impact of maternal heat stress during late...
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IMPACT OF MATERNAL HEAT STRESS DURING LATE GESTATION ON CALF
PERFORMANCE AND HEALTH
By
ANA PAULA ALVES MONTEIRO
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
2013
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© 2013 Ana Paula Alves Monteiro
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To my family for their endless love and support
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ACKNOWLEDGMENTS
First of all, I would like to thank God for my life and all the blessings and good things
that always happen to me.
I would like to express my gratitude to my advisor Dr. Geoffrey E. Dahl, for giving me
the opportunity of pursuing my Master of Science degree at the University of Florida and for his
guidance and support during these two years.
I extend my appreciation to my other committee members, Dr. John Driver and Dr. Klibs
Galvão, for their contribution with great suggestions and for their willingness to help.
I would like to thank all the staff and faculty at the Department of Animal Sciences,
specially Dr. Lokenga Badinga, Dr. Charles Staples and Dr. Jose Santos for sharing their
laboratories and for general assistance. I also want to thank Dr. Donovan for the great
suggestions and for taking care of the calves’ health. Special thanks for Mrs. Joyce Hayen, Mrs.
Joann Fischer and Mrs. Debra Sykes for all their help.
Special thanks to my labmates, Sha Tao and Izabella Thompson. I am very lucky to have
such awesome people around me. They helped me with everything and they taught me a lot since
I arrived. Above of all their friendship is very important for me.
I would like to thank all the staff of the Dairy Unit of University of Florida, especially
Eric Diepersloot, Sherry Hay and Grady Byers for taking care of the animals and for all their
help during my experiment. It was such a great time.
I also want to express my appreciation to all the volunteers who helped me with the farm
work, Alexis Taylor, Maureen Thieme, Glorian Pombo, Barbara Bearden, Rachel Corlett, Emily
Ferrell, Lisette Perez and Briana Nixon. I would not be able to complete my experiment without
their help.
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Thanks to all my friends in the Department of Animal Sciences, specially Anna Carolina
Denicol, Bianca Artiaga, Izabella Thompson and João Bittar for all the good times and helping
me whenever I needed. I specially thank Eduardo de Souza Ribeiro for making the initial contact
and helping me to get an internship at the University of Florida and subsequently get into the
master’s program. I also would like to extend my appreciation to all my friends in Brazil for their
support.
I thank all my family, specially my parents Eclair and Luiz Carlos and my brother Tiago
for their endless love and for being the best family anyone could have.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES ...........................................................................................................................8
LIST OF FIGURES .........................................................................................................................9
LIST OF ABBREVIATIONS ........................................................................................................11
ABSTRACT ...................................................................................................................................12
CHAPTER
1 LITERATURE REVIEW ........................................................................................................15
Immune System Development in the Newborn Calf ..............................................................15
Importance of Feeding High Quality Colostrum to Newborn Calves .............................17
Colostrum Composition and its Impact on the Development of Calf Immune
System ..........................................................................................................................18
Passive Immune Transfer ................................................................................................21
Factors Influencing Colostrum Quality and Passive Immune Transfer ..........................23
Colostrum Heat-Treatment ..............................................................................................25
Effects of Heat Stress During the Dry Period .........................................................................25
Effects on the Dam ..........................................................................................................26
Effect on Colostrum ........................................................................................................27
Effects of Heat Stress in Utero During Late Gestation on the Fetus ......................................28
Effects on Fetal Thermoregulation ..................................................................................28
Heat Stress Induced Intrauterine Growth Restriction ......................................................30
Effects on the Immune Function of the Offspring ...........................................................33
Summary .................................................................................................................................34
2 EFFECT OF HEAT STRESS DURING THE DRY PERIOD ON IMMUNE
FUNCTION AND GROWTH PERFORMANCE OF CALVES RESULTING FROM
ALTERED COLOSTRAL AND CALF FACTORS ..............................................................35
Materials and Methods ...........................................................................................................36
Animals and Experimental Design ..................................................................................36
Experiment 1 ............................................................................................................36
Experiment 2 ............................................................................................................37
Growth Measures and Blood Collection .........................................................................38
Health Scoring .................................................................................................................38
Ovalbumin Challenge ......................................................................................................38
IgG Analysis ....................................................................................................................39
Peripheral Blood Mononuclear Cell (PBMC) Isolation and Proliferation ......................40
Whole Blood Proliferation ..............................................................................................41
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Cortisol and Insulin Analysis ..........................................................................................42
Statistical Analysis ..........................................................................................................42
Results.....................................................................................................................................43
Cow Performance ............................................................................................................43
Colostrum IgG Concentration .........................................................................................43
Growth Performance .......................................................................................................44
Health Scores and White Blood Cells Count ..................................................................44
Hematocrit, Total Plasma Protein and Serum IgG Concentration ..................................45
Ovalbumin Challenge Response ......................................................................................46
Serum Cortisol and Insulin ..............................................................................................46
PBMC and Whole Blood Proliferation ............................................................................47
Discussion ...............................................................................................................................47
Conclusions.............................................................................................................................53
3 EFFECT OF HEAT STRESS IN UTERO ON CALF PERFORMANCE AND HEALTH
THROUGH THE FIRST LACTATION ................................................................................69
Materials and Methods ...........................................................................................................69
Animals and Data Collection ...........................................................................................69
Statistical Analysis ..........................................................................................................70
Results.....................................................................................................................................71
Growth Performance .......................................................................................................71
Mortality ..........................................................................................................................71
Reproductive Performance and Milk Production ............................................................72
Discussion ...............................................................................................................................72
Conclusions.............................................................................................................................75
4 GENERAL DISCUSSION AND SUMMARY .......................................................................80
LIST OF REFERENCES ...............................................................................................................83
BIOGRAPHICAL SKETCH .........................................................................................................98
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LIST OF TABLES
Table page
2-1 Chemical composition of starter ........................................................................................54
2-2 Calf health scoring criteria .................................................................................................54
2-3 Growth performance of calves born to cows exposed to either heat stress or cooling
during the dry period ..........................................................................................................55
2-4 Growth performance of calves born to cows under thermoneutral conditions during
the dry period and fed frozen colostrum from cows exposed to either heat stress or
cooling during the dry period .............................................................................................55
3-1 Effect of maternal heat stress or cooling during late gestation on calf survival ................76
3-2 Effect of maternal heat stress or cooling during late gestation on reproductive
performance of heifers in the first lactation .......................................................................76
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LIST OF FIGURES
Figure page
2-1 Schematic description of experiments 1 and 2 ..................................................................56
2-2 Effect of heat stress or cooling during the dry period on pre-weaning growth
performance of the offspring .............................................................................................57
2-3 Effect of heat stress or cooling during the dry period on hematocrit and total plasma
protein of the offspring during the first 35 d of age ...........................................................58
2-4 Effect of heat stress or cooling during the dry period on total serum IgG
concentrations of the offspring during the first 28 d of age ...............................................59
2-5 Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on hematocrit and total plasma protein of calves during the first 56 d of age ...................60
2-6 Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on total serum IgG concentration of calves during the first week of age ..........................61
2-7 Effect of heat stress or cooling during the dry period on the response of the offspring
to ovalbumin challenge ......................................................................................................62
2-8 Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on the response of calves to ovalbumin challenge .............................................................63
2-9 Effect of heat stress or cooling during the dry period on serum cortisol
concentrations of the offspring during preweaning period ................................................64
2-10 Effect of heat stress or cooling during the dry period on serum insulin concentrations
of the offspring during preweaning period ........................................................................65
2-11 Effect of heat stress or cooling during the dry period on the peripheral blood
mononuclear cell proliferation of the offspring during the preweaning period .................66
2-12 Effect of heat stress or cooling during the dry period on whole blood proliferation of
the offspring during preweaning period .............................................................................67
2-13 Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on whole blood proliferation of the offspring during preweaning period .........................68
3-1 Effect of maternal heat stress or cooling during late gestation on body weight of the
offspring up to one year of age ..........................................................................................77
3-2 Effect of maternal heat stress or cooling during late gestation on milk production in
the first lactation ................................................................................................................78
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3-3 Effect of maternal heat stress or cooling during late gestation on body weight in the
first lactation ......................................................................................................................79
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LIST OF ABBREVIATIONS
AEA Apparent efficiency of absorption
CONA Concanavalin A
CPM Count per minute
CV Coefficient of variation
DIM Days in milk
DMI Dry matter intake
IG Immunoglobulin
LSM Least squares means
PBMC Peripheral blood mononuclear cell
PI-IUGR Placental insufficiency intrauterine growth retardation
PRL Prolactin
PRLR Prolactin receptor
SCC Somatic cell count
SD Standard deviation
SEM Standard error of the mean
SI Stimulation index
WH Withers height
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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
IMPACT OF MATERNAL HEAT STRESS DURING LATE GESTATION ON CALF
PERFORMANCE AND HEALTH
By
Ana Paula Alves Monteiro
August 2013
Chair: Geoffrey E. Dahl
Major: Animal Sciences
Calves born to and fed with colostrum from cows exposed to heat stress during the dry
period have compromised passive transfer and cell-mediated immune function compared with
calves born to cows under cooling. However, it is unknown if this compromised immune
response is caused by calf or colostrum intrinsic factors.
The objective of the first study was to evaluate the effect of maternal heat stress during
the dry period on calf specific factors related to immune response and growth performance.
Cows were dried off 46 d before expected calving and randomly assigned to a heat stress (HT, n
= 18) or HT and cooled (CL, n = 17) environment. After calving the cows were milked and their
colostrum was frozen for a subsequent study. Colostrum from cows exposed to a thermoneutral
environment during the dry period was pooled and stored frozen (-20°C). Within the first 4 h of
life, 3.8 L of the pooled colostrum was fed to calves from both HT and CL treatment groups.
Day of birth was considered study d 0. Subsequently, all the calves were exposed to the same
management and weaned at d 50. Blood samples were collected before colostrum feeding, 24 h
after birth and twice weekly up to d 32. Total serum IgG concentrations were determined. Body
weight was recorded at birth and at d 15, 30, 45 and 60. Relative to CL calves, HT calves were
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lighter at birth (38.3 vs. 43.1 kg) and at weaning (67 vs. 76 kg), but no differences in weight gain
were observed up to d 60. Additionally, HT calves had lower apparent efficiency of IgG
absorption (26.04 vs. 30.24 %), but no differences were observed for total IgG concentration.
In the second study the objective was to evaluate the isolated effect of the colostrum from
HT cows on calf immune response and growth performance. The experimental design was very
similar to the first study, but all cows were under thermoneutral conditions during the dry period.
At birth calves were blocked by gender and birth weight and then randomly assigned to one of
two treatments, which meant they received colostrum from HT cows or CL cows. No treatment
effect was observed on passive immune transfer, cell mediated immunity and postnatal growth.
Thus, data from these first two studies suggest that heat stress during the last 6 wks of gestation
negatively impacts the ability of the calf to acquire passive immunity regardless of colostrum
source.
The third study evaluated the effect of heat stress during late gestation on growth, fertility
and milk production in the first lactation of the offspring. Data of animals obtained from
previous experiments conducted during five consecutive summers were pooled and analyzed.
The experimental design in those studies was similar to that described for the first study, but
calves were not fed pooled colostrum, instead they were fed fresh or frozen colostrum from a
single cow. Birth weight and survival of 146 calves (HT=74; CL=72) and body weight and
growth rate from 72 heifers (HT=34; CL=38) were analyzed. Additionally, fertility and milk
production in the first lactation from 38 heifers (HT=17; CL=21) were analyzed. CL heifers were
heavier (P < 0.05) up to one year old, but had similar (P = 0.44) weight gain (305.8 ± 5.9 vs.
299.1 ± 6.3 kg) compared with HT heifers. No differences in age at first AI or age at first
parturition was observed, but HT heifers had a greater number of services per conception than
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CL heifers (2.6 ± 0.3 vs. 1.8 ± 0.3, P = 0.03). Additionally, HT heifers tended to produce less
milk up to 30 weeks of the first lactation compared with CL heifers (26.4 ± 2.1 vs. 30.9 ± 1.7 kg,
P = 0.11). Data from this third study suggest that heat stress during the last 6 weeks of gestation
negatively impacts fertility and milk production up to and through the first lactation of the
offspring.
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CHAPTER 1
LITERATURE REVIEW
Immune System Development in the Newborn Calf
Developing and newborn calves are subject to several immunomodulatory effects. During
gestation the placenta produces hormones, such as progesterone and prostaglandin E2, and
cytokines, such as IL-4 and IL-10 that suppress cell mediated and memory (TH1) responses and
promote TH2 responses, which encourage antibody production, particularly IgE responses, and
enhance eosinophil proliferation and function (Morein et al., 2002). Additionally, the dam
produces immunosuppressive hormones before parturition, such as estrogen and cortisol (Jacob
et al., 2001) and the calf also produces high levels of cortisol, while undergoing birth, that
remain elevated for the first week of life (Mao et al., 1994).
The fetus is protected primarily by the innate immune system, but its phagocytic activity
is not fully developed until late in gestation (Barrington, 2001). Although below the levels of
those found in adults, humoral elements such as complement are present in calves (Chase et al.,
2008). Unless infected in utero, bovine fetuses are agammaglobulinemic (Barrington, 2001) and
are immunologically naïve at birth. The calf will have all essential immune components at birth,
but most of them are not functional until at least 2 to 4 weeks of age and some will continue to
develop until puberty (Reber et al., 2006). Starting at about one month before parturition,
peripheral blood T cells decrease from approximately 60% to 30% at birth, as they traffic to
lymphoid tissues of the fetus. The normal range of B cells in mature calves (i.e., ~2 mo of age) is
10% to 20% of total lymphocytes against only 1 to 2 % in the fetus and 4% at one week after
birth (Kampen et al., 2006; Senogles et al., 1979). This shift in B cell frequency coupled with
the calves’ endogenous corticosteroids and absorbed maternal hormones results in the lack of
any endogenous antibody response until 2 to 4 weeks of age, even with the more favorable TH2
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response in neonates. In this context, the ingestion of colostrum is of extreme importance to
provide immunologic defense to the calf during the first 2 to 4 weeks of life (Chase et al., 2008).
Calves that do not receive colostrum start to produce IgM only after 4 days of life and do not
achieve satisfactory levels of circulating immunoglobulins until 16 to 32 days after birth
(Husband and Lascelles, 1975). Despite the lower proportion of B cells, T cell subsets in the
neonatal calf are present at similar levels compared with that in adult animals (Kampen et al.,
2006; Wilson et al., 1996). Total T cells account for 28 to 34% of total lymphocytes. Of these
~20% are helper T cells (CD4) and ~10% are cytotoxic T cells (CD8) (Kampen et al., 2006).
Gamma-delta T cells represent approximately 25% of the total lymphocytes during the first week
but decrease to approximately 16% by 19 to 21 weeks of age. For these reasons, parenteral
vaccines at birth induce predominantly a T cell mediated response rather than a B cell response,
despite the presence of all immune cell types. However, mucosal immunity and immune
responses seem to be adequate in the newborn calf (Chase et al., 2008).
In addition to humoral components of the adaptive immune system being suppressed,
components of the innate immune arm do not function very well. Interferon activity in the
epithelial cells of neonates appears normal, but the production of type 1 interferon by leukocytes
is decreased (Firth et al., 2005). Natural Killer cell frequency is low during the first week of life,
changing from 3 to 10% of total lymphocytes by 6 to 8 weeks of age (Kampen et al., 2006).
Dendritic cells are also reduced in number and their ability to present antigens to lymphocytes is
limited (Morein et al., 2002). The number of neutrophils circulating in the newborn calf is about
four times higher than at 3 weeks of age. Neutrophils and macrophages have reduced phagocytic
capacity, but this is increased after the ingestion of colostrum (Menge et al., 1998).
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Importance of Feeding High Quality Colostrum to Newborn Calves
During gestation there is no transplacental transfer of antibodies from maternal to fetal
circulation in the bovine, consequently calves are agammaglobulinemic at birth (Barrington,
2001). Therefore, colostrum feeding has a major role in providing immunoglobulins, primarily
IgG, to the calf during the early postnatal period, until the calf’s own immune system has
matured. Although the half-life of colostrum derived immunoglobulins is about 21 days,
carryover effects of failure of passive immune transfer can be observed for the first 4 to 6 mo of
age (Donovan et al., 1998). In contrast, adequate colostrum transfer has been recognized to have
a beneficial effect not only on calf immune responses during early life, but also longer-term
effects on growth and development (Furman-Fratczak et al., 2011). Low levels of serum total
protein is a risk factor for the occurrence and severity of diseases such as pneumonia and
septicemia, and also increases mortality during the preweaning period and up to 3 months
(Donovan et al., 1986) or 6 months of age (Donovan et al., 1998).
It has been demonstrated that a minimum of 4 L of colostrum with an Ig concentration of
at least 50g/L is enough to guarantee adequate passive immune transfer (McGuirk and Collins,
2004; Morin et al., 1997). Despite these data, 60% of colostrum produced in the U.S. fails to
meet the minimum standard of 50g/L of IgG content (Morrill et al., 2012) and 20% of calves on
U.S. farms fail to acquire the minimum standard of 10 g/L for serum IgG (NAHMS, 2007).
Perhaps not surprisingly, 7.8% of preweaned calves die, primarily due to diarrhea (NAHMS,
2007). Moreover, Wells et al. (1996) observed that up to 31% of the mortality during the first 21
days of life in dairy heifers in the United States could be prevented by changes in first colostrum
feeding method, timing and volume. For these reasons, feeding an adequate volume of high
quality colostrum should be a major goal on dairy farms. Therefore, testing colostrum quality
should be routinely performed by using a colostrometer or refractometer. An important
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difference between these methods is that the results obtained using a colostrometer are
influenced by colostrum temperature at the time of measurement, whereas a refractometer is not
thermally influenced (Quigley et al., 2013).
Colostrum Composition and its Impact on the Development of the Calf Immune System
Bovine colostrum contains a wide spectrum of important immune and nutritional
components, such as immunoglobulins, which are transported from the maternal serum into the
mammary gland. The three major classes of immunoglobulins present in the colostrum are IgG,
IgM and IgA. The IgG subtype IgG1 accounts for over 75% of the immunoglobulins in colostral
whey, followed by IgM, IgA and IgG2 (Larson et al., 1980). The IgG concentration in colostrum
can vary dramatically among cows and may range from 9 to 186 g/L (Swan et al., 2007). A few
days after parturition immunoglobulin concentrations decrease to a total of ~0.6 mg/mL (Foley
and Otterby, 1978). Another component of the immune response present in colostrum is the
complement system, which also plays a role in passive immunization of the calf. The occurrence
of hemolytic or bactericidal complement activity in bovine colostrum and milk has been
demonstrated in several studies (Korhonen et al., 2000; Reiter and Brock, 1975). Other colostral
bioactive components besides immunoglobulin, such as live immune cells, cytokines, growth and
metabolic factors and microbes may also aid in improving IgG transfer through the intestinal
tract of neonates (Sangild, 2003), as well as enhancing the long-term performance of the heifer,
but their roles are not yet well understood.
Most of the cells present in colostrum are leukocytes and range in concentration between
1 x 106 and 3 x 10
6 cells/mL (Lee et al., 1980). The majority of these leukocytes are macrophages
(40 to 50%) and a smaller fraction consist of lymphocytes (22 to 25%) and neutrophils (25 to
37%) (Liebler-Tenorio et al., 2002; Reber et al., 2005). Yet, most of the lymphocytes are T
lymphocytes rather than B lymphocytes, which make up only 5 % of the total. Evidence exists to
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support the concept that maternal leukocytes cross the neonatal gut and circulate in the newborn
calf, but the impact of these cells on the development of neonatal immunity is not yet determined
(Donovan et al., 2007; Reber et al., 2006). Reber et al. (2006) observed that when feeding
maternal lymphocytes to newborn calves, only those cells exposed to an acellular colostrum, but
not medium alone, entered the circulation. The cells survived in the circulation for about 36 h
after ingestion, disappearing after that. That study indicates a role of the colostral environment in
facilitating the transfer of colostral cells to the neonate. The fate of maternal cells is uncertain,
but it appears that they home to secondary immune tissue in the central and mucosal
compartments of the body (Aldridge et al., 1998; Sheldrake and Husband, 1985). Donovan et al.
(2007) isolated pathogen-specific maternal T lymphocytes from neonatal calves with maximum
inducible proliferation at 1 day after birth. However, they were no longer detectable in
circulation at 7 days of age. Reber et al. (2008b) demonstrated that feeding colostrum containing
maternal cells accelerates the development and enhance activation of lymphocytes in the calf.
Calves fed whole colostrum had a higher percentage of lymphocytes expressing the activation
markers CD25 and CD26 at d 7 after birth and they expressed more MHC class I on their
surfaces when compared to lymphocytes from calves fed cell-free colostrum. Also, calves
receiving cell-free colostrum had a greater number of monocytes in the peripheral blood during
the first 2 weeks of life. However, these cells expressed lower levels of CD25 and MHC class I
compared to calves receiving whole colostrum (Reber et al., 2008a). Moreover, Reber et al.
(2005) demonstrated that feeding maternal colostrum containing leukocytes to the neonatal calf
enhances development of antigen-presenting function, which is very important for development
of an acquired immune response. Additionally, it seems that cell-mediated immune transfer to
neonates can be enhanced by maternal vaccination. Calves fed whole colostrum had enhanced
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responses to antigens against which the dams had previously responded, but not to antigens to
which the dams were naïve, suggesting that maternal memory cells circulate in the neonate and
retain their capacity to respond to antigens to which they were primed in the dam (Donovan et
al., 2007).
Cytokines present in the colostrum are very important for the development of the calf
immune system. A study with pigs demonstrated that colostral cytokines are absorbed and also
can be detected in the blood of the piglet (Nguyen et al., 2007). Whereas the source of the
colostral cytokines is not completely clear, they may be secreted in the mammary gland or
produced by leukocytes present in the colostrum (Chase et al., 2008). Among cytokines present
in bovine colostrum, interleukin 1-beta (IL-1β), IL-6, tumor necrosis factor- and interferon-γ
are associated with proinflammatory responses and may aid in the recruitment of neonatal
lymphocytes into the gut, thereby promoting normal immune development (Chase et al., 2008).
Menge et al. (1998) observed that ingestion of colostrum increased the percentage of
phagocytizing polymorphonuclear leukocytes and monocytes, further evidence of a role for
colostral derived factors that improve neonatal immune responses.
In addition to maternal factors, microbes present in the mammary gland may play a role
in calf immune development as well. Studies with heifers showed that more than 80% of them
had their mammary quarters colonized with bacteria, mostly Staphylococcus or Streptococcus
species, during the final days before birth (Oliver et al., 1992; Trinidad et al., 1990). Components
ubiquitous in microbes activate innate immune activity and those genus’ described above in
particular produce enterotoxins and virulence factors that nonspecifically activate lymphocytes.
This process of activation may prime and stimulate immune development in the calf, starting in
mucosal tissue, but more research is needed to prove this definitively.
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Passive Immune Transfer
The placentation of ruminants, sows and mares, known as epitheliochorial, does not
allow for transfer of antibodies between maternal and fetal circulation. However, in these species
the mammary gland concentrates large amounts of Ig during colostrum formation. The uptake
and transport of Ig across the mammary epithelial barrier is thought to occur primarily through
an Fc-receptor-mediated process (Butler and Kehrli, 2005; Cianga et al., 1999; Hunziker and
Kraehenbuhl, 1998; Larson, 1992). Immunoglobulins are thought to bind to receptors at the
basolateral surfaces of the mammary epithelial cell. These receptors are specific for the Fc
portion of the immunoglobulin molecule. The receptor-bound immunoglobulin is internalized via
an endocytic mechanism (He et al., 2008), transported to the apical end of the cell and released
into the alveolar lumen (Cervenak and Kacskovics, 2009). In the case of IgG, the receptor
responsible for transcytosis of IgG into colostrum is referred to as FcRn (Rodewald and
Kraehenbuhl, 1984). After birth, the newborn ingests colostrum and absorbs colostral proteins in
the intestine through an FcRn-independent and non-selective process (Baintner and Kocsis,
1984), mediated by “transport vacuoles”, as reviewed in Baintner (2007). Macromolecules so
transported are released into the lamina propria and then are absorbed into the lymphatic or
portal circulation. Some of the advantages of this FcRn- independent process includes the high
amount and diversity of colostral proteins being absorbed. Additionally, Castro-Alonso et al.
(2008) suggested that IgG absorption is mediated by apoptotic enterocytes. The absorptive
capacity begins to decrease after 6 to 12 hours of birth and ends by 48 hours probably as a result
of developmental processes occurring in the enterocytes (Sangild, 2003; Staley and Bush, 1985).
Neonatal corticosteroid levels must be high in order to increase colostral absorption (Sangild,
2003). Therefore, situations such as cold stress, dystocia, premature birth, cesarean section that
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inhibit release of cortisol by the neonate, also lead to inhibition of absorption of colostrum
(Chase et al., 2008).
Measuring serum Ig concentration 24 h after birth is an efficient way to determine if the
transfer of immunity through colostrum was successful. The recommendation is that calves
should have a serum IgG concentration of at least 10 g/L at 24 h after birth (Beam et al., 2009;
Swan et al., 2007). There are two tests that directly measure serum IgG concentration: radial
immunodiffusion and the enzyme-linked immunosorbent assay (ELISA). There are other tests,
such as serum total solids quantification by refractometry, sodium sulfite or zinc sulfate turbidity
tests, serum gamma-glutamyl transferase activity, and whole blood glutaraldehyde gelation,
which estimate serum IgG concentration based on concentration of total globulins or other
proteins (Laven, 2012; Tyler et al., 1999; Weaver et al., 2000). All of these practices are possible
because the passive transfer of those proteins is positively correlated with that of IgG.
Another method of evaluation is to calculate the apparent efficiency of absorption (AEA),
which takes into account the birth weight and intake of immunoglobulin during the first 12 to 24
h of life (Quigley and Drewry, 1998). The concentration (g/L) of total serum IgG at 24 h after
birth is multiplied by the birth weight (kg) and by 0.091 (considering the serum volume as 9.1%
of the birth weight). The value obtained is divided by the amount of IgG (g) fed to the calf and
then multiplied by 100, giving the percentage of absorption of IgG, which usually is only about
1/3 of total colostral IgG fed (Garry et al., 1996; Hopkins and Quigley, 1997; Morin et al., 1997).
Using the method of Evans’ blue dye, Quigley et al. (1998) concluded that an appropriate
estimate of plasma volume (PV) is 8.93% of BW in calves fed 1.5 to 4 L of colostrum and
sampled at 24 h of age; however the authors indicated that estimates of PV based on BW at birth
are subject to considerable variation.
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Factors Influencing Colostrum Quality and Passive Immune Transfer
Increasing IgG uptake reduces morbidity and mortality during the preweaning period,
improves growth and also increases future milk production (Faber et al., 2005; Weaver et al.,
2000). Because AEA usually is only about 1/3 of the mass consumed, many studies have focused
on the development of new strategies of colostrum and newborn management with the objective
of increasing the AEA. Many factors including immunoglobulin concentration of the colostrum,
amount of colostrum fed, timing of colostrum ingestion, route of feeding, parity of dam and
dystocia (Donovan et al. 1986) and also colostrum bacteria levels (Poulson et al., 2002) have
been shown to impact the optimization of Ig absorption.
Environmental factors are known to have a major effect on immunoglobulin absorption
by calves. Donovan et al. (1986) observed lower plasma Ig concentration in calves during
summer months in Florida and higher mortality of those same calves up to 6 months of age. In
fact, reducing heat stress in prepartum cows through a cooling system during summer months
improves IgG uptake by the calf (Tao et al., 2012a). The time elapsed from calving until
colostrum is removed from the udder also affects the concentration of IgG in colostrum, which
decreases 3.7% for every hour past calving (Morin et al., 2010). Additionally, the newborn is
only able to absorb intact proteins (i.e. immunoglobulins) and other components present in the
colostrum for the first 48 hours of life (Besser et al., 1985; Staley and Bush, 1985). Therefore,
colostrum must be expressed and fed to the calf as soon as possible using a nipple bottle or
esophageal feeder, to optimize Ig absorption. It is recommended that 3.8 L of good quality
colostrum be provided to calves either at one time or split into two feedings, and that the second
feeding occur within 12 h after birth (Faber et al., 2005; Morin et al., 1997). By taking these
steps farmers can ensure that adequate amounts of high quality colostrum reach the small
intestine of the newborn calf soon after birth. However, there is some data suggesting that IgG
24
uptake may also be improved if the calf remains with its dam (Stott et al., 1979), which may be
explained by a reduction in stress experienced by the calf. For most dairies, allowing the calf to
stay with the dam is not possible for practical management considerations, but there may be
other ways to reduce stress and enhance IgG uptake. Calves born by caesarean section and
stimulated physically have better respiratory parameters (i.e., diminished acidosis) and improved
passive immune transfer compared with unstimulated calves (Uystepruyst et al.; 2002).
However, stimulation of healthy newborn calves, delivered without dystocia, to simulate dam
mothering behavior had no effect on passive immune transfer (Haines and Godden, 2011).
Some studies demonstrate a positive effect on IgG uptake from colostrum from additives
such as trypsin and sodium bicarbonate (Morrill et al., 2010; Quigley et al., 1995). Bovine
colostrum contains large amounts of trypsin inhibitor (TI) to protect Ig from proteolytic cleavage
and to allow absorption of the intact molecule (Sandholm and Honkanen-Buzalski, 1979).
Quigley et al. (1995) demonstrated a positive effect of adding TI on IgG uptake of calves, but
these results were not observed in a study with goat kids (Ramos et al., 2010). Morrill et al.
(2010) found that calves receiving 30g of sodium bicarbonate added to a colostrum-based
colostrum replacer had higher serum IgG concentrations and AEA than calves that did not
receive the additive. However, Cabral et al. (2011) observed that feeding increasing levels of
sodium bicarbonate had negative linear effects on IgG concentration and AEA. Moreover, there
may be also a relationship between enhanced IgG uptake and the dam’s diet. A study with beef
cows (Hough et al. 1990) indicated that feeding a restricted energy diet (57% of NRC
requirements) for 90 days prepartum did not affect serum IgG concentration of calves born to
those cows, but calves fed colostrum from those cows tended to have lower serum IgG
concentration, although no effect on colostrum IgG was observed.
25
Colostrum Heat-Treatment
Contamination of colostrum is of major relevance on dairy farms because all the steps,
from collection through storage, are potential points of contamination. There are several studies
showing evidence of a negative relationship between colostral bacteria counts and passive
absorption of colostral IgG through the small intestine of neonatal calves (James and Polan,
1978; James et al., 1981; Staley and Bush, 1985), although the exact biological mechanism
behind this phenomenon is not known. A possible solution available for dairy farms to reduce
microbial contamination in colostrum without negatively affecting colostral IgG concentration is
heat-treatment (Donahue et al., 2012). A temperature of 60°C for 60 min reduces total plate
count (TPC) and total coliform count (TCC) in colostrum without increasing viscosity. Heating
colostrum to 60oC also reduced or eliminated specific inoculated pathogens, including
Mycoplasma spp., Listeria spp., Escherichia coli, Salmonella spp. and Mycobacterium avium
subsp. paratuberculosis (Godden et al., 2006). However, heating reduces the viability of
leukocytes present in the colostrum, but the importance of those cells is not known. One study
demonstrated that calves fed heat-treated (HT) colostrum had a greater efficiency of IgG
absorption and higher serum IgG concentrations when compared to calves fed fresh (FR)
colostrum (Godden et al., 2006). A large field study demonstrated that feeding HT colostrum
reduces calf morbidity during the preweaning period (Godden et al., 2012). Compared with
calves fed FR colostrum, calves fed HT colostrum were at 25 and 32% reduced odds of being
treated for scours and for any disease, respectively. However, it is still not clear if there are any
long-term benefits to cow health or lifetime productivity for calves fed HT colostrum at birth.
Effects of Heat Stress During the Dry Period
In dairy cattle the normal gestation length is about 280 days and the dry period or non-
lactating interval before parturition is the final 30-60 days of gestation, in which the cow is
26
allowed to reset from the last lactation and prepare for the next (Capuco et al., 1997). This period
is important for mammary involution and mammary gland growth (Capuco et al., 1997) and
different dry period lengths influence milk production in the subsequent lactation (Grummer and
Rastani, 2004). The dry period is also important for fetal growth and colostrum production. The
regulation of nutrient partitioning involves homeorhetic controls from the conceptus to assure
appropriate growth, not only of the fetus, but also fetal membranes, the gravid uterus and the
mammary gland (Bauman and Currie, 1980). As observed in lactating cows (Bernabucci et al.
2010; Kadzere et al., 2002), environmental factors such as photoperiod and temperature also can
influence production and health of dairy cattle during the dry period (Collier et al., 2006; Dahl
and Petitclerc, 2003).
Effects on the Dam
There are well documented negative effects of heat stress in dairy cows during the dry
period, specifically on DMI (Adin et al., 2009), immune function (do Amaral et al., 2010, 2011),
mammary gland development (Tao et al., 2011) and milk production in the subsequent lactation
(do Amaral et al., 2009, 2011; Wolfenson et al., 1988), as reviewed in Tao and Dahl (2013). A
study comparing cows dried off in hot months (June, July, and August) versus cool months
(December, January and February) in the northern hemisphere found that cows dried off in hot
months had greater incidence of health disorders in early lactation and poorer reproductive
performance compared with those dried in cool months (Thompson and Dahl, 2012). However,
some studies found no correlation between late gestation heat stress and reproductive
performance in the next lactation (Avendaño-Reyes et al., 2010; Moore et al., 1992). The greater
blood prolactin (PRL) concentrations (Collier et al., 1982a; do Amaral et al., 2009) and
consequently decreased gene expression of PRL receptor (PRL-r) in the liver and lymphocytes
(do Amaral et al., 2010, 2011) observed in cows exposed to heat stress during the dry period
27
have been related to the impaired mammary development and lower milk production in the
subsequent lactation. Additionally, heat stress decreases mammary blood flow when compared to
cows in a thermoneutral environment (Lough et al., 1990), which may also contribute to the
reduced mammary growth.
Effect on Colostrum
The dry period, in which mammary gland growth occurs, is also important for colostrum
production. A high air temperature (HAT) during late pregnancy affected the colostrum
composition of primiparous cows, however it did not affect colostrum yield (Nardone et al.,
1997). Heifers exposed to HAT produced colostrum with lower mean concentrations of IgG and
IgA, total protein, casein, -lactalbumin, fat and lactose, lower content of short- and medium-
chain fatty acids, lower energy, lower tritratable acidity and higher pH, yet no difference in
lactoglobulin or IgM was detected. Nardone et al. (1997) also observed that heifers under HAT
conditions underwent a smaller decline in total plasma Ig during the last two weeks of gestation
relative to those under thermal comfort conditions. Those authors concluded that the lower
percentages of casein and -lactalbumin might be a consequence of the deficits in protein and
energy during the dry period due to the lower DMI. Additionally, Nardone et al. (1997)
hypothesized that heat stress impairs the transfer of IgG from the blood to the mammary gland
and the production of IgA by plasma cells in the mammary gland. Moreover, cows under heat
stress are subject to hyperventilation, which reduces the carbonate content of colostrum, which is
probably the reason for the lower tritratable acidity. Quigley et al. (2000) observed no effect of
changes in pH within a range of 5 to 7.5 on IgG absorption from a colostrum supplement.
Therefore, pH per se does not seem to affect efficiency of absorption.
28
Effects of Heat Stress in Utero During Late Gestation on the Fetus
In addition to the impact on the overall performance of the dam, maternal heat stress
during late gestation also affects fetal growth and postnatal development. Heat stress during late
gestation decreases not only gestation length and birth weight of the offspring, but also placental
weight (Bell et al., 1989). The greatest accumulation of fetal mass (about 60% of its birth
weight) occurs late in pregnancy, during the dry period (Bauman and Currie, 1980). Due to the
redirection of blood flow, heat stress retards placental development, which impairs fetal nutrition
and oxygenation, leading to fetal growth retardation (Dreiling et al., 1991). Further, the
decreased concentration of placental hormones, such as estrone sulfate (Collier et al., 1982a),
placental lactogen (Bell et al., 1989), and pregnancy-associated glycoprotein (Thompson et al.,
2013) in heat-stressed animals relative to that of cooled cows is another indicator of
compromised placental function. Additionally, decreased energy intake is a hallmark of the heat
stress response in animals and malnutrition is a factor in reduced fetal growth (Wu et al., 2006).
In beef cows, severely restricted energy intake in the last trimester of gestation significantly
decreased calf birth weight (Tudor, 1972). However, a moderate decrease in energy intake during
late gestation had no effect on calf birth weight (Hough et al., 1990; Janovick and Drackley,
2010).
Effects on Fetal Thermoregulation
Mechanisms to dissipate heat or to avoid heat load accumulation are initiated upon
changes in temperature and humidity, with thermoneutral temperature range varying with
species, physiological state and age. During gestation, the fetus has to deal with thermoregulation
from a different perspective. The fetus is exposed to the environment of its dams uterus, hence it
is subjected to her thermoregulation, whereas its own thermoregulation is inhibited during this
period. However, there is a relationship of thermal protection between the fetus and its mother in
29
sheep and goats (Faurie et al., 2001; Laburn, 1996), which seems to be true in other mammals as
well (Adamsons and Towell, 1965; Hart and Faber, 1965; Morishima et al., 1975), in which the
fetus is protected from deviations in the dams body temperature. This mechanism avoids large
variations in fetal body temperature, which would be particularly beneficial during exposure to
hot environments, considering the possible teratogenic effects of heat (Dreiling et al., 1991;
Edwards, 1986). Under thermo-neutral conditions, fetal body temperature is about 0.5°C higher
than the dam in sheep (Laburn, 1996), goats (Faurie et al., 2001) and other mammals, including
humans (Adamsons and Towell, 1965; Morishima et al., 1975; Cefalo and Hellegers, 1978). The
high metabolic rate of the calf and in the placenta is responsible for a large amount of heat
generation, which seems to explain in part the higher body temperature of the fetus (Asakura,
2004; Laburn et al., 2000). When pregnant sheep or goats are exposed to heat or cold, the change
in body temperature observed in the fetus is attenuated relative to that of their dams (Laburn et
al., 1992, 2002; Faurie et al., 2001), significantly decreasing feto-maternal temperature
difference. The fetus exchanges heat primarily through fetal-placental circulation (~85%), but
can also exchange heat through fetal membranes, amniotic fluid and the uterine wall (Asakura,
2004; Laburn et al., 2000). The mechanism whereby fetal changes in temperature are attenuated
when the mother is subjected to thermal stress includes an increase or decrease in uteroplacental
blood flow during mild heat or cold exposure, respectively (Laburn, 1996; Laburn et al., 1992).
However, that is not true for cases of severe or prolonged heat stress (Bell, 1987) and fever
(Laburn et al., 1992). It has been reported that maternal heat stress compromises uterine blood
flow in sheep (Alexander et al., 1987; Brown and Harrison, 1984; Dreiling et al., 1991), hence
impairing fetal heat loss.
30
Heat Stress Induced Intrauterine Growth Restriction
Intrauterine hyperthermia is associated with congenital defects when it occurs in early
pregnancy (Edwards, 1986) and with intrauterine growth restriction (IUGR) when it occurs
during late gestation (Dreiling et al., 1991). The decrease in blood flow caused by heat stress
potentially compromises fetal and placental nutrition, oxygenation, and metabolic waste
disposal. A decrease in fetal metabolic rate with the objective to reduce fetal hyperthermia will
also compromise fetal growth. Placental insufficiency (PI) is a consequence of an impairment in
placental growth and development, and results in poor nutrient supply, fetal hypoxemia and
hence IUGR (Platz and Newman, 2008). As reviewed in Yates et al. (2011), fetal adaptive
responses have been studied in an ovine model of hyperthermically induced PI-IUGR (Barry et
al., 2008). Interestingly, heat stress induced IUGR in sheep during late gestation is independent
of maternal nutrition (Brown et al., 1977). Dreiling et al. (1991) hypothesized that the increase in
core body temperature in those animals, due to the high ambient temperature, could be the source
of a vasopressin-mediated reduction in maternal blood flow to the uterus. Indeed, exposure of
ewes to heat stress during gestation is associated with reduced placental weight (Bell et al.,
1989), primarily during late gestation, in which the difference in mass may be as much as 66% at
term (Galan, et al., 1999). The smaller placenta is explained by a reduction in size and total
tissue of the placentomes, rather than a reduction in number of placentomes (Alexander and
Williams, 1971; Thureen et al., 1992). Also, Early et al. (1991) verified decreased total placental
DNA, RNA and protein content, but concentrations were similar to that of placentas from
animals under thermoneutrality, indicating a reduction in total cell number rather than cell size.
Regnault et al. (2002) also verified a poor vascular organization in the cotyledons (the region of
fetal tissue in the placentome), which also contributes to reduced oxygen exchange. This
scenario can be explained by the disruption in the vasculogenesis subsequent to the abnormal
31
hypoxia caused by decreased blood flow. During normal gestation, angiogenesis is achieved by
the release of vascular endothelial growth factor (VEGF) stimulated by the need of oxygen
within the growing placenta (Arroyo and Winn, 2008). However, in the PI-IUGR model there is
an abnormal hypoxia at an early stage of the placental development, leading to a premature spike
in VEGF (Lyall et al., 1997). This may culminate in a period of placental hyperoxia (Regnault et
al., 2002), prematurely terminating the secretion of angiogenic factors and resulting in
underdevelopment of the placental vasculature structure (Kingdom and Kaufmann, 1997).
Umbilical vein oxygen content and glucose concentrations are diminished by as much as
50% in PI-IUGR fetuses during late gestation (Limesand et al., 2007; Wallace et al., 2005). In
contrast to oxygen, glucose is transported actively from maternal to fetal circulation, but in the
PI-IUGR model the expression of some glucose transporter transcripts is reduced (Limesand et
al., 2004; Wallace et al., 2005). Heat stress also diminishes umbilical uptake of amino acids,
likely due to the reduced placental mass, surface area and transporter expression (Vrijer et al.,
2004; Anderson et al., 1997; Ross et al., 1996). Under this scenario of chronic hypoxemia and
hypoglycemia, the fetus has to adapt, altering its physiological profile to support the most vital
organs at the expense of normal growth and development (Hales and Barker, 1992, 2001).
Additionally, adaptations are mediated by endocrine responses such as secretion of
catecholamines, norepinephrine and epinephrine from the adrenal medulla (Jellyman et al.,
2005). Catecholamines have a major role in maintaining glucose supply in the PI-IUGR through
suppression of insulin secretion (Leos et al., 2010) and increasing glucagon (Limesand et al.,
2006). Thus, insulin independent tissues (i.e. neural tissues) have an advantage over those that
require insulin in order to take up glucose. Skeletal muscle cells increase insulin receptor
concentration to restore glucose uptake (Limesand et al., 2006) primarily for anaerobic
32
metabolism, rather than oxidative energy production (Yates et al., 2011). In this way, alternative
substrates for oxidative energy production, such as amino acids, are utilized, which results in
restricted protein accretion and muscular growth in the PI-IUGR fetus (Anderson et al., 1997;
Limesand et al., 2009). Another characteristic of PI-IUGR is the premature development of
hepatic gluconeogenesis, using lactate, amino acids and other substrates as precursors. Stress
hormones appear to upregulate two important gluconeogenic enzymes, glucose-6-phosphatase
(G6P) and phosphoenolpyruvate carboxykinase (PEPCK), enhancing hepatic glucose production
(Fowden et al., 1993; Gentili et al., 2009), in part through increased levels of relevant
transcriptional factors and coactivators (Gentili et al., 2009; Limesand et al., 2007). Added to the
metabolic changes, fetal blood flow is also altered in PI-IUGR fetuses. Near the end of gestation,
there is an increase in blood flow to the brain and heart of 50 and 8%, respectively, at the
expense of flow to the liver, lungs, small intestine, pancreas, and adipose tissue (Alexander et al.,
1987; Walker et al., 1995).
Postnatally, the PI-IUGR offspring are at greater risk for metabolic complications than
controls, explained by altered sensitivities of tissues to stress hormones and other stimulants
(Yates et al., 2011). Among the negative outcomes, chronically elevated fetal catecholamines
may desensitize adipose tissue by downregulation of β2 adrenergic receptors, which was found
to impair fat mobilization and promote adiposity in PI-IUGR lambs (Chen et al., 2010).
Additionally, hypersensitivity to glucose stimulation (oversecretion of insulin) and enhanced
sensitivity to insulin in skeletal muscle due to chronic hypoinsulinemia (Limesand et al., 2006,
Thorn et al., 2009) seem to increase the propensity for glucose to be stored as fat during
compensatory growth (Greenwood et al., 1998; Chen et al., 2010). In dairy heifers greater fat
deposition during the prepubertal period is negatively correlated with mammary parenchymal
33
DNA at puberty and correlated to a higher BCS at breeding and both are related to lower milk
production in the first lactation (Silva et al., 2002; Whitlock et al., 2002). The pancreas is also
affected by PI-IUGR, in that β-cells may be reduced in mass by as much as 75% due to lower
replication rate (Limesand et al., 2005) and the fetal islets contain 80% less insulin content and
have decreased ability to induce glucose oxidative metabolism (Limesand et al., 2006;
Greenwood et al., 1998). Similar altered metabolic responses were also observed in dairy calves
that experienced heat stress in utero during late gestation. Calves born to cows heat-stressed or
cooled during the dry period had similar circulating insulin concentrations before colostrum
feeding, however, calves from heat-stressed dams had higher insulin concentrations at one day
after birth when compared to calves from cooled dams (Tao et al., 2012a). Also, similar to the
PI-IUGR sheep model (Chen et al., 2010), calves born to cows heat-stressed or cooled during the
dry period had a similar overall postnatal growth rate.
Effects on the Immune Function of the Offspring
There is evidence that passive immune function is compromised by maternal heat stress.
Studies performed in sows (Machado-Neto et al., 1987) and in dairy calves (Tao et al., 2012a)
observed a higher IgG concentration in the animals born to cooled dams when compared to heat-
stressed dams during late gestation and fed colostrum from their respective dams. As reviewed
in Merlot et al. (2008), prenatal stressors can modify T- and B- cell function of the offspring.
Couret et al. (2009) showed that prenatal social stress in pregnant sows increased the in vitro
lymphocyte proliferative response to a mitogen in piglets in early life. Tao et al. (2012a)
observed low PBMC proliferation in calves from heat-stressed dams, indicating impaired
lymphocyte function in those animals. However, in that study, the production of IgG in response
to an ovalbumin challenge was the same between treatments, indicating that humoral immunity
was not influenced by prenatal heat stress.
34
Summary
The negative effect of heat stress during dry period on dairy cattle is well established.
Heat stress during the last 45 days of gestation decreases milk production in the subsequent
lactation and compromises immune function during the transition period. In addition, late-
gestation heat stress also affects the offspring, where it decreases birth weight and seems to
compromise passive immune transfer and cellular immunity.
However, some questions arise regarding factors of the isolated effects of maternal heat
stress in the colostrum and in the calf. Additionally, it is not clear if maternal heat stress during
late gestation in cattle has carry over effects on growth, health and future milk production of the
heifers. The experiments described in Chapters 2 and 3 were designed with the aim to clarify
these questions.
35
CHAPTER 2
EFFECT OF HEAT STRESS DURING THE DRY PERIOD ON IMMUNE FUNCTION AND
GROWTH PERFORMANCE OF CALVES RESULTING FROM ALTERED COLOSTRAL
AND CALF FACTORS
It is well known that heat stress during the dry period affects general cow performance,
such as compromised mammary gland development prepartum (Tao et al., 2011) and lower milk
production in the subsequent lactation (Tao et al., 2011; 2012b), impairment of immune function
(do Amaral et al., 2011) and greater disease incidence (Thompson and Dahl, 2012). Additionally,
there is evidence that heat stress during late gestation causes adverse effects in the offspring.
Previous studies show that heat stress during gestation decreases uterine blood flow (Oakes et al.,
1976) placental weight (Alexander and Williams, 1971) and birth weight of the offspring (Collier
et al., 1982b; Tao et al., 2012a), which suggests compromised fetal growth. Additionally, Tao et
al. (2012a) observed decreased total plasma protein and hematocrit, compromised cellular
immune function and passive immune transfer in calves born to heat stressed cows during the
dry period relative to calves born to cooled dams. However, it is not clear if the results obtained
in that study were due to a calf effect or a colostrum effect, because the calves were fed with
colostrum from their own dam. Data on the effects of heat stress during the dry period on the
colostrum of dairy cows is limited. Nardone et al. (1997) found that high air temperature during
late pregnancy decreases the concentration of IgG and IgA, total protein, casein, -lactalbumin,
fat and lactose in the colostrum of primiparous dairy cows. Stott et al. (1976) reported that high
ambient temperatures impair colostral antibody absorption and increase mortality in the neonatal
calf. Additionally, Donovan et al. (1986) observed that serum total protein concentration is lower
for calves born during summer months in Florida. However, the impact of maternal heat stress
during late gestation on the health and postnatal growth of the calves is not well understood.
36
Therefore, the first experiment described herein was designed to evaluate the effect of maternal
heat stress during the dry period on calf specific factors related to immune response and growth
performance. The second study was performed with the objective of evaluating the isolated
effect of colostrum from cows under heat stress during the dry period on calf immune response
and postnatal growth.
Materials and Methods
Animals and Experimental Design
Experiment 1
The experiment took place at the Dairy Unit of the University of Florida (Hague, Florida)
during the period of July, 2011 to November, 2012. All the treatments and procedures received
approval from the Institutional Animal Care and Use Committee of University of Florida.
Multiparous Holstein cows were dried off 46 d before expected calving date and blocked on
mature equivalent milk production of the previous lactation, and then randomly assigned to one
of two treatments, heat stress (HT) or cooling (CL) environment. After dry off all cows were
housed in a free-stall barn that provided shade, but the stall areas for CL cows were equipped
with sprinklers and fans whereas those for HT were not (Thompson et al., 2013). Therefore, the
calves born to those cows were heat stressed (HT, n = 18) or cooled (CL, n = 18) in utero during
the final ~46 d of gestation. Three bull calves from each treatment were slaughtered by 7 d of age
for a pilot study. Thus, due to an unbalanced gender distribution between treatments, only data
from heifer calves (HT: n = 12; CL: n = 14) were used for all analyses in this experiment except
for birth weight and apparent efficiency of absorption AEA. After calving, cows were milked
and their first colostrum was stored frozen (-20°C) for the subsequent study (Figure 2-1). Also, a
sample was collected for future IgG analysis and stored frozen until the assay was performed.
Pooled colostrum from cows exposed to thermo-neutral conditions during the dry period,
37
previously collected and stored frozen, was fed to all the calves. Thus, the colostrum
composition was identical between treatment groups. For practical reasons two different pools of
colostrum were used during the experiment and were evenly distributed between both treatments.
Pooled colostrum 1 was fed to calves born during the first month of the experiment and pooled
colostrum 2 during the second month. Calves were fed 3.8 L of colostrum by esophageal feeder
within the first 4 h of birth. Day of birth was considered study d 0. After d 1 all calves were
individually housed in hutches, and water and starter (Table 2-1) were provided. Pasteurized
milk was fed twice a day according to BW, with a daily volume varying from 3.8 to 7.6 L.
Calves were weaned gradually, starting at day 42 and ending at d 49. After weaning, calves were
kept in the hutches for 10 d more before being turned out to group pens.
Experiment 2
The experiment took place at the Dairy Unit of the University of Florida (Hague, Florida)
during the period of December, 2011 to May, 2012. All the treatments and procedures received
approval from the Institutional Animal Care and Use Committee of University of Florida. The
colostrum stored from the first experiment was pooled and fed to calves born to cows that were
maintained under a thermo-neutral environment during the dry period. At birth calves were
blocked by gender and birth weight and then randomly assigned to one of two treatments. Calves
weighing 38.5 kg or more were considered heavy; calves weighing 38 kg or less were considered
light. Calves were fed pooled colostrum from cows that were heat stressed (HT, n = 17) or
cooled (CL, n = 16) during the final ~46 d of gestation. Calves were fed 3.8 L of colostrum by
esophageal feeder within the first 4 h of birth. Day of birth was considered study d 0. After d 1
all calves were managed as explained in Experiment 1.
38
Growth Measures and Blood Collection
In both studies calves were weighed at birth before colostrum feeding and then, BW and
withers height (WH) were measured every fifteen days until day 60. Preweaning BW gain and
height increase was calculated by subtracting data at birth or at d 15, respectively, from d 60
values. Blood samples were collected via jugular venipuncture at birth before colostrum feeding,
24 h after birth and at d 4, 7, 11, 14, 18, 21, 25 and 28. Sodium-heparinized tubes (Vacutainer,
Becton Dickinson, Franklin Lakes, NJ) were utilized to collect blood to determine hematocrit
and total plasma protein. Samples were placed in ice immediately after collection and parameters
were assessed within 3 h. To determine total IgG concentration, blood samples were collected
with Vacutainer tubes without anticoagulant and placed at room temperature (27oC) until clotted,
then serum was separated through centrifugation, aliquoted and stored frozen until time of assay.
Health Scoring
Health scores were recorded three times a week in the first study and daily in the second
study. Health events were evaluated based on a chart (Table 2-2) adapted from the School of
Veterinary Medicine, University of Wisconsin-Madison calf health scoring chart (found at:
http://www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_health_scoring_chart.pdf).
Respiratory score was calculated combining observed scores for cough, ear or eye (the highest
one), nasal discharge and rectal temperature. Calves were diagnosed with diarrhea when they had
a fecal score of at least 2. In the first study temperatures were measured only during the first 28 d
of age for a subset of animals (HT: n = 6; CL: n = 7).
Ovalbumin Challenge
In both studies an ovalbumin solution containing albumin from chicken egg white (0.5
mg/mL, Sigma-Aldrich, St. Louis, MO) and adjuvant Quil A (0.5 mg/mL, Accurate Chemical &
Scientific Corp., Westbury, NY) was prepared and 1 mL of the solution was injected s.c. at d 28
39
and 42 in the first study and at d 7, 28 and 42 in the second study. To determine concentrations
of IgG produced against ovalbumin, blood samples for serum were collected at d 28, 35, 42, 49
and 56 in the first study and at d 7, 18, 28, 32, 35, 39, 42, 45, 49 and 56 in the second study.
Serum was aliquoted and stored frozen until assay was performed.
IgG Analysis
For both studies, the total IgG concentration of colostrum and serum samples was
measured by the single radial immunodiffusion test (SRID, Triple J Farms, Bellingham, WA)
according to the manufacturer’s protocol. Briefly, the wells in the plates containing anti-bovine
IgG antibody in agarose gel were filled with 5 µL of diluted colostrum or serum sample and
incubated for 27 h at room temperature in the absence of light. After the end of incubation, the
diameter of the precipitin ring was measured and the total IgG concentration was calculated
based on the linear relationship between ring diameter squared and total IgG concentration. The
inter-assay CV for serum and colostrum samples was 5.6% and 3.6% in the first study, and 1.9%
and 2.7% in the second study, respectively. To calculate the AEA, a formula described by
Quigley and Drewry (1998) was used. The concentration (g/L) of total serum IgG at 24 h after
birth was multiplied by the birth weight (kg) and by 0.091 (assuming that the serum volume is
consistently 9.1% of the birth weight). The value obtained was divided by the amount of IgG (g)
fed to the calf and then multiplied by 100, giving the percentage of absorption of IgG.
The concentration of IgG anti-ovalbumin was measured by ELISA as described by
Mallard et al. (1997). Briefly, flat-bottom, 96 well high binding affinity plates (Immulon 2 HB,
Fisher, Pittsburgh, PA) were coated with sodium carbonate-bicarbonate buffer (pH 9.6)
containing 1.4 mg/mL ovalbumin and incubated at 4oC for 48 h. Following incubation, plates
were washed 4 times in a plate washer (ELX50, Biotek instruments, Inc., Winooski, VT) with
wash buffer solution (pH 7.4) containing PBS and 0.05% Tween-20 detergent solution (Fisher).
40
A blocking solution, containing PBS pH 7.4, 3% Tween-20 and 1% BSA (Sigma-Aldrich), was
then added and plate incubated for 1 h at room temperature. Plates were washed 4 times and
100µL of diluted control serum and sera samples (1:50 in washing buffer solution) was added to
the plate and incubated at room temperature for 2h. Positive and negative controls were run in
quadruplicates whereas test samples were run in duplicates placed in separate diagonal plate
quadrants. Next, plates were washed 4 times and 100µL of alkaline phosphatase conjugated
rabbit anti-bovine IgG (Sigma-Aldrich), diluted in Tris-buffer (TBS, pH 7.4, 0.05% Tween) to a
ratio of 1:38,000, was added to each well, followed by 1 h incubation at room temperature. After
an additional 4 washes, 80 µL of p-nitrophenyl phosphate liquid substrate (Sigma-Aldrich) was
added to each well and plates were incubated for 30 min at room temperature in the absence of
light. At the end of the incubation, plates were read immediately on an automatic ELISA plate
reader (MRX Revelation, Dynex Technologies Inc., Chantilly, VA) set at an absorbance of
450nm and 600nm background wavelength. The number of animals from each treatment was
balanced in each plate. The inter- and intra-assay CV were 3.5 and 8.9%, respectively. The data
is reported as the optical density (OD) of each sample divided by the OD of the positive control
from the respective plate.
Peripheral Blood Mononuclear Cell (PBMC) Isolation and Proliferation
This assay was performed only in the first study. Blood samples (30 mL) were collected
at d 7, 28, 42 and 56 (± 3 d) into sodium-heparinized tubes (Vacutainer, Becton Dickinson) and
immediately transported to the laboratory at ambient temperature. The procedure of PBMC
isolation and proliferation assessment is based on do Amaral et al. (2010). Briefly, tubes were
centrifuged at 1000 × g for 30 min at room temperature. The buffy coat was transferred to a tube
containing 2 ml of TCM-199 medium (Sigma-Aldrich) and totally mixed. The cell suspension
was carefully transferred on top of 2 ml of Fico/Lite LymphoH (Atlanta Biologicals,
41
Lawrenceville, GA) and centrifuged at 250 × g for 30 min at room temperature. Mononuclear
cells were collected from the Fico/Lite interface and washed once before the proliferation assay
began.
Concentrations of PBMC were determined using the Trypan blue dye exclusion method
and adjusted to 1×106 cells/mL by adding TCM-199 supplemented with horse serum (5%,
Atlanta Biologicals), penicillin (200 IU/mL, MP Biomedicals, Solon, OH), streptomycin (0.2
mg/mL, MP Biomedicals), glutamine (2 mM, Sigma-Aldrich) and β-mercaptoethanol (10-5
M,
Sigma-Aldrich). Adjusted PBMC suspensions were plated in triplicate (100 µL/well) and
stimulated or not with 10µg/mL of concanavalin A (ConA, 100µg/mL, Sigma-Aldrich) in a 96
well, flat-bottom sterile plate. ConA or Dulbecco’s PBS (Sigma-Aldrich) was added (20
µL/well) to the corresponding stimulated or control wells, respectively. Also, 80 µL of
supplemented TCM-199 was added to all the wells to reach a final volume of 200 µL. Plates
were incubated for 72 h at 37 0C with 5% CO2. After 48 h of incubation, 2 µL of
3[H]-Thymidine
(0.1 µCi/µL, MP Biomedicals) was added to each well. At the end of the incubation, cells were
harvested onto fiberglass filters using a cell harvester (Brandel, Gaithersburg, MD) and then read
using a liquid scintillation counter (Beckman LS6000, Inc., Fullerton, CA). Data were analyzed
based on the stimulation index (SI), which is the ratio of the average value of cpm of the
stimulated wells to the average cpm value of the control wells for each sample.
Whole Blood Proliferation
Blood samples (20 mL) were collected at d 7, 28, 42 and 56 (± 3 d) in the first study and
at d 7, 14, 28, 42 and 56 (± 3 d) in the second study and immediately transported to the
laboratory at ambient temperature. Sodium-heparinized tubes (Vacutainer, Becton Dickinson)
were used to collect the samples. Additionally, another blood sample (7 mL) was collected into
tubes containing EDTA and immediately transported under ambient temperature to be analyzed
42
for white blood cells content at the laboratory located in the Veterinary Medical Teaching
Hospital at the University of Florida. Briefly, modified RPMI (medium RPMI 1640 #11835
supplemented with 1% antibiotic-antimycotic, 100X; reagents from Gibco, Grand Island, NY)
was used to dilute blood samples. Diluted whole blood (1:15 in modified RPMI) was plated in
triplicate and stimulated or not with 5µg/mL of ConA (100µg/mL, Sigma-Aldrich) in a 96 well,
flat-bottom sterile plate. Diluted blood (100 µL/ well) was plated with 10 µL of diluted ConA
(1:9 in modified RPMI) or PBS (for control) and 90 µL of modified RPMI to reach a final
volume of 200 µL. The plate was incubated at 37oC and 5% CO2 for 72 h. After 48 h of
incubation, 10 µl of 3[H]-Thymidine (0.1 µCi/µL, MP Biomedicals) was added to each well. At
the end of the incubation, cells were harvested and read as explained for the PBMC proliferation
assay. Results were analyzed based on the number of lymphocytes and monocytes present in the
culture. Data is reported as net cpm (cpm of control wells subtracted from stimulated wells) for
each 100,000 cells.
Cortisol and Insulin Analysis
In the first study, serum samples collected at birth, 1, 4, 7, 14, 21, 28, 35, 42, 49 and 56 d
of age were analyzed for cortisol and insulin concentration. To determine cortisol concentrations
a radioimmunoassay (RIA) was performed using a commercial kit (Coat-A-Coat Cortisol Kit,
Siemens Healthcare Diagnostics, Deerfield, IL). The inter- and intra-assay CV were 5.1 and 10.4
%, respectively. The concentration of insulin was determined by RIA (Malven et al., 1987), and
the inter- and intra-assay CV were 8.3 and 5.7 %, respectively.
Statistical Analysis
Birth weight, weight at d 60, BW gain at d 60, colostrum production, colostrum IgG
concentration and AEA were analyzed by PROC GLM procedure of SAS 9.2 (SAS Institute,
Cary, NC) and least squares means ± standard error of the mean (LSM ± SEM) are reported.
43
Repeated measurements (BW and WH from d 15 to 60, health scores, hematocrit, total plasma
protein, total serum IgG, IgG anti-ovalbumin, cortisol, insulin, PBMC and Whole Blood
Proliferation) were analyzed by the PROC MIXED procedure of SAS 9.2 and LSM ± SE are
presented. The SAS model included fixed effects of treatment, time and treatment by time with
calf within the treatment as random effect.
Results
Cow Performance
Despite all the cows being exposed to similar thermal stress (temperature-humidity index
~77) during the dry period, the cows from HT treatment had greater rectal temperature both in
the morning (38.6 ± 0.04 vs. 38.3 ± 0.04 °C; P = 0.01) and in the afternoon (39.9 ± 0.05 vs. 39.4
± 0.05 °C; P < 0.01) and greater respiration rate (78.7 ± 2.4 vs. 45.0 ± 2.3 breaths per min; P <
0.01) compared with those in the cooling system (Thompson et al., unpublished). CL cows also
consumed more (P < 0.01) feed prepartum but not postpartum, gained more BW prepartum (P <
0.01), but lost more BW in lactation (P = 0.05), had greater (P = 0.01) BCS score prepartum and
a lower (P < 0.10) BCS postpartum (Thompson et al., 2012). Additionally, HT cows had 6 d
shorter gestation length (270.1 ± 1.3 vs. 276.1 ± 1.3 d; P < 0.01) and consequently shorter dry
period length (34.8 ± 1.4 vs. 39.8 ± 1.4 d; P = 0.01) compared with CL cows (Thompson et al.,
unpublished). Cows from CL treatment produced more milk during the first 15 wks in lactation
compared to HT cows (38.8 vs. 33.6 kg/d; P < 0.07), but milk composition did not differ
(Thompson et al., 2012). Moreover, plasma prolactin concentrations were greater for HT cows
compared with CL cows (21.0 ± 1.6 vs. 13.8 ± 1.6 ng/mL; P < 0.01) (Thompson et al., 2013).
Colostrum IgG Concentration
In the first study no differences were observed in the amount of colostrum produced by
cows from each treatment group (HT: 7.2 ± 1.0 vs. CL: 6.4 ± 1.1 L; P = 0.52), IgG concentration
44
in colostrum (HT: 86.8 ± 7.2 vs. CL: 94.7 ± 7.7 g/L; P = 0.46) and total IgG produced (HT:
627.8 ± 88.9 vs. CL: 549.2 ± 91.6 g; P = 0.54). However, among cows cooled during the dry
period only one (6.25%) produced colostrum with less than 50g/L of IgG content, whereas
among cows heat stressed in utero three (16.7%) produced colostrum with less than 50g/L. On
the other hand, 43.8% of the CL cows produced less than 4L of colostrum at first milking against
22.2% among HT cows.
In the first study, the IgG concentrations in the pools of colostrum 1 and 2 were 108.8
and 109.9 g/L, providing 411.1 and 413.3 g of IgG for the calves, respectively. In the second
study, the pooled colostrum from heat stressed or cooled cows had an IgG concentration of 109.4
and 96.3 g/L providing to the calves 413.7 and 364.1 g of IgG, respectively. In both studies
calves were fed between 1 and 4 h after calving. The average time between calving and
colostrum feeding was 2.5 h in the first study and 2.7 h in the second study, for both treatments.
Growth Performance
Calves heat stressed in utero weighed less (P < 0.01) at birth and at weaning than cooled
calves (Table 2-3). Additionally, calves cooled in utero were taller (P < 0.01) and heavier (P <
0.01) during the pre-weaning period than the calves under heat stress in utero (Figure 2-2), but
no differences in weight gain or height increase were observed from birth up to d 60 (Table 2-3).
In the second study, calves from both treatments had similar birth weight, as expected
(Table 2-4). Additionally, calves that received colostrum from HT cows did not differ in growth
performance compared to calves that received colostrum from CL cows, so both treatment
groups were weaned at similar BW and WH (Table 2-4).
Health Scores and White Blood Cells Count
In the first study no differences were observed between treatments for incidence of
diarrhea, but calves born to HT cows had more cases of significant (i.e. ≥ 4) respiratory score
45
(46.7 vs. 13.3 %). When analyzing only temperature, calves born to HT cows had a tendency (P
= 0.1) for overall increased rectal temperature during the first 28 d of life (39.1 ± 0.07 vs. 38.9 ±
0.06 °C) and HT calves had higher (P < 0.05) rectal temperature during the second week of life
(39.3 ± 0.1 vs. 38.9 ± 0.1 °C). Regarding WBC count, no effect of treatment was observed. The
average lymphocyte and monocyte count for HT and CL calves were 3,695 vs. 3,833 cells/µL (P
= 0.65) and 528 vs. 520 cells/µL (P = 0.69), respectively.
In the second study the incidence and severity of diarrhea was similar during the first
month of life, but during the second month a higher number of animals were stricken by severe
cases of diarrhea (score = 3) among HT calves (76.5 vs. 56.3%). Calves fed colostrum from HT
cows had more cases of high (i.e. ≥ 5) respiratory score during the first month of life (70.6 vs.
43.8%). Overall temperature was higher (P < 0.05) for HT calves compared to CL calves (38.9 ±
0.03 vs. 38.8 ± 0.04 °C).
Hematocrit, Total Plasma Protein and Serum IgG Concentration
In the first study, treatments did not affect the hematocrit (P = 0.9) and total plasma
protein (P = 0.34) from birth to d 35 (Figure 2-3). Total IgG concentration in serum was also not
affected (P = 0.66) by heat stress (Figure 2-4). Despite the lack of difference in the total serum
IgG concentration between treatments, calves exposed to HT in utero had lower AEA (P < 0.05)
compared to calves cooled in utero (26.0 ± 1.4 vs. 30.2 ± 1.4 %). There was an effect (P < 0.05)
of the pool of colostrum in the IgG concentration and AEA, as calves that received pooled
colostrum 1 had higher IgG concentration and AEA than those that received pooled colostrum 2
(2311.0 ± 87.7 vs. 1923.4 ± 89.3 mg/dL and 31.4 ± 1.4 vs. 24.9 ± 1.4 %, respectively). However,
there was no interaction between treatment and pooled colostrum in the IgG (P = 0.25) and AEA
(P = 0.96) analysis.
46
In the second study, treatments also did not affect the hematocrit (P = 0.46) and total
plasma protein (P = 0.68) from birth to d 56 (Figure 2-5). IgG concentration tended (P = 0.08) to
be lower in the calves that received colostrum from CL cows compared to those that received
colostrum from HT cows (Figure 2-6), but no treatment effect (P = 0.95) was observed for AEA
(HS: 27.5 ± 1.6 vs. CL: 27.6 ± 1.8 %). In both studies there was no case of failure of passive
immune transfer (i.e. total serum IgG concentration lower than 10 g/L).
Ovalbumin Challenge Response
In the first study there was no difference (P = 0.51) in the response to the ovalbumin
challenge, as IgG production was similar between treatment groups (Figure 2-7). In the second
study there was also no treatment effect in the production of IgG against ovalbumin (P = 0.92;
Figure 2-8). However, there was a treatment x time interaction, such as the calves that received
the pooled colostrum from CL cows had a greater response at d 28 (P < 0.001), before the second
injection, compared to calves that received colostrum from HT cows. In contrast, calves that
received colostrum from HT cows had a greater (P < 0.05) response at d 42, 49 and 56 compared
to calves that received colostrum from CL cows.
Serum Cortisol and Insulin
Overall serum cortisol concentration during the preweaning period was higher (P < 0.05)
for calves born to CL cows compared to those born to HT cows (1.62 ± 0.11 vs. 1.25 ± 0.12
µg/dL; Figure 2-9). Differences were observed at d 4 (1.67 ± 0.27 vs. 0.74 ± 0.29; P < 0.05), d 7
(1.91 ± 0.33 vs. 0.97 ± 0.35; P < 0.1) and d 14 (0.51 ± 0.12 vs. 0.16 ± 0.13; P < 0.1). Heat stress
during the dry period did not affect insulin concentration (3.09 ± 0.68 vs. 2.81 ± 0.63 ng/mL,
respectively; P = 0.75) of calves compared with cooling (Figure 2-10).
47
PBMC and Whole Blood Proliferation
In the first study, no treatment effect (P = 0.3) was observed for PBMC proliferation
(HT: 66.6 ± 31.1 vs. CL: 98.0 ± 33.2 fold; Figure 2-11). Regardless of treatment, a time effect (P
< 0.05) was observed; calves had greater PBMC proliferation at d 7 than at other time points.
There was no effect of treatment (P = 0.99) on whole blood proliferation (HT: 129,289 ± 21,101
vs. CL: 149,433 ± 18,488 net cpm/100,000 cells), but HT calves had a greater response at d 7 (P
< 0.05) compared with CL calves (124,822 ± 50,102 vs. 45,118 ± 44,355 net cpm/100,000 cells,
respectively) and CL calves had a greater (P < 0.01) response at d 42 compared with HT calves
(282,854 ± 34,882 vs. 105,035 ± 44,039 net cpm/100,000 cells, respectively; Figure 2-12). In the
second study no difference (P = 0.49) was observed for whole blood proliferation between calves
that received colostrum from HT cows or from CL cows (190,029 ± 42,112 vs. 234,668 ± 41,209
net cpm/100,000 cells, respectively; Figure 2-13).
Discussion
The effectiveness of the evaporative cooling system utilized in the first study for the dry
cows can be verified by the lower rectal temperature and respiration rate observed in CL cows
when compared to those cows without the cooling system, despite being exposed to a similar
THI (do Amaral et al., 2011; Tao et al., 2012b). As described in other studies, HT cows had
lower DMI and BW gain during dry period relative to CL cows (Tao et al., 2011), shorter dry
period and gestation length (do Amaral et al., 2009, 2011; Tao et al., 2011), lower calf weight at
birth (Adin et al., 2009; do Amaral et al., 2011), greater prolactin concentrations in plasma (do
Amaral et al., 2010, 2011) and lower milk production in the next lactation (do Amaral et al.,
2009; Tao et al., 2011). All these observations provide evidence about the success of the heat
stress model in the present experiment, thus it was appropriate to investigate the effects of heat
stress or cooling during the dry period on factors related to calf and colostrum.
48
As demonstrated in other studies with dairy cows (Collier et al., 1982b; Tao et al., 2012a)
and sheep (Brown et al., 1977), newborns from HT dams were lighter at birth compared with
those from CL dams. This compromised fetal growth in calves from HT dams can be explained
by several factors, such as the shorter gestation length and decreased DMI during the dry period.
As discussed by Tao et al. (2012a), the shorter gestation length does not explain all the difference
observed in birth weight between treatments. In the present study, the gestation length was 6 d
shorter in HT dams and their calves were 4.8 kg lighter at birth. Considering that a fetus of a
Holstein dairy cow gains about 0.5 kg/d during the last week of gestation (Muller et al., 1975), in
the current study the shorter gestation length observed in HT cow accounts for about 62.5 % of
the lower birth weight of their calves. The reason for the additional reduction in body weight is
likely to be related to the overall effect of heat stress during the whole dry period rather than only
during the last week of gestation. Inadequate maternal nutrition is one of the major factors that
impair fetal growth (Wu et al., 2006). A study with Hereford cows showed that sub-maintenance
levels of nutrition during the last trimester reduced calf birth weight (Tudor, 1972). However, the
animals in that experiment were severely energy restricted and cannot be compared with the
animals in the present study, and other studies in which the decrease in DMI in heat-stressed
cows is only about 10 to 15 % (Adin et al., 2009; do Amaral et al., 2009; Tao et al., 2011).
Another study with beef cattle restricted intake for both protein and energy by 43% of NRC
requirements during the last 90 d of gestation and did not report any effect on gestation length,
birth weight or dystocia score (Hough et al., 1990).
The nutrient uptake by the fetus can be determined by both blood flow rate and nutrient
concentration in the arterial and venous blood (Reynolds et al., 2006). It is known that heat stress
during gestation decreases utero-placental blood flow and also reduces placental and fetal growth
49
(Reynolds et al., 2005), which is associated with decreased placental transport of oxygen and
nutrients from the dam to the fetus (Wallace et al., 2002). Thus, thermal stress per se leads to
intrauterine growth restriction (IUGR) in animals (Reynolds et al., 1985, 2005; Wallace et al.,
2005), which may account for the majority of the decreased birth weight in HT calves relative to
CL calves. In contrast to other studies (Nardone et al., 1997; Adin et al., 2009), in the present
study there was no effect of heat stress during the dry period on colostrum IgG concentration.
However, the result in the present study is in accordance with that found in Tao et al. (2012a).
The observed lower BW and WH in HT calves compared to CL calves during the first 2
months can be interpreted as a consequence of the lower birth weight, because no differences in
weight gain or height increase were observed until this age. The same result was obtained in a
previous study (Tao et al., 2012a). In the second study the lack of difference in birth weight
between treatments proves the efficacy of the blocks, so we can ensure that any treatment effect
was not influenced by differences in birth weight. Because treatment did not have an effect on
BW and WH up to d 60, we conclude that colostrum from heat stressed dams did not impair
preweaning growth compared with colostrum from cooled dams.
In the first study no differences in hematocrit, total plasma protein and total serum IgG
concentration was observed during the first month of age, as opposed to a previous study in
which dairy calves heat stressed in utero during the dry period had lower hematocrit levels at
birth before colostrum feeding and lower total plasma protein and total serum IgG after 1 d of
age and during the following 28 d, when compared to calves cooled in utero (Tao et al., 2012a).
In that study calves received fresh colostrum from their own dams, instead of pooled colostrum,
which may explain the differences in total plasma protein and total IgG after colostrum was fed.
Because colostrum was previously frozen in the present study, whole live cells were absent, but
50
this does not affect IgG availability and absorption by the calf (Holloway et al., 2001). It was
demonstrated that maternal leukocytes in colostrum are absorbed by the calf at the small intestine
and stay in the peripheral circulation for about 24 h (Reber et al., 2006), during which they home
to both neonatal peripheral non-lymphoid and secondary lymphoid tissues (Aldridge et al.,
1998). Because live whole cells are not present in frozen colostrum, the calves in the present
study did not have the contribution from colostral cells to enhance overall protein concentration
in plasma, such as immunoglobulins produced by B cells. Clearly, the difference in hematocrit at
birth observed in Tao et al. (2012a) is not related to the type of colostrum fed. The authors
hypothesized that the lower hematocrit in HT calves was a postnatal adaptation to the
intrauterine hypoxia. Additionally, the lower total plasma protein and hematocrit found in that
study during the first month of age of HT calves could be a result of a complementary effect
between colostrum from heat stressed cows and calves heat stressed in utero. In the second
study, when calves born to cows under thermo-neutral conditions were fed frozen pooled
colostrum from either heat stressed or cooled cows, no differences in hematocrit or total plasma
protein was observed during the first month of age. These data suggest that frozen colostrum
from HT cows per se does not seem to affect those hematological parameters.
Passive immune transfer was compromised in calves born to heat stressed cows, as
verified by their lower apparent efficiency of IgG absorption (AEA) compared with those calves
born to cooled cows. The same effect was observed in the study by Tao et al. (2012a), however
in the present study HT and CL calves received the same colostrum, eliminating a possible
influence of colostrum from HT cows on the efficiency of IgG absorption. One is not able to
conclude from this experiment if the lower absorption is due to a decrease in the ability of the
small intestine to absorb Ig or due to its smaller contact surface area, as a consequence of the
51
lower birth weight of those calves. There are studies showing the negative effect of
environmental heat stress on the serum IgG concentration and efficiency of absorption, however
data relating heat stress prepartum to passive immune transfer in dairy calves is limited. A study
designed in sows (Machado-Neto et al., 1987) observed a lower IgG concentration in the serum
of piglets from heat-stressed sows during late gestation, but the concentration of IgG tended to be
lower in the colostrum from heat-stressed sows. Thus, the mechanisms involved in the impaired
IgG absorption by heat stress in utero are not clear. In the second study, serum total IgG
concentration tended to differ between treatments. Part of this can be explained by the difference
in IgG concentration between the pooled colostrum fed to each treatment (i.e. colostrum from CL
cows provided about 50g less IgG to the calves than did colostrum from HT cows). However,
when AEA is analyzed, there is no difference between treatments, so in fact frozen colostrum
from heat stressed cows during the dry period per se does not impair the uptake of IgG by the
small intestine in calves when compared with colostrum from CL cows.
Heat stress in utero during late gestation did not affect PBMC proliferation during the
preweaning period when compared with cooling in utero, which contrasts with results obtained
by Tao et al. (2012a). Transfer of maternal cells with colostrum has been demonstrated to
enhance lymphocyte development in calves (Reber et al., 2008b). The colostrum fed to calves in
the present study was previously frozen, hence it lacked viable maternal cells, which may explain
why we did not observe differences in PBMC proliferation. However, despite no differences in
the PBMC assay, CL calves had a greater response at d 42 in the whole blood proliferation assay.
The reason of the greater response at this time point is unknown, but may be related to the timing
of the ovalbumin challenge, which started at d 28, so by d 42 there were more activated
lymphocytes in the peripheral circulation of the calves and it is possible that lymphocytes from
52
CL calves had greater proliferative activity at that time compared to those from HT calves. On
the other hand, HT calves had a greater proliferation at d 7. In the second study only whole blood
proliferation was performed because it has been shown that they give similar results when
compared with the PBMC proliferation assay (de Groote et al., 1992; Yaqoob et al., 1999). The
lack of difference between treatments for whole blood proliferation suggests that colostrum from
HT cows previously frozen does not seem to affect cellular immunity of calves during the
preweaning period when compared to colostrum from CL cows.
The ovalbumin challenge performed in the first study led to the production of similar
amounts of IgG antiovalbumin by both treatment groups, suggesting that heat stress in utero does
not impair the humoral immune response in calves from 28 d of age to weaning, as previously
reported by Tao et al. (2012a). Interestingly, in the second study, in which the ovalbumin
challenge started at d 7, calves that received colostrum from CL cows responded to the first
injection of ovalbumin, whereas those that received colostrum from HT cows only produced IgG
antiovalbumin after the second injection. There is some data indicating that transfer of maternal
cells with colostrum accelerates the development of lymphocytes in calves. Reber et al. (2008b)
demonstrated that calves fed whole colostrum had more active lymphocytes at d 7 after birth that
expressed more MHC class I compared with lymphocytes from calves fed cell-free colostrum.
However, in the present study, where maternally derived immune cells were lacking, the factors
in the colostrum that could enhance lymphocyte development are unknown.
Calves from CL dams had greater overall serum cortisol concentration, different from
what was observed in Tao et al. (2012a), wherein differences were observed only at birth. In that
study differences were attributed to a higher sensitivity to the stress of birth. In fact, in Tao et al.
(2012a) the difference in birth weight between HT and CL calves was greater (~6 kg vs. ~5 kg in
53
the present study) and the average birth weight of HT calves was 1.8 kg lower than in the present
study. This may explain the lower cortisol concentration at birth in that study and lack of
statistical difference in the present experiment, where differences were observed only after 4
days of age. In the current study, the higher cortisol levels detected in CL calves during the first
two weeks of age is intriguing, as the animals were managed in the same way after birth. Higher
levels of cortisol in the neonate are necessary to enhance colostral absorption (Sangild, 2003)
and conditions such as cold stress and dystocia inhibit the release of cortisol by the neonate and
decrease colostral absorption (Chase et al., 2008). Thus, the lower cortisol concentrations in HT
calves may have contributed to the impaired passive immune transfer observed in those calves.
Different from what Tao and Dahl (2013) observed, there was no effect of heat stress in utero on
insulin concentration. In that study HT calves had higher insulin concentration relative to CL
calves at d 1 after birth. The authors ascribed the difference to enhanced pancreatic insulin
secretion or impaired insulin clearance rather than an effect of diet. However, in the present
study in which the colostrum was the same for both treatments, no treatment effect was
observed.
Conclusions
Maternal heat stress during late gestation decreased calf birth weight and weaning weight
and compromised the passive IgG transfer regardless of colostrum source. Feeding colostrum
from heat stressed cows during the dry period does not impact AEA or growth performance
during the preweaning period, but seems to impair humoral immune response during the first
month of life. Further studies are necessary in order to understand the effects of heat stress in
utero, such as those observed in this experiment; mechanism of heat stress induced intrauterine
growth retardation, lower apparent efficiency of IgG absorption and impaired humoral immune
response by feeding colostrum from HT cows.
54
Table 2-1. Chemical composition of starter1
Chemical composition % DM
Crude protein (min) 18.00
Crude fat (min) 3.25
Crude fiber (max) 8.00
Acid detergent fiber (ADF) (max) 10.00
Calcium (Ca) (min - max) 0.8 – 1.2
Phosporus (P) (min) 0.64
Salt (NaCl) (min - max) 0.4 – 0.9
Selenium (Se) (min) 0.33 PPM
Vitamin A (min) 13,245 IU/kg
Vitamin D3 (min) 5,519 IU/kg
Vitamin E (min) 26 IU/kg 1 Purina
® calf starter (Purina Mills, LLC, St. Louis, MO)
Table 2-2. Calf health scoring criteria1
Score
Clinical sign 0 1 2 3
Cough None Induce single
cough
Induced repeated
coughs or
occasional
spontaneous
cough
Repeated
spontaneous
coughs
Nasal
discharge
Normal serous
discharge
Small amount of
unilateral cloudy
discharge
Bilateral, cloudy,
or excessive
mucus discharge
Copious bilateral
mucopurulent
discharge
Eye scores Normal Small amount of
ocular discharge
Moderate amount
of bilateral
discharge
Heavy ocular
discharge
Ear scores Normal Ear flick or head
shake
Slight unilateral
droop
Head tilt or
bilateral droop
Fecal Scores Normal Semi-formed,
pasty
Loose, but stays
on top of bedding
Watery, sifts
through bedding
Rectal
temperarure
(°C)
37.8 – 38.3 38.4 – 38.8 38.9 – 39.4 ≥ 39.5
1 Adapted from the School of Veterinary Medicine, University of Wisconsin-Madison calf health
scoring chart (found at:
http://www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_health_scoring_chart.pdf).
55
Table 2-3. Growth performance of calves born to cows exposed to either heat stress or cooling
during the dry period
Parameter Heat Stress
LSM ± SE
Cooling
LSM ± SE
P-value
Birth Weight (kg) 38.3 ± 1.1 43.1 ± 1.2 < 0.01
Weaning Weight (kg)1 67.0 ± 2.5 76.0 ± 2.4 < 0.01
Preweaning BW Gain (kg)2 30.4 ± 2.0 34.2 ± 1.9 0.17
Avg. Daily Gain (kg/d) 0.51 ± 0.15 0.57 ± 0.08 0.3
Weaning Withers Height (cm)1 83.1 ± 0.7 85.7 ± 0.6 < 0.01
Preweaning Height Increase (cm)2 5.7 ± 0.9 6.0 ± 0.9 0.84
1Weaning weight and weaning height were measured at d 60 of age.
2Preweaning BW gain and height increase was calculated by individually subtracting data at d 60
of age by data at birth or at d 15 of age, respectively.
Table 2-4. Growth performance of calves born to cows under thermoneutral conditions during
the dry period and fed frozen colostrum from cows exposed to either heat stress or
cooling during the dry period
Parameter Heat Stress
LSM ± SE
Cooling
LSM ± SE
P-value
Birth Weight (kg) 38.8 ± 1.4 39.2 ± 1.5 0.8
Weaning Weight (kg)1 68.4 ± 2.5 64.8 ± 2.6 0.4
Preweaning BW Gain (kg)2 29.6 ± 2.3 25.6 ± 2.4 0.3
Avg. Daily Gain (kg/d) 0.49 ± 0.7 0.43 ± 0.8 0.2
Weaning Withers Height (cm)1 84.3 ± 0.8 83.0 ± 0.9 0.4
Preweaning Height Increase (cm)2 7.8 ± 1.1 6.2 ± 1.0 0.3
1Weaning weight and weaning height were measured at d 60 of age.
2Preweaning BW gain and height increase was calculated by individually subtracting data at d 60
of age by data at birth.
56
Figure 2-1. Schematic description of experiments 1 and 2. In experiment 1 multiparous Holstein
cows were dried off 46 d before expected calving date and randomly assigned to one
of two treatments, heat stress (HT) or cooling (CL) environment. Therefore, the
calves born to those cows were heat stressed (calves I) or cooled (calves II) in utero
during the final ~46 d of gestation. After calving, cows were milked and their first
colostrum was stored frozen (-20°C) for the subsequent study. Pooled colostrum from
cows exposed to thermoneutral conditions during the dry period, previously collected
and stored frozen, was fed to all the calves. Thus, the colostrum composition was
identical between treatment groups. In experiment 2 the colostrum stored from the
first experiment was pooled and fed to calves born to cows that were under a
thermoneutral environment during the dry period. Therefore, calves were fed a pooled
colostrum from cows that were heat stressed (pooled colostrum I) or cooled (pooled
colostrum II) during the final ~46 d of gestation.
57
Figure 2-2. Effect of heat stress or cooling during the dry period on pre-weaning growth
performance of the offspring. Solid bars represent calves born to cows exposed to
cooling during the dry period and open bars represent those born to cows in heat
stress. Calves born to cows exposed to cooling were heavier (P < 0.01) and taller (P <
0.01) during the pre-weaning period (58.1 ± 1.3 vs. 51.9 ± 1.4 kg and 82.85 ± 0.5 vs.
80.4 ± 0.6 cm, respectively) compared to those born to heat stressed cows.
58
Figure 2-3. Effect of heat stress or cooling during the dry period on hematocrit and total plasma
protein of the offspring during the first 35 d of age. Solid diamonds (♦) represent
calves born to cows exposed to cooling during the dry period and open squares (□)
represent those born to cows in heat stress. Heat stress during the dry period did not
affect (P = 0.9) the hematocrit (26.8 ± 1.3 vs. 26.6 ± 1.4 %, respectively) and also did
not affect (P = 0.34) total plasma protein (5.43 ± 0.06 vs. 5.51 ± 0.06 g/dL,
respectively) of calves compared with cooling.
59
Figure 2-4. Effect of heat stress or cooling during the dry period on total serum IgG
concentrations of the offspring during the first 28 d of age. Solid diamonds (♦)
represent calves born to cows exposed to cooling during the dry period and open
squares (□) represent those born to cows in heat stress. Heat stress during the dry
period did not affect (P = 0.66) the total serum IgG concentration (2,089.2 ± 89.6 vs.
2,145.21 ± 89.6, respectively) of calves compared with cooling.
60
Figure 2-5. Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on hematocrit and total plasma protein of calves during the first 56 d of age. Calves
were born to cows exposed to thermo neutral conditions during the dry period. Solid
diamonds (♦) represent calves fed colostrum from cows exposed to cooling during the
dry period and open squares (□) represent those fed colostrum from heat stressed
cows. Feeding colostrum from heat stressed cows did not affect (P = 0.46) the
hematocrit (26.5 ± 1.2 vs. 27.7 ± 1.3 %, respectively) and also did not affect (P =
0.68) total plasma protein (5.82 ± 0.08 vs. 5.79 ± 0.08 g/dL, respectively) of calves
compared with colostrum from cooled cows.
61
Figure 2-6. Effect of feeding colostrum from cows heat stressed or cooled during the dry period
on total serum IgG concentration of calves during the first week of age. Calves were
born to cows exposed to thermoneutral conditions during the dry period. Solid
diamonds (♦) represent calves fed colostrum from cows exposed to cooling during the
dry period and open squares (□) represent those fed colostrum from heat stressed
cows. Feeding colostrum from heat stressed cows tended (P = 0.08) to increase total
serum IgG concentration (2,688.9 ± 133.3 vs. 2,399.6 ± 142.3 mg/dL) of calves
compared with colostrum from cooled cows.
62
Figure 2-7. Effect of heat stress or cooling during the dry period on the response of the offspring
to ovalbumin challenge. One milliliter of ovalbumin solution containing albumin
from chicken egg white (0.5 mg/mL) was injected s.c. at d 28 and 42 of age. Solid
diamonds (♦) represent calves born to cows exposed to cooling (CL) during the dry
period and open squares (□) represent those born to cows in heat stress (HT). HT
calves had similar (P = 0.51) IgG antiovalbumin production relative to CL calves
(0.40 ± 0.02 vs. 0.42 ± 0.02 optical density (OD), respectively).
63
Figure 2-8. Effect of feeding colostrum from cows heat stressed (HT) or cooled (CL) during the
dry period on the response of calves to ovalbumin challenge. Calves were born to
cows exposed to thermo neutral conditions during the dry period. One milliliter of
ovalbumin solution containing albumin from chicken egg white (0.5 mg/mL) was
injected s.c. at d 7, 28 and 42 of age. Solid diamonds (♦) represent calves fed
colostrum from cows exposed to cooling during the dry period and open squares (□)
represent those fed colostrum from heat stressed cows. Results are expressed in
optical density (OD). No treatment effect (P = 0.92) was observed on the overall
production of IgG against ovalbumin (HS: 0.61 ± 0.02 vs. CL: 0.61 ± 0.02 OD).
However, production of IgG antiovabumin was greater (P < 0.001) for CL calves at d
28 (0.45 ± 0.03 vs. 0.12 ± 0.03 OD). In the other hand, HT calves had a greater (P <
0.05) response at d 42 (0.87 ± 0.03 vs. 0.75 ± 0.03), 49 (0.92 ± 0.03 vs. 0.82 ± 0.03)
and 56 (0.95 ± 0.03 vs. 0.84 ± 0.03) compared to calves that received colostrum from
CL cows. * * * P < 0.001; * P < 0.05; † P < 0.10.
64
Figure 2-9. Effect of heat stress or cooling during the dry period on serum cortisol
concentrations of the offspring during preweaning period. Solid diamonds (♦)
represent calves born to cows exposed to cooling during the dry period and open
squares (□) represent those born to cows in heat stress. Heat stress during the dry
period decreased (P < 0.05) overall cortisol concentration (1.25 ± 0.12 vs. 1.62 ± 0.11
µg/dL, respectively) of calves compared with cooling. * P < 0.05; † P < 0.10.
65
Figure 2-10. Effect of heat stress or cooling during the dry period on serum insulin
concentrations of the offspring during preweaning period. Solid diamonds (♦)
represent calves born to cows exposed to cooling during the dry period and open
squares (□) represent those born to cows in heat stress. Heat stress during the dry
period did not affect (P = 0.75) insulin concentration (3.09 ± 0.68 vs. 2.81 ± 0.63
ng/mL, respectively) of calves compared with cooling.
66
Figure 2-11. Effect of heat stress or cooling during the dry period on the peripheral blood
mononuclear cell (PBMC) proliferation of the offspring during the preweaning
period. Solid bars represent calves born to cows exposed to cooling during the dry
period and open bars represent those born to cows in heat stress. No treatment effect
(P = 0.3) was observed (HT: 66.6 ± 31.1 vs. CL: 98.0 ± 33.2 fold). Regardless
treatment, calves had greater (P < 0.05) stimulation index at d 7 compared with other
time points.
67
Figure 2-12. Effect of heat stress (HT) or cooling (CL) during the dry period on whole blood
(WB) proliferation of the offspring during preweaning period. Solid bars represent
calves born to cows exposed to CL during the dry period and open bars represent
those born to HT cows. An effect of treatment was not observed (P = 0.99) for the
overall WB proliferation (HT: 129,289 ± 21,101 vs. CL: 149,433 ± 18,488 net cpm/
100,000 cells), but HT calves had a greater response at d 7 compared with CL calves
(124,822 ± 50,102 vs. 45,118 ± 44,355 net cpm/100,000 cells, respectively) and CL
calves had a greater response at d 42 compared with HT calves (282,854 ± 34,882 vs.
105,035 ± 44,039 net cpm/100,000 cells, respectively). * * P < 0.01; * P < 0.05.
68
Figure 2-13. Effect of feeding colostrum from cows heat stressed (HT) or cooled (CL) during
the dry period on whole blood proliferation of the offspring during preweaning
period. Calves were born to cows exposed to thermo neutral conditions during the dry
period. Solid bars represent calves fed colostrum from CL cows and open bars
represent those fed colostrum from HT cows. No difference (P = 0.49) between
treatments was observed (190,029 ± 42,112 vs. 234,668 ± 41,209 net cpm/100,000
cells, respectively).
69
CHAPTER 3
EFFECT OF HEAT STRESS IN UTERO ON CALF PERFORMANCE AND HEALTH
THROUGH THE FIRST LACTATION
Calves born to cows exposed to heat stress during the dry period have lower birth weight
(Collier et al., 1982b; Tao et al., 2012a), weaning weight and compromised passive immune
transfer (Tao et al., 2012a) compared with those born to dams that are cooled. Previous studies
show that heat stress during gestation decreases uterine blood flow (Oakes et al., 1976) and
placental weight (Alexander and Williams, 1971) suggesting an impairment of fetal growth.
Additionally, Tao et al. (2012a) observed decreased total plasma protein and hematocrit,
compromised cellular immune function and passive immune transfer in calves born to heat
stressed cows during the dry period relative to calves born to cooled dams.
However, the impact of heat stress in utero during late gestation on future performance of
the heifer related to milk production and reproduction is still not known. This study was designed
to investigate possible carryover effects of maternal heat stress during late gestation on growth,
health, fertility and milk production in the first-lactation.
Materials and Methods
Animals and Data Collection
Data of animals obtained from five previous experiments conducted during five
consecutive summers were pooled and analyzed. The experiments took place at the Dairy Unit of
the University of Florida (Hague, Florida) during the period of 2007 to 2011. Multiparous
Holstein cows were dried off ~46 d before expected calving date and blocked on mature
equivalent milk production of the previous lactation, and then randomly assigned to one of two
treatments, heat stress (HT) or cooling (CL) environment. After dry off CL cows were housed in
a free-stall barn with sprinklers, fans and shade, whereas only shade was provided to HT cows.
70
Therefore, the calves born to those cows were heat stressed or cooled in utero during the final
~46 d of gestation. Within 4 hours after birth, 3.8 L of colostrum was fed to calves from both
groups of cows. All calves were individually housed in hutches and water and starter was
provided. Pasteurized milk was fed twice a day according to BW, with a daily volume varying
from 3.8 to 7.6 L. Calves were weaned gradually by reducing the volume of milk fed, starting at
day 42 and ending at d 49. After weaning, calves were kept in the hutches for more 10 d before
being turned out to group pens. Observations for the current study were collected from
AfiFarmTM
Herd Management records. Birth weight and pre-weaning mortality of 146 calves
(HT=74; CL=72) and body weight and growth rate from 72 heifers (HT=34; CL=38) were
analyzed. Additionally, fertility and milk production in the first lactation from 38 heifers
(HT=17; CL=21) were analyzed. Most of the calves were from artificial insemination (AI), but
some of them were conceived by in vitro fertilization (IVF) and those were evenly distributed
among treatments (HT = 17; CL = 13).
Statistical Analysis
Birth weight, BW gain at 12 months of age, BCS at calving, number of services per
conception, age at first AI, age at first pregnancy and first parturition were analyzed by PROC
GLM procedure of SAS 9.2 (SAS Institute, Cary, NC) and least squares means ± standard error
of the mean (LSM ± SEM) are reported. Repeated measurements (BW and WH up to 12 mo of
age and during lactation, milk production and milk composition) were analyzed by the PROC
MIXED procedure of SAS 9.2 and LSM ± SEM are presented. The SAS model included fixed
effects of treatment, year, time and treatment by time with calf within the treatment as random
effect.
71
Results
Growth Performance
HT calves were lighter (P < 0.001) at birth than CL calves (39.1 ± 0.7 vs. 44.8 ± 0.7 kg).
Additionally, CL heifers were heavier (200.2 ± 3.4 vs. 190.9 ± 3.7 kg; P < 0.05; Figure 3-1) and
taller (111.8 ± 0.6 vs. 110.0 ± 0.7; P = 0.03) up to one year old, but had similar (P = 0.44) weight
gain from birth to one year old (305.8 ± 5.9 vs. 299.1 ± 6.3 kg) compared with HT heifers.
Mortality
The DOA (dead on arrival) rate was 4.1% among HT calves, whereas there was no DOA
cases among CL calves. Mortality was higher for HT calves when compared to CL calves (Table
3-1). Among bull calves, 10% of HT calves died before 4 months of age, against only 3.2% of
CL calves. However, most of bull calves were sold between birth and 5 months of age, therefore
there is not a true mortality rate for bull calves. Among heifer calves the difference was even
higher, as 20.5% of the HT calves left the herd before puberty due to death, sickness or growth
retardation, versus only 4.9% of the CL calves. Moreover, 77.8 % of CL heifers completed the
first lactation, compared with 53.1% of HT heifers.
The more common reasons for death or euthanasia before one month of age were
septicemia, navel infection, growth retardation, diarrhea and pneumonia. There was a negative
interaction between IVF calves and HT treatment, as 66 % of the HT heifers that left the herd
before puberty due to death, sickness or growth retardation, were IVF calves and most of them
were less than one month old. There were three cases of euthanasia or culling motivated by
growth retardation before puberty, all in the HT treatment. Only one of these calves was from
IVF.
72
Reproductive Performance and Milk Production
No differences were observed between CL heifers and HT heifers for age at first AI (13.3
± 0.3 vs. 13.5 ± 0.3 months, respectively; P = 0.63) or age at first parturition (24.2 ± 0.5 vs. 24.9
± 0.5 months, respectively; P = 0.32), but the number of services until first pregnancy was
confirmed was greater (P = 0.03) for HT heifers than for CL heifers (2.6 ± 0.3 vs. 1.8 ± 0.3, P =
0.03). Age at first pregnancy tended (P = 0.06) to be greater for HT calves compared to CL
calves (15.8 ± 0.5 vs. 14.5 ± 0.4 mo). Reproductive data is summarized in Table 3-2. At calving
HT and CL heifers had similar body condition score (3.47 ± 0.13 vs. 3.48 ± 0.17, respectively).
Compared with CL heifer, HT heifers tended (P = 0.11) to produce less milk up to 30
weeks of the first lactation (26.4 ± 2.1 vs. 30.9 ± 1.7 kg), but no difference in protein, fat and
somatic count cell count (SCC) was observed. Additionally, no difference (P = 0.49) in body
weight was observed during lactation (HT: 565.4 ± 12.0 vs. CL: 554.1 ± 11.0 kg; Figure 3-3).
Discussion
As expected and in accordance with other studies (Collier et al., 1982b; Tao et al.,
2012a), HT calves were lighter at birth and it was evident that they did not have any
compensatory growth after calving, as they remained smaller and lighter up to one year of age.
Because treatment groups did not differ in body weight gain up to 12 months old, it can be
concluded, similar to Tao et al. (2012a), that the difference in BW observed during the first year
after birth is a consequence of the lower birth weight rather than a difference in growth rate. The
greater morbidity and mortality observed in HT calves is strong evidence of the negative effects
of maternal heat stress on the health of the offspring. Indeed, Tao et al. (2012a) found that calves
born to cows heat-stressed during the dry period had lower PBMC proliferation during the
preweaning period and compromised passive immune transfer when compared to those born to
cows cooled when dry. It is known that calves born during summer months in Florida have
73
greater morbidity and mortality compared with those born during months of milder temperatures
(Donovan et al., 1998). However, since both treatment groups were managed under the same
circumstances after birth, the differences in morbidity and mortality between treatments is likely
related to the prenatal heat stress. The interaction observed between HT calves and calves
originating from IVF in the prepubertal period morbidity and mortality is not easily interpreted.
But, since the number of IVF calves was evenly distributed in both treatments, it is concluded
that heat stress in utero exacerbated the already known problems related to calf development,
such as growth retardation and malformation, associated with the in vitro fertilization technique
(Hansen, 2006).
Although no difference between treatments was observed in age at first parturition, the
differences observed in number of services per conception and hence age at first pregnancy is
evidence of an effect of maternal heat stress on fertility before the first lactation. A possible
explanation for this difference could be the direct effect of BW on fertility, as larger cows seem
to become fertile earlier. Archbold et al. (2012) observed that BW of the heifer at “mating start
date” (MSD) influenced the proportion of heifers pubertal at the start of the breeding season, as
greater BW at MSD increased rate of puberty. It is known that lifetime performance is influenced
by early development. Soberon et al. (2012) and Faber et al. (2005) found that greater
preweaning growth rate is related to greater first lactation milk yield. However, Warnick et al.
(1995) found no association between calf morbidity and milk production in the subsequent first
lactation. The fact that CL heifers were larger than HT heifers at one year old could explain the
differences in milk production, as BW at start of breeding season is associated with subsequent
milk solids yield potential (Archbold et al., 2012). However, because no difference in BW was
observed during lactation and actually the BW of HT cows was numerically greater than that of
74
CL cows, the difference in milk production is more likely to be explained by differences in
mammary gland development and altered metabolic efficiency as a consequence of changes in
the metabolism of those cows that experienced heat stress in utero.
Heat stress has been shown to induce PI-IUGR in a sheep model (Yates et al., 2011).
Offspring from these animals have major alterations in postnatal metabolism that probably also
exist in the offspring from heat stressed cattle during late gestation (Yates et al., 2011). The PI-
IUGR offspring express reduced β2 adrenergic receptors in the perirenal adipose tissue, caused
by elevated catecholamine exposure in utero (Chen et al., 2010). Additionally, PI-IUGR lambs
have difficulty mobilizing fat and develop greater levels of adipose tissue (Chen et al., 2010).
Moreover, PI-IUGR lambs are hypersensitive to glucose stimulation and insulin (Limesand et al.,
2006, Thorn et al., 2009), which further increases the probability that glucose will be stored as
fat during compensatory growth (Greenwood et al., 1998; Chen et al., 2010). Similar altered
metabolic responses were also observed in dairy calves. Tao et al. (2012a) observed that calves
born to cows heat stressed or cooled during the dry period had similar circulating insulin
concentrations before colostrum feeding, however calves from heat-stressed dams had higher
insulin concentrations at d 1 after birth when compared to calves from cooled dams. In calves, it
has been demonstrated that increased fat deposition during the prepubertal period and a higher
BCS at breeding are related to lower milk production in the first lactation (Silva et al., 2002).
Also, similar to the PI-IUGR sheep model (Chen et al., 2010), in Tao et al. (2012a) both
treatment groups of calves had similar overall postnatal growth rate. In the present study the HT
heifers never reached the BW of CL calves before puberty, however there was no difference in
BW during lactation, indicating that at some point during pregnancy HT heifers did reach CL
75
heifers BW. The BCS at breeding was not available, but no difference was observed in BCS at
calving, suggesting that fat deposition was similar among treatments at that time.
Conclusions
Maternal heat stress during late gestation decreases calf birth weight, BW and WH up to
one year of age. Additionally, these data suggest that heat stress during the last 6 weeks of
gestation negatively impacts fertility and milk production up to and through the first lactation of
offspring. Further studies with a larger number of animals are necessary in order to fully
understand the effects of heat stress in utero on morbidity and mortality in the prepubertal phase.
Also, special attention to the metabolism and mammary gland development of those heifers heat-
stressed in utero is necessary to understand the impact on fertility and milk production in the first
lactation.
76
Table 3-1. Effect of maternal heat stress (HT) or cooling (CL) during late gestation on calf
survival
Parameter CL %1 HT %
1 Total
Bull calves (n) 31 - 30 - 61
Heifer calves (n) 41 - 44 - 85
DOA rate2
0 0 3 4.1 3
Males mortality by 4 months of age 1 3.2 3 10.0 3
Heifers leaving herd before puberty 6 14.6 10 22.7 15
Due to death, sickness or growth retardation 2 4.9 9 20.5 11
Heifers leaving herd after puberty, before 1st lactation 1 2.4 3 6.8 4
Heifers completing first lactation3
21 77.8 17 53.1 38 1
Percentage out of total animals in the respective treatment 2
DOA = Dead on arrival 3
Out of 59 animals (CL = 27; HT = 32)
Table 3-2. Effect of maternal heat stress (HT) or cooling (CL) during late gestation on
reproductive performance of heifers in the first lactation
Parameter CL HT P
Age at first AI, mo 13.3 ± 0.3 13.5 ± 0.3 0.63
Number of AIs until first pregnancy 1.8 ± 0.3
2.6 ± 0.3 0.03
Age at first pregnancy, mo 14.5 ± 0.4 15.8 ± 0.5 0.06
Age at first parturition, mo 24.2 ± 0.5 24.9 ± 0.5 0.32
77
Figure 3-1. Effect of maternal heat stress or cooling during late gestation on body weight of the
offspring up to one year of age. Data from five consecutive years were analyzed.
Solid bars represent calves born to cows exposed to cooling during the dry period and
open bars represent those born to cows in heat stress. Calves born to cows exposed to
cooling were heavier (P < 0.05) compared to those born to heat stressed cows up to
12 months of age.
78
Figure 3-2. Effect of maternal heat stress or cooling during late gestation on milk production in
the first lactation. Data from five consecutive years were analyzed. Solid diamonds
(♦) represent heifers born to cows exposed to cooling (CL) during the dry period and
open squares (□) represent those born to cows in heat stress (HT). Compared with CL
heifer, HT heifers tended (P = 0.11) to produce less milk up to 30 weeks of the first
lactation (26.4 ± 2.1 vs. 30.9 ± 1.7 kg).
79
Figure 3-3. Effect of maternal heat stress or cooling during late gestation on body weight in the
first lactation. Data from five consecutive years were analyzed. Solid diamonds (♦)
represent heifers born to cows exposed to cooling (CL) during the dry period and
open squares (□) represent those born to cows in heat stress (HT). No difference (P =
0.49) in body weight during lactation was observed (HT: 565.4 ± 12.0 vs. CL: 554.1
± 11.0 kg) up to 30 weeks postpartum.
80
CHAPTER 4
GENERAL DISCUSSION AND SUMMARY
The importance of passive transfer of maternal immunity to the newborn calf through
colostrum feeding is well recognized. It protects the calf from acquiring infection during the
preweaning period and also improves productivity and longevity. However, the rate of failure to
attain adequate passive immune transfer is about 20% of all dairy heifers and almost 8% of
preweaned calves die due to diarrhea on US dairy farms (NAHMS, 2007). These data reveal a
great opportunity for improvement. Additionally, efficiency of absorption is usually only 1/3 of
the total IgG ingested, therefore factors that may affect AEA, and hence serum IgG
concentrations of the calves, are subject of many studies. Maternal heat stress during late
gestation is another factor that may impair passive immune transfer in newborn calves.
Heat stress during late gestation is associated with compromised placental function and
fetal growth retardation. In dairy cows, heat stress during the dry period compromises milk
production in the subsequent lactation and decreases birth weight of the offspring (Collier et al.,
1982b; do Amaral et al., 2009, 2011). Moreover, Tao et al. (2012a) observed that calves born to
cows exposed to heat stress during the dry period had lower serum IgG concentration and AEA
compared with calves born to cows under cooling. In that study calves were fed with colostrum
from their own dams. Factors influencing passive immune transfer related to the calf and the
colostrum were analyzed separately in the studies described in Chapter 2. In the first study,
calves born to cows heat-stressed or cooled during the dry period were fed pooled colostrum
from cows housed under thermoneutral conditions during the dry period. Those studies
demonstrated that calves heat stressed in utero during late gestation have compromised passive
immune transfer when compared with calves cooled in utero, regardless of colostrum source. In
the second study calves born to cows under thermoneutral conditions during the dry period were
81
fed pooled colostrum from the cows of the first study (heat-stressed or cooled during the dry
period). In this study there was no effect on passive immune transfer. However, calves that
received colostrum from cooled cows responded earlier to ovalbumin challenge, suggesting that
colostrum from cooled cows aid in the development of lymphocytes, specifically B cells,
positively influencing humoral adaptive immune response. Besides immunoglobulins, colostrum
has other components, such as cells and cytokines that may improve the passive immune transfer
and the development of the calf’s immune system (Sangild, 2003). Indeed, feeding colostrum
containing maternal cells accelerated the development and enhanced activation of lymphocytes
in the calf (Reber et al., 2008b). In the present study the pooled colostrum fed to calves was
previously frozen (i.e., did not have live cells), therefore the factor(s) present in the colostrum
that may play a role in the effect on acquired immune function observed in the present study are
not known.
With the objective to study the possible carry over effects from heat stress in utero during
late gestation on postnatal growth and future milk production, data from previous experiments
was analyzed, and those results are described in Chapter 3. Heifers born to cows under heat
stress during the dry period without a cooling system were lighter and smaller up to one year of
age compared with heifers born to cows cooled in utero, however the growth rate was similar
between treatments. Additionally, fertility of heifers heat stressed in utero was impaired.
Although no difference was observed in age at first parturition, heat stressed heifers required
more inseminations to become pregnant and hence were older at first pregnancy. Moreover,
heifers cooled in utero tended to produce more milk in the first lactation when compared with
heifers heat stressed in utero. These data suggests that, in addition to the compromised fetal
82
growth, heat stress during late gestation alters fetal metabolic and endocrine function and the
fetus is programmed to become less productive later in life.
In summary, the results presented in this dissertation indicate that heat stress during late
gestation per se negatively influences passive immune transfer. Additionally, feeding colostrum
from cows cooled during the dry period seems to accelerate lymphocyte development compared
with colostrum from heat stressed cows. Moreover, heat stress in utero during late gestation is
likely to have carryover effects on fertility and future milk production of the heifers.
83
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BIOGRAPHICAL SKETCH
Ana Paula Alves Monteiro was born in São José, Santa Catarina, Brazil in 1989. In
March 2006 she began her studies in the School of Veterinary Medicine at the Center of
Agroveterinarian Sciences at Santa Catarina State University (CAV/UDESC). She graduated in
Veterinary Medicine in December 2010. In 2011 she moved to Gainesville, Florida, USA and
joined the Animal Sciences program of University of Florida as a master’s student under the
supervision of Dr. Geoffrey Dahl in the fall of 2011 and graduated in the summer of 2013.