E FFEC TS OF MATERNAL DIETS WITH NEGATIVE DCAD PRE PARTUM ON CALF GROWTH, HEALTH AND METABOLISM By CAROLINA COLLAZOS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017
2017 Carolina Collazos
To my mother
4 ACKNOWLEDGMENTS I, specially and sincerely, thank my advisor Dr. Jimena Laporta because she encouraged me to go the extra mile by giving me the opportunity to pursue a thesis under her guidance. She dedicated her time and patience while working with me throughout this project and helped me overcome some of my weaknes s es like writing and public speaking. I will always be grateful for her confidence in me. I ext end my appreciation to my committee members, Dr. Jos Santos and Dr. Corwin Ne lson. Since day one, they encouraged me to think outside the box and always took the time and effort to help me while sharing their knowledge and ideas. I am thankful to my fellow graduate students and friends, who have helped me throughout this project. Thanks to Camilo Lopera for dedicating time to collect calf samples and assisting me in gathering intel. Thanks to Michael Poindexter for helping with my calcium and magnesium analyses, I know it was quite an adventure. Thanks to Anderson Veronese for exp laining and walking me through the biochemical analyzer whenever I needed his expertise. Thanks to Marcos Zenobi for having the patience to help me understand the world of statistics and analyzing my data. With their support, I was able to achieve as much as I did. Thanks to Leslie, Andrea and Nikia, who helped me with data collections at the farm. I wish to thank my lab mate, Bethany Dado, for being awesome and, willingly, listen to my practice presentations. I would like to thank the faculty and staff at the Department of Animal Sciences for their hard work and the staff at the University of Florid a Dairy and Calf Unit for taking care of the animals. I want to extend my appreciation to my frien ds, Ana, Karly, Sam, Sossi, Matt and Emily who have been there when I needed them, helped motivate me, proof read my papers, and accepted my silliness. They made my adventure at the University of Florida a greater experience.
5 I dedicate a special thanks to my beautiful family, they always support me and give me words of encouragement. A huge thanks to my Tias and Tios, who are so loving towards me. Thanks Ca ta for being my number one fan. Thanks to my brother, Sebi, who makes me laugh every time I see him. A very special thanks to my parents, Alexandra and William, they show me unconditional love and support. My parents are instrumental in my successes, perso nal growth and are my every day inspiration, I am eternally grateful for them. I thank God for all that I have to be thankful for. Words cannot express how truly appreciative I am for all my experiences and my life because it is more than a blessing to be here guided by love.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................................ ... 11 CHAPTER 1 LITERATURE REVIEW ................................ ................................ ................................ ....... 13 Maternal Effects on the Offspring ................................ ................................ .......................... 13 Maternal Nutrients During Gestation that Impact the Offspring ................................ ..... 14 The Neonatal Period ................................ ................................ ................................ ........ 18 Perinatal Mortality ................................ ................................ ................................ ........... 19 Acid Base Balance and Buffer Systems ................................ ................................ ................. 20 Evaluating Acid Base Status ................................ ................................ ........................... 21 Acid Base Imbalances ................................ ................................ ................................ ..... 22 Acid Base Imbalances During the Neonatal Period ................................ ........................ 23 Diet induced Acidosis ................................ ................................ ................................ ..... 24 Transition Period in Dairy Cows ................................ ................................ ............................ 25 Peri partum Paresis Hypocalcemia ................................ ................................ .................. 26 Strategies to Prevent Hypocalcemia in Dairy Cows ................................ ........................ 27 Dietary Cation Anion Difference ................................ ................................ .................... 28 Maternal Metabolic Acidosis in Dairy Cows and Effects on Offspring ......................... 29 Summary ................................ ................................ ................................ ................................ 30 2 EFFECTS OF THE LEVEL AND DURATION OF MATERNAL DIETS WITH NEGATIVE DCAD PREPARTUM ON CA LF GROWTH, IMMUNITY, MINERAL AND ENERGY METABOLISM ................................ ................................ ........................... 31 Summary ................................ ................................ ................................ ................................ 31 Introductory Remarks ................................ ................................ ................................ ............. 32 Materials and Methods ................................ ................................ ................................ ........... 34 Animal s and Experimental Design ................................ ................................ .................. 34 Cows, Housing and Prepartum diets ................................ ................................ ............... 34 Calf Management ................................ ................................ ................................ ............ 35 Growth and Health Parameters ................................ ................................ ........................ 35 Blood Minerals and Acid Base Balance ................................ ................................ .......... 36 Energy Metabolism ................................ ................................ ................................ ......... 36 Efficiency of Immunoglobulin Absorption ................................ ................................ ..... 37
7 Hematology Analysis ................................ ................................ ................................ ...... 37 Statistical Analysis ................................ ................................ ................................ .......... 38 Results ................................ ................................ ................................ ................................ ..... 38 Growth and Health Parameters ................................ ................................ ........................ 38 Mineral Metabolism ................................ ................................ ................................ ........ 39 Acid Base Balance ................................ ................................ ................................ .......... 40 Energy Metabolism ................................ ................................ ................................ ......... 40 Efficiency of Immunoglobulin Absorption ................................ ................................ ..... 41 Hematology Analysis ................................ ................................ ................................ ...... 41 Discussion ................................ ................................ ................................ ............................... 42 Conclusions ................................ ................................ ................................ ............................. 48 3 GENERAL DISCUSSI ON AND SUMMARY ................................ ................................ ...... 5 8 LIST OF REFERENCES ................................ ................................ ................................ ............... 61 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 71
8 LIST OF TABLES Table page 2 1 Ingredient composition and nutrient profile of diets fed to cows ................................ ...... 50 2 2 Body weight (BW) at birth, 21, 42 and 62 d and calf hip height at 21, 42, and 62 d from calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ................................ ................................ ................................ ............ 51 2 3 Ionized calcium (iCa), sodium (Na), potassium (K), total calcium (tCa) and magnesium (Mg) concentrations of calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ................................ ........................... 52 2 4 Bicarbonate (HCO 3 ), pH, and partial pressure of carbon dioxide (pCO 2 ) in calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ................................ ................................ ................................ ........................... 53 2 5 Immunoglobulin G and apparent efficiency of absorption in calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ............... 53 2 6 Hematology parameters in calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ................................ ................................ ............... 54 2 7 Neutrophil percentage and count, lymphocyte percentage and count, monocyte percentage and count, basophil percentage and count, and eosinophil percentage and count of their calves born to Holstein dams fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the l ast 42 d (long, L) prepartum ................................ ................................ ....................... 55
9 LIST OF FIGURES Figure page 2 1 Effects of exacerbating the level (Lev; 70 vs. 180 mEq/kg) and extending the duration (Dur; 21 d, Short vs. 42 d, Long) of maternal negative DCAD diets du ring hydroxybutyric acid (BHBA), non esterified fatty acids (NEFA), glucose and total protein (TP) of their calves ................................ ............ 57
10 LIST OF ABBREVIATIONS BHBA Beta hydroxybutyric acid BW Body weight CO 2 Carbon dioxide d Day DCAD Dietary cation anion difference HCO 3 Bicarbonate iCa Ionized c alcium IGF Insulin like growth factor IgG Immunoglobulin G K Potassium L Long duration of feeding a diet with negative DCAD LSM Least squares means Mg Magnesium Na Sodium NEFA Non esterified fatty acids pCO 2 Partial pressure of carbon dioxide pO 2 Partial pressure of oxygen S Short duration of feeding a diet with negative DCAD SD Standard d eviation SEM Standard error of the mean tCa Total calcium
11 ABSTRACT Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF MATERNAL DIETS WITH NEGATIVE DCAD PREPARTUM ON CALF GROWTH, HEALTH AND METABOLISM By Carolina Collazos May 2017 Chair: Jimena Laporta Major: Animal Science s The objective of Chapter 1 is to review research that has linked maternal nutrition to fetal programming in various species. Human epidemiolog ical studies have provided extensive scientific information that allowed researchers to correlate adult diseases, such as diabetes and obesi ty, with fetal growth. Furthermore, malnutrition either under or over nutrition during early, shown that nutrient deficiency and nutrient supplementations duri ng late gestation can impact the programming within livestock species, the limited available research in dairy cattle will be discussed in this chapter. The dairy i ndustry utilizes several management and nutritional strategies to alleviate the metabolic stress associated with the onset of lactation in the transition period as well as to mitigate disorders such as hypocalcemia. This literature review summarize s the va rious strategies that are currently used to mitigate hypocalcemia, more specifically, it focus es on the use metabolism and immunity.
12 Chapter 2 describe s and discuss the findings of my experiment designed to evaluate the effects of maternal supplementation of acidogenic salts in growth, immunity, hematology parameters, energy and mineral metabolism of calves. The experimental design was a randomized block desi gn with a 2x2 factorial arrangement of two negative DCAD levels, 70 or 180 mEq/kg and two feeding duration s, the last 21 days (d; short ) or 42 d (long ) prepartum. After birth, all calves were transported to the University of Florida Calf Unit and manage d according to the University of Florida standard operating procedures. At birth and weaning, calves born to dams fed diets with negative DCAD for 42 d prepartum weighed less compared with calves born to dams fed the same diets with negative DCAD for 21 d (40 .0 vs 42.8 kg 0.8 and 76.7 vs 81.5 1.8 kg, respectively). However, calf body weight at 3 or 6 months of age did not differ with treatments Calves born to dams fed 180 mEq/kg DCAD had greater ionized calcium (iCa) concentrations from birth to 3 d of age than calves born to dams fed 70 mEq/kg DCAD. At birth, calves born to 180 DCAD dams experienced a more defined metabolic acidosis compared with calves born to 70 DCAD dam s, and by 3 d of age all calves there were no differences in the measures of acid base balance. Calves born to 180 DCAD dams had smaller concentration of BHBA compared with calves born to 70 DCAD dams. Plasma immunoglobulin G (IgG) concentrations and apparent eff iciency of absorption of IgG did not differ with maternal dietary treatments, thus, passive immune transfer was not impacted. Our data show that in spite of slight alterations in calf growth, acid base balance, mineral and energy metabolism during the neo natal period, feeding 70 and 180 mEq/kg DCAD diets during late gestation to dairy cows did not greatly impact the overall health and performance of the offspring
13 CHAPTER 1 LITERATURE REVIEW Maternal Effects on the Offspring Development of the concep tus involves crucial periods of rapid cell division that occurs at different times of gestation and in various parts of the body which are essential for growth and maturation of tissues and organs (Barker, 1993 ). During these crucial developmental periods, maternal nutrition and stressors may result in epigenetic changes possibly leading to lifelong 2000). Dr. Barker proposed the fetal programming theory during his pivot al study on fetal and infant growth in the Hertfordshire cohort study, which demonstrated that diseases and metabolic fetal origins H uman epidemiolog ical studies concept of f etal programming. For example, the study done on the 1944 Dutch Famine of World War II demonstrated that malnutrition during periods of gestation can have consequences on fetal development, leading to coronary disease, insulin resistance and gl ucose intolerance (Roseboom et al., 2006). Additionally, t his study reported that famine during the last trimester of gestation lead to a greater risk of infant mortality compared with those exposed during early and mid gestation indicating that malnutrit ion has different e ffects depending on the period of gestation (Roseboom et al., 2006 ). Researchers reported that rats born to dam s fed a nutrient restricted diet led to alterations in the postnatal metabolism resulting in high systolic blood pressure (Lan gley Evans et al., 1996) hyperinsuli nism, hyperleptinemia and hyperphagia (Vickers et al., 2000), all of which may play key roles in hy pertension and obesity Similar to humans and rodents, the number of research studies in livestock species exploring th health and productivity are increasing. Nutrients available for the offspring, in utero is largely
14 determined by the way the cow partitions nutrients to support embryonic placental, and fetal development in concert with her growth and performance, which can be determined by her production level and energy status ( Banos et al., 2007 ) The placenta plays a key role in regulating fetal growth, thus, manipulation of maternal nutrition is an important factor that may influence the development and funct ion of the fetal organ systems and metabolism The transport of nutrients from the maternal surface of the placenta to the fetal surface of the placenta is a complex process, which controls the transport of nutrients with varies mechanisms such as a bidirectional or unidirectional simple diffusion or highly regulated active transport system Developmental physiological events occur systematically during gestation and therefore effects of nutrition during different stages of gestation on the offspring var y greatly Maternal Nutrients D uring Gestati on that Impact the Offspring I n cows the first half of gestation is focused mainly on placental vascularization, placentome formati on and fetal organogenesis processes that are essential for normal conceptus development (Funston et al., 2010) Maternal nutrition may affe ct the embryo even during early to mid gestation regardless of the reduced embryo size during that period Barker and colleagues found that maternal undernutrition in humans during the first half of gestation, followed by adequate nutrition from mid gestat ion to term, resulted in infants of normal birth body weight to be proportionally longer and thinner than normal (Godfrey and Barker, 2000). In addition disproportionate body size observed at birth was associated with increased risk of obesit y, diabetes and coronary heart disease (Godfrey and Barker, 2000) Correspondingly, researchers have shown that when ewes are undernourished during the first 100 d ays of gestation, is altered which led to postnat al hypertension in lambs (Hawkins et al., 2000) During mid gestation, 28 to 72 d ays of gestation, ewes subjected to a nutrient restriction diet of 50% of NRC requirements (NRC, 1985) had lambs with increased intramuscular
15 triglyceride content and a decreased l ean to fat ratio compared with ewes that were not restricted (Zhu et al., 2006) I n beef cows, nutrient restriction exclusively, during 30 to 125 days of gestation, affected placental angiogenesis, possibly disturbing the amount of nutrient s transport ing through the maternal fetal placenta (Vonnahme et al., 2007) One of the earliest events du ring embryonic and placental development is establishment of fetal and uteroplac ental circulation (Patten, 1964 ). Factors affecting uteroplacental blood flow will impact the placental efficiency and, consequently, fetal growth (Funston et al., 2010). Addit ionally, beef cows given a nutrient restricted diet of 70% of NRC requirements (NRC, 2000) from ea rly to mid gestation from 60 to 180 d ays of gestation produced calves with reduced skeletal muscle fibers, impacting long term growth performance, and marb ling quality (Du et al., 2010) Relative to the brain and heart, the skeletal muscles have a low er priority in nutrient partitioning (Bauman et al. 1982; Close and Pettigrew, 1989 ). Additionally, the fetal period is essential for skeletal muscle development because there is no net increase in the number of muscle fibers postnatally (Glore et al., 1982 ; Greenwo od el at., 2000). In ruminant species, m aternal nutrient restriction during early and mi d gestation can lead to long During the second half of gestation, in the cow the growth of the placenta slows while its function and metabolism increases drama tically with a substantial increase in transplacental exchange of nutrients which is necessary for the exponential growth of the fetus and fetal organ maturation. It has been noted that nearly 75% of t he fetus growth occurs during that last 2 months of gestation (Robinson et al., 1977). E nergy and protein requirements increase significantly during the last 4 to 8 weeks of gestation to support t he increased growth rate of fetal tissues (Prior et al., 197 9). In a human study, pregnant women exposed to the Dutch famine during mid or late gestation had infants with reduced birth BW and short body length; these characteristics were associated with increased risk of obesity later in adult life (Roseboom et al. 200 6 ). During late
16 gestation prenatal exposure to famine i s associated with glu cose intolerance in adults (Ravelli et al., 1998) A study in ewes demonstrated that undernutrition during the second half of gestation ( 90 to 142 d of gestation ) resulted in decreased glucose and amino acid concentration s in the fetus, in addition to irreversible fetal growth retardation, which is linked with increased morbidity and mortality ( Mellor and Murray, 1982 ). E wes given restricted diets during late gestation (110 to 147 d of gestation) parallel with the ra pid period of fetal growth, resulted in reduced birth BW and had the greatest impact on the weight at birth compared with early and mid gestation (Gardner et al., 2007) Corah et al. (1975) demons trated that beef cows consuming a diet with only 65% of calories recommended by the NRC ( NRC, 1 970), during the last 100 d ays of gestation resulted in li ghter calves and lower neonatal survival Furthermore, the cows consuming a low energy diet had calves with a higher incidence of being treated for scours and had decreased weaning weights compared with calves born to cows consuming a diet with 100% of NRC recommendations ( Corah et al., 1975). Theoretically, similar effects of fetal programming would be expected to occur when dairy cows are nutrient restricted however data in support of such occurr ence is limited. It is worth mentioning that dairy cows are not as prone to suffer nutrient deficiency b ecause of the different nutritional management s trategies between the beef and dairy industries The l ate gestation period is critical for livestock be cause they need to prepare to trans ition into lactation while simultaneously provid ing the highest nutri ent demand to their growing fetus It is common for dairy cows to undergo a period of negative nutrient balance in which the cow is expending more calories than she is consuming. Therefore, during late gestation nutritional manipulations and management strategies, such as manipulations of the diet composition are implemented to alleviate the increased nutritional needs and enhance the cow ance. This may inadve rtently impact the fetus and lead to short term, long term or even permanent effects that could improve or wo rsen their performance and health postnatally. In Gao et al. (2012), dairy cows
17 consuming a diet considered to contain inadequ ate caloric content, 1.26 Mcal of net energy of lactation (NE L ) per kg of dry matter, during the last 21 d ays prepartum had calves with compromised immunity compared with calves born to cows fed diets with either moderate (NE L = 1.41 Mcal/kg dry matter) or high (NE L = 1.55 M cal /kg of dry matter ) caloric content A study that investigated protein supplementation in beef cows during the last trimester did not find differences betw een birth BW in calves born to dams given a protein supplementation and calves b orn to dams given a placebo supplementation (Martin et al., 2007). H owever, heifers born to dams given the protein supplementation had increased pre gnancy rates compared with calves born to dams not supplemented with protein (Martin et al., 2007) The possible mechanism that alter ed conception in those beef calves is unknown however it has been shown in rats that a low protein diets result s in persistent maternal hyperglycemia and contribute to changes in endocrine signaling leading to long (Fernandez Twinn et al., 2003) Beef steer progen y from protein supplemented dams were heavier at weaning, had heavier carcass weight and had increased intramuscular fat, resulting in greater meat quality compared w ith steer progeny not given protein supplements (Stalker et al., 2007; Larson et al., 2009). Holstein cows given selenium supplementation the last 60 days prepartum, had increased selenium concentrations rth and 42 days of age compared with calves born the Holstein dams not supplemented with selenium (Abdelrahman and Kincaid, 1995) This is important because se lenium deficiency can lead to myocardial degeneration and neonatal mor tality (Cawley et al., 1978) Furthermore selenium supplementation in dairy cows can reduce retained placenta (Trinder et al., 1973). Despite the different mechanisms controlling the tr ansport of nutrients from the mother to the fetus, high concentrations of minerals may significantly influence the offspring even after the neonatal period Moreover hypercalcemic ewes, induced with calcium infusions, had lambs with significantly increase d calcium concentrations compared with lambs
18 born to ewes not given th e same calcium infusions ( Abbas et al 198 7). This demonstrates that there is a carry over effect from that dams to their labs. The combination of fetal programming within different spe cies has led to a better comprehension of maternal nutritional influences on the developing fetus, in utero and postnatally. However, cow based experimental studies exploring the effects fetal programming ha ve on production and performance of their calves are limited. Nutrient deficiency often occurs in beef cattle because forage based diet availability varies because of seasonality of production forage quality and mismanagement. On the other hand, d airy cattle ar e predisposed to nutritional manipulations such as feeding acidogenic diets or other mineral supplementation s which are used with the intention of enhancing performance and health but growing fetus. I nvestigating the effects of maternal nutritional supplementations during late gestation on performance of the offspring will strengthen recommendations for improve ment in management strategies, growth efficiency and health in the dairy herd The Neonatal Period The neonatal period is considered to be the first 28 days of life The neonatal period is a dynamic state for the newborn because of the intrinsic adjustments to the extrauterine environment at the time of parturition Once the fetus separates from the umb ilicus during parturition, the neonate no longer has nutrient blood supply from the mother through the placenta and therefore causes a change from high pressure, low resistance to low pressure, high resistance in the neonatal respiratory and circulatory system. This causes asphyxia to rise, forcin g the newborn calf to initiate respiration and increase oxygenated blood by lung inflation leading to decreased pulmonary vascular resistance (Detweiler and Riedesel, 1993; Kasari, 1994). Through these physiologi cal changes, the neonate must be able to maintain adequate oxygen saturation, regulate acid base balance, engage endogenous metabolic pathways fo r energy production and maintain body
19 temperature within physiological limits (Kasari, 1994) Furthermore, during late gestation th e fetal calf utilizes glucose and lactate as energy sources (Comline and Silver, 1976). These sources may be used by the fetus as fuel for oxidation and source of carbon for net ti ssue accretion (Kasari, 1994). Perinatal Mortality If vital physiological functions are disturbed, this can lead to perinatal mortality. Perinatal mortality is defined as calves born alive, but die within 48 hours after birth Perinatal mortality in Holstein calves presents a reoccurring concern in the United States that has led to a loss of more than $125 million per year (Berger and Meyer, 2004) Factors that can lead to increased perinatal mortality in calves include : low calf birth BW to cow BW ratio, birth weights greater than 42 kg, and gestation lengths that are shorter than 273.1 d (Johanson and Berger, 200 3) In dairy cows it has been reported that heif ers born to cows with a gestation length ranging from 270 to 282 days live a short er or long er gestation length comp ared with the average gestation length (Vieira Neto et al., 2017) Normal gestation length is essential for the final stages of development in the calf, in addition, assist the calf in achieving a normal birth BW (40 5.7 kg) which can prevent dystocia and mortality in both the calf and dam (Johanson and Berger, 2003). Birth BW is instrumental to determining short and long term calf health, but it may not be the most efficient indicato r of other health parameters such as immunity, acid base status, and mineral and energy metabolism. Neonates are born agammaglobulinemic and achieve passive immunity through ingestion of colostrum. The macronutrients in colostrum are then transported thro ugh the intestinal epithelium, which remains permeable to large molecules for approximately 24 hours after birth (Staley et al., 1985; Stott et al., 1979). Therefore, neonates rely on colostrum nutrients such as immunoglobulins, proteins, and growth factor s, until the specific immune system of the neonate
20 matures. Blood immunoglobulin G (IgG) is an important indicator of passive immunity transfer in the neonate. For instance, a 24 hour old calf with blood IgG concentrations below 10 g/L (Quigley and Drewry, 1998) is highly susceptible to morbidity and mortality (Besser et al., 1994), and can affect l ong term calf performance (Wittum et al., 1995). Failure of passive immunity can be caused by ext ernal factors including decreased colostral IgG concentration (Morin et al., 1997) and calf stres s (Stott et al., 19 79 A cid Base Balance and Buffer Systems During the neonatal period, calves are predisposed to acid base imbalances because of t he rupture of the fetal membrane and the uterine contractions that occur during parturition, which alters respiratory components in t he acid base balance. Acid base equilibrium is essential to maintain balance b etween c hemical acids and bases, which are important for biological mechanisms such as enzymatic activity. For example, ranges outside of normal can denature proteins and increase loss of function in enzymes. Hence, the acid base balance is tightly regulated by buffer systems. A buffer system is a mixture of weakly dissociated acid and a salt of that acid designed to minimize changes in pH (Kasari 1999). There are three ba sic mechanisms that are used to correct imbalances; the chemical buffering system respiratory adjustment of blood carbon dioxide concentration s and excretion of hydrogen ions (H + ) or bicarbonate ions ( HCO 3 ) by the kidney s (Reece, 2009 ). The chemical buf fering system includes the bicarbonate system, phosphate buffer system, and protein peptide buffer system (Sherwood, 2012). The bicarbonate buffer system is extremely important in maintaining pH homeostasis in the blood because it is the first responder an d primary buffer in the extracellular fluid fo r noncarbonic acids (Reece, 2009 ). Nearly 80% of carbon dioxide ( CO 2 ) transport occur s in the form of HCO 3 and is regulated following this equation (CO 2 + H 2 2 CO 3 + + HCO 3 ; Reece, 2009 ). The hydration reaction equation in
21 plasma favors the left side because it accounts for minimal transport of CO 2 and is favored within erythrocytes because of the presence of carbonic anhydrase, catalyzing the formation of H + and HCO 3 Therefore, venous blood has a lower pH than arterial blood (Reece, 2009 ). Erythrocytes play a key role in transporting CO 2 because it contains hemoglobin, one of the most plentiful proteins that are available for buffering H + during the hydration equation (Reece, 20 09 ). H emoglobin releases CO 2 intracellularly to attract H + ions and then diffuses CO 2 back into plasma as bicarbonate (Kasari, 1999). When erythrocytes are oxygenated then hemoglobin releases H + ions and combine with bicarbonate to form carbonic acid, thus dissociating into CO 2 and H 2 O. Evaluating Acid Base Status Strategies to evaluate acid base status include co llecting venous or arterial blood and the parameters that are analyzed are pH, partial pressure of carbon dioxide ( pCO 2 ), HCO 3 partial pressure of oxygen and base excess Analyzing blood samples for the specific parameters such as pH, pCO 2 HCO 3 and oxygen in the blood allows to evaluate the severity, classification and probable cause of the aci dosis However, it is important to note tha t the functions of venous and arterial blood differ, therefore arterial blood is recommended to evaluate all the blood gases because the CO 2 and oxygen exchange from peripheral tissues can be assessed However, this method can be more strenuous on the anim al and not as practical. D ue to the nature of veins, in which they carry oxygen depleted blood to the heart, measuring oxygen levels to determine acid base status would be inconcl usive (Moore, 1969 ). Many stu dies have demonstrated that pH, pCO 2 and HCO 3 f rom venous blood are adequate to determine acid base balance when comparing to reference va lues from venous blood of control animals (Kasari, 1999; Moore 1969; Boyd, 1989). The normal physiological values in venous blood in a calf immediately postpartum fo r pH are 7.22 0.05 to 7.24 0.08; pCO 2 41.0 5.9 to 67.4 7.2 mmHg; HCO 3 24.2 2.7 to 28.2 4.4 mmol/L (Szenci, 1985). Most calves experience a partially compensated metabolic acidosis when
22 the pH values are from 7.2 0 to 7.44, pC O 2 from 38.5 to 8 8.5 mmHg and bicarbonate from 21.3 to 48 mmol/L (Boyd, 1989). Boyd (1989) observed a positive correlation between the age and plasma pH, pCO 2 and HCO 3 Analyzing the parameters to detect the acid base status are essential because there is a strong correl ation between perinatal mortality in the calf caused by metabolic or respiratory acidosis ( Szenci, 1985 ). Acid Base Imbalances An acid base imbalance, acidosis or alkalosis can develop from disturbances in respiratory or me tabolic control mechanisms (Carlson, 1997) Acidosis is commonly seen at birth, normally caused by the accumulation of excess acid or the removal of base from the extracellular fluid, while alkalosis, not commonly seen at birth, is an imbalance caused by excess base or loss of acid (Kasari, 1999). There are four general classifications of acid base disturbances: metabolic acidosis, metabolic alkalosis, respiratory acidosis and respiratory alkalosis Aci d base abnormalities in the blood or urine are quantified by pH, which is defined as the negative logarithm to the base 10 of hydrogen ion concentration in a solution and therefore pH is inversely related to hydrogen ion concentration (Butler et al., 1971) Soluti ons that have H + ions greater than 10 7 are acidic and those having H + ion activity of less than 10 7 are alkalotic (Kasari, 1999). When an animal develops metabolic acidosis, their pH is below normal levels because of a decrease in HCO 3 normally caused by impaired funct ion of the kidneys excessive intestinal loss of HCO 3 during diarrhea, or overproduction of acid in the blood In compensated metabolic acidosis, there is an inc reas e in ventilation forcing CO 2 to be released, causing the hydration equation (CO 2 + H 2 H 2 CO 3 + + HCO 3 ) to shift to the left, thus increasing pH (Sherwood, 2012). Compensation is the result of another acid base disturbance used to correct the original pH abnormality (Kasari, 1999). During uncom pensated metabolic acidosis, the pH is low and the HCO 3 concentratio ns are decreased while the CO 2 concentrations are increased. Animals experiencing acidotic condi tions
23 resulting from increased CO 2 in the blood, is referred to as respiratory acidosis bec ause the respiratory component, CO 2 and not the metabolic comp onent, HCO3 is the factor altering pH (Quigley and Drewry, 1998). Acid Base Imbalances D uring the Neonatal Period In a growing infant, acid base balance is partly maintained by while the i nfant kidney function to excrete acid (Quigley and Baum, 2004) T he neonatal period is a dynamic state involving variability in oxygen and CO 2 concentrations, colostral consumption, and changes between fetal hemoglobin and adult hemoglobin concentrations (Kasari, 1994). Because hemoglobin and erythrocytes play an important role in maintaining the normal acid base balance these variations could possibly impact the acid base status of the calf during the neonatal period Furthermore, imbalances in the acid base status can be caus ed by temporary anaerobic glycolysis initiated by poor perfused tissues attempting to maintain active energy metabolism during the transition between loss of maternal blood supply and establishment of respiratory function (Szenci, 1985). Diarrhea is one of th e most recognized causes of metabolic acid osis in calves because it results in intestinal loss of the HCO 3 which reduces buffering capacity in the extracellular fluid to counteract the production of organic acids, particularly lactic acid (Kasari, 199 9). P articularly during the neonatal period d iarrhea can be caused by viral, bacterial or protozoal organisms in addition to noninfectious conditions including consumpti on of poor quality milk all of which lead to predictable physiological and m etabolic events (Kasari, 1999). The cause of acidosis during the neonatal period varies and may be easily influenced by nutrition, infection or simply physiological changes that are disturbed during parturition. meta bolism. In newborn calves and lambs severe respiratory and metabolic acidosis may need to be treated or can lead to negative long term effects in health such as hypoxic ischemic encephalopathy (Gardiner, 1980). Besser et al. (1990) found that calve s with respiratory acidosis
24 had decreased IgG concentrations in the blood Also calves with postnatal respiratory acidosis ha d prolonged acidosis (> 8 h ours ) compared with calves diagnosed with postnatal metabolic acidosis (< 4 h ours ) which resulted in impaired IgG consumption in those calves ( Besser et al. 1990) Boyd (1989) found a negative correlation between severely acidotic calves and colostrum intake which led those calves to have decreased blood IgG concentrations, 24.5 g/L On the other hand, in moderat ely acidotic calves, with a blood pH greater than 7.2 0 th e c orrelation between colostral absorption and blood pH was not significant which resulted in calves with increased blood IgG concentrations, 37.9 g/L (Boyd 1989). P ersistent acidosis can be detri mental to pulmonary function because the pulmonary arterioles remain constricted; without correction, this can lead to death (Kasari, 1994). Diet induced Acid osis Acid induced diets causing acid base disequilibrium ha ve been shown to modulate molecular ac tivity including adrenal glucocorticoid, insulin like growth factor (IGF 1), adipocyte cytokine signaling, dysregulated cellular mechanism, and osteoclast activation (Robey, 2012) Studies in rodents have reported that cortisol concentrations in blood are enhanced by a transiently induced metabolic acidosis, suggesting that acidosis mediates cortisol activity through the pituitary adrenal c ortex renal glutaminase axis, possibly in response to lower bicarbonate concentrations (Welbourne, 1976) E pidemiolog ical studies in humans have correlated obesity with decreased cellular pH, along with the incidence of hypertension, insulin resistance and diabetes (Berkemeyer, 2009). Metabolic acidosis can lead to insulin resistance possibly because metabolic acidosis incre ases circulating glucose and adiponectin, a hormone secreted from mature adipocytes responsible for insulin sensitizing and anti inflammatory properties (Disthabanchong et al., 2011) Additionally, chronic metabolic acidosis has been shown to increase glucocorticoid response in humans (Sicuro et al., 1998) and in rodents (May et al., 1986). It was observed that
25 humans with a diet ind uced metabolic acidosis had increased higher plasma cortisol concentrations and cortisol secretion ; however no change was observed in adrenocorticotrophic hormone (Maurer et al., 2003). A cidogenic diet s consumed by humans result in a mild acidosis linked with excess cortisol that might play a role in bone turnover (Maure r et al. 2003) Osteoclast resorption and blood pH ar e negatively correlat ed because during acidification there is increased activity of carbonic anhydrase II ( Biskobing and Fan, 2000), the pumping of protons that solubilize bone mineral in osetoclasts (Nordstrom et al. 1997), and increased e nzyme s for orga nic matrix degradation (Brandao Burch, et al., 2003). Although acid osis induced diets present complic ations in human literature, in the dairy industry the use of acid osis induced diets to mitigate hypocalcemia specifically, are a wid ely adopted management practice that properly administered can result in benefits to cow health Transition Period in Dairy Cows In dairy cows, the transition period is defined as the three weeks before to three weeks after parturition. Thus, this period is challenging and cri of the major physiological changes that occur from involution of the mammary gland and supporting a fetus to producing colostrum and large quantities of milk The onset of lactation is characterized by the m ost substantial endocrine and metabolic changes during the lactation cycle of a cow. Cows need to adjust their metabolism to the dramatic increase in energy and nutrient requirements for lactation. M aternal tissues, particularly the liver, adipose tissue, the mammary gland and the bones, undergo numerous adaptations to support milk synthesis (Kovacs and Kronenberg, 1997) which may result in some cows having inability to adjust the homeorhetic mechanism s to maintain homeostasis which can lead to diseases. Disturbances in one or more metabolic processes is known as a metabolic disorder and is manifested when the cow cannot meet the metabolic demands ( Ametaj, 2010 ) Approximately 75% of disease in dairy cows occurs
26 during the first month after calving and are rooted with the impaired physiological immune functions and reduced feed intake during the 2 to 3 weeks prior to calving (LeBlanc et al., 2006) Peri partum P a resis Hypocalcemia Hypocalcemia is a metabolic disorder that can occur in high pro ducing dairy cows, primarily at the onset of lactation. Colostrum contains approximately 2.3 grams (g) of c a lcium per kilogram (kg), therefore approximately 23 g of calcium are needed to produce 10 kg of colostrum (Goff, 2008). In addition, calcium is also sequestered in the mammary gland before colostrum milking, which likely increases the calcium requirements with the onset of lactation. Mature milk requires 1.1 g of calcium per kg, therefore between early and mid lactation the high producing dairy cow loses about 30 to 50 g of calcium per day (DeGaris and Lean, 2008). The increased and extracellular fluid causes to a sudden decrease in blood calcium leading to the suscep tibility of hypocalcemia (Goff, 2008). Nearly 5 to 7% of the 9.2 billion dairy cow population in the U.S. will develop clinical hypocalcemia Clinical hypocalcemia is characterized by observable clinical signs such as tremors, hypersensitivity and recum bency when total calcium concentrations fall below 1.4 m M (Goff, 2008) Approximately 25% of periparturient primiparous cows and 50% of periparturient multiparous cows will suffer from subclinical hypocalcemia (USDA NA SS, 2012; Reinhardt et al., 2011) when total circulating calcium concentrations range between 1.4 and 2 m M (Goff, 2008). There are no visible signs of subclinical hypocalcemia making it challenging to diagnose. The incidence of hypocalcemia increases w ith the age of the cow and the risk of milk fever increases by 9% per lactation (Lean et al., 2006). Total calcium in blood is 50% bound to albumin, primarily, but also with salts of phosphate and lactate, and 50% in the ionized form. Normal circula ting to tal calcium concentrations are within 2.1 to 2.8 mM in dairy cow s (Reinhardt et al., 2011). Calcium is one of the most widespread
27 and ubiquitous second messenger ions (Parekh, 2006) Proper cell function requires calcium homeostasis to be a highly regulated process because of its versatility and critical role in biological processes such as cell signaling, blood coagulation, enzyme activity, membrane permeability and muscle contraction. The occurrence of clinical hypocalcemia has been shown to reduce muscle activity leading to increased susceptibility of dystocia, retained placenta and mastitis in Holstein dairy cows ( Curtis et al., 1983). Despite the lack of clinical si gns of subc linical hypocalcemia, it has been shown that it can reduce rumen contractions (Larsen et al., 2001 ), increase lipid accumulation in hepatocytes ( Chamberlin et al., 2013 ) and present a n increased risk of displaced abomasum compared with normoc alcemic cows (Massey e t al., 199 3). The varie ty of other diseases linked to clinical and subclinical hypocalcemia can lead to economic losses given the cost of treatments, culling and reduced productivity To put it in perspective, treatment co sts for clinical hypocalcemia are estimated to be $334 per cow and $125 per cow for subclinical hypocalcemia (Goff, 2008; Reinhardt et al., 2011). Strategies to Prevent Hypocalcemia in Dairy Cows At parturition, the cow must increase the po ol of plasma calcium to about 30 g of calcium per d ay to replace for the loss of calcium in colostrum through bone resorption and intestinal absor ption of dietary calcium (Horst et al., 1997) When the parathyroid gland senses a decrease in circulating iCa it secretes parathyroid hormone (PTH) to increase mobilization of calcium through indirectly activating vitamin D 3 in the kidneys The active vitamin D 3 will stimulate active transport of calcium across the in testinal epithelium and PTH increases renal tubular calcium reabsorption in addition continued secretion of PTH will initiate calcium mobilization from the bone. D ifferent strategies have been deve loped to mitigate the incidence of hypocalcemia Large doses of vitamin D supplementation can be given to cows about 7 days before calving ; however, this presents an issue because calving dates vary and increased doses of vitamin D can lead to
28 toxicity (Ka hn, 2005). Fe eding low calcium diets can be implemented into the prepartum diet to increase calcium absorption and bone resorption, but low calcium diets are difficult to formulate because most feeds contain calcium concentrations that result in positive c alcium balance, therefore, precluding the activation of homeorhetic mechanisms for maintaining blood calcium in absence of adequate intake. For instance, alfalfa, a common ingredient in formulating dairy feed, could not be used in the prepartum rations if a negative calcium balance is desired (Bethard et al., 1998) The negative dietary cation anion difference (DCAD) is another strategy in which acidogenic salts are added to the prepartum diet to induce a compensated metabolic acidosis. E ven though it is recomme nded for 21 d, the optimal duration to mitigate hypocalcemia is uncertain. One disadvantage of feeding diets with negative DCAD is that the metabolic acidosis itself or the excess of unpalatable salts might reduce feed intake during a crucial p eriod for th e dairy cow (O etzel and Barmore, 1993) Dietary Cation Anion Difference The negative DCAD is formulated by reducing strong cations, sodium and potassium, while adding more strong anions, chloride and sulfur. Ender et al. (1971) proposed the first equation to compute the DCAD for diet formulation as [mEq of Na + + mEq of K + ] [mEq of Cl mEq of S 2 ]. The goal of feeding diets with a negative DCAD is to manipulate the acid base status of the cow by inducing a compensated metabolic acidosis. In cattle, blood p H below 7.4 is considered acidic and may have been caused by a ccumulation of noncarbonic acids in the blood or decreased HCO 3 from the kidneys (Reece, 2009 ). The normal blood pH in cattle ranges from 7 .4 to 7.5 0 (Reece, 2009 ) Abu Damir et al. (19 94) fed prepartum cows either an alkalogenic or an acidogenic diet and estimated the fractional calcium absorption after correcting for endogenous calcium loss The authors showed that the negative DCAD diet increased calcium balance from 0.436 mol/day (17 .4 g/day) to 0.65 mol/day (26.0 g/day), which resulted in an increase in the
29 estimated fractional calcium absorption from 0.25 to 0.35. Nevertheless, the estimated increased intestinal absorption of calcium with acidogenic diets need to be confirmed with r adiolabeled Ca because mobilization from bone could have influenced those results. The recommended level of negative DCAD to induce a compensated metabolic acidosis range between 100 to 50 mEq/kg and should be fed for a recommended duration of 21 days prepartum (Oetzel, 2000) Feeding prepartum diets with n egative DCAD effectively reduces the incidence of clinical and subclinical hypocalcemia from 15% to app roximately 4 or 5 % when changing the DCAD from +200 to 100 mEq/kg (Charbonneau et al., 2006; Oetzel, 2000) T here is still a high percentage of dairy cows that suffer from hypocalcemia and therefore the optimal combination of feeding duration of negative DCAD and the level of the negative DCAD in the prepartum diet are uncertain. During the close up dry period dairy cows reduced their dry matter intake and it is possible that i ncreased feedi ng of anionic salts can further reduce feed intake because of unpalatability or acidosis. H aving a single prepartum ration for the entire dry period will void the uncertainty in calving date and provide flexibility in management strategies. Current researc h is investigat ing the optimal level and duration of negative DCAD feeding Maternal Metabolic Acidosis in Dairy Cows and Effects on Offspring It is possible that the maternal metabolic acidosis induced by feeding a cidogenic salts prepartum to the gestating dam could impact the neonate in utero because of the increased highly vascularized nutrient transfer system that occurs during the last trimester. Only two studies have evaluated the impact of maternal DCAD on the calf. Morrill et al. (201 0) found that the efficiency of immunoglobulin absorption and the blood concentrations of immunoglobulins of calves born to dams fed a negative DCAD ( 100 mEq/kg) were not different compared with calves born to dams fed a positive DCAD (+77 mEq/kg) Th ese author s fed a diet with negative DCAD for the last 21 d ays prepartum. On the other hand, Weich et al. (2013) found no differences in calf birth BW
30 when they compared two durations of 160 mEq/kg level of DCAD fed the last 21 or 42 days prepartum, or when compared with a con trol group fed a positive DCAD of +120 mEq/kg for the entire dry period It is likely that maternal negative DCAD evaluated in these studies might impact the offspring beyond their birth BW and absorption of immunoglobulins. The effect t hat diets with base balance ha ve been reported extensively (Charbonneau et al., 2006 ; Horst et al., 1997 ; Goff, 2008) ; however studies describing the potential carry over e ffects on the acid base balance, mineral and energy metabolism and performanc e of their offspring are limited Summary There are limited data in dairy cows exploring the impact of the use of anionic salts for the prevention of hypocalcemia on the programming of the fetus and the offspring postnatally. Animal scientists and cattle producers are beginning to acknowledge that the management and nutritional decision we make at the farm level during gest ation (i.e. late gestation) can impact the future generations of dairy cows. This, together with the urgent need to investigate the optimal DCAD level and duration during late gestation to more effectively mitigate hypocalcemia in dairy cows, motivated us to pursue this study. The objectives of the experiment are to evaluate different parameters such as growth, immunity, acid base balance, mineral and energy metabolism, in calves born to cows fed two different negative DCAD levels 70 vs. 180 mEq/kg, in the diet for two feeding durations 21 and 42 d It is hypothesized that growth, immunity and energy metabolism will not be greatly impacted, whereas acid base balance and mineral metabolism could be influenced by feeding cows the 180 mEq/kg for an extend ed feeding duration to 42 d. This thesis will contribute to the knowledge in this area and will directly benefit dai ry producers by assisting them to make integral decisions taking into account the health and wellbeing of both the cow and the offspring
31 CHAPTER 2 E FFECTS OF THE LEVEL AND DURATION OF MATERNAL DIETS WITH NEGATIVE DCAD PRE PARTUM ON CALF GROWTH, IMMUNITY, MINERAL AND ENERGY METABOLISM Summary The objectives were to investigate the effects that maternal diets containing negative dietary cation anion difference s ( DCAD ) fed prepartum may have on the acid base status, hematology, mineral and energy metabolism, growth and health of their calves postnatally. The experiment was a randomized block design with a 2 x 2 factorial arrangement of treatments, which consisted of two levels of negative DCAD, 70 or 180 mEq/kg; and two feeding durations, the last 21 d (short S ) or the last 42 d (long, L ) prepartum. A total of s ixty calves born to these dams were fed 3.8 L of pooled colostrum for thei r first feeding. Calf BW was recorded at birth and 21, 42, and 62 3 d of age. Blood was collected at birth bef ore colostrum feeding, and at 1, 2, 3, 21 and 42 d of age. Data was analyzed by ANOVA fitting mixed models C alve s born to L dams weighed 2.8 k g and 4.8 kg less at birth and 62 d, respectively, compared with calves born to S dams, however, at 3 and 6 months of age BW did not differ with treatments Calves born to 180 DCAD dams had increased blood concentrations of ionized calcium from birth to 3 d of age compared with calves born to 70 DCAD dams. At birth, calves born to 180 DCAD dams experienced a subtle and transient metabolic acidosis (pH = 7.28 0.02; pCO 2 = 59.2 1.7 mmHg; HCO 3 = 27.8 0.5 mmol/L) compared with calves born to 70 DCAD cows (pH = 7.33 0.02; pCO 2 = 52.9 1.7 mmHg; HCO 3 = 27.6 0.5 mmol/L). Calves born to 180 DCAD dams had smaller concentrations beta hydroxybutric acid and non esterified fatty acids compared with calves born to 70 DCAD dams. Calf p assive transfer of immunity and immunoglobulin G concentration s were not different between maternal treatments. Percentage of lymphocytes and neutrophils were altered by maternal treatments; however, health of the calf was not negativel y impacted by maternal dietary treatments
32 Extending the duration or exacerbating the level of maternal negative DCAD diets exerted a transient metabolic acidosis in the calves and slightly impacted measures of mineral, energy metabolism and growth. Intro duct ory Remarks Approximately 25% of periparturient primiparous cows and 50% of periparturient multiparous cows in the U. S. suffer from subclinical hypocalcemia and 5 to 7 % of cows in the U.S. will develop clinical hypocalcemia (Reinhardt et al., 2011) Hypocalcemia is a metabolic disorder that occurs when there are reduced circulati ng calcium concentrations in the blood. Hypocalcemia in Holstein dairy cows can lead to other disorders or diseases such as mastitis and displaced abomasum. Many dairy farms implement a diet with a negative DCAD to reduce the incidence of hypocalcemia. The typical recommendation is for the last 21 d prepartum and the recommended DCAD typically ranges from 50 to 100 mEq/kg. In some situations, dairy producers might prefer a single prepartum diet which would result in feeding dairy cows a diet with a negati ve DCAD for more than 21 d prepartum. The effects of extending a diet with a negative DCAD remain to be determined. Additionally, there is a need to investigate the proper level of negative DCAD in combination with the feeding duration to further reduce th e incidence of hypocalcemia. A diet with a negative DCAD prepartum induces a compensated metabolic acidosis in dairy cows which decreases the blood pH in the dairy cows and has been correlated with increased blood calcium concentrations. The effects anion ic salts fed to dairy cows during late gestation may have on the developing offspring are uncertain. However, it has been demonstrated that n utrient manipulations during the third trimester of gestation can influence fetal and postnatal development of the offspring such as maturation of organs, adipose accretion, muscle and skeletal development (Corah et al., 1975; Du et al., 2010 ; Gao et al., 2012) Acidosis in n eonates can occur as the result
33 of dystocia and hypoxia during the first hours after birth (Quigley and Baum, 2004) Postnatal respiratory or metabolic acidosis has been linked to reduced colostral immunoglobulin absorption leading to increased risk of mortality in dairy calves (B esser et al., 1990) It is possible that maternal metabolic acidosis induced by feeding anionic salts prepartum could impact the neonate in utero because of the highly vascularized nutrient transfer system that occurs during the last trimester. Limited research has explored whether the induced maternal compensated metabolic acidosis might influence the physiology of the calf postnatally. Morrill et al. (2010) found that the efficiency of absorption and the concentrations of immunoglobulins in calves born to dams fed a diet with DCAD of 100 mEq/kg did not differ from that of calves born to dams fed a diet with a DCAD of +77 mEq/kg during the last 21 d pre partum Weich et al. (2013) observed that reducing the DCAD from +120 to 160 mEq/kg, or extending the feeding the 160 mEq/kg from 21 to 42 d prepartum did not influence colostrum yield or calf birth BW The effect that negative DCAD base balance has been extensively reported (Charbonneau et al., 2006 ; Horst et al., 1997 ; Goff, 2008) ; however the effects of such dietary manipulations exert on the acid base balance, mineral and energy metabolis m and performance of the offspring remains mostly unknown The objectives of this experiment are to evaluate parameters of immunity, acid base balance, mineral and energy metabolism in calves born to cows fed two different levels of negative DCAD, 70 or 180 mEq/kg, for two durations, 21 or 42 d prepartum, and to determine the impact of these diets on their postnatal growth and health. It is hypothesized that growth, immunity and energy metabolism will not be greatly impacted, whereas acid base balance a nd mineral metabolism could be influenced by feeding cows the 180 mEq/kg for an extended feeding duration to 42 d.
34 Materials and Methods Animals and Experimental Design All procedures involving animals were approved by the University of Florida and Institutional Animal Care and Use Committee (protocol number 201509133). The experiment was conducted from January to June 2016 at the Dairy and Calf Research Units of the Univ ersity of Florida (Alachua, FL). The experiment was a randomized block design with a 2 x 2 factorial arrangement of treatments. Parous Holsteins cows were used in the experiment. Weekly cohort of cows were blocked by parity and 305 d milk yield and, within each block, they were randomly assigned to one of the four treatments with two levels of negative DCAD, 70 mEq/kg ( 70) or 180 mEq/kg ( 180) fed for two durations, the last 21 d of gestation which was designated as short (S) or the last 42 d of gestatio n which was designated as long period of feeding (L). Cows, Housing and Prepartum D iets Dams at 230 3 d of gestation were moved to a barn with individual feeding gates for treatment administration. Cows were trained for 2 d and treatments started at 232 3 d of gestation. Description of the diets is presented in Table 2 1. Cows in S were fed a diet with positive DCAD from 232 3 to 255 d of gestation, and then they were switched to diets containing negative DCAD starting at 255 d of gestation until calv ing. Cows in L were fed the respective negative DCAD treatments from 232 3 to calving. Diets were isonitrogenous and isocaloric and were formulated to differ in the concentrations of strong ions to manipulate the DCAD to achieve 70 or 180 mEq/kg. Sampl es of forages and concentrates were collected weekly and analyzed for their chemical composition to assure desired negative DCAD levels. Details of diet sampling and analyses is presented elsewhere (Lopera et al., 2015).
35 Calf Management Sixty calves born t o dams fed 70 S ( n = 9 heifers, n = 5 bulls), 70 L ( n = 12 heifers, n = 3 bulls), 180 L ( n = 11 heifers, n = 4 bulls) or 180 S ( n = 12 heifers, n = 4 bulls) were used in the experiment Twins and stillbirths were not incl uded and one dystocia case oc cu rred. G estation length of dams was calcu l ated and BW of calves was recorded at birth Day o f birth was considered experiment d 0 Calves were separated from their dams, had their navels dipped with 2% iodine to prevent in fection, and were fed 3.8 liters of pooled colostrum. Samples of the pooled colostrum fed to the calve s were collected three times per week and placed at 20 C until analysis. All calves were transported to the University of Florida Calf Unit and housed in individual h u tch es and calves rece ived ad libitum calf starter grain and water and the vaccination was according to the University of Florida standard operating procedures. Calves were fed pasteurized milk in two meals, 6:00 A.M. and 6:00 P.M. until 42 d, by bucket. In the first 21 d of life, calves were given 6 L of milk/d and then from 21 to 42 d of life, 8 L/d Milk allotment was reduced to 3 L /d before complete weaning at 49 d. Calves were moved to a group pen at 62 3 d of age. Growth and Health Parameters Calf BW, hip height, respiration rate and rectal tempe ratu re were recorded at 21 and 42 d of calf age. Additionally, BW at 62 3 d and at 3 and 6 months of age were recorded. Average daily gain from birth to 62 3 d was calculated. Health scores were determined using the Uni versity of Wisconsin (found at: http://www.vetmed.wisc.edu/dms/fapm/fapmtools/8calf/calf_health_scoring_chart.pdf ) to asse ss the physical health status of the calves at 21 and 42 d. Scores were determined by the same person throughout the experiment. Scores were assigned for the presence and severity of nasal and ocular discharge (0 to 3; 0 = normal and 3 = heavy discharge) a nd cough (0 to 3; 0 = none, 1 = induced and 3 = spontaneous cough). The total respiratory scores were calculated by adding the nasal,
36 cough and ocular scores; if total respiratory score exceeded 4 then calves required treatment. Calves that were treated fo r scours were documented on AfiFarm Dairy Farm Management Software (AfiMilk Ltd, Israel) and assigned a score of 1 if treated and a score of 0 if untreated. Blood Minerals and Acid Base Balance Blood samples were collected from calves via venipuncture of the jugular vein into 10 mL BD Vacutainer sodium heparin plasma tubes (Frankl in Lakes, NJ, USA) at birth before colostrum feeding, and at 1 (24 3 h), 2, 3, 21, and 42 d of age. Within 20 min of collection, plasma was separated by centrifugation at 2 80 0 rpm for 20 min and stored at 20 C until laboratory analysis. Additional bl ood samples were collected at birth and 3 d and analyzed, within 5 min of collection, for pH, partial pressure of carbon dioxide ( pCO 2 ), partial pressure of oxygen (pO 2 ), bicarbonate (HCO 3 ), sodium (Na), potassium (K) and ionized calcium (iCa) concentrations using a handheld biochemical analyzer (CG8+; VetScan iSTAT, Abaxis, Union City, CA). Plasma samples were analyzed for total calcium (tCa) and magnesium (Mg) concentrat ions using an atomic absorption spectrophotometer (AAnalyst 200, Perkin Elmer Inc. Waltham, MA) according to procedures previously described by Martinez et al. (2012). Intra assay coefficient variations wer e 9.9% for tCa and 4.8% for Mg. Energy Metabolism Plasma concentrations of non esterified fatty acids ( NEFA ) and beta hydroxybutric acid ( BHBA ) were measured by colorimetric and enzymatic methods, respectively (kit no. FA115 and RB1007, Randox Laboratories Ltd, UK). The inter assay coefficient of variati ons for NEFA and BHBA were 8.5% and 8.3%, respectively. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to measure concentrations of plasma glucose (Bran and Luebbe Industrial Method 339 19; Gochman and Schmitz, 1972). The int ra and inter assay coefficient of variations were 2% and 13 % respectively
37 Efficiency of Immunoglobulin Absorption Pooled colostrum samples and plasma samples collected 1 d after birth were used to measure total immunoglobulin ( IgG ) concentrations by r adial immunodiffusion assay (Triple J were diluted 1:2 and 1:5, respectively in 0.9% saline to fall within the linear range of the standard curve. The diluted sample s were pipetted into the bovine anti bovine IgG antibody plate, and incubated for 27 h in a flat surface protected from light. The diameter of the precipitin ring was measured using a 7x scale lupe (Peak, n 1975) and used to calculate the IgG concentrat ions. The inter assay CV of the radial immunodiffusion a ssay was 11%. To calculate the percentage of apparent efficiency of IgG absorption of the calves at 1 d of age (24 3 h) we used the equation entration at 1 d of age in plasma was multiplied by birth BW in kg and by 0.091, assuming the plasma volume is consistently 9.1% of the birth BW, then divided by IgG intake Colostrum IgG concentrations in grams were used to determine IgG intake Plasma to tal protein was assessed using a digital refractometer (Milwaukee Instruments; Rocky Mount, NC). Hematology Analysis For the assessment of blood hematology whole blood was collected via venipuncture of the jugular vein from calves into BD Vacutainer K 2 ED TA Tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at birth and before colostrum feeding and at 1, 2, 3, 21 and 42 d of age. Samples were carefully mixed, placed on ice and transported to the laboratory within 2 h of collection to be analyz ed using the IDEXX ProCyte Dx analyzer (IDEXX Laboratories, Inc., Westbrook, Maine, U.S.A.). The ProCyte Dx analyzer employs laser flow cytometry, optical fluorescence and laminar flow impedance technologies in combination with chemical reagents that lyse or alter the blood cells to enable the measurement of the complete blood count. The complete
38 blood count parameter s analyzed were red blood cell count, hematocrit, hemoglobin, percentage of reticulocyte and their counts, platelet count, white blood cell count, percentage of monocytes and their count, percentage of lymphocytes and their counts, percentage o f neutrophils and their counts, percentage of eosinophils and their coun ts, and percentage of basophils and their count s Stati stical Analysis Continuous data were analyzed by ANOVA with mixed model s using the MIXED procedure of SAS (SAS ver. 9.4, SAS Institute Inc., Cary, NC). The model included the fixed effects of level of DCAD, 70 or 180 mEq /kg, the feeding durations, S or L the sampling time or age of calf ( age ) and their interactions. The random effect was calf nested within DCAD level and duration. Body weight at birth, 62 3 d and average daily gain were analyzed using the same model without repeated measures. The Kenwa rd Roger method was used to calculate the denominator degrees of freedom for the F tests in the mixed models and the AR (1) or SP (POW) covariance structure was used as the covariate structure, depending on the variable analyzed. Data from bull calves was only included for the analysis of birth BW and gestation length. Normality of residuals and homogeneity of variance was assessed in all models before final analyses Whenever appropriate, t ransformation of data was performed to achieve normality or non par ametric tests were performed. Categorical variables were analyzed by logistic regression using the GLIMMIX procedure of SAS fitting binary distribution All results reported are least squares means (LSM) SEM. Differences with P tistically significant and between 0.05 < P 0.10 tending towards significance. Result s Growth and Health Parameters Dams fed DCAD for L duration had s horter ( P = 0.02) gestation lengths relative to S dams (274 vs. 277 0.85 d, respectively ). C onsequently, calves born to dams supplemented negative
39 DCAD prepartum for L duration weighed less ( P = 0 001) compared with those born to S dams (40 vs. 42.8 0.8 kg, respectively; Table 2 2). When gestation length was included as a covariate in the stati stical model for birth BW, there was still a significant difference between S and L DCAD duration in which calves born to L dams weigh ed less ( P = 0.04) compared with calves born to S dams (40.6 vs. 42.3 0.7 kg, respectively). As expected, males weighed more ( P = 0.01) than females at birth (42.9 vs. 39.7 0.81 kg, respectively) but there were no interaction s between gender of the calf, DCAD level or duration. There was a significant interaction betw een duration and age of the calves for BW at 21, 42 a nd 62 d of age. Calves born to L dams weighed less ( P = 0.01 ) at 62 d compared with calves born to S dams (76.7 vs. 81.5 1.8 kg, respectively; Table 2 2). There were no differences between DCAD level, duration or their interactions for BW at 3 and 6 mont hs of age and average daily gain from birth to 62 d of age There was a tendency for the interaction between DCAD level and duration for hip height C alves born to dams fed a 180 DCAD for an S duration tended to be taller (P = 0.08) than calves born to da ms fed a 70 DCAD for a L duration (Table 2 2) The total respiratory scores were not affected by treatments and no calves exceeded a total respiratory score of 4. The number of calves treated for scours was not different between maternal treat ments. There were three instances of respiratory problems however these were not attributed to maternal DCAD treatments prepartum There were no differences between total respiratory scores ( P > 0.9), and therefore calves were considered healthy througho ut the experiment. Mineral Metabolism Circulating concentration of K and Na were not affected by DCAD level, duration or their interactions (Table 2 3). There was a significant effect of age ( P < 0.001 ) of the calves for both minerals, in which K concent rations increased and Na c oncentrations decreased from birth to 3 d (Table 2 3). C irculating Mg concentrations were not affected by DCAD level, duration or their
40 interactions, but there was a sig nificant effect of age, in which Mg concentrations were eleva ted at birth and at 1 d of age and decreased thereafter ( P < 0.001, Table 2 3). T here was a significant interaction ( P = 0.05 ) between DCAD level and age of the calves for iCa, in which c al ves born to 180 dams had increased iCa concentrations compared wit h calves born to 70 dams at 3 d ( Table 2 3). C alves born to 180 dams tended ( P = 0.10) to have greater tCa concentrations compared with calves born to 70 dams (Table 2 3). Additionally, tCa concentrations were greater ( P = 0.002) on 0 d relative to 42 d of age (Table 2 3). Acid Base Balance Blood pH was significantly affected by the interaction ( P = 0.01) between DCAD level and age of the calves (Table 2 4) C alves born to 180 dams had a less acidic blood pH at birth compared with calves born to 70 da ms (7.33 vs. 7.28, respectively) ; however, at 3 d of age blood pH did not differ among treatments T here was a significant effect of age ( P < 0.001) of the calves for HCO 3 conce ntrations ( Table 2 4), in which concentrations of HCO 3 increased from b irth to 3 d of age (27.8 vs. 34.1 mmol/L ) Similar to pH, there was a significant interac tion ( P = 0.01) betwee n DCAD level and age of the calves for pCO 2 ( Table 2 4). At birth calves born to 180 dams had less pCO 2 compared with calves born to 70 dams, but at 3 d pCO 2 levels w ere not different DCAD levels Energy Metabolism There was a significant interaction ( P = 0.04) between DCAD level and age of the calves for BHBA concentrations (Figure 2 1 A) Calves born to 180 dams had lower BHBA concentrations compared with calves born to 70 dams specifically at 1 and 42 d of age Plasma concentration of NEFA tended to be less ( P = 0.07) in calves born to 180 dams compared with those born from 70 dams (Figure 2 1 B). A t birth, calves had greater ( P < 0.001) plasma NEFA
41 concentrations compared with later days, possibly because they were not fed until after the first blood sample was collected (Figure 2 1 B). Glucose concentrations did not differ between treatments, however there was a signi ficant effect of age of the calves ( P < 0.001 ) in which glucose concentrations increase d markedly from birth to 2 d of age (80 to 140 mg/dL respectively ) then decreased and remained steady thereafter ( 106.9 6.1 mg/dL ; Figure 2 1 C). Efficiency of Immunoglobulin Absorption Plasm a IgG concentrations at 1 d of age (24 3 hours) did not differ between DCAD level or duration and t he average IgG concentrations was 24.8 2.7 g/L (Table 2 5) T reatments or the interaction between tre atment and age did not influence efficiency of IgG absorption, which average d 3 3 % (Table 2 5) Total protein concentrations were not affected by level of maternal DCAD, however, at 3 d of age calves born to L dams had d ecreased ( P = 0.04) total protein compared with calves born to S dams and concentrations were not statistically different thereafter (9.3 vs. 9.7 0.15, respectively ; Figure 2 1 D). Hematology Analysis There were no differences ( P > 0.11) between DCAD level, duration or their interactio ns for red blood cells and hematocrit ( Table 2 6 ). There was a significant effect ( P < 0.001) of age of the calves for hematocrit and red blood cells in which both parameters decreased from birth to 3 d, and then increased at 21 and 42 d ( Table 2 6 ). Hemo globin concentrations did not differ ( P > 0.12) between maternal treatments ( Table 2 6 ). R eticulocyte co unts peaked at 3 d of age and then drastically decreased ( P < 0.001 ) at 21 and 42 d, but there were no differences between treatments ( Table 2 6 ). There was a significant interaction ( P = 0.05) between DCAD duration and ag e of the calf for platelet count s, in which c alves born to L dams had decreased platelet count s compared with calves born to S dams specifically at birth ( Table 2 6 ).
42 Overall, w h ite blood cell counts increased ( P < 0.001) from birth to 42 d of age, but there were no differences ( P > 0.39) between DCAD level, duration or their interaction s There was only an age effect for lymphocyte counts in which calves at 1, 2 and 3 d of age ha d l ess counts compared with calves at 21 and 42 d of age ( P < 0.001) However, c alves born to L dams had increased ( P = 0.02 ) percentage of lymphocytes at birth and at 1 d of calf age c ompared with calves born to S dams ( Table 2 7 ). N eutrophil counts were not affected ( P > 0.34) by DCAD level, duration or their interactions H owever, there was an interaction ( P = 0.03 ) between DCAD duration and age for the perc entage of neutrophils, in which calves born to L dams had less at birth and at 1 d of age compared with calves born to S da ms ( Table 2 7 ) There were no differences between DCAD level, duration or their interactions for monocytes, basophils, and eosinophils counts Similarly, there were no differences between DCAD level, duration or their interactions for the percentage of monocytes and the percentage of basophils. There was a tendency ( P = 0.10) for an interaction between DCAD level and duration for percentage of eosinophils ( Table 2 7 ), in which c alves born t o dams fed 70 DCAD for an S duration had a decreased percentage of eosinophil s relative to calve s born to dams fed 180 DCAD for an S duration Discussion The nutritional management of dairy cows during gestation not only influences cow productivity but c an also influence offspring health and productivity. S everal studies have shown that m aternal nutrition manipulations during the last trimester in cattle can have long term impacts on the developing offspring It is well established that when intrauterine condition s are poor, or not optimal, the progeny can experience complications later in life (Barker et al., 2002) Integral dairy cow and calf management decisions are needed in order to assure a healthy herd. There is l imited data investigating the effects that maternal nutrition manipulation might have on the offspring postnatally The use of diets with negative DCA D is a recommended dietary intervention and
43 widely adopted management practice in dairy farms to prevent the occurrence of hypocalcemia during early lactation (Reinhardt et al., 2011) Lowering the level of negative DCAD in the diet and supplying it for 21 days prepartum have proved to reduce the incidences of hypocalcemia by inducing a compensated metabolic acidosis, but the optimal duration and level are still under investigation Th e main objective of th e present study was to examin e whether extending the duration and exacerbating the level of the maternal negative DCAD prepartum may impact the metabolism, health and performance of the offspring postnatally Our results show that e xte nding the duration of supplemented DCAD from the recommended 21 d to 42 d in the dam reduced the calf a t birth from 42.9 to 4 0 kg. Th e normal Ho lstein calf birth weight ranges from 38.2 to 41.7 kg and weight s outside this range can lead to perinatal mortality (Johanson and Berger, 2003). Some factors shown to increase perinatal mortality in dairy calves are birth weights greater than 42 kg gestation lengths shorter than 275 d and higher ratio of dam weight to calf birth BW (Johanson and Berger, 2003) Contrary to my experiment Weich et al. (201 3 ) did not find differences in birth weight whe n 160 mEq/kg DCAD was extended from 21 to 42 d prepartum and compared with a control group fed +120 mEq/kg for 42 d. Calf BW can be a predictor of calving ease and perinatal mortality, and weights below or above normal can present complications for both t he offspring and dam such as d ystocia and retained placenta (Johanson and Berger, 2003) In our study, there wer e no cases of perinatal mortali t y even though calves born to dams given negative DCAD for a shorter duration had increased birth weigh ts (42.9 kg) relative to calves born to dams with an extended duration of negative DCAD (40 kg) Notably, calves born to dams fed a negative DCAD for a shorter duration continued to have greater BWs at weaning compared with calves born to dams with an extended negative DCAD duration. H owever at 3 and 6 month of age the BW of the heifers was not different between DCAD durations Although calf BW is highly correlated with gestation length,
44 maternal nut rition is a major influence on fetal growth and may trigger parturition ( Warnes et al., 1998; Tudor, 1972) In our present study, extending the negative DCAD significantly reduced gestation length compared with cows given a shorter duration by approxima tely 3 days. The 3 day difference may seem negligible but it has been shown that gestation lengths of 268, 273, 284 and 290 d yield probabilities of calf mortality of 5.5, 3.9, 3.1, 3.1 and 3.6%, respectively (Johanson and Berger, 2003). Changes in circula ting hormone levels in both the maternal and the fetal circulations at the end of pregnancy impacts the timing of parturition (Kota et al., 2006) I t could be speculated that t he prolonged acidosis of the dam stimulated f etal hormones of the hypothalamic pituitary adrenal axis resulting in shorter gestation length s. R egardless of th e shorter gestation lengths a nd the reduced birth BW e xtending the maternal negative DCAD did not detrimentally impact calf BW after weaning. The duration of maternal DCAD diets but not the negative level of maternal DCAD seem to have a significant impact on calf BW and growth during early life. The primary goal of the impl ementation of a diet with negative DCAD prepartum in dairy cows is to calcium mobilization before the onset of lactation to prevent the sudden and steep decrease in circulating calcium at calving It has been suggested that calcium ions are actively pumped across the placenta from mother to fetus but that the fetus is capable of independently controlling its own calcium homeostasis (Delivoria Papadopoulos et al., 1967; Care, 1989) Specifically, i n sheep, pigs and guinea pigs, it has been shown that increased calcium in the pregnant dam does not have a significant effect on fetal plasma calcium levels (Bawden and Wolkoff, 1967; Abbas et al., 1987 ; Greeson et al., 1968 ) In our study, t his could potentially explain the lack of difference in circulating iCa c oncentrations in the calves immediately after birth, despite the increased iCa concentrations in the dams fed an exacerbated negative DCAD prepartum (data not shown). Interestingly, we observed that at 3 d of age calves born to 180
45 DCAD fed dams had highe r iCa compared with calves born to 70 DCAD fed dams. C alves begin to depend on absorption of calcium through the intestines and skeletal calcium stores at approximately 3 d of age (Kovacs and Kronenberg, 1997) Whet her t he origin of the increased iCa in calves born to 180 DCAD fed dam s was a result of increased absor ption efficiency or an increase in feed intake is unknown because colostrum intake was not recorded after the first feeding. Additionally, milk and grain intake was not recorded A n in vi tro study observed that an acidic environment, decrease s calcium binding to proteins, mainly albumin, and increases iCa (Wang et al., 2002) This could possibly explain the decrease in circulating tCa in calves born to dams fed that 70 DCAD fed dams compared with calves born to dams fed that exacerbated negative DCAD It is known that feeding negative DCAD prepartum induces a compensated metabolic acidosis in cows (Goff, 2008), but it is unknown whether the maternal acid base status can i nfluence the developing offspring postnatally Metabolic acidosis is characterized by imbalances in the HCO 3 buffer system, while respiratory acidosis is cha racterized by imbalances in pCO 2 and both imbalances can cause blood pH to fall below a normal range of 7. 35 to 7.5 in cows. These imbalances in HCO 3 and pCO 2 reflect kidney and lung function (Kasari, 1999) During comp ensated metabolic acidosis, the lungs assist in removing t he excess acid in the blood by increasing respiration rate thus, increasing pH to be within the normal range In the present study, the acid base status of th e calves was determined by measuring the pH, pCO 2 and HCO 3 from their venous blood. According to Boyd (1989), calves with a mean pH of 7.24, mean pCO 2 of 67.4 mmHg and mean HCO 3 of 28.3 mmol/L are diagnosed with mixed (respiratory or metabolic ) acidosis. If the mixed acidosis persisted 24 h after birth in addition to reduced pCO 2 then calves are diagnos ed with respiratory acidosis. H owever, if the calves recovered after 24 h they are diagnosed with metabolic acidosis (Boyd, 1989) Here, c alves born to 70 DCAD dams had a more
46 evident metabolic acidosis because they had decreased pH and their pCO 2 was greater compared with calves born to dams fed the exacerbated negative DCAD. It is worth mentioning that all the calves had some degree of metabolic acidosis at birth It has been reported that acidosis may be common at birth when blood pH and plasma HCO 3 concentrations are lower and the pCO 2 are greater; then with increasing age pCO 2 decreases and blood pH increases as a result of improved respiratory function (Moore, 1969) By 3 d of age, all the calves from the present study achieved pH, pCO 2 and HCO 3 values that no longer reflected metabolic acidosis. Overall, neither the level n or the duration of maternal negative DCAD fed prepartum induce d a noticeable uncompensated metabolic acidosis in their calves. Scours have been linked to metabolic acidosis (Kasari, 1999) however, m aternal dietary treatments did not affect the number of calve s that had to be treated for diarrhea during the pre weaning period. F urthermore we did not observe differences blood electrolytes, Na and K, which could be indicat iv e of gastrointestinal tract inflammation and loss of buffers (Sobiech et al., 2013) There were only two cases of respiratory problems and one case of pneumonia, but none of them were associated with the maternal negative DCAD treatments. Additionally, regardless of the transient acido sis observed at bi rth in calves born to dams fed the exacerbated negative DCAD the plasma concentrations of IgG and the apparent efficiency of IgG absorption in the calves at 1 d of age did not differ. Boyd (1989) reported a negative correlation between extremely acidotic calves (with blood pH < 7.15) and reduced colostrum intake which led to decreased IgG concentrations in the calves, however they did not find any correlations between moderately or normal calves and their IgG concentrations (Boyd, 1989) C alves are born agammaglobunemic and therefore rely on c olostrum supply to provide vital IgG in addition to other immune proteins and nutrient s that support the newborn calf while transitioning from its nave state to acquired immune system. Here, extending the duration or exacerbating th e level of negative DCAD fed to the dams
47 prepartum did not impact the calf intrinsic ability to absorb IgG. I t is s till unknown whether the extended duration of negative DCAD or exacerbated negative DCAD affect s the quality and/or the quantity of colostr um produced by th e dam, in this study we feed the newborn calves with pooled colostrum to avoid any confounding effects of maternal colostrum It is possible that slight alterations of the base balance and mineral metabolism due to the maternal diets could have alter ed the intracellular signaling of cells (i.e. adipocytes) Parameters such as glucose, NEFA and BHBA are commonly used as indicators of energy balance in dairy cows, thus, we set out to determine if maternal DCAD t reatments could have affected the metabolism of the calves by decreasing circulating concentrations of b oth BHBA and NEFA without alteration s in circulating glucose concentrations during the preweaning period It is important to note that in the present experiment we did not evaluate measures of intracellular signaling to confirm this. In a young calf, BHBA concentrations are indi cators of rumen development with incre asing calf starter or grain intake (Quigley et al., 199 1 ) Decreased NEFA concentrations ar e associated with increased nutrient consumption. I t could be speculated that the decrease in both NEFA and BHBA concentrations in calves born to 70 dams, could h ave been a response to increased feed consumption or in creased efficiency absorption of nutr ients Despite subtle differences seen in BHBA a nd NEFA concentrations, the energy metabolism of the was within normal range. Moreover, g lucose concentrations did not diff er between maternal treatments and the patterns of glucose concentrations in calves varied with their age and are similar to previous studies ( Lents et al., 1998; Knowles et al., 2000) The complete blood count of the calves was not greatly impacted by maternal dietary treatments fed prepartum and no hematologic abnormalities were detecte d. However, as expected, both the erythrocytes and leukocytes were impacted by the age of the calf. This is not surprising
48 given the young calf is actively developing immunological maturation (i.e. increased erythropoiesis, replacing fetal hemoglobin with adult hemoglobin and increase d B cells and T helper cells due to the introduction of environmental antigens during the first month of life ; Reece, 2009 ) Calves born to dams fed the negative DCAD for an extended duration had less platelet counts compared w ith calves born to dams fed the negative DCAD for a shorter duration. Platelets are necess ary for the coagulation process; therefore, d ecreased platelets are associated with failure of clot retraction; however, platelet blood indices such as mean platelet volume and pl atelet hematocrit did not suggest susce ptibility of illness in the calves with less platelet counts When the negative DCAD duration was extended the percentage of neutrophils decreased whil e the percentage of lymphocytes increased In additio n, monocyte, basophil, eosinophil counts and their respective percentages were expected to change as the calves undergo imm unological maturation, but similarly, these parameters were not impacted by the duration or level of maternal DCAD Regardless of the se hematological differences, the health of the calves was not compromised. Conclusions E xtending the duration of feeding from the recommended 21 d to 42 d, and exacerbating the level of negative DCAD from 70 to 180 mEq/kg fed pre partum impac ted the growth and energy metabolism, which does not support our hypothesis. However, the acid base status and mineral m etabolism during the first 3 d of life and the pre weaning period were impacted by the more negative DCAD in the diet G estation len gth and the growth of the calves was affected primarily by the extended duration of maternal DCAD and not by the level of the negative DCAD. Despite the transient acidosis observed in calves born to 70 DCAD fed dams, these calves were able to recover comp letely by 3 d of age, suggesting that it was a compensated acidosis. R egardless of the subtle differences in measures of innate immunity observed in this study the hematology parameters were all within the normal ranges of healthy calves. In fact,
49 there w as a sudden death of unknown cause and instances of scours and respiratory acidosis observed herein, but overall all the heifers were healthy and the lag in the growth of the calves born to 180 DCAD dams was compensated by 3 and 6 months of age. It is imp ortant to note that we did not gather data from calves that were born to dams fed an alkalogenic diet, therefore we only investigated the effects a maternal diet with negative DCAD may have on the calves. However, t he results obtained from this study are v aluable for dairy producers because it would allow them to have more flexibility on the feeding management of anionic salts pre partum to prevent hypocalcemia, without compromising t he health and performance of the offspring postnatally. Further research is needed to evaluate the long term performance of the heifer s during their first lactation, when the mineral and energy metabolism are greatly challenged.
50 Table 2 1. Ingredient composition and nutrient profile of diets fed to cows Diet Ingredient, % dry matter Positive DCAD 70 mEq/kg 180 mEq/kg Corn silage 34.2 34.2 34.2 Triticale silage 20.4 20.4 20.4 Bermuda hay 6.7 6.7 6.7 Straw 13.8 13.8 13.8 Citrus pulp 7.7 7.1 6.7 Soybean meal 13.1 8.5 5.8 Prepartum mineral 4.2 4.2 4.2 Bio Chlor 1 0 5.2 8.3 Nutrient content, mean SD Crude protein, % 14.9 0.8 14.7 0.4 14.6 0.6 Acid detergent fiber % 29.4 1.4 28.9 1.2 29.1 1.1 Neutral detergent fiber, % 43.1 1.7 43.7 1.5 43.8 1.5 Forage neutral detergent fiber, % 39.3 1.7 39.3 1.7 39.3 1.7 Nonfib rous carbohydrates, % 31.7 1.3 31.1 1.6 31.1 1.9 Starch, % 12.3 0.4 12.6 0.5 12.9 0.6 Fat, % 2.8 0.2 2.8 0.1 2.8 0.1 Ca lcium % 0.67 0.07 0.64 0.05 0.62 0.05 Phosphorus, % 0.33 0.01 0.33 0.02 0.33 0.03 M agnesium % 0.44 0.06 0.47 0.06 0.48 0.03 Potassium % 1.54 0.10 1.49 0.09 1.46 0.09 Sulfur, % 0.29 0.03 0.40 0.03 0.47 0.03 Sodium % 0.08 0.03 0.11 0.03 0.13 0.04 Chloride, % 0.50 0.07 0.86 0.07 1.11 0.03 DCAD 2 mEq/kg +109 35 66 17 176 20 1 Bio Chlor anion source (Arm & Hammer Animal Nutrition, Inc.) 2 DCAD = d ietary c ation a nion d ifference and calculated as follows: DCAD = [(mEq of K) + (mEq of Na)] [(mEq of Cl) + (mEq of S)].
51 Table 2 2. B ody weight ( BW ) at birth, 21 42 and 62 d and calf hip height at 21, 42, and 62 d from calves born to Holstein dams fed two differe nt levels of negative DCAD 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S ) or the last 42 d (long, L ) prepartum ; (n = 9 to 12 calves per treatment) DCAD level DCAD duration P values Item 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age BW, kg Birth Â§ 41.6 41.3 42.9 40.0 0.81 0.80 0.001 0.31 21 d 53.6 51.9 54 .0 51.5 1.8 0.47 0.38 <0.001 0.29 0.95 0.01 42 d 69.5 67.9 67.6 69.7 62 d 79.6 78.7 81.5 76.7 Hip height, cm 21 d 33.6 33.6 33.8 33.4 0.29 0.74 0.77 <0.001 0.08 0.83 0.48 42 d 35.7 35.8 35.7 35.8 62 d 36.9 37.2 37.1 37.1 Data is presented as least squares means SEM; P Age = 21, 42 and 62 days (d) after birth DCAD = d ietary c ation a nion d ifference. Lev = DCAD level. Dur = DCAD duration. The triple interaction DCAD level*duration*age, was not significant for any of the parameters estimated (P > 0.10). Â§ Birth BW includes data from bulls and heifers.
52 Table 2 3. Ionized calcium (iCa), sodium (Na), potassium (K), total calcium (tCa) and magnesium (Mg) concentrations of calves born to Holstein dams fed two different levels of negati ve DCAD, 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S) or the last 42 d (long, L ) prepartum; ( n = 9 to 12 calves per treatment ) DC AD level DCAD duration P value Item Age, d 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age iCa, mmol/L 0 1.30 1.28 1.28 1.30 0.02 0.62 0.63 0.25 0.53 0.05 0.66 3 1.29 1.33 1.31 1.31 Na, mmol/L 0 139.4 139.7 139.9 139.2 0.31 0.47 0.85 <0.001 0.51 0.96 0.47 3 136.2 136.5 136.4 136.3 K, mmol/L 0 4.42 4.28 4.33 4.38 0.06 0.53 0.63 <0.001 0.36 0.30 0.95 3 4.77 4.80 4.76 4.8 0 tCa, mmol/L 0 3.00 3.05 2.98 3.07 0.07 0.10 0.25 0.002 0.28 0.84 0.31 1 2.91 3.03 2.88 3.05 2 2.99 2.99 3.01 2.96 3 3.12 3.17 3.05 3.24 21 2.78 2.97 2.91 2.84 42 2.83 2.91 2.86 2.88 Mg, mmol/L 0 0.65 0.63 0.63 0.64 0.02 0.50 0.81 <0.001 0.30 0.88 0.44 1 0.69 0.69 0.69 0.69 2 0.57 0.57 0.57 0.56 3 0.54 0.55 0.54 0.55 21 0.54 0.58 0.56 0.55 42 0.54 0.55 0.56 0.54 Data is presented as least squares means SEM; P Age = 0, 1, 2, 3, 21, 42 days (d) after birth. DCAD = d ietary c ation a nion d ifference. Lev = DCAD level. Dur = DCAD duration. The triple interaction DCAD level*duration*age was not significant for any of the parameters estimated (P > 0.10).
53 Table 2 4. Bicarbonate ( HCO 3 ), pH, and partial pressure of carbon dioxide ( pCO 2 ) in calves born to Holstein dams fed two differe nt levels of negative DCAD 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S ) or the last 42 d (long, L ) prepartum ; (n = 9 to 12 calves per treatment ) DCAD level DCAD duration P value Item Age, d 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age pH 0 7.28 7.33 7.32 7.29 0.02 0.62 0.20 <0.001 0.31 0.01 0.45 3 7.53 7.50 7.52 7.50 HCO 3 mmol/L 0 27.8 27.6 28.0 27.4 0.53 0.36 0.76 <0.001 0.12 0.56 0.44 3 34.4 33.7 34.0 34.3 pCO 2 mmHg 0 59.2 52.9 54.7 57.5 1.74 0.23 0.26 <0.001 0.85 0.01 0.61 3 41.8 43.9 42.2 43.4 Data is presented least squares means SEM; P Age = 0 and 3 days (d) after birth. DCAD = d ietary c ation a nion d ifference. Lev = DCAD level. Dur = DCAD duration. The triple interaction DCAD level*duration*age was not significant for any of the parameters estimated (P > 0.10). Table 2 5. Immunoglobulin G and apparent efficiency of absorption in calves born to Holstein dams fed two differe nt levels of negative DCAD 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S ) or the last 42 d (long, L ) prepartum ; (n = 9 to 12 calves per treatment ) DCAD Level DCAD duration P value Item 70 180 S L SEM Level Duration Lev*Dur Immunoglobulin G, g/L 24.82 24.75 24.89 24.68 2.67 0.99 0.96 0.94 Apparent efficiency of absorption, % 35.90 29.40 36.70 28.60 0.04 0.24 0.15 0.70 Data is presented least squares means SEM; P DCAD = dietary cation anion difference. Lev = DCAD level. Dur = DCAD duration.
54 Table 2 6 Hematology parameters in calves born to Holstein dams fed two differe nt levels of negative DCAD 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S ) or the last 42 d (long, L ) prepartum ; ( n = 9 to 12 calves per treatment) DCAD level DCAD duration P value Item Age d 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age RBC, M/ L 0 8.1 8.4 8.2 8.4 0.25 0.15 0.47 <0.001 0.62 0.17 0.82 1 7.1 7.4 7.1 7.4 2 6.7 6.9 6.8 6.8 3 6.3 6.9 6.6 6.6 21 7.4 8.2 7.7 8.0 42 8.4 8.7 8.4 8.8 Hematocrit, % 0 35.9 37.2 35.4 37.8 1.24 0.11 0.24 <0.001 0.61 0.28 0.97 1 29.3 31.3 29.3 31.3 2 26.9 27.8 26.7 28.0 3 23.7 26.5 24.5 25.7 21 25.9 30.1 27.3 28.7 42 28.4 30.5 28.7 30.2 Hemoglobin, g/dL 0 3.8 3.7 3.6 3.9 0.22 0.30 0.19 0.002 0.74 0.12 0.99 1 3.4 3.1 3.1 3.4 2 3.1 3.6 3.2 3.4 3 3.0 3.2 3.0 3.2 21 2.7 3.2 2.8 3.1 42 3.0 3.5 3.1 3.4 Reticulocyte, K/ L 0 6.8 3.2 5.4 4.6 1.26 0.98 0.47 <0.001 0.99 0.96 0.99 1 5.9 4.0 4.6 5.4 2 5.1 4.4 4.5 4.9 3 16.8 11.8 12.9 15.8 21 0.7 1.0 0.7 1.0 42 0.5 0.7 0.4 0.8 Platelet, K/ L 0 331.4 314.7 388.2 257.9 30.50 0.34 0.24 <0.001 0.97 0.35 0.05 1 266.6 339.4 295.0 311.0 2 247.4 285.9 273.6 259.7 3 302.4 331.9 304.2 330.0 21 561.1 535.9 575.2 521.9 42 462.3 515.7 505.5 472.5 Data is presented as least squares means SEM; P RBC = r ed blood cells = RBC. Age = 0 and 3 days (d) after birth. DCAD = d ietary c ation a nion d ifference. Lev = DCAD level. Dur = DCAD duration. The triple interaction DCAD level*duration*age was not significant for any of the parameters estimated (P > 0.10).
55 Table 2 7 N eutrophil percentag e and count lymphocyte percentage and count monocyte percentage and count basophil percentage and count, and eosinophil percentage and count of their calves born to Holstein dams fed two differe nt levels of negative DCAD 70 or 180 mEq/kg, for two different durations, the last 21 d (short, S ) or the last 42 d (long, L ) prepartum ; (n = 9 to 12 cal ves per treatment ) DCAD level DCAD duration P value Item Age, d 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age Neutrophil, % 0 54.5 54.5 58.5 50.5 2.81 0.56 0.03 <0.001 0.65 0.37 0.92 1 65.0 62.4 67.0 60.4 2 59.9 66.2 64.6 61.5 3 50.0 54.7 54.9 49.9 21 39.3 41.0 42.5 37.8 42 39.1 36.9 39.4 36.6 Neutrophil, K/ L 0 4.3 5.1 5.5 3.9 0.66 0.72 0.35 0.08 0.68 0.63 0.80 1 5.6 6.4 6.2 5.8 2 5.3 4.9 5.4 4.9 3 4.5 4.8 4.7 4.6 21 4.7 5.3 5.3 4.7 42 4.9 4.3 4.8 4.4 Lymphocyte, % 0 44.3 42.2 38.5 48.1 2.80 0.66 0.02 <0.001 0.87 0.31 0.75 1 33.7 35.5 31.0 38.2 2 35.5 31.1 32.2 34.4 3 46.8 41.5 41.6 46.7 21 54.3 53.7 51.4 56.6 42 50.7 55.2 51.3 54.6 Lymphocyte, K/ L 0 3.5 3.5 3.6 3.4 0.19 0.35 0.44 <0.001 0.98 0.29 0.48 1 2.7 3.5 2.7 3.5 2 2.7 2.3 2.6 2.4 3 3.5 3.4 3.3 3.6 21 5.9 6.6 6.0 6.5 42 5.7 6.0 5.8 5.8 Monocyte, % 0 0.15 0.22 0.20 0.17 0.74 0.51 0.21 <0.001 0.56 0.65 0.80 1 0.21 0.35 0.22 0.34 2 0.03 0.16 0.18 .0001 3 0.58 0.83 1.33 0.08 21 3.27 1.24 2.94 1.57 42 8.00 6.97 8.25 6.73
56 Table 2 7. Continued DCAD level DCAD duration P value Item Age, d 70 180 S L SEM Level Duration Age Lev*Dur Lev*Age Dur*Age Monocyte, K/L 0 0.0005 0.033 0.029 0.009 0.10 0.47 0.21 < 0.001 0.69 0.76 0.83 1 0.008 0.047 0.037 0.018 2 0.004 0.031 0.035 0.001 3 0.07 0.05 0.11 0.005 21 0.22 0.18 0.22 0.18 42 0.98 0.76 1.01 0.73 Basophil, % 0 0.63 1.56 0.87 1.32 0.76 0.94 0.45 <0.001 0.63 0.43 0.79 1 0.66 0.81 0.47 0.99 2 4.30 2.54 4.02 2.81 3 1.98 2.50 2.70 1.78 21 3.18 3.73 4.05 2.86 42 0.37 0.24 0.33 0.28 Basophil, K/L 0 0.054 0.134 0.12 0.07 0.08 0.57 0.90 < 0.001 0.60 0.59 0.58 1 0.045 0.087 0.09 0.04 2 0.36 0.18 0.23 0.31 3 0.26 0.26 0.20 0.32 21 0.36 0.48 0.39 0.46 42 0.03 0.03 0.03 0.03 Eosinophil, % 0 0.75 1.53 0.78 1.50 0.42 0.97 0.5 <0.001 0.10 0.4 0.35 1 0.46 1.29 0.62 1.13 2 0.23 0.40 0.29 0.34 3 0.52 0.71 0.67 0.56 21 0.20 0.19 0.21 0.18 42 1.66 0.82 1.76 0.73 Eosionphils, K/L 0 0.06 0.11 0.12 0.05 0.08 0.52 0.23 < 0.001 0.17 0.43 0.75 1 0.04 0.13 0.12 0.05 2 0.02 0.02 0.02 0.03 3 0.02 0.06 0.05 0.04 21 0.02 0.02 0.02 0.02 42 0.32 0.08 0.05 0.32 Data is presented as least squares means SEM; P DCAD = Dietary cation anion difference The triple interaction DCAD level*duration*age, was not significant for any of the parameters estimated (P > 0.10).
57 Figure 2 1. Effects of exacerbating the level ( Lev; 70 vs. 180 mEq/kg) and extending the duration (Dur; 21 d, Short vs. 42 d, Long) of maternal negative d ietary c ation a nion d ifference ( DCAD ) hydroxybutyric acid (BHBA), non esterified fatty acids (NEFA), glucose and total protein (TP) of their calves ( n = 9 to 12 per treatment) at birth (0 h), 24 h, 48 h, 72 h, 3 and 6 weeks after birth. Calves born to da ms fed the 180 DCAD had decreased BHBA concentrations compared to calves born to dams fed 70 DCAD specifically at 1 and 42 d of age ( 0.059 and 0.082 0.008 mmol/L vs. 0.087 and 0.122 0.008 mmol/L, respectively ; P = 0.01). Calves born to dams fed the 180 DCAD tended to have decreased NEFA concentrations compared with calves born to 70 DCAD fed dams ( 0.27 vs. 0.31 0.03 mmol/L, respectively; P = 0.07). Furthermore, there was an age effect for NEFA concentrations ( P < 0.001). There no treatment effect for glucose and total protein (P > 0.13); only an age effect both for glucose ( P < 0.001) and total protein ( P < 0.001). Triple interactions were not significant (P > 0.10) *denotes statistical difference, P
58 CHAPTER 3 GENERAL DIS CUSSION AND SUMMARY It is well recognized that maternal nutrient restriction at different time periods of gestation may lead to permanent alternations in development possibly resulti ng in adult diseases However, consumption of excess supplementation of nu trients such as minerals, energy rich nutrients and proteins can either negatively or positively influence or growth, immunity, energy metabolism and reproduction. There are limited data that explore fetal programming in dairy cows, spe cifically the effects of negative DCAD for the prevention of hypocalcemia. The negative DCAD diet has been shown to mitigate hypocalcemia, a metabolic disorder that will affect nearly 5 to 7% of U.S. dairy cows (Reinhardt, 2011). Fetal programming in dairy cattle will help researchers understand the relationships between maternal nutrition during late gestation and postnatal calf health This knowledge will improve managemen t and nutritional strategies of future generatio ns of high producing dairy cows thu s, improving the performance and health of the dairy herd. C alves born to the cows that were given the negative DCAD for an extended duration had reduced birth BW. B irth BW above or below 40.3 kg can lead to dystocia, which increases the risk of neonatal mortality defined as death within 28 d after birth (Johanson and Berger, 2003). In th e present study, we observe d a reduction of 3 d in the gestation lengths of the cows that were given the negative DCA D for an extended duration. H uman studies have show n that a gestation length of 40 weeks is essential for the maturation of fetal organs and tissues. For instance, preterm infants born at 37 weeks have an incre ased risk of neonatal mortality and increased respiration (McIntire and Leveno, 2008) Additionall y, in Holstein dairy cows it has been reported that heife rs born to cows with normal gestation length ranging from 270 to 282 days lived longer and had improved reproductive perfo rmance compared with heifers born to dams
59 with a short er or long er gestation length compared to the average (Vieira Neto et al., 2017) T he fetus initiates the act of parturition through a cascade of endocrine signaling from the hypothalamus pituitary adrenal axis (Mcmillen et al., 19 95) but maternal factors such as nutrition and stressors may influence the timing of parturition. Limited studies have investigated maternal factors that may alter mechanisms in the fetal hypothalamus pituitary adrenal axis and endocrine signaling. In the current study, t here were no evaluations measuring endocrine signaling or concentration differences between the two groups, however it would be worthwhile to further investigate the impacts over acidification may have on gestation length and the potent Moreover, exploring the conception rate of calves born to dams fed the negative DCAD for an extended duration compared with the shorter duration would provide insight on alterations that may have oc curred in utero The physiological changes that occur during parturition may lead the newborn calf into a respiratory or metabolic acidosis. In the present study, all the calves experienced some degree of metabolic acidosis, but the calves born to 70 dam s had a more defined case Notably, by 3 d of age all the calves were able to recover from metabolic acidosis, therefore t he maternal DCAD did not have long lasting negative effect s. s iCa concentration at 3 d of age, i n addition to differences i n NEFA and BHBA concentrations. In addition, t he rumen development in a calf is dependent on the initiation and amount of consumption of grain, therefore the ability to absorb nutrients may vary depending on the grain co nsumption of each individual calf (Meyer and Canton, 2016) As previously discussed, the mechanism explaining these differences are uncertain future stud ies measuring the feed intake of the calves may provide more insights into these physiological respons es.
60 In summary, this thesis demonstrate d that extending the duration or exacerbating the level of maternal DCAD during late gestation impact the offs as well as their acid base status, mineral and energy metabolism during early life and that these effects are not long lasting Our results indicate that r egardless of subtle differences in measures of innate immunity the health of the calves born to cows consuming different combinations of DCAD levels and durations was not greatly impacted
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71 BIOGRAPHICAL SKETCH Carolina Collazos is Colombian American and was born in Miami, Florida, USA in 1993. In August 2011, she began her studies in biological s ciences at Florida State University and graduated in May 2015 with her b achelor s degree In August 2015, she moved to Gainesville, Florida, USA, to join the Animal Sciences program of the University of Florida as a M aster of Science student and graduated in the Spring of 2017 under the supervision of Dr. Jimena Laporta.