GL increased, so did
yields of fat and true protein. Jenkins et al. (2016) evaluated productive performance of
cows in seasonally calving dairy herds and found that GL category was a predictor for
yields of milk, fat, and protein. Cows with GL within the shortest 5% of the study
population and those with GL within the longest 5% of the study population were less
productive with lower yields of milk and milk components compared with cows with
intermediate duration of gestation. The current study also showed that cows with SGL
or EGL produced less milk than herdmates with AGL; however, the impact of GL on
production depended on parity of cows. In multiparous cows, those with SGL had
reduced milk yield, whereas those with EGL had increased milk yield. For primiparous
cows, both SGL and EGL reduced milk yield. A short duration of gestation in
multiparous might have implications for the length of the dry period and exposure of the
transition group to dietary manipulations. Furthermore, during the last 2 weeks of
gestation there is an increase in plasma concentrations of estrone and estrone sulfate
concurrent with a sharp decrease in concentrations of progesterone (Thatcher et al.,
1980). Eley et al. (1981) showed that concentrations of prolactin were positively
associated with concentrations of estrone and negatively associated with those of
progesterone; therefore, the shift in steroidogenesis influences prolactin production,
which is important for galactopoiesis. Cows with abnormal GL likely had these shifts in
steroidogenesis occurring in different times of gestation and relative to parturition, which
could have affected the peak of prolactin at the onset of lactation, thereby influencing
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subsequent galactopoiesis. Also, the combined increased incidence of diseases, many
of which are known to reduce milk yield, with the short dry period and less exposure to
transition group management might explain the reduction in milk yield in multiparous
cows with duration of gestation inferior to 270 d. On the other hand, extending GL in
primiparous might have resulted in larger calves and more calving difficulty which,
combined with the increased morbidity, might have caused the decrease in milk yield.
Because of the observational nature of this study, further research is needed to better
understand the implications of duration of gestation on subsequent production in dairy
cows.
Gestation length category was not associated with impairment in reproductive
performance during the first insemination in dams. That was unexpected because dams
with SGL or EGL had increased morbidity, particularly diseases that affect the
reproductive tract, and those diseases are known to affect reproduction in dairy cows
(Ribeiro et al., 2016). Cows with SGL left the herd earlier than cows with AGL, and cows
removed early are normally those with health disorders and less prompt to adequate
reproduction. It is possible that early removal from the herd might have underestimated
the impact of SGL on P/AI and pregnancy loss at first postpartum insemination. The
reduced number of cows with SGL that received at least one AI by 300 DIM support this
hypothesis. Nonetheless, the proportion of cows pregnant by the end of the 300 days
observation showed that SGL compromised reproduction in dairy cows, however EGL
did not. It seems that the harm of abnormal GL on reproduction of dairy cows is not
timely, and carries over the entire lactation. In agreement with our findings, Norman et
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al. (2011) also showed that cows with either short or extended GL had increased days
open suggesting reduced reproductive performance.
The associations between GL and health, survival and reproduction were also
established for female offspring. Heifer mortality increased in both SGL and EGL. This
negative impact was observed during the postweaning periods, which resulted in larger
proportions of SGL and EGL calves dead by 300 days of age compared with calves
born from AGL dams. In fact, the rate of removal of heifers from the herd was greatly
increased when the duration of gestation deviated from that of dams with AGL.
As parturition approaches, the fetal lungs undergo biochemical, physiologic and
morphologic changes to adapt to the postnatal life. One of those adaptations is the
increase in production of pulmonary surfactants essential to prevent alveolar collapse
and to maintain bronchiolar patency during respiration in extrauterine life (Rooney,
1985). Calves born before 270 days of gestation are at a high risk of development of
respiratory distress syndrome (Eigenmann et al., 1984). It is possible that calves born
from cows with SGL may have had increased mortality because of the greater
susceptibility to pulmonary disorders as their lungs were immature. Similar processes
could have occurred in the digestive and immune system, which could have affected
colostrum absorption and the ability to fight gastrointestinal infections common of the
preweaning period. Premature calves have been shown to have immature small
intestine compared with full-term calves (Bittrich et al., 2004). Therefore, SGL calves
may also have increased respiratory and digestive disorders predisposing them to
greater mortality than calves with AGL (Virtala et al., 1996). Nogalski and Piwczyński
(2012) reported that there is a linear relationship with calf birth weight and GL; therefore
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EGL calves probably were heavier at birth. Johanson and Berger (2003) showed a
linear relationship between calf birth weight and perinatal mortality exist, and heavier
calves had increased mortality. The increased calving difficulty associated with larger,
heavier calves and increased stress suffered during parturition may compromise
transfer of passive immunity and increase susceptibility to development of diseases
and, consequently, mortality.
Reproductive performance of female offsprings was associated with GL. Of the
3,623 heifers born alive, a larger portion of SGL and EGL never received an AI by 500
days of age compared with AGL. Approximately 67% of the non-inseminated heifers
had left the herd by 300 days of age either because of death or culling. The remaining
33% were not inseminated by 500 days of age either because they left the herd
between 300 and 500 days of age, were not observed in estrus, or were moved to a
group of heifers with natural service. Of the heifers that received at least 1 AI, the P/AI
at first insemination was only reduced in those with EGL, which eventually resulted in a
smaller proportion of EGL pregnant by 500 days of age compared with SGL.
Unfortunately, incidence of diseases in calves was not available for this study, but the
greater mortality suggests increased morbidity in SGL and EGL, and calves with
diseases in early life have increased age at pregnancy (Waltner-Toews et al., 1986). In
addition, increased morbidity is associated with less BW gain, which can influence time
to reach puberty and subsequent reproduction. It is interesting that heifers born from
SGL dams that received AI had reproductive performance similar to those from AGL
dams. Perhaps the greater rate of removal from the herd in SGL and EGL heifers
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compared with AGL heifers attenuated the negative consequences to reproduction
because those most affected either died or were culled prematurely.
Final Remarks
Abnormal GL, based on deviations of at least 1 SD from the population mean,
was associated with impaired health, productive and reproductive performance in
Holstein cows. Cows with SGL had increased incidence of dystocia, stillbirth, morbidity
in the first 90 DIM, and rate of removal from the herd, and reduced proportion pregnant
by 300 DIM compared with cows with AGL. Primiparous cows with SGL and EGL
produced less milk than primiparous cows with AGL. Similarly, multiparous cows with
SGL produced less milk than multiparous cows with AGL; however, multiparous cows
with EGL produced more milk than multiparous cows with AGL. Cows with extended GL
had increased mortality and reduced rate of pregnancy compared with cows with AGL.
Offsprings from dams with abnormal GL had increased mortality during the postweaning
period resulting in increased rate of removal from the herd compared with heifers from
dams with AGL. Smaller proportions of heifers from dams with SGL and EGL received
at least one AI compared with heifers from dams with AGL. Collectively, these results
indicate that deviations larger than 6 days from the mean GL of 276 days in Holstein
cows are associated with depressed dam health, production, and reproduction. The
negative effects of either shortened or extended GL were also observed in the female
calves born alive. Offsprings from dams with AGL survived longer and had improved
reproductive performance.
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Table 4-1. Association between gestation length category and incidence of diseases, culling and mortality in Holstein cows
Gestation length category1
Item AGL SGL EGL P-value
Cows, n 6,181 762 1,152 --- Dystocia, % 25.5a 30.3b 26.3 0.12 Stillbirth, % 7.0a 16.0b 8.3a < 0.01 Retained fetal membranes, % 5.1aA 26.0b 6.7B < 0.01 Metritis, % 22.9a 46.0b 25.1a < 0.01 Mastitis by 90 DIM, % 4.2 3.9 4.8 0.43 Lameness by 90 DIM, % 6.7 5.4A 7.5B 0.20 Morbidity by 90 DIM2, % 37.4b 55.2a 39.9b < 0.01 Culling by 300 DIM, % 20.1a 23.9b 18.4a 0.02 Death by 300 DIM, % 2.2a 2.5 3.3b 0.05
a,b Values with distinct superscripts in the same row differed (P < 0.05). A,B Values with distinct superscripts in the same row tended to differed (P < 0.10). 1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d). 2Morbidity includes at least one the following conditions: retained fetal membranes, metritis, mastitis, lameness, and other miscellaneous diseases such as milk fever, displaced abomasum, pneumonia, and bloat.
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Table 4-2. Cox’s regression model for time to diagnosis of disease up to 90 DIM in Holstein cows
Item (cows) Adjusted hazard ratio 95% CI P-value
Gestation length1 < 0.01 AGL (6,181) Reference --- --- SGL (762) 1.56 1.40 - 1.74 < 0.01 EGL (1,152) 1.11 1.00 - 1.23 < 0.05
Parity Primiparous (3,242) Reference --- --- Multiparous (4,853) 0.53 0.50 - 0.57 < 0.01
Season of calving Hot (3,408) Reference --- --- Cool (4,687) 0.82 0.77 - 0.88 < 0.01
Gender of calf < 0.01 Twin (359) Reference --- --- Male (3,612) 0.27 0.24 - 0.31 < 0.01 Female (4,124) 0.20 0.18 - 0.24 < 0.01
Farm 1 (1,841) Reference --- --- 2 (6,254) 0.93 0.85 – 1.01 0.07
1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
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Table 4-3. Cox’s regression model for time to removal from the herd up to 300 DIM in Holstein cows
Item (cows) Adjusted hazard ratio
95% CI P-value
Gestation length1 < 0.01 AGL (6,181) Reference --- --- SGL (762) 1.38 1.17 - 1.62 < 0.01 EGL (1,152) 1.09 0.96 - 1.24 0.17
Parity Primiparous (3,242) Reference --- --- Multiparous (4,853) 2.56 2.29 - 2.86 < 0.01
Season of calving Hot (3,408) Reference --- --- Cool (4,687) 1.16 1.05 - 1.27 < 0.01
Gender of calf < 0.01 Twin (359) Reference --- --- Male (3,612) 0.77 0.63 - 0.93 < 0.01 Female (4,124) 0.71 0.59 - 0.87 < 0.01
Farm 1 (1,841) Reference --- --- 2 (6,254) 1.18 1.05 - 1.32 < 0.01
1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
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Table 4-4. Association between gestation length category and milk production in Holstein cows
Item (cows) Milk production, kg/d SEM P-value
Gestation length1 < 0.01 AGL (6,181) 38.5 0.2 --- SGL (762) 36.6 0.3 <0.01 EGL (1,152) 37.7 0.3 < 0.01
Parity Primiparous (3,242) 34.3 0.3 --- Multiparous (4,853) 40.9 0.2 < 0.01
Gestation length x parity2 < 0.01 Primiparous - AGL (2,456) 35.4 0.2 --- Primiparous - SGL (475) 34.6 0.4 0.04 Primiparous - EGL (311) 33.0 0.4 < 0.01 Multiparous - AGL (3,725) 41.6 0.2 --- Multiparous - SGL (287) 38.6 0.4 < 0.01 Multiparous - EGL (841) 42.4 0.3 < 0.01
Season of calving Hot (3,408) 37.8 0.2 --- Cool (4,687) 37.3 0.2 < 0.01
Gender of calf 0.20 Twin (359) 37.2 0.4 --- Male (3,612) 37.8 0.2 0.09 Female (4,124) 37.8 0.2 0.07
Farm 1 (1,841) 37.7 0.3 --- 2 (6,254) 37.5 0.2 0.43
Gestation length x farm2 < 0.01 Farm 1 – AGL (1,412) 38.3 0.2 --- Farm 1 – SGL (175) 36.6 0.5 <0.01 Farm 1 – EGL (254) 38.2 0.4 0.75 Farm 2 – AGL (4,769) 38.6 0.2 --- Farm 2 – SGL (587) 36.7 0.3 < 0.01 Farm 2 – EGL (898) 37.2 0.3 < 0.01
1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d). 2 For interactions between GL and parity or GL and farm, the P-values representing comparisons among AGL are depicted for within parity and within farm pairwise comparisons.
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Table 4-5. Reproductive performance in Holstein cows according to gestation length category
Gestation length category1
Item AGL SGL EGL P-value
Cows, n 6,181 762 1,152 Inseminated by 300 DIM, % 89.6a 83.7b 89.7a < 0.01 DIM at first AI, LSM ± SEM 65.4 ± 0.3 65.0 ± 0.5 65.9 ± 0.4 0.21 Pregnant at first AI, %
Day 32 38.8 38.0 37.2 0.60 Day 60 33.8 33.3 33.7 0.97
Pregnancy loss, % 11.8 12.7 11.1 0.86 Pregnant 300 DIM, % 77.8a 69.5b 77.1a 0.03
a,b Values with distinct superscripts in the same row differ (P < 0.05). 1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
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Table 4-6. Cox’s regression model for time to pregnancy up to 300 DIM in Holstein cows
Days to pregnancy
Item (cows) Median (95% CI) LSM ± SEM AHR1 (95% CI) P-value
Gestation length2
0.03
AGL (6,181) 100a (98 to 103) 128.4 ± 1.1 Reference --- SGL (762) 99ab (93 to 104) 126.3 ± 3.0 0.97 (0.88 to 1.06) 0.45 EGL (1,152) 111b (103 to
116) 137.2 ± 2.6 0.91 (0.84 to 0.98) 0.01
Parity Primiparous 96 (92 to 99) 122.5 ± 1.4 Reference --- Multiparous 106 (103 to 110) 135.0 ± 1.3 0.85 (0.81 to 0.89) < 0.01
Season of calving
Hot 99 (97 to 102) 124.2 ± 1.3 Reference --- Cool 103 (101 to 107) 133.7 ± 1.3 0.89 (0.85 to 0.94) < 0.01
Gender of calf 0.10 Twin 116 (107 to 129) 140.4 ± 4.8 Reference --- Male 103 (101 to 106) 130.8 ± 1.3 1.08 (0.95 to 1.23) 0.25 Female 98 (95 to 101) 126.6 ± 1.4 1.13 (0.99 to 1.29) 0.08
a,b Values with distinct superscripts in the same row differ (P < 0.05). 1 AHR = adjusted hazard ratio. 2 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
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Table 4-7. Survival and reproduction of Holstein heifers born live according to gestation length
Gestation length category1
Item AGL SGL EGL P-value
Heifers, n 2,880 337 406 --- Death, %
Preweaning 1.8 3.6 4.1 0.28 Postweaning 3.2b 6.3a 5.1a < 0.01 Birth to 300 days of age 5.0a 10.1b 9.5b < 0.01
Culled by 300 days of age, % 1.6A 1.4 3.0B 0.16 Left herd by 300 days of age, % 7.5a 10.2 13.6b 0.01 Inseminated, % 89.5a 84.7b 83.2b < 0.01 Days of age at first AI, LSM ± SEM
412.2 ± 0.3a 413.7 ± 1.0a 406.8 ±
1.0b < 0.01
Pregnant first AI, % 65.8 70.0a 61.3b 0.09 Pregnant by 500 days of age, % 82.3 79.2 73.9 0.24
a,b Values with different superscripts differed (P < 0.05). A,B Values with different superscripts tended to differ (P < 0.10). 1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
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Table 4-8. Cox’s regression model for time to removal from the herd up to 300 days of age in Holstein heifers
Item (heifers) Adjusted hazard ratio 95% CI P-value
Gestation length1 < 0.01 AGL (2,880) Reference --- --- SGL (337) 1.84 1.31 to 2.60 < 0.01 EGL (406) 1.97 1.43 to 2.70 < 0.01
Parity of dam Primiparous (1,409) Reference --- --- Multiparous (2,214) 0.85 0.66 to 1.08 0.19
Season of birth Hot (1,486) Reference --- --- Cool (2,137) 0.70 0.55 to 0.89 < 0.01
Farm 1 (825) Reference --- --- 2 (2,798) 1.61 1.25 to 2.07 < 0.01
1 Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
131
Figure 4-1. Survival curves for time to diagnosis of disease up to 90 DIM (A) and time to removal from the herd up to 300 DIM (B) in Holstein cows according to gestation length category. Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
300250200150100500
100
90
80
70
60
50
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Figure 4-2. Survival curves for days postpartum to pregnancy in Holstein cows up to 300 DIM according to gestation length category. Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
300250200150100500
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Figure 4-3. Survival curves for age at removal from the herd up to 300 days of age in heifers according to gestation length category of their dams. Gestation length was categorized as average (AGL; population mean ± 1 SD; mean = 276 d, range 270 to 282 d), shortened (SGL; at least 1 SD below the population mean; mean = 266 d, range 256 to 269 d), and extended (EGL; > 1 SD above the population mean; mean = 285 d, range 283 to 296 d).
300250200150100500
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CHAPTER 5 CONCLUSIONS
The experiment presented in Chapter 3 in this thesis document the role of
calcitriol during the immediate postpartum period on mineral metabolism, prevalence of
subclinical hypocalcemia (SCH), plasma metabolites, productive parameters, urinary
loss of minerals, blood cell count, and measures of innate immune function, and provide
a possible strategy to reduce the prevalence of SCH during the early postpartum period.
Subclinical hypocalcemia is still highly prevalent in dairy cows, and has been associated
with negative effects on energy metabolism and immune status. This subclinical
metabolic disorder has been described as an important risk factor for different diseases
during the postpartum period, including those that affect the reproductive tract and,
therefore, have implications beyond the first week of lactation. The implications of SCH
and the subsequent diseases result in economic losses to dairy producers. The study
presented in Chapter 4 established associations between the duration of gestation in
dairy cows with health, productive and reproductive performance in dams. Cows with
shortened and prolonged gestation length (GL) had impaired health and performance
with subsequent impacts on their offsprings. Cows with GL deviating more than 1
standard deviation (SD) from the population mean, in this case 6 days, had increased
morbidity and reduced production and proportion pregnant by 300 days postpartum
compared with those with GL within 1 SD of the population mean. Furthermore,
deviations in GL not only affected the dams, but also survival and reproduction of their
female offsprings.
The results presented in Chapter 3 provide evidences that the use of calcitriol
within 6 h after parturition was an efficacious and a safe approach to increase
135
concentrations of iCa and tCa in blood of early postpartum dairy cows. Administration of
300 μg of 1α,25-dihydroxyvitamin D3 rapidly increased concentrations of iCa, tCa, and
tP in blood of dairy cows, thereby reducing the prevalence of SCH in the first 3 days
postpartum. Nevertheless, administration of 1α,25-dihydroxyvitamin D3 induced a more
exacerbated decreased in plasma tMg probably because of increased urinary loss. In
fact, not only urinary loss of Mg increased, but also that of Ca because of the transient
hypercalcemia. The increase in blood iCa and tCa did not seem to be mediated by
increased bone resorption, at least based on the marker investigated. The
concentrations of CTX-1 did not differ between treatments and cows treated with 1α,25-
dihydroxyvitamin D3 had increased urinary loss of Ca thereby suggesting that increases
in blood iCa and tCa were mediated likely by increased absorption from the
gastrointestinal tract. Results also indicated improvements in neutrophil function based
on increased intensities of phagocytosis and oxidative burst and increased percentage
of neutrophils with oxidative burst activity. Collectively, the reduced prevalence of
subclinical hypocalcemia and improved measures of innate immune function suggest
that treatment with 1α,25-dihydroxyvitamin D3 might benefit postpartum health in dairy
cows. Nevertheless, treatment with 1α,25-dihydroxyvitamin D3 did not affect lactation
performance or influence measures of energy metabolism such as energy balance and
plasma concentrations of nonesterified fatty acids and glucose. In fact, concentrations
of 3-hydroxybutyrate tended to be greater in cows receiving 1α,25-dihydroxyvitamin D3.
Because SCH can depress dry matter intake and blunt insulin release, it was
hypothesized that improvements in Ca homeostasis in early lactation would benefit
energy metabolism. Further research is warranted to verify if use of 1α,25-
136
dihydroxyvitamin D3 to reduce SCH plays any role in the risk of clinical and subclinical
ketosis in dairy cows. In summary, results from Chapter 3 indicate that 1α,25-
dihydroxyvitamin D3 might be a viable alternative to attenuate SCH by enhancing
concentrations of iCa and tCa in blood of dairy cows at the onset of lactation and
minimize their negative effects on innate immune function. Further experiments are
needed to elucidate the effects of 1α,25-dihydroxyvitamin D3 treatment on incidence of
postpartum diseases and production disorders, as well as the carry over effects on
reproductive performance in dairy cows.
The study presented in Chapter 4 was designed to evaluate the association
between the duration of gestation in dairy cows and health, productive and reproductive
performance in dams, and survival and reproductive performance of their female
offsprings. Results revealed that deviations from the population mean GL of at least 1
SD, 6 days, was associated with impaired health, productive and reproductive
performance in Holstein cows. Cows with shortened GL had increased incidence of
dystocia, stillbirth and morbidity in the first 90 DIM, and rate of removal from the herd in
the first 300 days postpartum. Cows with GL shorter than 1 SD from the population
mean were less likely to be pregnant by 300 DIM compared with cows with average GL.
Primiparous cows with short and extended GL had reduced milk production compared
with primiparous cows with average GL. Moreover, cows with extended GL had
increased mortality, and reduced rate of pregnancy compared with cows with average
GL; multiparous cows with shortened GL produced less milk than multiparous cows with
average GL, whereas multiparous cows with extended GL produced more milk than
multiparous cows with average GL. Offsprings from dams with shortened or extended
137
GL had increased mortality during the postweaning period resulting in increased rate of
removal from the herd compared with heifers from dams with average GL. Smaller
proportions of heifers from dams with shortened or extended GL received at least one
AI compared with heifers from dams with average GL. Collectively, these results
indicate that deviations larger than 6 days from the mean GL of 276 days in Holstein
cows were associated with depressed dam health, production, and reproduction. The
negative effects of either shortened or extended GL were characterized by reduced calf
viability and imprinted depressed performance in the female offsprings born alive.
Offsprings from dams with average GL survived longer and had improved reproductive
performance. The mechanisms involved in abnormal GL, either shortened or extended,
and the subsequent consequences to offsprings are not fully understood, therefore,
investigation in the physiological processes occurring at the end of abnormal GL needs
further research.
138
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BIOGRAPHICAL SKETCH
Achilles Vieira Neto was born in Itajaí, state of Santa Catarina, located in the
south of Brazil. From a young age, during his time spent in the family farm, he knew he
liked animals and wanted to be involved with veterinary medicine and animal
agriculture. In the winter of 2008, he began his studies in the School of Veterinary
Medicine at Santa Catarina State University, in Lages, Santa Catarina, Brazil.
Throughout his undergraduate work he was involved with research and in his senior
year he was awarded a scholarship by the Brazilian National Council for Scientific and
Technological Development to study abroad. He moved to the University of Florida and,
from July of 2012 to July of 2013, he worked under the supervision of Dr. Klibs Galvão
in the Food Animal Reproduction and Medicine Service (FARMS) of the College of
Veterinary Medicine. During this time, he studied the development and treatment of
uterine diseases including metritis, and clinical and subclinical endometritis in dairy
cows. Upon finishing his externship at the University of Florida, he returned to Brazil to
complete his final year in veterinary medicine and then graduated in July of 2014. He
returned to the University of Florida in the fall of 2014 to begin his Master of Science
program in Animal Sciences under the supervision of Dr. José Eduardo P. Santos. His
research focus has been on dairy cattle health and reproduction. Upon completion of his
Master of Science in Animal Sciences, Achilles will initiate the PhD program in Animal
Molecular and Cellular Biology at the University of Florida under the supervision of Dr.
Santos.