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1 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 REQUIRE MENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Ana Paula Alves Monteiro
3 To my family for their endless love and support
4 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 o f 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 Galvo, for their contribution with great suggestions and for their willing ness 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 fo r the great sugges tions and for taking care of the Spec ial 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 arou nd 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 Pom bo Barbara Bearden Rachel Corlett Emily Ferrell Lisette Perez and Briana Nixon I would not be able to complete my experiment without their help.
5 Thanks to all my friends in the Department of Animal Sciences, specially Anna Carolina Denicol Bianca A rtiaga Izabella Thompson and Joo 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 a nd subsequently g et into the m aster 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.
6 TABLE OF CONTENTS p age 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 C omposition and its Impact on the Development of Calf Immune System ................................ ................................ ................................ .......................... 18 Passive Immune Transfer ................................ ................................ ................................ 21 Factors Influencing Colostrum Q uality 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 D uring 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 Challen ge ................................ ................................ ................................ ...... 38 IgG Analysis ................................ ................................ ................................ .................... 39 Peripheral Blood Mononuclear Cell (PBMC) Isolation and Proliferation ...................... 40 Whole Blood Proliferation ................................ ................................ .............................. 41
7 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 Productio n ................................ ............................ 72 Discussion ................................ ................................ ................................ ............................... 72 Conclusions ................................ ................................ ................................ ............................. 75 4 GENERAL DISCUSSION AND SU MMARY ................................ ................................ ....... 80 LIST OF REFERENCES ................................ ................................ ................................ ............... 83 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 98
8 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 cond itions during the dry period and fed frozen colostrum from cows exposed to either heat stress or cooling during the dry period ................................ ................................ ............................. 55 3 1 Eff ect of maternal heat stress or cooling during late gest ation on calf survival ................ 76 3 2 Effe ct of maternal heat stress or cooling during late gestation on reproductive performance of heifers in the first lactation ................................ ................................ ....... 76
9 LIST OF FIGURES Figure page 2 1 Schematic des cription of experiments 1 and 2 ................................ ................................ .. 56 2 2 Effect of heat stress or cooling during the dry period on pre weaning grow th performance of the offspring ................................ ................................ ............................. 57 2 3 Effect of heat stress or cooling during the dry period on hematocrit and total plasma pro tein 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 th e first 56 d of age ................... 60 2 6 Effect of feeding colostru m from cows heat stressed or cooled during the dry period on total serum IgG concentration of cal ves 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 colostr um from cows heat stressed or cooled during the dry period on the response o f calves to ovalbumin challenge ................................ ............................. 63 2 9 Effect of heat stress or cooling during t he dry period on serum cortisol concentrations of the offspring d uring 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 peripher al blood mononuclear cell proliferation of the offspri ng during the preweaning period ................. 66 2 12 Effect of heat stress or cooling during th e dry period on whole blood proliferation of the offspring during preweaning period ................................ ................................ ............. 67 2 13 Effect of feeding colostrum from cows h eat st ressed 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 wei ght of the offspring up to one year of age ................................ ................................ .......................... 7 7 3 2 Effect of maternal heat stress or cooling during late gestation on milk production in the first lactation ................................ ................................ ................................ ................ 78
10 3 3 Effect of maternal heat stress or cooling during late gestation on body weight in the first lactation ................................ ................................ ................................ ...................... 79
11 LIST OF ABBREVIATIONS AEA Apparent efficiency of absorption CONA Concanavalin A CPM Count per minute CV Coefficient of variation DIM Days in m ilk DMI Dry matter intake IG Immunoglobulin LSM Least squares means PBMC Peripheral blood mononuclear cell PI IUGR Placental i nsufficiency intrauterine growth retardation PRL Prolactin PRLR Prolactin r eceptor SCC Somatic cell count SD Standard deviation SEM Standard error of the mean SI Stimulation i ndex WH Withers h eight
12 Abstract of Thesis Presented to the Graduate School of the University of Florida in P artial 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 Cal ves 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 dri ed 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 th ermoneutral environment during the dry period was pooled and stored frozen ( 20C). 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 a t d 15, 30, 45 and 60. Relative to CL calves, HT calves were
13 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 absorpt ion (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 des ign 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 colostru m 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 poo led 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 producti on 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
14 CL heifers (2.6 0.3 vs. 1.8 0.3, P = 0.03). Additionally, HT heifers tended to produce less milk up to 3 0 weeks of the first lactation comp ared 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.
15 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, a nd 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 (M ao 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 com plement are present in calves (Chase et al., 2008). Unless infected in utero, bovine fetuses are agammaglobulinemic (Barrington, 2001) and are immunologically nave 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 traff ic 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). Th is shift in B cell frequency coupled with result s in the lack of any endogenous antibody response until 2 to 4 weeks of age even with the more favorable TH2
16 response in neonates In this c ontext, the ingestion of colostrum is of extreme importance to provide immunologic defense to the calf duri ng 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 no t 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 adu lt 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 presen ce 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 t he p roduction of type 1 interferon by leukocytes is decreased (Firth et al., 2005). Natural Killer cell frequency i s low during the first week of lif e, 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 circulati ng in the newborn calf is about four times higher than at 3 week s of age. Neutrophils and macrophages have reduced phagocytic capacity but this is increased after the ingestion of colostrum (Menge et al., 1998).
17 Importance of Feeding High Quality Colostru m 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 passiv e 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 response s during early life, but also longer term effe cts 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 le ast 50g/L is enough to guarantee adequate passive immune transfer (McGuirk and Colli ns, 2004; Morin et al., 1997) Despite th e se 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 se rum 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 S tates could be prevented by changes in first colostrum feeding method, timing and volume. For these reasons, feeding an adequate volume of high quality col ostrum should be a major goal on dairy farms. Therefore, testing colostrum quality should be routine l y performed by using a colostrometer or refractometer. An important
18 difference between th e se methods is that the results obtained using a colostrometer are influenced by colostrum temperature at the time of measurement, whereas a refractometer is not therm ally 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 comp onents, such as immunoglobulins which are transpor ted 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 concentration s 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 b een 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 10 6 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
19 support the concept that maternal leukocytes cross the neonatal gut and circulate in the newborn calf, but the impact of thes e 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 t he circulation. The cells survived in the circulation for about 36 h after ingestion, disappearing after that. That study indicates a rol e of the colostral environment i n facilitating the transfer of colostral cells to the neona te. 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 ce lls 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 t heir 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 leve ls 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
20 responses to antigens a gainst which the dams had previously responded, but not to antigens to which the dams were nave, 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., 2 007). 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 1 IL 6, tumor necrosis factor and interferon are associated with proinflammatory responses and may aid in the recruitment of neonatal lymphocytes into the gut, thereby promoting normal immun e development (Chase et al., 200 8). Menge et al. (1998) obse rved 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, microbe s 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 d ays 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 lymphoc ytes. 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
21 Passive Immune Transfer Th e placentation of ruminants, sows and mares known as ep itheliochorial, 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 epi thelial 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 r eleased 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 abs orbs colostral proteins in the intestine through a n FcRn independent and non selective process (Baintner and Kocsis, 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 include s 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
22 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 con centration of total globulins or other proteins (Laven, 2012; Tyler et al., 1999; Weaver et al., 2000) All of these practice s are possible because the passive transfer of those proteins is positively correlated with that of IgG. Another method of evaluat ion 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) 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 2 4 h of age; however the authors indicate d that estimates of PV based on BW at birth are subject to considerable variation.
23 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 stud ies 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 bacteri a 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 immunoglo bulin absorption by calves. Donovan et al. (1986) observed lower plasma Ig concentration in calves during summer months in Florida and higher mort a lity of those same calves up to 6 months of age In fact, reducing heat stress in prepartum cows through a co oling system during summer months improves IgG uptake by the calf (Tao et al., 2012a). T he time elapsed from calving until colostrum is removed from the udder also affects t he concentration of IgG in colostrum which decreas es 3.7% for every hour past calv ing (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, colostru m 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, a nd 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. Howe ver, there is some data suggesting that IgG
24 uptake may also be improved if the calf remains with its dam (Stott et al., 1979), wh ich 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 c a esarean section and stimulated physically have better respiratory parameters ( i.e., diminished acidosis) and imp roved 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 effec t 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 addit ive. 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 relation ship th 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 lo wer serum IgG concentration, although no effect on colostrum IgG was observed.
25 Colostrum Heat Treatment C ontamination of co lostrum is of major relevance on dairy farms because all the steps, from collection through storage, are potential points of conta mination. 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 mecha nism behind this phenomen on is not known. A possible solution available for dairy farms to reduce microbial contamination in colostrum without negatively affecting colostral IgG concentration is heat treatmen t (Donahue et al., 2012). A temperature of 60C for 60 min reduces total plate count (TPC) and total coliform count (TCC) in colostrum without increasing viscosity. Heating colostrum to 60 o C also reduced or eliminated specific inoculated pathogens, includi ng 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 demonst rated 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, respe ctively. 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 d ays 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 invo lution 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 regula tion of nutrient partitioning involves homeorhetic controls from the conceptus to assure appropriate growth, not only of the fetus, but also fetal membranes, the gravid ute rus and the mammary gland (Bauman and Currie, 1980) As observe d 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 Th ere 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 su bsequent 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 hemis phere 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 be tween late gestation heat stress and reproductive performance in the next lactation (Avendao 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 decreas ed 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 subs equent 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 mam mary 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 yie ld (Nardone et al., 1997). Heifers expo sed to HAT produced colostrum with lower mean concentrations of IgG a nd 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 differen ce 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 a uthor s 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 t ransfer 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 rea son 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 Hea t 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 decreas es 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 a nimals relative to th at of cooled cows is another indicator of compromised placental functio n. 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 g estation signifi cantly decreased calf birth wei ght (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 dis sipate 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 fro m 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 b etween 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 protecte d from deviatio ns 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; Edwa rds, 1986). Under thermo neutral conditions, fetal body temperature is about 0.5C 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 H ellegers, 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 f etus is attenuated relative to that of their dams (Laburn et al., 1992, 2002; Faurie et al., 2001), significantly decreasing feto maternal temperature difference. The fe tus 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 at tenuated 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 p rol onged heat stress (Bell, 198 7 ) 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 los s.
30 Heat Stress Induced Intrauterine Growth Restriction Intrauterine hyperthermia is associated with co ngenital defects when it occurs in early pregnancy (Edwards, 1986) and with intrauterine growth re striction (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 wi ll als o compromise fetal growth. P lacental insuffic iency (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 in duced 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 reduc tion 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 concentr ations 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 achie ved 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 placenta l 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 vascul ature 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; Walla ce et al., 2005). In contrast to oxygen, glucose i s transported actively from maternal to fetal circulation, but in the PI IUGR model the expression of some glu cose transporter transcripts is reduced (Limesand et al., 2004; Wallace et al., 2005). Heat stress also diminishes u mbilic al 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 physiologic al 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 fr om 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 glucago n (Limesand et al., 2006). Thus insulin indepe ndent 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 concentra tion to restore glucose uptake (Limesand et al., 2006) primarily for anaerobic
32 metab olism, 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 fe tus (Anderson et al., 1997; Limesand et al., 2009) Another characteristic of PI IUGR is the premature development of hepatic gluconeogenesis, us ing lactate, amino a cids and other substrates as precursor s 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 co activators (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 2 adrenergic receptors, which was found to i mpa ir 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 (Limes and 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 e t al., 2010). In dairy heifers greater fat deposition during the prepubertal period is negativel y 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 t hat 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 concentrat ions 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), calv es 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 perfor med 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 o f the offspring. Couret et al. ( 2009 ) showed that prenatal social stress in pregnant sows increased the in vitro lymphocyte pro liferative response to a mitogen in p iglets 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 th e 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 dec reases milk production in the subsequent lactation and compromises immune function during the transition period. In addition, late gestation heat stress also affect s the offspring, where it decreases birth weight and seem s to compromise passive immune tran sfer 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 o ver effects on growth, health and future milk production of the heifers. The experiments described in C hapters 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 mi lk production in the subsequent lactation (Tao et al., 2011; 2012b), impairment of immune function (do Am aral et al., 2011) and greater disease incidence (Thompson and Dahl, 2012). Additionally, there is evidence that heat stress during late gestation caus es 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 result s obtained in that study were due to a calf effect or a colostrum effect, beca use the calves were fed with colostrum from their own dam Data on the effects of heat st ress 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 a nd IgA, total protein, casein, lactalbumin, fat and lactose in the colos trum 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 concentratio n 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 desi gned 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 p eriod 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 a reas 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. Thr ee 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 exce pt for birth weight and apparent efficiency of absorption AEA. After calving, cows were milked and their first colostrum was stored frozen ( 20C) for the subsequent study (Figure 2 1). Also, a sample was collected for future IgG analysis and stored froze n 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 group s 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) w ere provided. Paste urized 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 gro up 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 we ight 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. C alves 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 calve s were managed as explained in E xperiment 1.
3 8 Growth Measures and Blood Collection In both studies calves were weighed at birth before colostrum fe eding 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 birt h 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, Frankl in 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 collect ed with Vacutainer tubes without anticoagulant and placed at room temperature (27 o C) 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 so lution w as 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 immunodiff usion test (SRID, Triple J Farms, Bellingham, WA) bovine IgG antibody in agarose gel were filled with 5 L 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 concent ration. 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) o f 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 multiplie d 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) wer e coated with sodium carbonate bicarbonate buffer (pH 9.6) containing 1.4 mg/mL ovalbumin and incubated at 4 o C for 48 h. Following incubation, plates were washed 4 times in a plate washer (ELX50, Biotek instruments, Inc., Winooski, VT) with wash buffer sol ution (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% B SA (Sigma Aldrich ), was then added and plate incubated for 1 h at room temperature. Plates were washed 4 times and 100L 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 qua druplicates w h ereas test samples were run in duplicates placed in separate diagonal plate quadrants. Next, plates were washed 4 times and 100L of alkaline phosphatase conjugated rabbit anti bovine I gG (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, f ollowed by 1 h incubation at room temperature After an additional 4 washes, 80 L of p nitrophenyl phosphate liquid substra te (Sigma Aldrich ) was added to each well and plates were incubated for 30 min at room temperature in the absence of light. At the e nd 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 bala nced 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 T CM 199 medi um (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 co llected 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 110 6 cells/mL by adding TCM 199 supplemented with horse serum (5%, Atlant a Biologicals ), penicillin (200 IU/mL, MP Biomedicals, Solon, OH), streptomy cin (0.2 mg/mL, MP Biomedicals ), glu tamine (2 mM, Sigma Aldrich mercaptoethanol (10 5 M, Sigma Aldrich ). Adjusted PBMC suspensions were plated in triplicate (100 L/well) and stimulated or not with 10g/mL of concanavalin A (ConA, 100g/ mL, Sigma Aldrich ) in a 96 well, flat bottom sterile plate. ConA or Dulbecc BS (Sigma Aldrich ) was added (20 L/well) to the corresponding stimulated or control wells, respectively. Also, 80 L of supplemented TCM 199 was added to all the wells to reach a final volume of 200 L. Plates were incubated for 72 h at 37 0 C with 5% CO 2 After 48 h of incubation, 2 L of 3 [H] Thymidine (0.1 Ci/L, 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 heparin ized tubes (Vacutainer, Bect on 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 conten t 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 dilu te blood samples. Diluted whole blood (1:15 in modified RPMI) was plated in triplicate and stimulated or not with 5g/mL of ConA ( 100g/mL Sigma Aldrich ) in a 96 well, flat bottom sterile plate. Diluted blood (100 L/ well) was plated with 10 L of dilute d ConA (1:9 in modified RPMI) or PBS (for control) and 90 L of modified RPMI to reach a final volume of 200 L. The plate was incubated at 37 o C and 5% CO 2 for 72 h. After 48 h of incubation, 10 l of 3 [H] Thymidine ( 0.1 Ci/L, MP Biomedicals ) was added t o 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 contr ol 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. T o 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 a nalyzed 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., unpublishe d). 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 great er ( P = 0.01) BCS score prepartum and a lower ( P < 0.10) BCS postpartum (Thompson et al., 2 012) Additiona lly, HT cows had 6 d shorter gestation length (270.1 1 .3 vs. 276.1 1 .3 d ; P < 0.01 ) and consequently shorte r 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 f rom CL tre atment 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 wer e 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 colostru m 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. O n 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 Ig G 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 < 0.0 1) 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 bi rth 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.
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 f or HT and CL calves were 3,695 vs. 3,833 cells/L ( P = 0.65) and 528 vs. 520 cells/L ( 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 43.8%). Overall temperature was higher ( P < 0.05) for HT cal ves 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. 1 923.4 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 hematoc rit ( 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 co lostrum 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 betw een treatment groups (Figure 2 7 ). In the second study there was also no treatment effect in the production of IgG against ovalbumi n ( 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 colost rum 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 duri ng 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 v s. 1.25 0.12 g/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 c ompared with cooling (Figure 2 10 ).
47 PBMC and W hole Blood Proliferation In the first study, no treatment effect ( P = 0.3) was observed for PBMC proliferation (HT: 66.6 31.1 vs. C L: 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 po ints. 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 1 2 ). 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 ). Discus sion 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 b eing exposed to a similar THI (do Amaral et al., 2011; Tao et al., 2012 b ). 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 e t 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 co lostrum.
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 fro m 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. (2012 a ), the shorter gestation length does not explain all the difference observed in birth weight betwe en treatments. In the present stu dy, 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. I nadequate 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 nutriti on 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 he at 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 r eport any effect on gestation length, birth weight or dysto cia 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 (Wal lace 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 cal ves. 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 f ound 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 unti l 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 differenc es 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 befo re 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 t heir 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 fro zen 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 hom e 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 colostr al cells to enhance overal l 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 pro tein 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 the ir 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, eliminat ing a possible influence of colostrum from HT cows on the effic iency of IgG absorption. One is not able to concl ude 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 c ontact 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 prepa rtum 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 sow s during late gestation, but the concentration of IgG tended t o 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 c an 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 di fference 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 ges tation 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 de velopment 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 differ ences 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 start ed 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 h ad 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., 199 9 ). The lack of difference between treatments for whole blood proliferation suggests that colostrum from HT cows previously frozen does not seem to affect cellular immun ity of calves during the preweaning period when compared to colostrum from CL cows. The ovalbum in 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 pre viously 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 fr om 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 whol e colostrum had more active lymphocytes at d 7 after birth th at 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 diff erences were observed only at birth In that study d ifferences were attributed to a higher sensitivity to the stress of birth. In fact, in Tao et al. (2012a) the difference in birt h weight between HT and CL calves was greater ( ~ 6 kg vs. ~5 kg in
53 the present study ) and the averag e birth weight of HT calves w as 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. H igher levels of cortisol in the neonate are necessary to enhance colostral absorption (Sangild, 20 03) and conditions such as cold stress and dystocia inhibit the release of cortisol by the neonate and decrease colostral absorption (Chase et al., 2008). Th us the lower cortisol concentrations in HT calves may have contributed to the impaired passive imm une 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 obser ved. 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 g rowth 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 t his 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 com position % 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 D 3 (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 criteria 1 Score Clinical sign 0 1 2 3 Cough None I nduce 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 m ucopurulent 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 Sem i 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 1 Adapted from the School of Veterinary Medicine, University of Wisconsin Madison calf health scoring chart (found at: http://www.vetmed.wisc.edu/dms/f apm/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 In crease (cm) 2 5.7 0.9 6.0 0.9 0.84 1 Weaning weight and weaning height were measured at d 60 of age. 2 Preweaning 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, respectivel y. 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 Coo ling 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 1 Weaning weight and weaning height were measured at d 60 of age. 2 Preweaning 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 m ultiparous 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) environmen t. Therefore, the calves born to those cow s 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 ( 20C) for the subsequent study. Pool ed 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 store d 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 peri od 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 calves born to cows 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 durin g the dry period on total serum IgG concentrations of the offspring during the first 28 d of represent calves born to cows exposed to cooling during the dry period and open 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 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 calve s 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 the rmoneutral conditions during the dry period. Solid cows. Feeding colostrum from heat stress ed 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 calves had similar ( P = 0.51) IgG antiovalbumin production relative to CL calv es (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. represent those fed colostrum from heat stressed cows. Results are expressed in optical density (OD). No treatment effect ( P = 0.92) w as 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 ha d 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 P < 0.10.
64 Figure 2 9. Effect of heat stress or cooling during the dry period on serum cortisol represent calves born to cows exposed to cooling during the dry period and open se 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 g/dL, respectively) of calves compared with cooling. P P < 0.10.
65 Figure 2 10. Effect of h eat stress or cooling during the dry period on serum insulin represent calves born to cows exposed to cooling during the dry period and open 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) proliferatio n 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 o ffspring 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 proliferatio n 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 h eat 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 hea t 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 hematocri t, 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 o f 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 lacta tion. 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 accordi ng to BW with a da il y 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 AfiFarm TM 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 SA S 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 p rocedure of SAS 9.2 and LSM SE M 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 th an 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 bir th 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 (Tabl e 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 hei fer 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 5 3.1% 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 O nly one of these cal ves 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 a t 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 pregn ancy 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 differe nce ( 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 smalle r 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 conclude d similar to Tao e t al. ( 2012a), that the differenc e 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 nega tive effects of maternal heat stress on the health of the offspring. Indeed, Tao et al. (2012 a ) found that calves born to cows heat stressed during the dry period had lower PBMC proliferation during the preweaning period and compromised passive immune tran sfer 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 fro m 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 ca lf 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 serv ices 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 ferti le earlier. Archbold et al. (201 greater BW at MSD increased rate of puberty. It is known that l ifetime 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 yiel d. However, Warnick et al. (1995 ) found no association between ca lf 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. H eat stress has been shown to induce PI IUGR in a sheep model (Yates et al., 2 011) O ffspring 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 2 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 adipos e tissue (Chen et al., 2010). Moreover, PI IUGR lambs ar e hypersensitiv e 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 alte red metabolic responses were also observed in dairy calves Tao et al. ( 2012a) observed that c alves 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 prepuber t al 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 observ ed in BCS at calving, suggesting that fat deposition was similar among treatments at that time. Conclusions Maternal heat stress during late gestation decrease s 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 larg er 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 t he 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 rate 2 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 1 st lactation 1 2.4 3 6.8 4 Heifers completing first lactati on 3 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 r eproductive 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 cal ves 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 (CL) during the dry period and (HT) Compared with CL hei fer, 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 lact represent heifers born to cows exposed to cooling (CL) during the dry period and (HT) No difference ( P = 0.49) in body w eight 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 fee ding 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 affec t AEA, and hence serum IgG concentrations of the calves, are subject of many studies. M aternal heat stress during late gestation is another factor that may impair passive immune transfer in newborn calves. Heat stress during late gestation is associated w ith 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 c oncentration 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 separate ly in the studies described in C hapter 2. In the first study, calves born to cows heat stressed or cooled dur ing the dry period we re 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 tra nsfer 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 th e cows of the first study (heat stressed or cooled during the dry period). In t his study t here 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, positi vely influencing humoral adaptive immune response. Besides immunoglobulins, colostrum has other components, such as cel ls and cytokines that may improve th e passive immune transfer and the develop ment of the calf immune system (Sangild, 2003). Indeed, fe eding colostrum conta ining maternal cells accelerated the development and enhance d 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), t herefore 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 ge station on postnatal growth and future milk production, data from previous experiments was analyzed, and those results are described in C hapter 3. Heifer s born to cows under heat stress during the dry period without a cooling system were lighte r and smalle r 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 re quired 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 uter o. Th e s e 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 presen ted 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 ly mphocyte development compared with c olostrum 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.
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98 BIOGRAPHICAL SKETCH Ana Paula Alves Monteiro was born in So J os, 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 m aster student under the supervision of Dr. Geoffrey Dahl in the fall of 2011 and graduated in the summer of 2013.