Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1-1-2003
Placental relationships to parturition in dairy cattle
Allison Loraine Riddle Iowa State University
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Recommended Citation Riddle, Allison Loraine, "Placental relationships to parturition in dairy cattle" (2003). Retrospective Theses and Dissertations. 20009. https://lib.dr.iastate.edu/rtd/20009
This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Placental relationships to parturition in dairy cattle
by
Allison Loraine Riddle
A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Major: Animal Physiology
Program of Study Committee: Howard D. Tyler (Major Professor) Carolyn Komar Michael D. Kenealy
Iowa State University
Ames, Iowa
2003 ii
Graduate College Iowa State University
This is to certify that the master's thesis of
Allison Loraine Riddle has met the thesis requirements of Iowa State University
Signatures have been redacted for privacy iii
TABLE OF CONTENTS
LIST OFTABLES V
LIST OF FIGURES vi
ACKNOWLEDGEMENTS Vll
CHAPTER 1. GENERAL INTRODUCTION 1 Thesis Organization 1 Placental Impacts on Adaptations from Fetal to Neonatal Life: A Review 1 Introduction 1 Placental Development 3 Placental Structure 5 Placental Function 7 Respiration 7 Nutrition 9 Hormone Production 10 Placental Circulation 11 Fetal Patterns of Blood Flow 11 Neonatal Patterns of Blood Flow 12 Placental Transfusion 13 Umbilical Cord Rupture 15 Blood Volume Changes 21 Maternal Blood Volume 21 Fetal-Neonatal Blood Volume 22 Residual Placental Blood Volume 23 Placental Expulsion 24 Placental Retention 24 Literature Cited 27
CHAPTER 2. FACTORS AFFECTING RESIDUAL PLACENTAL BLOOD VOLUME FOLLOWING PARTURITION IN DAIRY CATTLE 41 Abstract 41 Introduction 42 Materials and Methods 44 Animal Management 44 Placental Analysis 45 Statistical Analysis 46 Results 47 Placental Blood Volume 47 Placental Expulsion Time 4 7 Correlations 4 7 Discussion 55 Literature Cited 57 iv
CHAPTER 3. RELATIONSHIPS BETWEEN PLACENTAL CHARACTERISTICS, CALF CHARACTERISTICS, DELIVERY PARAMETERS AND PLACENTAL RETENTION IN DAIRY CATTLE 60 Abstract 60 Introduction 61 Materials and Methods 63 Animal Management 63 Placental Parameters 64 Statistical Analysis 64 Results 67 Placental Expulsion Time 67 Duration of Calving 67 Calving Ease Score 67 Calf Weight 71 Correlations 71 Discussion 83 Literature Cited 85
CHAPTER 4. GENERAL CONCLUSIONS 91 V
LIST OF TABLES
Table 1. The fetal membranes of farm animals. 5
Table 2. Classification of chorioallantoic placentae. 6
Table 3. Values of manually drained residual placental blood volume vs. calculated residual placental blood volume. 48
Table 4. Descriptions of calculated parameters. 65
Table 5. Factors affecting duration of calving, placental expulsion, calving ease score and calf weight in Holstein cattle. 66
Table 6. Variables included in the model for placental expulsion time. 68
Table 7. Variables included in the model for duration of calving. 70
Table 8. Percent of total calvings in association with calving ease score. 72
Table 9. Variables included in the model for calving ease score. 78
Table 10. Variables included in the model for calf weight. 79 vi
LIST OF FIGURES
Figure 1. Correlation between calf weight and placental blood volume in dairy calves. 49
Figure 2. Correlation between assisted birth on placental blood volume in dairy calves. 50
Figure 3. Correlation between total cotyledonary hemoglobin and placental blood volume in dairy calves. 51
Figure 4. Correlation of calf weight and placental expulsion time in dairy calves. 53
Figure 5. Correlation between number of cotyledons and placental expulsion time in dairy calves. 54
Figure 6. Effect of parity on duration of calving. 69
Figure 7. Color index of cotyledons in the center of placenta in relation to calving ease score. 73
Figure 8. Color index of cotyledons at tips of placenta in relation to calving ease score. 74
Figure 9. Diameter of umbilical stump in relation to calving ease score. 75
Figure 10. Cotyledon number in relation to calving ease score. 76
Figure 11. Umbilical cord break point in relation to calving ease score. 77
Figure 12. Correlation between umbilical cord efficiency and duration of calving. 80
Figure 13. Correlation between calf sex and calf weight. 81
Figure 14. Relationship between weight of the placenta and calf weight. 82
Figure 15. Correlation between total length of the umbilical cord and placental expulsion time. 84 vii
ACKNOWLEDGEMENTS
There are many people I would like to give my sincere appreciation to for all of their hard work, guidance, support, and friendship. First of all, I would like to thank Dr. Howard Tyler for all of his support, friendship and most of all patience. I am forever indebted to him for his friendship during my undergraduate and graduate career at Iowa State University. I would like to thank the remaining committee members, Dr. Kenealy and Dr. Komar. Thank you so much for taking the time and effort to play such an important role in my career. I would also like to thank all of the graduate students including Carrie Hammer, Josie Booth, and Sylvia Wawrzyniak for all of their endless guidance, support, and cheerfulness. I wish them all the best of luck with all of their goals and dreams.
I would like to thank all of those who helped during all hours of the day for my research projects. Liz Norris, Kate McKenna, Amber Hermann, and most of all Becca Ritson and Jenice Jim.
Without all of you my Master's project would not be complete.
I was fortunate to meet so many wonderful friends and co-workers through out my undergraduate and graduate career that it would be impossible to name everyone that has influenced me. I would like to take this time to mention one of them. Jay Wenther, my soon to be husband.
Without his love, support and friendship I would have not made it through graduate school. Thank you for all of your guidance.
Lastly, I would like to thank my parents and remaining family. They have given me the world, and for that I am forever grateful. CHAPTER 1
GENERAL INTRODUCTION
Thesis Organization
The following thesis is organized into four chapters. Chapter One is a review of the literature covering some relevant adaptations from fetal to neonatal life. Chapter Two is a summary of research conducted to develop a novel technique for determining placental blood volume. Chapter Three includes research conducted to determine relationships between placental characteristics and placental retention. General conclusions from these experiments are in Chapter Four. The research summarized in this thesis will be submitted for publication, and co-authors will include Howard D. Tyler.
Placental Impacts on Adaptations from Fetal to Neonatal Life
Introduction
Parturition is the final step in the reproductive process. The parturition process is initiated by the fetus and involves a cascade of endocrine events that lead to myometrial contractions, dilation of the cervix, expulsion of the fetus, and finally expulsion of the placenta (Senger, 1999). As the fetal mass reaches the uterine limitations it is believed that the fetus becomes stressed and releases adrenocorticotropic hormones (ACTH) from the fetal anterior pituitary into the fetal circulation. Elevated ACTH stimulates the fetal adrenal cortex to produce corticoids. This elevation of fetal corticoids initiates changes to the condition of the dam such as the removal of the myometrial progesterone block, enabling myometrial contractions to begin. Fetal corticoids also stimulate an increase in reproductive tract secretions, including an increase in mucous production. Fetal corticoids induce placental synthesis of PGF2a that also aids in the removal of the progesterone block. 2
Increases in estradiol and prostaglandins allow the myometrium to become contractile therefore aiding in the expulsion of the fetus. The contracting uterus pushes the fetus toward the cervix, stimulating cervical dilation. Pressure on the cervix also causes a release of oxytocin into the maternal circulation leading to additional myometrial contractions.
Cervical dilation is also partly a response to increased concentrations of relaxin. Relaxin is produced by the placenta in bovines. Production of relaxin is stimulated by PGF2a, and as more relaxin is released, cervical dilation is further enhanced. Oxytocin-induced myometrial contractions continue and eventually result in expulsion of the fetus.
There are many crucial physiological changes that occur during the transition from fetal to neonatal life. Many of these changes are dependent on the dramatic transformations occurring in the circulatory system during this period. At birth, patterns of blood flow must be immediately altered from fetal circulation patterns to those characteristic of the neonate to ensure survival. Changes in circulatory patterns also play a key role in the transitions in respiration, thermoregulation and intestinal function that occur during the transition to extrauterine life. Timing of umbilical cord rupture is an important consideration for maximizing feto-maternal transfusion of blood to the neonate via the umbilical cord.
Premature rupture of the umbilical cord in humans may decrease neonatal blood volume and increase incidence of retained placenta (Thomas et al., 1990).
The transition from fetal to neonatal life is one of the most difficult and traumatic stages for an animal. Therefore, it is critical to understand the normal physiological adaptations occurring in the perinatal period to ensure optimal health for both the neonate and dam. This review focuses on selected aspects of placental functions focusing on placental circulation and the circulatory changes that occur during parturition. 3
Placental Development
Fetal placentae provide many critical services to developing fetuses (Collins, 1993).
Because placentae are the sole sites through which nutrients, wastes, and respiratory gases are transported between maternal and fetal systems, their primary function is to supply metabolic substrates to support fetal growth and development. Proper placental implantation, growth and function are all necessary for normal fetal growth and development (Collins,
1993). Additional factors that affect fetal growth, such as maternal and paternal genotype, increased number of fetuses, uterine surface area, maternal nutrient deprivation, or environmental stress, typically also affect placental size and development (Walton and
Hammond, 1938; Ebbs et al., 1942; McKeown and Recoed, 1953; Echstein et al., 1955;
Hunter, 1956; Jouber and Hammond, 1958; Alexander, 1964; Alexander and Williams, 1971;
Turman et al., 1971; Rattray et al., 1974; Corah et al, 1975; Sreenan and Beehan, 1976;
Knight et al., 1977; Thompson et al., 1982; Ferrell, 1991; Alexander, 1964; Knight et al.,
1977).
The chorion, amnion, vitelline sac (yolk sac) and allantoic sac are the four different membranous structures involved in placental development (Mossman, 1987). The chorion is an epithelial layer derived from the outer wall of the blastocyst, or trophoblast. During the course of implantation the placenta is completed by an inner layer of mesenchyme that originates from the embryo. Shortly after implantation, the mesenchyme develops its own capillary system. The chorionic epithelium represents the outermost layer of the fetoplacental unit which encloses the fetus. Fetal membranes act as an exchange barrier between the fetus and dam. The amnion, another epithelial layer, is formed by folding of the extraembryonic ectodern and somatic mesoderm that encloses the embryo in a double-walled 4 sac. This occurs by the eighteenth day of pregnancy in cattle (Greenstein and Foley, 1958).
The amnion is filled with a clear, watery fluid that holds the embryo suspended. It forms a protective cushion for the embryo and also prevents the embryo from adhering to surrounding membranes. During parturition, the amnion acts as a wedge to dilate the cervix, aiding in the parturition process (Salisbury et al., 1978). The yolk sac (vitelline sac) is part of the primitive gut, but is not included in the body when the edges of the embryo form the gut (Table 1). As the embryo folds, the yolk stalk extends below the embryo to the yolk sac, which supplies nutrients to the embryo. Soon thereafter, blood vessels develop in the walls of the yolk sac and carry materials absorbed from uterine fluids to the embryo. The yolk sac within the bovine specie is only functional for approximately 3 wk and then is replaced by the allantois (Leiser and Kaufmann, 1994). And finally, the allantois is an additional extra embryonic membrane that is formed as an outpouching of the hindgut. By day 23 of pregnancy, the bovine embryo has a well-developed allantois, and by day twenty-six this membrane is larger than the embryo. The allantois fills the space between the amnion and the serosa. The allantois is connected to the bladder by means of the urachus, that runs through the umbilical cord. This then functions as the urinary receptacle for the embryo.
Over time, the allantois expands and merges with the serosa to form the chorion, a four layered membrane, which now completely surrounds the embryo, amnion, and allantoic cavity. With complete development and attachment of the placenta to the uterine wall, the embryo is now classified as a fetus. Gases and nutrients are now able to exchange between blood vessels of the fetus and blood vessels of the dam (Leiser and Kaufmann, 1994 ). 5
Table 1. The fetal membranes of farm animals (Senger, 1999).
Membrane Origin Functions
Yolk Sac Early entodermal layer Vestigial
Amnion Cavitation from inner cell mass Encloses fetus in a fluid-filled cavity
Allantois Diverticulum of hindgut Blood vessels connect fetal with placental circulation
Fuses with chorion to form the chorioallantoic placenta
Chorion Trophoblastic capsule of blastocyst Encloses embryo and other fetal membranes
Intimately associated with lining of uterus to form placenta
Umbilical cord Amnion wraps about the yolk stalk Encloses allantoic vessels and acts as the vascular link
Placental Structure
Placentae may be classified according to morphology, characteristics of the maternal fetal barrier, and loss of maternal tissue at birth (Jainudeen and Hafez, 1980). The gross shape of the placenta is determined by distribution of villi over the chorionic surface (Table
2). 6
Table 2. Classification of chorioallantoic placentae {Senger, 1999).
Classification
Species Chorionic Villous Maternal-fetal Barrier Loss of Maternal Pattern Tissue at Birth
Pig Diffuse Epitheliochorial None
Mare Diffuse and Micro- Epitheliochorial None
Cotyledonary
Cow Cotyledonary Epitheliochorial None
Goat, Sheep Cotyledonary Epitheliochorial None
Ruminants have a cotyledonary placenta. Cotyledons are defined as portions of the placenta of trophoblastic origin that consist of blood vessels and connective tissue. Cattle placentae have 70 to 120 cotyledons (Senger, 1999; Jainudeen and Hafez, 1980) and these cotyledons reach a maximum weight around the ninetieth day of gestation (Barcroft and Kennedy,
1939). A placentome is defined as the point of interface in the cotyledonary placenta that include both fetal cotyledons and convex maternal caruncles originating from the caruncular regions of the uterus. Caruncles are points of attachment in the uterus and serve to exchange nutrients and gases to the fetus. Typically, there are approximately 200 caruncles located in irregular fashion throughout the two horns and body of the uterus. They measure 10-15 mm in diameter in the nonpregnant cow (Sorensen, 1979). At day 25-30 of pregnancy the chorion attaches to the caruncles in the uterus in cattle (King, 1993). During the formation of placentomes, chorionic villi located on the surface of the chorion, some 10 mm in length, protrude into crypts in the caruncular tissue (Senger, 1999; Echkstein and Kelly 1977;
Marshall and Halman, 1945; Guillomot et al., 1981; Wooding and Staples, 1981). In cattle, 7
attachment is established by day 40 of pregnancy. Cotyledons also serve as points of nutrient exchange for the fetus.
Placental Function
The importance of placental function is probably best exemplified by the close relationship between fetal weight, placental size, and uterine and umbilical blood flows in many mammalian species (Ibsen, 1928; Warwick, 1928; Hammond, 1935; Alexander, 1964;
Oh et al., 1975; Wooton et al., 1977; Christenson and Prior, 1978; McDonald et al., 1979;
Prior and Laster, 1979; Hard and Anderson, 1982; Coaton et al., 1984; Ford et al., 1984;
Reynolds et al., 1984; Metcalfe et al., 1988; Ferrell, 1989; Ferrell and Reynolds, 1992).
Placentae perform many functions, including those of the gastrointestinal tract, lung, kidney,
liver, and endocrine glands prior to birth (Ramsey, 1982; Faber and Thornburg, 1983).
Placentae also physically separate the fetal and maternal organisms.
Respiration
Prior to birth, the placenta is the site of gas exchange for the fetus. At birth, neonates must transition from a fluid-filled uterus and adapt to breathing air. For adequate gas
exchange to occur, pulmonary fluid secreted throughout fetal life must be absorbed and replaced with air. In fetal ruminants, gas exchange occurs through cotyledons. Both nutrients and gases enter the embryonic (or fetal) circulation by diffusion, facilitated
diffusion or active transport through the placenta, after which they enter blood vessels in the
allantois which ultimately form the umbilical cord (Salisbury and VanDemark, 1961). The delivery of oxygen from the placenta to the fetus and oxygen uptake by the fetus are greatly
affected by the magnitude of umbilical blood flow (Dawes and Mott, 1964). Umbilical
arteries carry deoxygenated blood from the fetus to the placenta, while the umbilical veins 8 carry oxygenated blood in the reverse direction. Silver et al. (1973) and Comline and Silver
(1975) reported positive correlations between the diameter of umbilical vessels and placental efficiency of gas exchange.
There are several factors that affect placental transfer of oxygen, including the relative shapes of maternal and fetal oxyhemoglobin dissociation curves, transplacental diffusion distance and total surface area for diffusion, rate of oxygen use by placental tissue, spatial relationships between maternal and fetal exchange vessels, and maternal and fetal blood flow rates (Longo, 1972; Comline and Silver, 1974). The fetal dissociation curve for oxyhemoglobin is left shifted relative to the maternal curve in cattle. This means that at any given oxygen tension, fetal blood has a higher affinity for oxygen than maternal blood. In the cow, the minimum P02 gradient between fetus and dam is about 20 mm Hg. This high affinity of fetal hemoglobin for 0 2 may be related to a specific hemoglobin type (Hbp) that has a high 0 2 affinity or to the presence of high 2, 3-BPG (bisphosphoglycerate).
Survival of the fetus depends on efficient oxygen transport. Alignment of maternal and fetal vessels is crucial for maximizing oxygen transport. When microscopically examined, the blood flow pattern in humans most closely fits a countercurrent flow pattern
( defined as the vessels being parallel to each other with blood flow in opposite directions).
With this pattern the venous side of the fetal capillary bed is aligned with the arterial side of the maternal capillary bed to maintain the highest possible concentration gradients for oxygen and other nutrients (Comline and Silver, 1974).
Carbon dioxide diffuses freely from the fetal to the maternal circulation across the placenta, primarily in the molecular form. This transfer is facilitated by maternal and fetal
CO2-hemoglobin dissociation curves. These curves show that fetal hemoglobin has a lower 9
affinity for CO2 than maternal hemoglobin during placental oxygen transfer. This affinity gradient aids in the diffusion of CO2 from fetal to maternal blood (J ainudeen and Hafez,
1980).
Nutrition
Prior to birth, nutrients are supplied to the fetus by the dam. The placenta permits transport of sugars, amino acids, vitamins and minerals to the fetus as substrates for fetal metabolism and growth. Placentae also serve as storage organs for glycogen as well as certain nutrients such as iron.
The placenta also is freely permeable to water and electrolytes (Steven, 1975). Large quantities of sodium and other electrolytes cross the placenta by simple diffusion. In addition to the exchange that occurs across the placenta, circulation of water and exchange of solutes also occurs between the fetus and the amniotic and allantoic sacs.
Other minerals are also transported across the placenta (Steven, 1975). Iron tends to be more abundant in the fetus than in the dam. In the fetus, iron and copper are primarily stored in the liver, spleen and bone marrow. Calcium and phosphorus are also crucial for fetal development. These two minerals cross the placenta and enter the fetal circulation against a concentration gradient.
Glucose is the major source of energy for the bovine fetus. The majority of glucose is derived from the maternal circulation and is actively transported across the placenta.
Glucose is the principal fetal circulating carbohydrate in humans. In ruminants, fructose is the main circulating carbohydrate in fetal blood and placenta; however, it is not utilized as an energy source. Fructose is synthesized by the placenta and varies inversely with glucose concentrations (Edwards and Powers, 1967; Tyler and Ramsey, 1993). Concentrations of 11
Placental progesterone is necessary for early embryonic development and provides the stimulus for elevated secretion of progesterone by endometrial glands (Barnes et al.,
1975). High levels of progesterone function to inhibit myometrial contractions (Edgerton and Erb, 1972). Progesterone increases in the blood of pregnant females and peaks at different stages of pregnancy depending on the specie (for example 6-8 mo of gestation in cattle) (Hunter et al., 1970). During the latter half of gestation, an increased rate of estrogen production occurs in the placentae of mares, cows, sows and sheep (Hunter et al., 1970). The placenta relies on the fetus to provide precursors for the synthesis of estrogens, whereas fetal precursors are not required for the synthesis of progesterone.
Placental Circulation
Fetal Patterns of Blood Flow
The fetal heart is responsible for circulating blood through both the fetus and the placenta. Two umbilical arteries carry carbon-dioxide rich blood from the fetus into the placenta, where most carbon dioxide is exchanged for oxygen. Oxygen-rich blood returns to the fetus via two umbilical veins. Not all species contain two umbilical arteries and two umbilical veins. For example, in humans two umbilical arteries are present along with only a single umbilical vein. Umbilical venous blood flow in human fetuses ranges between 75 and
115 ml per minute per kilogram of fetal weight (Gill et al., 1981). Indik and Reed (1990) reported that umbilical arterial flow was related to umbilical venous flow, implying an interdependence between fetal cardiovascular blood flow and placental blood flow.
Hemoglobin in umbilical venous blood is 80% saturated with 0 2 (Dawes et al., 1954). The umbilical veins, which pass near the liver, separate into several vessels. Some veins go to the liver, but one large branch, the ductus venosus, goes directly to the posterior vena cava. By 10 fructose decrease after birth in lambs (Alexander et al., 1955; Alexander et al., 1970) and in calves (Kurz and Willett, 1991).
Some compounds cannot be transported across the placenta. Most maternal proteins do not cross the placental barrier, although amino acids are actively transported. The fetus is able to synthesize most proteins from amino acids contributed by the dam. In humans and other primates, maternal immunoglobulins are transplacentally transported to the fetus, although this does not occur in ruminants (such as cattle, sheep, goats, deer), horses, or pigs.
In species lacking transplacental IgG transfer, maternal-fetal antibody transfer occurs via ingestion of colostrum. Differences in transplacental IgG transfer can be explained by structural differences between various placental types.
Lipids are also unable to cross the placenta. Instead, the placenta hydrolyzes triglycerides and maternal phospholipids and synthesizes new lipid materials to be utilized by the fetus. Lipid soluble vitamins, such as vitamins A, D, and E, are also poorly transported across the placenta, while water soluble vitamins (B and C) are transported across the placenta with relative ease.
Hormone Production
Placentae secrete many bioactive peptides and hormones, including tropic
(somatotropins and gonadotropins) hormones that are released into fetal as well as maternal circulations. The suffix "tropin" (the singular form of the word of tropic) means "having affinity for". Thus, gonadotropins have an affinity for the gonads (the ovary and testis).
Some of these hormones enter amniotic fluid or are reabsorbed by the fetus or dam (Senger,
1999). 12
this point, 0 2 saturation of the hemoglobin in blood is reduced to 67% (Dawes et al., 1954;
Dawes, 1962). Next, the posterior vena cava along with the anterior vena cava collect venous blood from all parts of the body and direct it to the right atrium. From there, blood crosses the foramen ovale (a temporary opening) into the left atrium where 02 saturation of hemoglobin is further reduced to about 58% (Dawes et al., 1954) the remainder of blood flows into the right ventricle. The portion of blood directed to the left atrium, which ultimately supplies the heart and brain, mixes with blood from the pulmonary veins. This mixed blood passes into the left ventricle and then is pumped into the aorta. The aorta carries the blood to all parts of the body (except the lungs) and ultimately re-enters the placenta again via the umbilical arteries (Dawes et al., 1954). The remainder of blood from the right atrium enters the right ventricle and is pumped into the pulmonary circulation.
Neonatal Patterns of Blood Flow
Following rupture of the umbilical cord and constriction of umbilical arteries and veins, patterns of circulation change. Fetal heart rate and pulmonary arterial pressure both decrease, while systemic arterial pressure increases (Dawes et al., 1953). The ductus venosus' function is then completed by the portal vein (Dawes et al., 1953). Thus, the hepatic vein and posterior vena cava carry only low oxygen, high-carbon dioxide blood. This blood enters the right atrium. The foramen ovale closes in response to decreasing vascular resistance of the pulmonary artery (Assali et al., 1963). Therefore, all blood from the right atrium is directed into the right ventricle and is pumped to the lungs where it becomes oxygenated if the calf has begun to breathe. A large uninterrupted venous return through the umbilical vein that occurs during the first breath (a long, powerful gasp) may influence the success of the establishment of pulmonary ventilation (Cort, 1962). During breathing, 13 venous flow rate varies because of fluctuations in intrathoracic pressure in the fetus (lndik and Reed, 1990). The ductus arteriosus also closes which prevents blood from the right ventricle and the pulmonary artery being re-directed into the aorta (Assali et al., 1962).
Placental Transfusion
Studies of isolated placentae have shown that the placental blood volume increases with increasing umbilical blood flow (Bissonnette, 1975; Schroder et al., 1981). Distension of placental vessels results in increased vascular capacity. Placental transfusion is the transfer of blood from the placenta to the fetus during parturition. Placental transfusion has been widely studied in human infants. The amount of placental transfusion is typically measured by determining residual placental blood volume and(or) neonatal blood volume following severage of the umbilical cord. Linderkamp (1982) suggested that methods for determining residual placental blood volume may underestimate true placental blood volume by 9 ml/kg by not accounting for blood remaining in smaller vessels within the placenta.
Relative diameter of the umbilical arteries and umbilical veins play a key role in placental transfusion along with force of uterine contractions, effect of gravity, and breathing of the neonate (Yao et al., 1967). An imbalance of umbilical arterial and venous blood flow causes redistribution of blood between the fetus and placenta at birth. Haselhorst (1929) measured venous pressure in the umbilical vein and found that pressure increased from 47 to
123 mm Hg during uterine contractions. During parturition, increases in catecholamines and prostaglandins enhance transfer of blood from the placenta to the fetus by inducing vasoconstriction of placental vessels.
In human neonates, postnatal transfer of blood from the placenta to the fetus is complete within the first 3 min after birth (Sisson, 1973). According to studies, blood 14 volume in human infants increases by ten percent to thirty percent and red cell mass increases by twenty-five percent to sixty percent following late cord clamping when compared to the same parameters in early-clamped infants (Sisson, 1973). Lind (1965) reported that 40 ml of blood enters infants within 15 sec and the remaining 80 ml is transferred within 60 sec after birth. In these studies, birth was defined as the delivery of the buttocks. Placental transfusion is also thought to be enhanced by uterine contractions.
During the first 10 to 15 sec of uterine contractions after birth there is a 15 percent increase in blood volume transferred to the fetus and another 20 percent increase between 45 to 60 sec
(Yao et al., 1968).
It has been theorized that infants delivered by cesarean sections have decreased total blood volume and leave behind an increased amount of residual placental blood. It also has been suggested that the high neonatal mortality of infants born by cesarean section in the
1930's and 1940's was caused by excessive blood remaining in the placenta, which is not compressed by uterine contractions during cesarean section deliveries. Early recommendations included either suspension of the placenta or lowering of the neonate after birth to allow for gravitational forces to aid in the flow of placental blood to the neonate
(Duckman et al., 1953; Landau et al., 1950). Along with utilizing gravitational forces in this manner, it was thought that stripping or milking of the umbilical cord would also aid in increasing the amount of blood received by the neonate (Siddall et al., 1952).
The rate of placental transfusion is greatly influenced by position of the delivered infant in relation to the placenta (Yao and Lind, 1969). Gravity is thought to be responsible for this increase in the rate of placental transfusion. Infants that are held 50 to 60 cm above the placenta do not receive any placental transfusion within 3 min after birth. However, 15 infants that are either held 10 cm above or held below the placenta receive full transfer of placental blood within 3 min of delivery (Yao and Lind, 1969).
There are conflicting reports regarding the effect of the onset of neonatal respiration on placental transfusion. Usher et al. (1963) and Yao et al. (1968) found no relationship between postnatal blood volume and onset of the first breath. However, other authors report that little or no placental transfusion occurs when the umbilical cord is clamped prior to the onset of breathing, and that clamping after the onset of respiration increased neonatal blood volume by 10 ml/kg in human infants (Philip and Teng, 1977).
Umbilical Cord Rupture
The umbilical cord is structurally very simple. In humans, it is composed of two arteries that carry deoxygenated blood from the fetus to the placenta, and one vein that carries oxygenated blood from the placenta to the fetus, as well as the urachus and Wharton's jelly. Wharton's jelly functions to provide a flexible structure, helping to prevent accidental compression of the vasculature. The umbilical vasculature and the urachus enter the abdominal cavity through the inner umbilical ring, an oval opening in the abdominal wall.
The umbilical veins become a simple structure at the level of the inner umbilical ring.
Unlike the umbilical arteries and urachus, the umbilical vein tightly adheres to surrounding tissue. The umbilical vein ascends slightly to the right, passing through the liver to join the left branch of the portal vein. The umbilical vein carries oxygenated and nutrient rich blood from the placenta through the ductus venosus directly into the caudal vena cava. The umbilical arteries originate near the pelvic inlet from the left and right internal iliac arteries.
They pass along the sides of the bladder to the umbilicus, and carry deoxygenated blood from the fetus to the placenta (Fisher, 1932). In humans, the umbilical cord attains its full length 16 by the beginning of the eighth month of pregnancy (Malpas, 1964) and umbilical cord lengths range from as short as 22 cm to as long as 130 cm (Purola, 1968). No studies have been conducted regarding umbilical cord length in other species, although it is known that there are tremendous variations. The bovine species tends to have shorter umbilical cords that may potentially play a significant role in the amount of placental transfusion to the neonate due to the early timing of cord rupture during the delivery process. The length of umbilical cord does not appear to be related to the insertion point into the placenta.
However, variations in mode of attachment of umbilical vessels are associated with gestational age and placental weight (Addai et al., 1994).
In mammals, the timing of umbilical cord severage varies considerably based on species and delivery conditions. Umbilical cord severage can be caused by movement of the neonate, movement of the dam, chewing of the umbilical cord by the dam, or manual tearing
(Yao et al., 1977). In humans, breaking strength of the umbilical cord ranges between 2-12 kg of pressure (Morris and Hunt, 1966). At birth, the umbilical cord breaks, and the amniotic sheath remains as the only visible structure at the umbilicus. Within 3-4 d the umbilicus becomes dry, and after 2 wk the umbilical stump falls off leaving a scab that remains until the third or fourth week of life (Stober, 1990). The umbilical arteries and the urachus typically retract into the abdomen immediately after umbilical cord rupture. Closure of the umbilical arteries begins within 5 sec of birth; this is accomplished by numerous local circular contractions of both arteries. Within 45 sec of birth complete closure of the umbilical arteries has occurred (Moinian et al., 1969). Closure of umbilical veins is initiated by local vasoconstrictors during the first 15 sec of birth. Functional closure of umbilical veins occurs within 3 min after birth (Meyer et al., 1978). Ultrasonography of umbilical 17 structures in calves shows that umbilical veins start as oval, thin-walled structures with a wide center (vertical diameter of 11 mm, and a horizontal diameter of 19 mm), and decrease in diameter during the first week of life to 5-8 mm. Umbilical arteries are round thick-walled vessels with a mean diameter of approximately 8 mm. In that study, the urachus was unable to be seen via ultrasonography (Lischer and Steiner, 1993).
Important mediators of umbilical constriction are small, biologically nona-peptides called bradykinins produced in the lungs in response to increased oxygen tension (Regoli et al., 1998). Bradykinins are potent mediators of inflammation and smooth muscle contraction. Two bradykinin receptors have been identified in umbilical vessels: B1 and B2
(Regoli et al., 1998). Bradykinin B2 receptors are constitutively present in most tissues while bradykinin B1 receptors are only expressed in injured tissues, such as the ruptured umbilical cord, as an inflammatory response (Marceu et al., 1998; Sardi et al., 1997; Sardi et al., 1998; Sardi et al., 2000). By inducing constriction of the umbilical arteries, bradykinins stimulate an increase in umbilical arterial-venous pressure difference and umbilical vascular resistance, resulting in a decrease in umbilical blood flow (Berman et al., 1978). These effects are more pronounced when bradycardia is present; these effects could result in a transfusion of blood from the placenta to the fetus (Berman et al., 1978). Serotonin (White,
1989) and endothelin (Haegerstrand, 1989) are also important mediators of umbilical cord closure in human infants; however, dopamine and histamine appear to have no effect on umbilical cord blood flow and local vascular resistance (Berman et al., 1978). Prostaglandin
(PG) endoperoxides PGE2, PGF2u, PGA2, PGB2 and thromboxane A2 are all contractors of human umbilical vessels in vitro; POii and PGE1 cause relaxation of umbilical arteries at low concentrations but cause contraction at higher concentrations (Berman et al., 1978; Cassin et 18 al., 1979; Novy et al., 1974; Rankin and Phernetton, 1976). High concentrations of epinephrine, norepinephrine, PGE and PGF are present in the neonate immediately after birth, with the highest concentrations present during fetal distress (Broughton and Symonds,
1977; Lewis et al., 1982; Messow-Zahn et al., 1978; William et al., 1977). To further understand placental transfusion, the effects of infusion of epinephrine and norepinephrine lamb fetuses have been studied. Infusions of these compounds cause an increased fetal arterial pressure, increased umbilical venous pressure, increased pressure gradient between umbilical arteries and veins, increased umbilical-placental blood flow, and increased total fetal vascular resistances. However, total cardiac output and umbilical vascular resistance are not changed and blood flow to the total fetal body decreases (Berman et al., 1978; Lorijn and Longo, 1980; Novy et al., 1974). An increase in oxygen tension plays a key role in the constriction of placental vessels along with umbilical vessels (Creasy et al., 1972).
Umbilical cord compression also results in the reduction of oxygen delivery to the fetus during delivery. Acute umbilical cord compression, which is one of the most common causes of fetal distress, produces hypertension and bradycardia, as does acute hypoxemia. As a result, umbilical-placental blood flow decreases while fetal blood flow is maintained. In response to experimentally induced acute uterine artery compression, umbilical-placental blood flow is maintained while blood flow to the fetal body decreases. The differences in these fetal responses may be due to chemoreceptor stimulation and activation of neurohormonal response mechanisms during uterine artery compression (Iwamoto et al.,
1991; Stembera et al., 1968).
In human medical practices there is considerable controversy as to whether the umbilical cord should be clamped immediately after birth or whether clamping should be 19 delayed to allow maximal placental blood volume to be transfused into the fetus. In the late
1800's, early ligation of the umbilical cord was a method utilized by many doctors and midwives to minimize loss of blood from the mother. However, immediate cord clamping leads to hypovolemia, hypotension, and anemia in infants. This may be especially harmful in premature infants that are at greater risk to develop severe respiratory distress when their blood volume at birth is low (Linderkamp et al., 1978). Delayed cord clamping is also associated with a reduced volume of residual placental blood (Colozzi, 1954; De Marsh et al., 1942; Nyberg and Westin, 1958; Philip and Teng, 1977; Yao et al., 1969). Late cord clamped infants have higher central venous pressure, higher left and right arterial pressures during the first hour of life, higher systolic blood pressures, higher pulmonary arterial pressure during the first 9 h of life, and higher systemic arterial pressures for as long as 6 hr after birth when compared with early cord-clamped infants (Arcilla et al., 1966a; Arcilla et al., 1966b; Buckles and Usher, 1965; Burnard and James, 1963; Oh et al., 1966a). These responses are all thought to result from the increased quantity of blood transferred to the infant following late clamping. Late clamping also results in an increased number of erythrocytes, which range from 1/2 million cells to 1 1/2 million cells, and an increase in total hemoglobin from 1 to 4 g above values seen in early clamped infants (Siddall and
Richardson, 1953). One study suggests that one-fourth to one-third of placental blood remains in the placenta if the cord is clamped early (Lanzkowsky, 1960). Hematocrit values are higher in infants following delayed cord clamping, increasing from an estimated 48% at birth to 59% by the end of a 30 min period, and continuing to increase to 64% within 4 h postpartum (Bernstine and Ludmir, 1959; Usher et al., 1963; Linderkamp et al., 1992). Early clamped infants initiate breathing more quickly after birth than late-clamped infants because 20 of earlier severance of oxygen supply (Oh et al., 1967; Baier, et al., 1990; Adamson et al.,
1991), although clamping the umbilical cord either before or after the onset of respiration has no apparent effect on the incidence of respiratory distress (Spears et al., 1966). Spears
(1966) suggested that closing of the umbilical circulation before aeration of the lungs has occurred is a highly unphysiological measure that should be avoided in humans. Early cord ligation or clamping has been theorized to increase rates of retained placenta, however, studies on placental drainage of cord blood in relation to incidence rates of retained placenta in humans have shown no correlation (Thomas et al., 1990). In addition, umbilical cord ligation was theorized to have an effect on thermoregulation by affecting total blood volume and oxygen uptake, however, timing of umbilical cord ligation also does not affect the ability of neonatal lambs to thermoregulate. All lambs show reduced rectal temperatures after delivery regardless of timing of umbilical cord ligation (Sack et al., 1976). These data suggests that umbilical cord ligation had no effect on thermoregulation. However, Mahaffey
(1961) reported a decrease in rectal temperatures in lambs with immediate umbilical cord ligation. Temperatures fell from 104 °F to as low as 94 °F compared to late cord ligation lambs with temperatures never dropping below 100°F.
In human studies, umbilical cord pulsation is often used as a good parameter for determining timing of cord ligation. In early studies, red blood cell (RBC) counts of babies following late cord ligation (less cord pulsation) were higher than those recorded from infants in the early cord ligation group (Frischkorn, 1939; Rucker, 1949). Pulsation of the cord continues for between one and thirty minutes after a normal birth (Barclay et al., 1957).
In difficult births, pulsation of the umbilical stump has been recorded to last anywhere from forty minutes up to thirteen hours (Desmond et al., 1959). Cessation of pulsation in 21 umbilical vessels begins at the placenta and proceeds toward the umbilicus; when this has been completed, the vessels collapse. A 30 sec delay in cord clamping is feasible in both vaginal and cesarean births, but does not lead to the predicted difference in infant hematocrit
(McDonnell and Henderson-Smart, 1997). The prediction estimated that cesarean section delivered infants would have lower hematocrit levels than vaginally delivered infants due to reduced uterine contractions. Delaying cord clamping to 45 sec after delivery is recommended for preterm infants below 33 weeks of age (Rabe et al., 2000). Many pre-term infants are born with under developed lungs and have an increased risk of respiratory disorders. The additional 15 sec is thought to provide adequate time for additional transfusion to maximize neonatal blood volume and therefore increase oxygen carrying capacity.
Blood Volume Changes
Maternal Blood Volume
Many maternal circulatory changes accompany pregnancy. One of the major changes is an increase in maternal blood volume. Blood volume in pregnant women increases from between 20 and 100% as gestation proceeds (Pritchard, 1965). Maternal blood volume near term ranges between 73 and 96 ml/kg (Rovinsky and Jaffin, 1965). Variations in blood volume measurements between studies may be in part caused by variability in techniques used to determine maternal blood volume. In addition, several factors affect maternal blood volume such as physical condition of the mother and weight of the fetus (Hytten and Paintin,
1963). The volume of blood lost during vaginal delivery in women is approximately 500 to
600 ml; following a cesarean section this amount may be doubled (Pritchard, 1965). 22
Deland (1976) reported that maternal blood volume tended to rise with increasing weight of the human infant; for example, a woman carrying a 3.25 kg baby would have a mean maternal blood volume of 5.6 liters (Deland, 1976). Maternal hematocrit increased by
5.2 percent in the mothers that delivered vaginally but declined following cesarean section by
5.8 percent (Deland, 1976). Six weeks after delivery there were no differences in blood volume between mothers that delivered vaginally and the mothers that delivered their infants via cesarean section. However, hematocrit was consistently lower in those patients delivered via cesarean section (Deland, 1976). Maternal blood volume in humans averages 5.05 liters at full term (Deland, 1976; Berlin et al., 1953; Bhatt, 1965).
Fetal-Neonatal Blood Volume
Some studies have been conducted using fetal or the neonatal lamb to determine blood volume (Barcroft and Gorsev, 1937; Gotsev, 1939; Barcroft and Kennedy, 1939).
These studies have provided an improved understanding of blood volume through the use of radioisotope methodology. Blood volume in fetal lambs averaged 156.1 ml/kg (Creasy et al.,
1970). Blood volume strongly influences placental blood flow in lambs, with a reported change of 1% in blood volume associated with a change of 1.7% in placental blood flow
(Faber et al., 1972). Fetoplacental blood volume is also positively correlated with gestational age (Nicolaides et al., 1987).
Yao et al. (1967) reported that infants born via cesarean section had decreased blood volume, red cell volume and plasma volume (66.4 ml/kg, 31.2 ml/kg, and 35 ml/kg, respectively) compared to values following vaginal delivery (90.2 ml/kg, 44.4 ml/kg, and
45.8 ml/kg, respectively). They theorized that since the mothers were under general anesthesia, the uterus is relatively relaxed and therefore does not plays a significant role in 23 the feta-placental blood transfusion process. However, high blood volumes were recorded for asphyxiated infants. It was theorized that the asphyxia stimulated compensatory mechanisms that resulted in increased fetal cardiac output and umbilical flow in an effort to achieve more oxygen exchange or oxygen uptake through the placental circulation (Born et al., 1956). Umbilical cords from asphyxiated infants have greater degrees of vasoconstriction in arteries but not in the vein. This, along with increased umbilical flow may result in greater blood volume in the fetus.
Blood volume in fetal humans and fetal lambs have been measured using radioactive iodinated human serum albumin (1251-RISA) (Creasy et al., 1970; Deland, 1976). Slight differences within these results have been noted due to differences in delivery techniques.
Some differences in techniques would include position of the baby after delivery, and timing of umbilical cord clamping.
Residual Placental Blood Volume
The placenta after delivery of the fetus is clearly not a closed organ, and estimates of vascular volumes may depend upon the degree of blood loss from the placenta during expulsion of the organ. Since measurement of residual blood is so inaccurate, few studies have been conducted in humans or other mammals. However, in studies that have been conducted, the amount of residual placental blood volume is positively correlated with timing of umbilical cord clamping. Late umbilical cord clamping results in less residual placental blood volume, while early umbilical cord clamping results in greater residual volumes (Moss and Monset-Couchard, 1967). 24
Placental Expulsion
The third stage of labor involves continued contractions of the uterus and delivery of the placenta. The bovine placenta is normally released from the uterus between 2 and 6 h postpartum (Kennedy, 1947; Eiler and Hopkins, 1992). The human placenta is expelled within a 10 to 20 min period. These differences in expulsion time between species are due to differences in the mode of attachment to the uterine wall and differences in placental type. In cattle placentae are released from the uterine wall partly in response to contraction and retraction of myometrial fibers. During pregnancy, the myometrial fibers increase in length and each fiber develops contraction and retraction properties that are essential for delivery of placentae postpartum (Walker, 1975). These postpartum uterine contractions, along with vasoconstriction of placental vessels, loosen the remaining attachments of the chorionic villi allowing these attachments to become dislodged from the crypts of the caruncle and ultimately leading to the expulsion of the membranes (Barcroft and Gotsev, 1937).
Frequently, following abortions, difficult calvings, premature births and (or) twinning, placentae are not released and expelled in a normal manner due to a deficiency of myometrial contractions.
Placental Retention
Failure of the placenta to be expelled during the third stage of labor is a common complication in ruminants, primarily due to failure of release of fetal chorionic villi from maternal uterine crypts. Approximately 11 % of cows fail to expel the placenta within 12 h postpartum (Gunnert, 1980). Dairy cattle tend to have a higher rate of retained placenta than beef cattle, although the reasons for this are unknown. The retained placenta often remains 25 attached to the uterine wall for over a week and leads to negative effects on health and milk production as well as reproductive efficiency (Lona-D and Romero-R, 2001).
The risk of placental retention increases as a result of premature birth, caesarian section, pharmacological induction of parturition (Inskeep, 1973), twinning (Morrison and
Erb, 1957), endocrine imbalances (Chew et al., 1978), vitamin E and selenium deficiencies
(Harrison et al., 1984), or trauma and hormonal imbalances (Marion et al., 1968; Takagi et al., 2002). The etiology of retained placenta is not completely understood, although some studies have shown that failure of collagen (a protein involved in placental development and postpartum uterine involution) breakdown appears to be related to placental retention (Eiler and Hopkins, 1992). In these cases, a lack of cotyledon proteolysis (collagenolysis) appears to be the underlying cause of retained fetal membranes; if placenta-anchoring systems are not enzymatically degraded, fetal membranes are retained (Eiler and Hopkins, 1992). In addition, higher plasma progesterone and lower plasma estrogen concentrations in late gestation cows increase the risk of retention of fetal membranes, possibly due to impaired uterine contractions (Chew et al., 1972). Another theory suggests that fetal placentae are recognized as foreign tissues and rejected by the immune system after parturition, and this process leads to expulsion of the placenta. Impaired neutrophil function (Kimura, et al., 2002) and impaired leukocyte activity (Gunnick, 1984) are both associated with increased risk of placental retention. Other researchers have suggested that drainage of placental blood facilitates delivery of the placenta, but there is currently no scientific evidence to support this theory. Due to differences in collection techniques, accurate measurements of placental drainage is difficult (Thomas et al., 1990). 26
The adverse impact of retained placenta on health, reproductive functions, and milk production result in extensive economical loss to the dairy industry. In a study that included
160,000 calvings, the relative economic impact of retained placenta was categorized into four major categories: decreased milk production (40% ), increased veterinary services (32% ), increased culling rates (19%), and increased calving interval (9%) (Joosten et al., 1988). It is estimated that total economic loss per affected cow varied from $100 to $200. For problem farms, with and incidence rate for retained placenta of 30%, the losses were estimated to total
$2139 per year based on 100 head (Joosten et al., 1988)
The appropriate treatment for retained placenta remains controversial amongst veterinarians. Manual removal of the placenta is one technique used. Manual removal is performed by detaching maternal caruncles from fetal cotyledons. This procedure is often carried out on day 3 after delivery. However, there is controversy whether manual removal will lead to further uterine infection, uterine wall damage and trauma to the endometrium (De
Bois, 1982). Along with manual removal, antibiotic therapy is often prescribed.
Most often, pharmacological means are used in the active management of retained placenta to encourage placental separation. Since the condition is associated with a lack of myometrial contractions, pharmacological agents that stimulate these contractions are often prescribed. These pharmacological agents, such as oxytocin are most effective when applied one to two days before calving (Arthur et al., 1982); however, even though some studies failed to show any benefit from the use of oxytocin, it is still frequently used in combination
with PGF2a for the treatment of cows with retained placenta (Carroli et al., 1998; Hickey et al., 1984). Some studies report that the use of oxytocin immediately following delivery of human infants significantly shortens the third stage of labor and reduces blood loss 27
(Heinonen and Pihkala, 1985; Mollo et al., 1997; Sorbe, 1978; Walker, 1975). Umbilical cord injections of collagenase are also effective treatments for retained placenta (Eiler and
Hopkins, 1993) and reduce the incidence of retained placenta by 30%.
In conclusion, understanding the mechanisms that may impact the incidence of retained placenta as well as placental transfusion are crucial aspects of the parturition process that can either enhance or detract from the health of both the mother and her offspring.
However, estimating the efficiency of placental transfusion and determining residual blood volume is currently a difficult process. The development of an accurate measurement technique would allow researchers to pinpoint optimal delivery management practices.
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CHAPTER2
FACTORS AFFECTING RESIDUAL PLACENTAL BLOOD VOLUME FOLLOWING PARTURITION IN DAIRY CATTLE
A paper, a portion of which will be submitted to Journal of Dairy Science
A.L. Riddle and H.D. Tyler
ABSTRACT
The objective of this study was to determine factors affecting the volume of blood retained in the placenta following delivery of the calf. In addition, a technique was developed for accurately measuring the volume of blood retained in the placenta following delivery. Optimal delivery conditions can improve both the short term and long term health status of the calf. Fifteen Holstein cows and heifers were placed in a maternity barn approximately 3 to 4 d prior to their estimated delivery date. An electronic birth monitoring system was used to determine the initiation of stage 2 labor. The umbilical cord was clamped during the delivery process. Calves were separated into two groups: those with umbilical cords clamped prior to the first breath or simultaneous with the first breath (n = 7) and those with umbilical cords clamped approximately one-minute after the first breath (n =
8). The first breath was considered as the first inspiration (gasp) of air. Placentae were evaluated within 12 h after expulsion. Cotyledon color, cotyledon number, hemoglobin concentration from all cotyledons, placental weight, and cotyledon weight were recorded.
Blood remaining within the placenta was calculated using an algorithm that included cotyledonary weight, cotyledonary [Hb], and calf blood [Hb]. Multiple regression analysis was used to identify explanatory variables associated with each response variable. Response 42 variables included placental blood volume and placental expulsion time. Mean and standard deviation of calculated residual placental blood volume was recorded at 204.81 ml and 98.10.
Factors that were associated with residual placental blood volume included cotyledon hemoglobin concentration (P < 0.001), placental weight (P < 0.01), and calf hemoglobin concentration (P < 0.01). The only factor that significantly affected placental expulsion time was the weight of the placenta (P < 0.01). These data suggest that placental blood transfer does not appear to affect placental expulsion time in cattle.
INTRODUCTION
In the human neonate, postnatal transfer of blood from the placenta to the newly delivered fetus occurs within the first 3 min after birth (Sisson, 1973); neonatal blood volume increases by ten percent to thirty percent and red cell mass increases by twenty-five percent to sixty percent as a response to delayed umbilical cord clamping (Sisson, 1973). Lind
(1965) reported that 40 ml of blood enters the infant within the first 15 sec after birth and blood volume increases by another 80 ml by 60 sec after birth. In foals, premature rupture of the umbilical cord has been estimated to result in a loss of up to 1500 ml or 30% of the potential neonatal blood volume (Mahaffey, 1961). Pinpointing the ideal time of umbilical cord clamping along with developing good management practices during delivery would maximize placental transfusion and enhance neonatal health.
Residual placental blood volume is typically measured by simple drainage of blood from the expelled placenta (Chau and Ackerman, 1973; Klebe and Ingomar, 1973; Nelson et al., 1980; Ogata et al., 1977; Philip and Teng, 1977; Yao and Lind, 1969; Yao and Lind,
1972; Yao and Lind, 1974a; Yao and Lind, 1974b; Yao et al., 1969; and Yao et al., 1967).
Simple placental drainage may not be an accurate measurement of residual placental blood 43 volume due to significant amounts of blood remaining in small vessels located throughout the placenta. Another method of collecting residual placental and umbilical cord blood in humans uses an airtight acrylic vacuum chamber. Umbilical stumps are punctured with collection needles, and a negative pressure up to 50 cm is created within the chamber, drawing blood into a collection bag containing anticoagulants (Bifano et al., 1994). There are several potential sources of error in estimates of residual placental blood volume obtained using this technique. For example, there is the possibility of collecting extracellular fluid along with residual placental blood, therefore overestimating the true blood volume. In addition, vacuum developed by this system may collapse major vessels, thus leaving blood remaining within smaller placental vessels and underestimating the true value.
An alternative approach to determining the extent of placental transfusion at birth is by measuring changes in neonatal blood volume postnatally. Measuring placental transfer via changes in neonatal blood volume may also lead to inaccuracies due to dramatic changes in total body water and fluid distribution that occur immediately following parturition (Cheek et al., 1984). Cheek et al. (1984) reported that human infants normally show an increase in intracellular volume and a decrease in extracellular volume after birth. Due to these fluctuations in fluid balances, increases in hematocrit, hemoglobin or red blood cell counts occur that complicate estimates of the true placental blood transfer.
Therefore, an improved technique for measuring residual placental blood volume might enhance our estimates of placental transfusion and improve perinatal management practices to ensure maximal survivability of neonates. The objective of this study was to determine factors affecting the volume of blood retained in the placenta following delivery of 44 the calf. In addition, a technique was developed for more accurately measuring the volume of blood retained in the placenta following delivery.
MATERIALS AND METHODS
Animal Management
Fifteen Holstein cows and heifers were placed in a maternity barn approximately 3-4 d prior to their estimated delivery date. An electronic birth monitoring system was used to determine the initiation of stage 2 labor (FOALERT ™). Umbilical cords were clamped during the delivery process. Cows and heifers were separated into two groups: those with umbilical cords clamped prior to the first breath or simultaneous with the first breath (n=7) and those with umbilical cords clamped approximately 1 min after the first breath (n=8). The first breath was considered as the first inspiration (gasp) of air. All umbilical arteries and veins were manually clamped using 2 ½ inch umbilical clamps (Emergency Medical
Products, Waukesha, WI). Umbilical cords were clamped as far as possible from the navel of the neonate. All umbilical cords that did not naturally tear were cut approximately 1 cm from the umbilical clamp on the neonate side. Delivery parameters measured included total time of calving (from the initial sighting of the calves' front hooves to the time of the delivery of the last rib), time from initial breath until the umbilical cord was broken or clamped, and level of assistance provided during delivery. Calves were removed from the dam immediately after delivery and an initial jugular blood sample was collected for total hemoglobin (tHb) analysis. 45
Placental Analysis
After delivery all cows were monitored for expulsion of their placentae and the time of expulsion was recorded. Placentae that were not expelled within 12 h after delivery were excluded from the data set due to the potential for excessive autolysis of placental tissue.
Incomplete placentae were also excluded. Following expulsion, all placentae were washed and refrigerated (38°F) until further analysis could be performed. No placentae were refrigerated for longer than 12 h. All placentae were analyzed for total weight (kg), cotyledon weight (kg), number of cotyledons, manual drainage of placental blood and total hemoglobin concentration of homogenized cotyledons. Also, any obvious placental abnormalities were recorded. Umbilical cord clamps were removed and umbilical cord blood was manually drained into a beaker and the volume of collected blood was recorded.
Cotyledons were manually dissected from the placenta, counted and weighed (kg), and placed into a Waring Blender. All samples were processed for 15 sec and blended contents were collected into plastic bags. Three average samples from the total blended cotyledons were sifted through a cheesecloth prior to analysis. Total hemoglobin concentration was determined via oximetry using an OSM3 Hemoximeter (Radiometer, Copenhagen).
Residual placental blood volume was determined using an algorithm that included cotyledonary weight, cotyledonary [Hb], and calf blood [Hb]. In previous studies, cotyledon hemoglobin concentration was found to be positively correlated with cotyledon color. Also, umbilical cord hemoglobin was positively correlated with calf hemoglobin concentration.
Calculations are as follows: 46
100 ml of cotyledon = 102.3 g
Total cotyledon weight (g) = 102.3 (g) = Total volume (ml) of cotyledon x 100 ml
Total volume (ml) of cotyledon + Total volume (ml) of manually = Uncorrected drained placental and umbilical total volume (ml) cord blood of residual placental blood volume
Cotyledon [Hb] (g%) = ____....;;x-=------= Total 100 ml Uncorrected total volume (ml) placental [Hb l of residual placental blood volume (g%)
Calf [Hb] (g%) = Total cotyledonary [Hb] = Total volume (ml) 100 ml x of residual placental blood volume
Statistical Analysis
Data were analyzed using the OLM procedure of SAS (SAS, 1996). Multiple regression analysis was used to identify explanatory variables (parity, weight of the calf, sex of the calf, assisted birth, duration of calving, umbilical cord clamp time, cotyledon weight, number of cotyledons, calf jugular hemoglobin, cotyledon hemoglobin) associated with each response variable. Response variables included calculated residual placental blood volume and placental expulsion time. Repeated iterations of the OLM procedure were performed, and following each iteration the explanatory variable with the highest p-value was eliminated until all remaining explanatory variables had p-values of< 0.05. In addition, all data were analyzed using PROC CORR of SAS (SAS, 1996). 47
RESULTS
Placental Blood Volume
Mean calculated residual placental blood volume for placentae in this study was
204.81 ml. The mean volume of residual placental blood collected via simple drainage from placentae in this study was 390.53 ml (Table 3). No differences were found between early and late umbilical cord clamping so all data is analyzed as combined data. Factors that affected placental blood volume included placental weight (p < 0.01), cotyledon hemoglobin concentration (p < 0.001), and calf hemoglobin concentration (p < 0.01). No other factors identified in this study affected placental blood volume.
Placental Expulsion Time
The mean placental expulsion time in this study for those placentae collected within the 12 h time limit was approximately 7 1/2 h. The only factor that significantly affected placental expulsion time was the weight of the placenta (p < 0.01).
Correlations
Simple correlations were performed to determine any relationships between placental blood volume and all other parameters measured in this study. Simple correlations were also performed on time of placental expulsion in correlation to all other parameters measured in this study. Birth weight of calves was positively correlated (r = 0.69) with total placental blood volume (p < 0.01) (Figure 1). Assistance during delivery was correlated (r = -0.66) with higher placental blood volume (p < 0.01) (Figure 2). Total cotyledonary hemoglobin concentration was positively correlated (r = 0.79) with placental blood volume (p < 0.01)
(Figure 3). 48
Table 3. Values of manually drained residual placental blood volume vs. calculated residual placental blood volume.
Manual drained residual blood volume (ml) Calculated residual placental blood volume (ml)
250 396.60
110 246.44
25 180.94
50 140.62
50 312.36
1000 306.70
500 294.45
400 93.74
350 270.66
1100 41.66
350 171.29
400 177.74
200 87.85
350 198.24
340 152.89 49
Figure 1. Correlation (r = 0.69) between calf weight (kg) and placental blood volume (ml) (p < 0.01) in dairy calves (n = 15).
::::- 400 • ,§_ 350 Cl) E ::I 300 0 > 250 "C 0 200 m0 150 ca 100 C • • -Cl) 0 50 ca • ii: 0 30 35 40 45 50 55 60 Weight of Calf (kg) 50
Figure 2. Correlation (r = -0.66) between assisted birth on placental blood volume (ml) (p < 0.01) in dairy calves (n = 15).
:::- 330 E -G) E 280 :, 0 > "C 230 0 0 in 180 cu C -G) () 130 as a: G) 80 C) ...as G) > 30 Figure 3. Correlation (r = 0. 79) between total cotyledonary hemoglobin (g%) and placental blood volume (ml) (p < 0.01) in dairy calves (n = 15). :::::- 400 §. 350 G) E 300 ::::I ~ 250 ,:, 200 0 0 m 150 «i 100 C -G) 50 (,) ca • a: 0 +------.------,------,-----.-----~ 0 2 3 4 5 Total Cotyledonary Hemoglobin (g%) 52 Calf birth weight was also positively correlated (r = 0.67) with time of placental expulsion (p < 0.01) (Figure 4). And finally, total number of cotyledons was negatively correlated (r = -0.66) with time of placental expulsion (p < 0.01) (Figure 5). 53 Figure 4. Correlation (r = 0.67) of calf weight (kg) and placental expulsion time (min) (p < 0.01) in dairy calves (n = 15). 1200 Cl) • E j:: 1000 C ·u;0 800 ::::, C --C. ·- 600 w->< E 'ii 400 C -Cl) 200 "C'CI a: 0 30 35 40 45 50 55 60 Weight of Calf (kg) 54 Figure 5. Correlation (r = -0.66) between number of cotyledons and placental expulsion time (min) (p < 0.01) in dairy calves (n = 15). 1200 Cl) • E t= 1000 C ·;;0 800 • ::I C • --0. ·- 600 w->< E • cu 400 C • -Cl) (,) 200 • ca C: 0 20 40 60 80 100 120 140 Number of Cotyledons 55 DISCUSSION With the percentage of calves receiving assistance during parturition increasing each year (Berger, 1992) there is a need to understand the factors that determine optimal delivery times for improved survivability. In a study where assistance was provided during all deliveries, calving difficulty scores increased 2-fold (Berger, 1992). This data suggests that inappropriate assistance hinders the calving process; in cattle, early assistance also prematurely ruptures the umbilical cord. Hammer (1998) reported that calves receiving premature assistance that ruptured the umbilical cord earlier during delivery had low Po2 and high Pco2 values postnatally; these effects were theorized to be due to poor pulmonary perfusion because of decreased neonatal blood volume. Strawn et al. (1996) also reported low Po2 and decreased [Hb] in calves following assisted deliveries. Premature rupture of umbilical cords in calves occurs in part due to relatively short umbilical cord lengths. In humans, the umbilical cord attains its full length by the beginning of the eighth month of pregnancy (Mal pas, 1964) and umbilical cord lengths range from as short as 22 cm to as long as 130 cm (Purola, 1968). The umbilical cord remains intact even after delivery is complete. No studies have been conducted regarding umbilical length in bovines, although it is known that during normal deliveries, the umbilical cord ruptures as the hind legs are expelled from the birth canal. This short umbilical cord may potentially play a significant role in the amount of placental transfusion to the neonate due to the stretching that occurs during delivery and the early timing of rupture during the delivery process. The data showed no effect of the timing of umbilical cord clamping on residual placental blood volume. In contrast, studies in humans have reported delayed cord clamping was associated with a reduced volume of residual placental blood (Colozzi, 1954; De Marsh 56 et al., 1942; Nyberg and Westin, 1958; Philip and Teng, 1977; Yao et al., 1969). One study suggests that one-fourth to one-third of placental blood remains in the placenta if the cord is clamped early compared to late cord clamping (Lanzkowsky, 1960). Ultimately, the residual blood residing within the placenta due to early cord clamping would better aid the neonate by increasing total neonatal blood volume and increasing neonatal survivability. The lack of relationship between the timing of umbilical cord clamping and residual placental blood volume in this study, may be due to an early placental blood transfusion during the early stages of parturition in response to umbilical cord stretching and vasoconstriction. Stretching of the cord during the birth process also triggers placental blood transfusion (Sardi et al., 1997). Differences in umbilical cord lengths between species may play a significant role in timing of placental transfusion during parturition. Further studies are needed to verify the mechanisms responsible for these apparent species differences. In many human studies, residual placental blood volume is measured by simple drainage of blood from the expelled placenta (Chau and Ackerman, 1973; Klebe and Ingamar, 1973; Nelson et al., 1980; Ogata et al., 1977; Philip and Teng, 1977; Yao and Lind, 1969; Yao and Lind, 1972; Yao and Lind, 1974a; Yao and Lind, 1974b; Yao et al., 1969; and Yao et al., 1967). This study verifies that simple placental drainage is not an accurate measurement of residual placental blood volume due to significant amount of blood remaining in small vessels located throughout the placenta and dilution of placental blood with extravascular fluids following delivery. These inaccuracies are eliminated using the novel technique described in this study. By determining the total hemoglobin remaining in the placenta, and adjusting for dilution by using the calf's blood hemoglobin concentration (obtained immediately following birth) as a reference, the true residual placental blood 57 volume can be calculated. No relationship was found between the length of time of placental retention and the amount of "dilution" that occurs. Our data suggests that simple drainage techniques overestimate true residual placental blood volume in some cases and underestimate this value in other cases. For this reason, simple drainage cannot be considered an accurate technique and should not be used to estimate efficiency of placental transfusion. LITERATURE CITED Barker, G., N. Cunliffe, W.G. Bardsley, S.W. D'Souza, P. Donnai and R.D.H. Boyd. 1988. Fetal and maternal blood volumes in shed human placentae: Discrepant results comparing morphometry and haemoglobin content. Placenta. 9:289. Berger, P.J. 1996. Summary of calving ease score data. Mimeo. Iowa State University. Ames. Berman, W., R.C. Goodlin and M.A. Heymann. Effects of pharmacologic agents on umbilical blood flow in fetal lambs in utero. Biol. Neonate. 33:225. Bifano, E.M., R.A. Dracker, K. Lorah and A. Palit. 1994. Collection and 28-day storage of human placental blood. Pediatric Research. 36(1):90. Cheek, D.B. J. Wishart, A MacLennan and R. Haslam. 1984. Regulation of extracellular fluid volume in neonates. Early Hum. Dev. 34:179. Chou, P.J. and B.D. Ackerman. 1973. Perinatal acidosis and placental transfusion. Acta Paediatr. Scan. 62:417. Colozzi, A.E. 1954. Clamping of the umbilical cord. New Eng. J. Med. 250:629. De Marsh, Q.B., W.F. Windle and H.L. Alt. 1942. Blood volume of newborn infant in relation to early and late cord clamping of the umbilical cord. Am. J. Dis. Child. 63:1123. Hammer, C.J. 1998. Effect of obstetrical assistance on Jersey calves. Masters. Ames. Klebe, J.G., and C.J. Ingomar. 1974. The fetoplacental circulation during parturition: Evidence from residual placental blood volume. Pediatrics. 54:213. 58 Lind, J. 1965. Physiological adaptation to the placental transfusion. Canad. Med. Assoc. J. 93:1091. Lanzkowsky, P. 1960. Effects of early and late clamping of the umbilical cord on infant's hemoglobin. Brit. Med. J. 2:1777. Mahaffey, L.W. 1961. Pulmonary syndrome in newborn foals. In: Somatic Stability in the newly born. G.E.W. Wolstenholme and M. O'Conner (eds). Little, Brown, and Company. Boston. Malpas, P. 1964. Length of the human umbilical cord at term. Brit. Med. J. 1:673. Moinian, M., W.W. Meyer and J. Lind. 1969. Diameters of umbilical cord vessels and the weight of the cord in relation to clamping time. Arn. J. Obstet. Gynecol. 105:604. Nelson, N.M., M.W. Endkin and S. Saigal. 1980. A randomized clinical trail of the Leboyer approach to childbirth. N. Engl. J. Med. 302:655. Nyberg, R. and B. Westin. 1958. On the influence of uterine contractions on the blood pressure in the umbilical vein at birth. Acta. Paediatr. Scand. 47:350. Ogata, E.S., J.A. Kitterman, F. Kleinberg, L. Dong, M. Willis, J. Mates and R.H. Phibbs. 1977. The effect of time of cord clamping and maternal blood pressure on placental transfusion with cesarean section. Arn. J. Obst. Gynec. 128:197. Philip, A.G.S. and S.S. Teng. 1977. Role of respiration in effecting placental transfusion at cesarean section. Biol. Neonate. 31:219. Purola, E. 1968. The length and insertion of the umbilical cord. Annales Chirurgiae et Gynaecologia Fenniae. 57:621. Regoli, D., S. Nsa Allogho, A. Rizzi and F.J. Gobeil. 1998. Bradykinin receptors and their antagonists. Eur. J. Pharmacol. 348: 1. Sardi, S.P., H. Perez, P. Antunez and R.P. Rothlin. 1997. Bradykinin B1 receptors in human umbilical vein. Euro. J. Pharrnacol. 321:33. Sisson, T.R., S. Knutson and N. Kendall. 1973. The blood volume of infants. Arn. J. Obstet. Gynecol. 117:351. Strawn, K.M, H.D. Tyler, M.A. Faust and B.J. Nonneche. 1996. Effects of birth stress in calves. J. Dairy Sci. 79: 130. Yao, A.C., A. Wist, J. Lind. 1967. The blood volume of the newborn infant delivered by caesarean section. Acta Paediatr. Scand. 56:585. 59 Yao, A.C. and J. Lind. 1969. Effect of gravity on placental transfusion. Lancet. 2:505. Yao, A.C., M. Moinian and J. Lind. 1969. Distribution of blood between infant and placenta after birth. Lancet. 2:871. Yao, A.C. and J. Lind. 1972. Blood volume in the asphyxiated term neonate. Biol. Neonate. 21:199. Yao, A.C. and J. Lind. 1974a. Placental transfusion. Am. J. Dis. Child. 127:128. Yao, A.C. and J. Lind. 1974b. Blood flow in the umbilical vessels during the third stage of labor. Biol. Neonate. 25: 186. 60 CHAPTER3 RELATIONSHIPS BETWEEN PLACENTAL CHARACTERISTICS, CALF CHARACTERISTICS, DELIVERY PARAMETERS AND PLACENTAL RETENTION IN DAIRY CATTLE A paper, a portion of which will be submitted to Journal of Dairy Science A.L. Riddle and H.D. Tyler ABSTRACT Retained placenta and dystocia are increasing problems in the dairy industry. Optimal delivery conditions can improve overall health status of the calf and dam, along with reducing the incidence of retained placenta. Calves (n =70) and placentae (n =44) were obtained from Holstein cattle following parturition. Delivery parameters include calving ease scores, duration of parturition, calf weight and parity. Placental characteristics evaluated after expulsion included color index (1-light to 5-dark) of cotyledons located at center and tips of placenta, cotyledon number, placental weight and length of umbilical stump. After delivery, calves were weighed, blood samples were collected to evaluate hematocrit, and both length and diameter of umbilical cords were measured. Multiple regression analysis was used to identify explanatory variables associated with each response variable. Response variables included placental expulsion time, duration of calving, calf weight and calving ease scores (CES). The only factor that significantly affected placental expulsion time was umbilical cord break point (p < 0.05). Factors that affected duration of calving included parity (p < 0.01), diameter of umbilical stump (p < 0.01), calf weight (p < 0.0001), total length of umbilical cord (p < 0.05) and calf umbilical cord efficiency (calf weight/diameter of umbilical stump) (p < 0.0001). Factors affecting CES included color 61 index in center of placenta (p < 0.01), color index in tips of placenta (p < 0.01), diameter of the umbilical stump (p < 0.01), cotyledon number (p < 0.05), and umbilical cord break point (p < 0.01). Finally, the only factor that affected calf weight was weight of the placenta (p < 0.05). The data strongly reflects the relationship between placental factors, delivery parameters and calf outcomes. INTRODUCTION The importance of placental function is probably best exemplified by the close relationship between fetal weight, placental size, and uterine and umbilical blood flows in many mammalian species (Ibsen, 1928; Warwick, 1928; Hammond, 1935; Alexander, 1964; Oh et al., 1975; Wooton et al., 1977; Christenson and Prior, 1978; McDonald et al., 1979; Prior and Laster, 1979; Hard and Anderson, 1982; Coaton et al., 1984; Ford et al., 1984; Reynolds et al., 1984; Metcalfe et al., 1988; Ferrell, 1989; Ferrell and Reynolds, 1992). The placenta performs many functions and substitutes for the fetal gastrointestinal tract, lung, kidney, liver, and endocrine glands (Ramsey, 1982; Faber and Thornburg, 1983). Additional factors that effect fetal growth, such as maternal genotype, increased number of fetuses, maternal nutrient deprivation, or environmental stress, typically have similar effects on placental size and development (Walton and Hammond, 1938; Ebbs et al., 1942; Echstein et al., 1955; Hunter, 1956; Joubert and Hammond, 1958; Alexander, 1964; Alexander and Williams, 1971; Turman et al., 1971; Rattray et al., 1974; Corah et al., 1975; Sreenan and Beehan, 1976; Knight et al., 1977; Thompson et al., 1982; Ferrell, 1991). Placentae also play key roles in the induction of parturition. Beginning a few days prior to parturition, increasing concentrations of fetal cortisol result in reduced secretion of placental 62 progesterone and increased secretion of estrogen. Decreasing concentrations of progesterone in maternal blood allow an increased rate of both the synthesis and release of prostaglandin F2a (PGF2a)- Increased concentrations of PGF2a induce sensitivity of the uterine myometrium to oxytocin. This ultimately allows uterine contractions and expulsion of the fetus (Liggins et al., 1973). Dystocia is one factor that affects the ability of the calf to make the transition to an independent existence. The majority of calf deaths (57.4%) occur during the first 24 h after parturition, and it has been estimated that dystocia accounts for 69.6% of all deaths occurring during the first 24 h postpartum (Patterson et al., 1987). Defects in both placental structure (Hammer et al., 2001) and function (Chew et al., 1972) increase the risk of dystocia in calves. Dystocia increases the risk of many problems in both the calf and dam, including increasing the risk of metabolic problems such as retained placenta. Failure of placentae to be expelled during the third stage of labor is a common complication of parturition in ruminants, primarily due to failure of release of fetal villi from maternal crypts. Approximately 11 % of cows fail to release the placenta within 12 h postpartum (Eiler, 1992). The risk of placental retention increases as a result of premature birth, caesarian section, pharmacological induction of parturition (Inskeep, 1973), twinning or large calf size (Morrison and Erb, 1957), endocrine imbalances (Chew et al., 1978), vitamin E and selenium deficiencies (Harrison et al., 1984), impaired immune function (Kimura, et al., 2002; Gunnick, 1984) or trauma and hormonal imbalances (Marion et al., 1968; Takagi et al., 2002). The number of cotyledons present and the placental type may play a key role in deterioration of the attachment and thus release of the placenta (Silver et al., 1973). It has also been theorized that drainage of placental blood facilitates delivery of 63 the placenta, but currently there is no scientific evidence supporting this theory (Thomas et al., 1990). Because of the dramatic increases in the rates of dystocia and stillbirth over the last 20 yin the dairy industry, as well as the increased risk of retained placenta associated with dystocia, it is crucial to fully understand the relationship between placental characteristics, calf characteristics, delivery parameters and placental retention. Therefore, the objective of this experiment was to determine placental factors that may be associated with calf characteristics, delivery parameters and retained placenta. MATERIALS AND METHODS Animal Management Seventy pregnant Holsteins cows and heifers were monitored at the Gallo Dairy Farm in Atwater, California. All animal management schemes were determined and conducted by facility personnel. Holsteins were moved from open feeder lots to individual maternity barn stalls either prior to or during delivery. Delivery parameters measured included total time of calving (from initial sighting of calves hooves to the initial breath (gasp)), time from initial breath until the umbilical cord was ruptured, and level of assistance provided during delivery. Calves were removed from dams immediately after delivery and placed in elevated individual calf crates. Calf sex, calf weight, and length and diameter of umbilical stump were recorded. An initial jugular blood sample was collected within 10 min of delivery for hematocrit analysis. Following delivery, dams were moved from individual maternity pens to a group holding pen. 64 Placental Parameters Placentae were collected and stored in coolers filled with ice. Expulsion time was recorded. All placentae were analyzed within 12 h after expulsion. Incomplete placentae and placentae that were not expelled within 12 h were excluded from the trial. Placentae were analyzed for cotyledonary color at both the center and tips of placentae, number of cotyledons, length of umbilical cord remnants attached to placentae and total placental weight. Any abnormalities were also recorded. Cotyledonary color was determined via a color chart ranging from 1 (lightest red in color) to 5 (darkest red in color). Statistical Analysis Data were analyzed using the GLM procedure of SAS (SAS, 1996). Multiple regression procedures were used to identify explanatory variables (parity, sex of the calf, unassisted or assisted birth, umbilical cord rupture time, diameter of umbilical stump, calf length of umbilical cord, weight of placenta, cotyledon color, number of cotyledons, calf jugular hematocrit, placental length of umbilical cord, color index of cotyledons at both tips and center of placenta, total length of umbilical cord, umbilical cord break point, placental weight by calf weight, combined total blood at tips and center of placenta, efficiency of placenta, average placental color index, calf efficiency, total volume of umbilical cord, and calf umbilical cord efficiency) associated with each response variable (Table 4) (Table 5). Response variables included placental expulsion time, duration of calving, calf weight and calving ease scores (CBS). Repeated iterations of the GLM procedure were performed, and following each iteration the explanatory variable with the highest p-value was eliminated until all remaining explanatory variables had p-values of< 0.05. In addition, all data were analyzed using PROC CORR of SAS (SAS, 1996). Table 4. Descriptions of calculated parameters. ~ Description Duration (min) Duration of calving (min) Parity(Cows) Parity (Heifers) Pulled % of Calves pulled BBUCB % of Calves breathing before umbilical cord was broken Hematocrit (%) Diameter of Umbilical Stump (mm) Calf Length Umbilical Cord (mm) Measurement of calf umbilical stump (mm) Calf Sex Calf Weight (kg) Expulsion (min) Time from end of parturition to time of placental expulsion (min) Weight of Placenta (kg) Total Number of Cotyledons Vt Placental Length of Umbilical Cord (mm) °' Color Index of Cotyledons ( center) Color Index Of Cotyledons (tips) Total Length of Umbilical Cord (mm) Total length of umbilical cord (calf length of umbilical cord + placental length of umbilical cord) U.C. Break Point Position of umbilical cord rupture (calf length of umbilical cord/total length of umbilical cord) Placental Weight by Calf Weight Placental weight (kg)/ calf weight (kg) Estimated Blood Tips of Placenta (ml) Cotyledon number x cotyledon color index at tips of placenta Estimated Blood Center of Placenta (ml) Cotyledon number x cotyledon color index at center of placenta Efficiency Weight of placenta (kg)/ total cotyledon number Avg Placental Color Index ((Color index at tip of placenta x .66) + (color index at center of placenta x .34)/2) x total cotyledon number Calf Efficiency Calf weight (kg)/ total cotyledon number Total Area of Umbilical Cord (mm) Diameter of umbilical stump (mm) x total length of umbilical cord (mm) Calf Umbilical Cord Efficiency Calf weight (kg)/ diameter of umbilical stump (mm) Table 5. Factors affecting duration of calving, placental expulsion, calving ease scores and calf weight in Holstein cattle. Model-- Parameter Mean Standard Deviation Duration of calving Placental Exuulsion CES Calf Weight Duration (min) 37.84 37.46 N/A N/A NS NS Parity (Cows) 72% NIA 0.01 NS NS NS Parity (Heifers) 28% NIA NS NS NS NS Pulled 58% NIA NS NS NS NS BBUCB 66% NIA NS NS NS NS Hematocrit (%) 37.86 8.32 NS NS NS NS Diameter of Umbilical Stump (mm) 25.48 25.54 0.01 NS 0.01 NS Calf Length Umbilical Cord (mm) 232.2 189.04 NS NS NS NS Calf Sex 47%Male NIA NS NS NS NS Calf Weight (kg) 46.8 12.04 0.0001 NS NS N/A Expulsion (min) 261.76 126.74 N/A N/A NS NS Weight of Placenta (kg) 5.57 2.49 NS NS NS 0.05 Total Number of Cotyledons 83.54 28.22 NS NS 0.05 NS Placental Length of Umbilical Cord (mm) 334.03 66.34 NS NS NS NS Color Index of Cotyledons (center) 3.46 1.09 NS NS 0.01 NS °' Color Index Of Cotyledons (tips) 4.06 0.78 NS NS 0.01 NS Total Length of Umbilical Cord (mm) 548.44 181.75 0.05 NS NS NS U.C. Break Point 34.35 19.78 NS 0.05 0.01 NS Placental Weight by Calf Weight 0.11 0.02 NS NS NS NS Estimated Blood Tips of Placenta (ml) 343.22 138.64 NS NS NS NS Estimated Blood Center of Placenta (ml) 286.51 132.93 NS NS NS NS Efficiency 0.16 0.08 NS NS NS NS Avg Placental Color Index 162.76 64 NS NS NS NS Calf Efficiency 1.39 0.67 NS NS NS NS Total Area of Umbilical Cord (mm) 14489.25 15354.65 NS NS NS NS Calf Umbilical Cord Efficiency 4.67 1.02 0.0001 NS NS NS 67 RESULTS Placental Expulsion Time Placental expulsion time was defined as the time elapsed from the end of parturition to complete release and expulsion of the placenta. Mean placental expulsion time was approximately 4.5 h for those placentae expelled within the 12 h limit; 26 placentae were either not expelled within the 12 h limit or not collected for other reasons. Many of the placentae (n = 27) from this trial were not collected due to the uncontrolled movement of the cows into holding pens located outside of the maternity barn. The minimum recorded placental expulsion time was 79 min and maximum time to placental expulsion was 662 min. The only factor that significantly affected placental expulsion time was umbilical cord break point (p < 0.05). No other factors identified in this study affected placental expulsion time (Table 6). Duration of Calving Duration of calving was defined as the time elapsed from first sighting of calves' front hooves to complete expulsion of the calf from the birth canal. Mean duration of calving was 37.84 min. The minimum recorded duration of calving was 2 min and maximal duration of calving was reported at 167 minutes. Factors that affected duration of calving included parity (p < 0.01) (Figure 6), diameter of umbilical stump (p < 0.01), calf weight (p < 0.0001), total length of umbilical cord (p < 0.05) and calf umbilical cord efficiency (p < 0.0001) (Table 7). Calving Ease Score Calving ease scores ranged from 1 (no assistance) to 5 (as the most difficult). Mean calving ease score was 2 in this study. Over 40% of calvings were assisted in this study, 68 Table 6. Variables included in the model for placental expulsion time. Explanatory Variable P < 0.05 Parity No Pulled No BBUCB No Calf Sex No Color Index Center No Color Index Tips No Duration of Calving No Calving Ease Score No Hematocrit No Diameter of Umbilical Stump No Calf Weight No Placental Weight No Cotyledon Number No Total Length of Umbilical Cord No Umbilical Cord Break Point Yes Placental Weight by Calf Weight No Total Blood Tips and Center No Efficiency No Average Placental Color Index No Total Volume Umbilical Cord No Calf Umbilical Cord Efficiency No 69 Figure 6. Effect of parity on duration of calving (n = 57). c 70 .§. 60 g> 50 ·s; ca 40 0 -0 30 C 20 0 i... 10 5 0 +----- Parity 2 and greater (n = 40) Parity 1 (n = 17) Parity 70 Table 7. Variables included in the model for duration of calving. Explanatory Variable p < 0.05 Parity Yes Pulled No BBUCB No Calf Sex No Color Index Center No Color Index Tips No Calving Ease Score No Hematocrit No Diameter of Umbilical Stump Yes Calf Weight Yes Placental Weight No Cotyledon Number No Total Length of Umbilical Cord Yes Placental Weight by Calf Weight No Total Blood Tips and Center No Efficiency No Average Placental Color Index No Total Volume Umbilical Cord No Calf Umbilical Cord Efficiency Yes Umbilical Cord Break Point No 71 although only 1% required extreme force (Table 8). Factors affecting CBS included color index in center of placenta (p < 0.01) (Figure 7), color index in tips of placenta (p < 0.01) (Figure 8), diameter of the umbilical stump (p < 0.01) (Figure 9), cotyledon number (p < 0.05) (Figure 10), and umbilical cord break point (p < 0.01) (Figure 11) (Table 9). Calf Weight All calves were weighed immediately after birth and prior to colostrum ingestion. Mean calf weight for calves in this study was 46.80 kg. The minimum and maximum calf weights were recorded as 31.81 kg and 61.36 kg. Finally, the only factor that affected calf weight in this study was weight of the placenta (p < 0.05) (Table 10). Correlations Simple correlations were performed to identify associations between placental expulsion time, duration of calving, calving ease score, calf weight and all other parameters measured in this study. Parity was negatively correlated (r = -0.43) with duration of calving (p < 0.01). Umbilical cord efficiency was also positively correlated (r = 0.29) with duration of calving (p < 0.05) (Figure 12). Umbilical cord efficiency was calculated as calf weight (kg) / diameter of umbilical cord stump (mm). Male calves were associated (r = 0.27) with higher calf weights (p < 0.05) (Figure 13). Weight of the placenta was positively correlated (r = 0.34) with calf weight (p < 0.05) (Figure 14). 72 Table 8. Percent of total calvings in association with calving ease score. % of total calvings 1 (Unassisted delivery) 58 2 (Slight assistance) 7 3 (Moderate assistance) 13 4 (Considerable force needed) 20 5 (Extreme difficulty) 1 73 Figure 7. Color index of cotyledons in the center of placenta in relation to calving ease score (n = 42). Cl) a.. 3 0 (.) en 2.5 Cl) u, ca w 2 a, C ·- 1.5 ~ ca 0 1 C ca Cl) 0.5 ~ 1 2 3 4 5 Color Index at Center of Placenta 74 Figure 8. Color index of cotyledons at tips of placenta in relation to calving ease score (n = 43). Cl) 3.5 a.. u0 ti) 3 Cl) tnca 2.5 w C) 2 ·s:C ca 1.5 0 C 1 ca Cl) :!!: 0.5 1 2 3 4 5 Color Index at Tips of Placenta 75 Figure 9. Diameter of umbilical stump in relation to calving ease score (n = 67). ca 27 .5:? ·-.c E 25 ::, - -E 23 ...o E s -Cl. 21 a, E E ::::, 19 i5as en- G) CJ) 17 ...as G) 15 > <( 1 2 3 4 5 Calving Ease Score 76 Figure 10. Cotyledon number in relation to calving ease score (n = 43). tn 85 C ,,o 80 G) ~ 75 8 70 0 65 a:; 60 ~ 55 :i 50 Cca 45 ~ 40 1 2 3 4 5 Calving Ease Score 77 Figure 11. Umbilical cord break point in relation to calving ease score (n = 37). 60 -0 0~ 50 --C: ·-0 .c - 40 a.~ -en ca c: G) .! 30 m... "C... 0 "C... (,) o- 20 C.> ca 0 'ii 10 .2 - :c E 0 :::, 1 2 3 4 Calving Ease Score 78 Table 9. Variables included in the model for calving ease score. Explanatory Variable p < 0.05 Parity No BBUCB No Calf Sex No Color Index Center Yes Color Index Tips Yes Duration No Hematocrit No Diameter of Umbilical Stump Yes Calf Weight No Placental Weight No Cotyledon Number Yes Total Length of Umbilical Cord No Umbilical Cord Break Point Yes Placental Weight by Calf Weight No Total Blood Tips and Center No Efficiency No Average Placental Color Index No Total Volume Umbilical Cord No Calf Umbilical Cord Efficiency No 79 Table 10. Variables included in the model for calf weight. Explanatory Variable p < 0.05 Parity No BBUCB No Calf Sex No Color Index Center No Color Index Tips No Hematocrit No Diameter of Umbilical Stump No Placental Weight Yes Cotyledon Number No Total Length of Umbilical Cord No Total Blood Tips and Center No Efficiency No Average Placental Color Index No Total Volume Umbilical Cord No 80 Figure 12. Correlation (r = 0.29) between umbilical cord efficiency and duration of calving (p < 0.01) (n = 55). 160 • -·-C: • E 140 • • -C) 120 • ·s;C: (U 100 0 80 • • -0 60 C: •• 0 • -.• .:; 40 • I 1· • ...ca ••• • • ::::, 20 • • • .. C : .,, . • 0 • ·• 1 1 1.5 2 2.5 3 Calf Umbilical Cord Efficiency(%) 81 Figure 13. Correlation (r = -0.27) between calf sex and calf weight (p < 0.05) (n =69). '6, 48.5 c 48 -"E, 47.5 ~ 47 :!:: 46.5 ~ 46 8, 45.5 G)f! 45 ~ 44.5 --+----- Male Female Calf Sex 82 Figure 14. Relationship between weight of the placenta and calf weight (n = 43). 65 60 • C') -.:.i: 55 ... • • •• • • -.c 50 • I ... .2> • • • •I G) 45 • •y ••• I • 3: •••• • • m 40 - • • • ·- -0 • 35 • - 30 3 4 5 6 7 8 Weight of Placenta (kg) 83 Total length of the umbilical cord was negatively correlated (r = -0.37) with placental expulsion time (p < 0.05) (Figure 15). Finally, umbilical cord break point was negatively correlated (r = -0.34) with placental expulsion time (p < 0.05). DISCUSSION With the odds of unassisted birth decreasing each year in the dairy industry, it is essential to understand the relationships between delivery parameters, placental characteristics, calf characteristics, as well as their combined effects on placental retention. Our study found no association between calving ease score, duration of calving, or calf weight and placental expulsion time. In contrast, Eiler (1992) reported that the incidence of retained placenta increased as calving difficulty increased, and Varona et al. (1999) reported that increasing birth weight was associated with higher calving ease scores. In addition, other data shows that as calf weight increases and parity decreases, duration of calving increases (Bellows et al., 1988). Hammer (1998) suggested that it was partly the stress of a long labor, in addition to the type of assistance that was provided, that leads to detrimental effects often associated with dystocia. The low number of observations in our trial may have impacted our ability to detect these associations. In our study, umbilical cord diameter affected calving ease score. An increase in umbilical cord diameter is an indicator of increased size of the umbilical vessels within the cord. An increase in the size of vasculature may be a compensatory mechanism for an inefficient placenta. Hammer et al. (2001) reported that pregnancies that follow interspecies embryo transfer with !VF-derived embryos often exhibit similar problems. Placentae developed from these manipulated embryos often have fewer, larger, less vascular cotyledons 84 Figure 15. Correlation (r = -0.37) between total length of the umbilical cord and placental expulsion time (p < 0.05) (n = 40). a., 650 E • i= C 550 0 • ·u; 450 ::, C • • • --·e 350 w-e- «s 250 C -a., (,) 150 «s -c.. 50 250 450 650 850 1050 1250 Total Length of Umbilical Cord (mm) 85 and the calves have extremely difficult deliveries with high death losses. In addition, umbilical cords in cloned fetal calves or in interspecies pregnancies can reach diameters of up to 8 cm or more (Faber, D., TransOva Genetics, personal communication). Data from our study suggests that differences in umbilical cord size may indicate placental deficiencies that adversely affected the parturition process. 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Other methods described in the literature do not account for the potential addition of extravascular fluids to drained blood as well as ignoring blood retained in small vessels located throughout the placenta. By determining the total hemoglobin remaining in the placenta, and adjusting for dilution by using the calf's blood hemoglobin concentration (obtained immediately following birth) as a reference, the true residual placental blood volume can be calculated. In addition, our data showed no effect of the timing of umbilical cord clamping on the amount of residual placental blood volume. This lack of relationship may be due to an early placental blood transfusion in response to umbilical cord stretching and vasoconstriction during fetal expulsion rather than following delivery. Understanding differences in the anatomy physiology of the placenta between species would not only allow for a better method of determining residual placental blood volume but would allow a further understanding of the mechanisms of placental blood transfusion during delivery. One essential goal of this trial was to further understand the importance of umbilical cord stretching, size and diameter in relationship to calf health and parturition. In our trial, mean umbilical cord length was 548.45 mm. Mean diameter of the umbilical stump 92 was 25.48 mm. Interestingly enough, diameter and length of the umbilical cord significantly affected both duration of calving and calving ease scores. A better understanding of the relationships between umbilical cord length and assistance during delivery may lead to improved health of the calf by maximizing feta-placental transfusion during the delivery process. Other variables such as color indexes at both the center and tips of the placentae, cotyledon number and umbilical cord break point also significantly affected calving ease scores. This data suggests that these placental factors may play a key role in the parturition process, along with calf and dam health, to a far greater extent than previously theorized. Overall, the data strongly reflects the relationship between placental factors, delivery parameters and calf outcomes. Since the trials were conducted at two separate facilities that utilized different management practices, it allowed for further insights into the incidence of retained placenta and calving problems in relation to management practices. The incidence of retained placenta and calving ease scores were both much higher at the Iowa State University Dairy than at the Gallo Dairy. Management at the Gallo Dairy allowed more time for calving to occur naturally and provided less assistance. There is a vast amount of knowledge still awaiting discovery in relation to optimal delivery conditions that maximize placental blood transfusion and minimize placental retention. Further research is essential in these areas. Follow up projects may include investigating the response of the umbilical cord to bradykinins. Due to large umbilical vessels in cloned calves this data may be useful to stop excessive umbilical cord bleeding along with umbilical cord rupture near the body wall. A further understanding of placental characteristics is necessary for further developments in assisted reproductive technologies 93 such as interspecies transfers. Investigating this matter may lead to more viable pregnancies. Also, further investigation in the relationship between placental characteristics and retained placenta may lead to a better understanding of the mechanisms of retained placenta.