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Rectal Temperature, Calving-Related Factors, and the Incidence of Puerperal Metritis in Postpartum Dairy Cows


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RECTAL TEMPERATURE, CALVING-RELATED FACTORS, AND THE INCIDENCE OF PUERPERAL METRITIS IN POSTPARTUM DAIRY COWS By MAURICIO ESTEBAN BENZAQUEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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To my parents, Marta and Alberto who gave me the gift of life, of love and education.

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ACKNOWLEDGMENTS I wish to express my most sincere gratitude to Dr. Carlos A. Risco (my supervisory committee chair) for seeing in me what even I did not see, and for giving me the opportunity of a lifetime. It has been an incredible experience that I will never forget: a mixture of theriogenology, clinical knowledge, and research, blended with friendship. I thank Dr. Louis F Archbald for his invaluable advice and support, and for having the right words at the right moment. I thank Dr. William W. Thatcher for his invaluable advice, for his teaching, and for showing me the fourth dimension of statistical analysis. I thank Dr. Pedro Melendez for his support, advice and fellowship during these 3 years. I also thank Marie-Joelle Thatcher, Biological Scientist, for her hard work on my research and for improving my organizational skills. I thank Dr. Owen Rae and Dr. Arthur Donovan (of the Food Animal and Reproduction and Medicine Service) for their support and indulgence during this experience. I gratefully acknowledgeFlorida Dairy Check-Off for their economic support of my study. I thank Mr. Ingo Kreig, owner of Mecklenburg Dairy, for his cooperation with the project; particularly for the use of his cows and facilities. I thank Mr. Roger Rowe, farm manager at Mecklenburg Dairy for his time and assistance with the project. I thank Dr. Bronwyn Crane and Dr. Pablo Pinedo, fellow graduate students, for their friendship andsupport. I thank my fellow residents and interns for the extra effort they gave, which helpedme to complete this program. I thank God for allowing all of these people to cross my path. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.....................................................................................................i ii LIST OF TABLES................................................................................................................. vii LIST OF FIGURES...............................................................................................................vi ii ABSTRACT...........................................................................................................................x CHAPTER 1 INTRODUCTION..........................................................................................................1 2 LITERATURE REVIEW...............................................................................................4 The Bovine Uterus..........................................................................................................4 Uterine Involution...........................................................................................................6 Endocrine Changes Early Post Partum...........................................................................13 Uterine Defense Mechanisms.........................................................................................17 Innate Uterine Defense............................................................................................18 Anatomical barriers..........................................................................................19 Uterine and cervical secretions........................................................................20 Bacterial antagonism........................................................................................22 Innate cell component......................................................................................2 3 Humoral-mediated immunity...........................................................................24 Cell-Mediated Immunity.........................................................................................26 Fever Pathway during Infections....................................................................................26 General Review of Uterine Infections............................................................................29 Endometritis............................................................................................................3 0 Pathogenesis.....................................................................................................3 0 Histopathological disease definition................................................................3 1 Clinical disease definition................................................................................3 3 Puerperal Metritis....................................................................................................3 3 Pathogenesis ....................................................................................................3 3 Histopathological disease definition................................................................37 Clinical disease definition................................................................................38 Literature disease definitions...........................................................................39 Current research definition...............................................................................51 iv

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Risk Factors for Puerperal Metritis.........................................................................52 Retained fetal membranes................................................................................52 Stillbirth, multiple birth and dystocia...............................................................54 Parity................................................................................................................56 Season...............................................................................................................57 Hypocalcemia...................................................................................................58 Postpartum Health Monitoring.......................................................................................58 Attitude............................................................................................................. 59 Milk Production................................................................................................63 Rectal Temperature..........................................................................................66 Ketone Bodies..................................................................................................68 Evaluation of Uterine Discharge.....................................................................70 3 MATERIALS AND METHODS...................................................................................72 Cows and Herd Management..........................................................................................72 Study Design...................................................................................................................73 Data Management...........................................................................................................75 Statistical Analysis..........................................................................................................75 4 RESULTS.......................................................................................................................79 Final Sample...................................................................................................................79 Incidence of Diseases.....................................................................................................79 Rectal Temperature.........................................................................................................81 Reproduction...................................................................................................................85 5 DISCUSSION.................................................................................................................89 6 CONCLUSION...............................................................................................................96 LIST OF REFERENCES.......................................................................................................97 BIOGRAPHICAL SKETCH.................................................................................................112 v

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LIST OF TABLES Table page 2-1 Ketosis threshold, sensitivity and specificity for different tests...............................69 4-1 Incidence and risk factors for puerperal metritis in the first 13 days postpartum in lactating dairy cattle.............................................................................................80 4-2 Incidence and risk factors of clinical endometritis at 20 to 30 days postpartum in lactating dairy cattle.................................................................................................81 4-3 Mean (SEM), 25 quartile, median, 75 quartile, and population 95% confidence intervals rectal temperatures of cows with and without puerperal metritis......................................................................................................................82 th th 4-4 Logistic regression model of conception rate to first service and pregnancy rate at 150 day postpartum days postpartum by group, presence or not of metritis, parity, season and presence or not of endometritis..................................................87 4-5 Cox proportional model of days postpartum to first service and pregnancy rate at 150 day postpartum days postpartum by group, presence or not of metritis, parity, season and presence or not of endometritis..................................................88 vi

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LIST OF FIGURES Figure page 2-1 Changes in lochia volume during the first 20 days postpartum in dairy cows...........7 2-2 Uterine changes in weight, diameter and length during the first 20 days postpartum in cows ....................................................................................................8 2-3 Reduction of the cervical opening during the first 24 h post partum.......................10 2-4 Reduction of the cervical opening during the first10 days post partum...................10 2-5 Pooled within cows least-squares regressions of plasma progesterone (P4), estrone sulfate (E1SO4), estrone (E1), prolactin (Prl), and 13, 14-dihydro, 15-keto prostaglandin F2 (PGFM) during the periparturient period...........................14 2-6 Pathway of fever development in response to infection, inflammation, or trauma..28 2-7 Percentage of uteruses from postpartum cows in which bacteria were recovered in the first 60 days post partum................................................................................30 2-8 Overall incidence and degree of endometritis in uterine biopsy samples during the first seven weeks post partum............................................................................32 2-9 Daily mean feeding time of Holstein cows with acute metritis and Holstein cows without acute metritis from 12 d before calving until 19 d after calving.................62 2-10 Difference in activity and daily milk yield for cows with an occurrence of ketosis, left displaced abomasum, and general digestive disorders compared to cows without an incidence of a disease in the prebreeding stage during 10 to 10 days relative to disease diagnosis (day 0)................................................................65 4-1 LSM SEM of daily rectal temperatures of cows 5 days before and 5 days after the diagnosis of metritis in cows with and without puerperal metritis.....................83 4-2 LSM SEM of daily rectal temperatures of cows 5 days before and 5 days after diagnosis for cows with puerperal metritis and fever, puerperal metritis without fever and cows without puerperal metritis...............................................................84 4-3 Proportion of pregnant cows during cold or warm season by 150 days postpartum................................................................................................................85 vii

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4-4 Proportion of pregnant cows with or without puerperal metritis by 150 days postpartum................................................................................................................86 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RECTAL TEMPERATURE, CALVING-RELATED FACTORS, AND THE INCIDENCE OF PUERPERAL METRITIS IN POSTPARTUM DAIRY COWS By Mauricio Esteban Benzaquen August, 2006 Chair: Carlos A. Risco Major Department: Veterinary Medicine Science. The objectives of my study were to evaluate the association of calving status, parity and season on the incidence of puerperal metritis (PM) and clinical endometritis (CE) in lactating dairy cows; and examine the role of rectal temperature as a predictor for puerperal metritis, and document the effect of puerperal metritis on subsequent reproductive performance. This study was a prospective cohort design. Cows were classified as abnormal calving status (Ac), cows calving with dystocia, RFM with or without dystocia or twins and cows with a normal calving status (Nc), those without any calving related problems. Daily rectal temperature (RT) of all cows was taken early in the morning from days 3 to 13 postpartum, and health examinations were performed by the on-farm veterinarian. We evaluated a total of 450 calvings. Cows with Nc had a lower incidence of PM compared to cows with an Ac status (13% vs. 41%, respectively; P < 0.001). During the cool season, primiparous cows had the highest incidence of PM compared to primiparous cows in warm season or multiparous cows in either season (P < ix

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0.01). Cows with Ac were more frequently diagnosed with CE than those with Nc (AOR = 2.8, 95% CI 1.7-4.9, P < 0.001). A higher incidence ( 38.2%) of CE was found in cows diagnosed with PM (AOR = 2.2, 95% CI 1.1-3.9, P < 0.005). The RT in cows diagnosed with PM increased between 48 to 24 h before diagnosis of PM and continued to increase until reaching a maximum RT of 39.2C 0.05 on Day 0 (day of diagnosis). In cows with PM and fever at diagnosis the RT started to increase between 72 to 48 h before the diagnosis of PM, and continued to increase until reaching a maximum RT of 39.7C 0.09 on the Day 0 (day of diagnosis). In cows with PM and no fever at diagnosis there was not a significant daily increment of RT before the diagnosis of PM. Cows without metritis did not show any variation in RT during the first 13 days postpartum. There were no detected differences in accumulated pregnancy rate by 150 days post partum (mean= 50%) among normal cows and cows experiencing PM. A season effect was detected (Cool season [40 %] > than warm season [28 %; P < 0.02]). Occurrence of PM was higher in cows experiencing an Ac. Primiparous cows had a greater incidence of PM in the cool season for both normal and abnormal calvings. In contrast, multiparous cows showed no seasonality in the occurrence of PM. Evaluation of daily RT was able to distinguished PM with fever and PM without fever. Sequential increases in RT on two consecutive days prior to the actual diagnosis can serve as a predictor of PM with fever. Abnormal calving, and PM were risk factors for clinical endometritis. Pregnancy rates were comparable between cows with normal or abnormal calving status cows, regardless of the occurrence of PM. x

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CHAPTER 1 INTRODUCTION Puerperal metritis has multiple factors contributing to its etiology, severity, and duration. It occurs during the period from calving to when the anterior pituitary gland becomes responsive to gonadotrophin releasing hormone (GnRH) approximately 7 to 14 days postpartum (Olson et al., 1986). In puerperal metritis there is inflammation of all layers of the uterus and it is characterized by the presence of a fetid, watery reddish-brown vulvar discharge (Lewis, 1997). Sheldom et al. (2006) attempted to standardize the clinical definition of puerperal metritis. Their definition includes clinical symptoms such as decreased milk production, dullness, or other signs of toxemia with fever (> 39.5C) within the first 21 days postpartum. Factors that predispose cows to puerperal metritis have been reviewed previously (Curtis et al., 1985; Correa et al., 1993; Markusfeld 1984; Bartlett et al., 1986). However, in some cases puerperal metritis was classified as a disease complex without distinguishing clinical severity or presentation, making studies difficult to compare (Lewis, 1997). Prevention and early treatment of puerperal metritis is more economical than let this condition progress to a stage where treatment is not beneficial on cost effective (Bartlett et al., 1986). Thus the need to identify puerperal metritis early post partum and provide treatment, monitoring of attitude and fever during the first 10 days postpartum (Upham, 1996). Rectal temperature is an indicator of the core body temperature, and is used as a diagnostic method to determine whether the cow has fever. Fever is the result of 1

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2 a complex communication between the peripheral immune system and the brain in response to infection, inflammation and/or trauma, and is clinically characterized by a rise in body temperature (Leon, 2002). Fever can be initiated by bacterial lipopolysaccharides (LPS) acting directly as exogenous pyrogens; or indirectly by activating liver macrophages (Steiner et al., 2006) inducing the production of endogenous pyrogens such as interleukin (IL)-6, IL-1, and TNF(Luheshi, 1998). It has been difficult to establish a minimum rectal temperature to define fever in postpartum-health monitoring protocols because of the broad range of rectal temperature described in the literature (Smith et al., 1998; Upham, 1996; Drillich et al., 2001; Zhou et al., 2001; Sheldon et al., 2004), and the multiple factors that affect rectal temperature values (Rebhun, 1995; Rosenberger, 1979). Kristula et al. (2001) evaluated postpartum rectal temperature: 48% of cows that calved normally had at least one daily temperature above 39.1C compared to 93, 83, 100 and 78% for cows with retained placenta, mastitis, puerperal metritis and dystocia, respectively. Kristula et al. (2001) concluded that rectal temperature (per se) is not enough to determine whether antibiotic treatment is needed for postpartum cows. Despite the common use of rectal temperature of postpartum cows to evaluate health, there is a lack of research on the value or significance of rectal temperature and calving status as tools to diagnose puerperal metritis in dairy cows. Understanding the factors that predispose cows to puerperal metritis, and understands rectal temperature responses in cows at risk of developing puerperal metritis should aid in the formulation of appropriate postpartum health monitoring strategies. In addition, early diagnosis and

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3 treatment of puerperal metritis could improve later reproductive performance and productivity. Our study objectives were to evaluate the effect of abnormal calving, parity, season, and rectal temperature on the incidence of puerperal metritis within the first 13 days post partum: and to report the effect (if any) of puerperal metritis and clinical endometritis on reproductive performance.

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CHAPTER 2 LITERATURE REVIEW The Bovine Uterus The uterus consists of a tubular structure formed by the cervix, uterine body, and the uterine horns. The non gravid uterus is located, depending on factors such as age, breed, and parity of the cow, on the pelvic cavity dorsal to the urinary bladder and ventral to the rectum. The main function of the uterus is to accept a fertilized ovum which becomes implanted into the endometrium, and derives nourishment from blood vessels developed exclusively for this purpose. The uterus is derived from the embryonic mllerian or paramesonephric ducts, the tubes will develop into the gonaductal system, giving rise, in the case of the female, to the oviduct, uterus, cervix and cranial vagina. In mammals the mllerian ducts fuse from cranial to caudal. The female mllerian ducts start to fuse from the most cranial part, caudally to form the oviducts, uterus, the cervix and the anterior part of the vagina. The fusion of the ducts happens in the medial walls, forming the paramesonephric septum. This partition tends to disappear in time, fusing and forming a single tube. In the cow, the ducts fuse forming a septum that separates the two horns giving the characterization of a bipartite type uterus. Both sides of the uterus are attached to the pelvic and abdominal walls by the broad ligaments, from were the uterus receives its blood and nerve supply. The middle uterine artery, a branch of either the internal iliac artery or the external iliac artery, provides the blood supply to the uterus in the region were the fetus develops. The cranial uterine 4

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5 artery, a branch of the utero-ovarian artery, supplies blood to the ovary by the ovarian artery and to the anterior extreme of the uterine horns by the cranial uterine artery. The utero-ovarian artery runs closely along the surface of the corresponding veins. The artery terminates by giving rise to a small branch to the tip of the uterine horn and oviduct. All lymphatics of the uterus are in communication, the cervix drains towards sacral nodes while the body of the uterus and oviducts drain towards the external iliac nodes, also there is some drainage towards internal iliac nodes. The uterus can be divided in three layers. From the lumen to the abdominal cavity can be divided in endometrium, myometrium and perimetrum or serosa. The inner portion of the uterus is composed of the mucosa and submucosa. Both compose the endometrium, characterized by having many endometrial folds. Histologically the mucosa is characterized by a simple pseudostratified cylindrical cell surface during most stages of the cycle; few ciliated cells are present, especially in the multiparous cow, less than 1%. The submucosa is predominantly connective tissue and houses the uterine glands. The uterine glands develop in the mucosa, projecting to the submucosa where they coil. Their basic structure is similar to those of a mucosal epithelial surface (Marinov and Lovell, 1968). The surface of the endometrium of the cow is covered by non glandular areas denominated caruncles. The caruncles are highly vascularized areas that give rise to the maternal portion were the cotyledons will attach. The endometrium is covered by two smooth muscular layers that constitute the myometrium. In contact with the endometrium a circular smooth muscle layer is present, it is in this portion where the blood vessels penetrate the submucosa stratum. Up on this portion, the longitudinal layer of smooth muscle is present, easily recognizable for it

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6 creases, or small ridges, palpable during the early post partum of the cow. The outer layer of the uterus is the serosa or perimetrum, quite thin and almost transparent covering the entire uterus and continues dorsally and covers the mesosalpinx. This portion of the uterus will be in contact with the pelvic and abdominal cavity of the cow. Uterine Involution In the pregnant cow, during the time period 1 month before and 1 month after parturition, several metabolic and endocrine events take place. As the cow enters the transition period, the dam has to be prepared for the impending parturition, and the uterus and ovaries must return to a certain stage to be prepared for a new pregnancy (Kindahl, et al., 2004). Most of these processes are due to or reflected in endocrine changes. The uterine lumen holds approximately 70 kg at the time of parturition, including fluids, fetal membranes, and the fetus. Approximately half of this weight contains fluids and the other half includes the fetus and the fetal membranes (Mortimer et al., 1997). However, after parturition the uterus of the cow, is a large, flabby sac, nearly a meter long and 9Kg in weight (Gier and Marion, 1968). The rapid re-organization along with a fast reduction in the diameter of the organ constitute probably a protective mechanism against ascending infections (Mortimer et al., 1997). Involution of the uterus involves loss of intraluminal fluids, reduction in size, and endometrial repair. During the first two days after calving, the expelled fluids are serosanguineous, and change in character after caruncular dissolution begins (Olson et al., 1986; Rasbeck, 1950) describes the elimination of decidual detritus of caruncular tissue starting 3 to 4 days after parturition and increasing until the 9 th day, and gradually becomes mixed with blood originating from hemorrhages on the surface of the caruncles. Gier and Marion (1968) found a considerable quantity of blood in the uterus after

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7 parturition, becoming mixed with sloughed caruncular material after day four, which changed by day 12 post partum to a more lymph like fluid which decreases in quantity by day 23 postpartum (Figure 2-1) (Gier and Marion, 1968). This discharge is named uterine lochia, which consist of mucus, tissue, detritus, and blood, that commenced 3 to 4 days and decreasing until the ninth day post partum (Roberts, 1986). Lochia may assume different appearances from white, yellowwhite or a grey mucopurulent character toward the latter part of the puerperal period. This discharge is considered a normal process of Figure 2-1. Changes in lochia volum uterine involution (Roberts, 1986). e during the first 20 days postpartum in dairy cows (Gier and Marion, 1968). m period, re-organization and involution of the uterus occurs and a rst 05001000150015101520Days postpartumLocial Volumen (ml) During the post partu small opening in the cervix remains for elimination of uterine contents (Wehrend etal., 2003). Reduction in the uterine weight, diameter and length occurs in a decreasing logarithmic scale (Figure 2-2) (Gier and Marion, 1968). This reduction in size may be explained partially by peristaltic contractions at intervals of 3 to 4 minutes during the fi

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8 day postpartum and continuing through the second day (Gier and Marion, 1968). Bajcsy et al. (2005) evaluated the contractility of the postpartum uterus by recording and quantifying the frequency, amplitude and duration of intrauterine pressure changesbetween 12 hours to 48 hours after parturition. In that study (Bajcsy et al., 2005) it was reported that mean frequency of uterine ost partum with a range of 6 to 11 tions 010203040506070809010005101520Diameter and Len g th of Uterus ( cm ) 012345678910Wei g ht of Uterus ( K g) Figure 2-2. Uterine changes in -Weight; -Diameter and -Length during the first 20 days postpartum in cows (Gier and Marion, 1968). contractions were 8.9 contractions per hour at 12 h p contractions every hour. The mean contraction frequency decreased to 1.8 contracper hour at 48 h post partum. The largest drop in mean values occurred between 12 and 24 h post partum, and the frequency decreased by 46% of the initial mean value. Resultsfrom amplitude showed an initial individual mean value of 40 mmHg at 12 h. Changes inmean amplitude showed a similar pattern as frequency, with highest initial mean values at

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9 12 h postpartum (19.6 mmHg) and a reduction of 16% of the starting values by 48 h postpartum (3.2 mmHg). The most marked drop (42 %) in this response also occurred between 12 and 24 h post partum. Mean duration at 12 h was 89.8 seconds and variedbetween 102.5 at 36 h postpartum to 67.9 seconds at 48 h. In that study (Bajcsy et al., 2005), the relationship between uterine activity and blood calcium levels also was investigated. Neither frequency, amplitude and duration showed a significant relationshto blood Ca2+ levels in cows at any of the four recording times. Diameter of the cervix at a given time after parturition is influenced by the involution process of the reproductive tract (Oltenacu et al., 1983 ip ). Gier and Marion (1968iameter ion on e ) reported that involution of the cervix measured in slaughtered cows. The dof the cervix at day 2 after parturition was about 15 cm; 9 to 11 cm at 10 days, 7 to 8 centimeters by 30 days, and 5 to 6 cm by 60 days. Wehrend et al. (2003) reported results of cervical involution in vivo by measuring the cervix canal from the time after expulsof the calf up to the tenth day post partum. They showed that the cervical folds were in constant formation and were detected at the third day postpartum. In addition, the organization of cervical folds started from cranial and continued caudally. After the expulsion of the calf, a reduction of the opening from 26.9 1.3 cm to 1.9 0.3 cmthe seventh day post partum was observed (Figure 2-3 and 2-4). Furthermore, up to ththird day post partum, the lumen of the cervix was detected in all cows, disappeared fromthe fourth to the seventh day, and re-appeared at the tenth day after parturition. The authors concluded that this opening was related to allow further elimination of uterine fluid contents (Wehrend et al., 2003).

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10 Figure. 2-3. Reduction of the cervix opening during the first 24 h post partum (W ehrend et al., 2003). 05101520 25 30 012345678910Days post partumOpening degree (cm) Figure 2-4. Reduction of the cervix opening during the first10 days post partum (Wehrend et al., 2003). 05101520250246812161824Hours post partumOpening degree (cm) 30

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11 Reduction of the size of the uterus seemed to be produced by early constriction between 5 to 10 days and a secondary cervix opening relaxation produced at 10 days postpartum, at the time of final sloughing of the caruncular masses (Gier and Marion, 1968). Morrow et al. (1966), reported that slow regression of the uterus occurred during the first 4 to 9 days after parturition followed by an accelerated regression during the period 10 to 14 days after calving. In addition, the maternal portion of the placentome (caruncle) after the removal of chronic villi, remained as a loose mass of tissue approximately 70 mm long, 35 mm wide, and 25 mm thick (Gier and Marion, 1968). Caruncular blood vessels constricted rapidly and were nearly occluded within 2 days postpartum. However, blood continued to flow from protruding arterioles and contributed a least 10 days (Gier and Marion, 1968). the as s as of the i to the luminal fluids for Archbald et al. (1972) described the histological involution of the uterus during first 60 days post partum. During the first day postpartum, the caruncular epithelium wdegenerated, as well as the intercaruncular epithelium. The myometrium was edematouwith degenerated muscle fibers, and fetal chorionic cells were still present but some werenecrotic. By the fifth day post partum, the caruncular epithelium was regenerated, but started to be lost given the sloughing of the superficial layer of the caruncles. The intercaruncular epithelium was regenerated with the exemption of the basilar arecells. The endometrial glands were degenerated in the basilar area with some ducts dilated. Vacuolization of the myometrium was almost generalized. However, the nucleof the muscle cells were normal and the fetal chorionic cells were necrotic, mineralized and surrounded by macrophages (Archbald et al., 1972).

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12 At visual examination, most of the necrotic layer is removed by day 10 post partumand by 15 days post partum all of the caruncular mass that had been involved in the placentome are sloughed, leaving only stubs of blood vessels extending beyond the surface of the stratum compactum (Gier and Marion, 1968). By 19 days post partumarterioles within and beyond the stratum compactum disappeared (Gier and Marion, 1968). The histology showed by day 15 post partum that the caruncular epithelium wabsent in some areas, but a cuboidal or flat epithelium was present in other areas. The intercaruncular epithelium was regenerated with few areas of degeneration in the basilar area of the cell and the myometrium was shrunken in size the as with vacuolation of the muscle fibersle. trial nt l for completion of gross uterine involution for multiparious cows was 40 days, and the and fetal chorionic cells were not present. By day 19 post partum, the caruncular epithelium was covered by a columnar to cuboidal epithelium over the entire caruncuA cuboidal epithelium was also present over the entire intercaruncular area. Endomeglands were normal and non-secretory. The myometrium had few necrotic fibers and continued to shrink in size. By day 19 postpartum fetal chorionic cells were not prese(Archbald et al., 1972). The caruncles already reduced their size at 19 days postpartum to 15 to 20 mm indiameter, and by day 39 post partum, are reduced to smooth knobs of 10 to 15 mm in diameter composed of circular cones of 8 to 10 mm across the base and 4 to 6 mm across the crown by 50 to 60 days postpartum (Gier and Marion, 1968). The final gross involution of the uterus has been reported to be completed in the previously pregnant horn by day 25 post partum in normal cows, and 30 days post partumin cows with postpartum disease (Morrow et al., 1966). In contrast, the average interva

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13 interval was affected by parity, season, and stress factors (Marion et al., 1968). The histological characteristi cs of the uterus by day 31 to 45 days postpartum showed that the caruns he ea was covered by a columes derin lactin cular and the intercaruncular tissue had epithelium present over the entire structure(Archbald et al., 1972). The endometrial glands were still normal and non-secretory. Tmyometrium was normal, and regained the normal shape and sarcoplasm. By day 60 post partum the uterus regained its normal histology. The caruncular ar nar type epithelium somewhat pseudostratified. Many pigment-bearing histocytwere present in the stratum compactum and neutrophils were not observed. There were many plasma cells distributed through the stratum compactum and spongiosum. The intercaruncular area was covered by a single layer of epithelium and consisted of cuboidal and columnar cells. Numerous mast cells and histocytes containing hemosiwere in the stratum compactum. Numerous mast cells were in the stratum spongiosum, and the blood vessels of this layer appeared normal. The endometrial glands appeared normal and non-secretory. The myometrium and the subserosa layer appeared normal, but were infiltrated by numerous mast cells (Archbald et al., 1972). Endocrine Changes During Early Post Partum A balanced, coordinated endocrine system is important for normal reproductive function. A graphical representation of this complex process is presented in (Figure 2-5)These changes involve the gonadotrophin releasing hormone (GnRH) from the hypothalamus, follicle stimulating hormone (FSH), luteinizing hormone (LH), pro(PRL) from the adenohypophysis, prostaglandin F 2 from the uterus, progesterone (P 4 ) from the corpus luteum, and estrone sulfate (E 1 SO 4 ), and estrone (E 1 ), from McDonald (1980)

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14 Cortisol is recognized as a stress hormone and is also responsible for the regulatioof prostaglandin synthesis (Hafez, 1993). In the fetus prior to parturit n ion, adrenocorticotropind, stimulating the adl increases to a mean of 74 ng/ml on the day of calving (Hunter, et al., 1977). At 30 days pre partum, only 50-60% of the corticosteroids fraction is cortisol, whereas in the last 10 hormone (ACTH) is released from the pituitary glan renal glands to release cortisol. The fetal cortisol is central to the survival of the neonate, as well as for induction of lactation and parturition in the cow (Nathanielsz 1993). Fifure 2-5. Pooled within cows least squares regressions of plasma progesterone (P4), estrone sulfate (E1SO4), estrone (E1), prolactin (Prl), and 13, 14-dihydro, 15-keto prostaglandin F2 (PGFM) during the periparturient period (Eley et al., 1981). As parturition approaches, the fetal adrenal cortex becomes increasingly sensitive to adrenocorticotropin (ACTH). Mean fetal levels of corticosteroids are within 5.0 ng/mat 20 days to 9.3 ng/ml at 10, and 25 ng/ml 4 days pre partum, and the level progressively

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15 days lf rectal compounds (Hoedermarker et al., 1990). Progesterone is secreted by luteal cells of the corpus luteum and by the placenta, and it secretion is primarily induced by LH (Hafez, 1993). The function of progesterone is to prepare the endometrium for the implantation of the embryo and for the maintenance of pregnancy. In contrast to sheep and horses that exhibit placental progesterone production, the corpus luteum of the cows maintains pregnancy by luteal cells P4 production which fluctuates between 6 to 15 ng/ml through gestation (Knickerbocker et al., 1986). During the peripartum period there is approximately a 20% reduction of the levels of progesterone within 4 to 1 week before parturition (Edqvist et al. 1978). A second phase of reduction can be distinguished with a more abrupt decrease of progesterone during the last 2 days prior to parturition, and this is interpreted as pre-partal luteolysis (Edqvist et al. 1978). Maternal blood progesterone falls towards term, l (Hunter et al., 1977; Eley et al., 1981), and it is not until day 16 post partum that cows increase their P4 levels over 1 ng/ml (Eley et al., 1981). The rise in fetal corticosteroids during the last month of gestation is reflected by the increment of estrogens levels (Knickerbocker et al., 1986). of gestation the proportion of cortisol rise to over 90% (Hunter, et al., 1977). Dystocia may affect levels of cortisol. Severe dystocia resulted in lower catemperature, reduced serum cortisol and increased serum glucose (Bellows and Lammoglia 2000). Close to parturition the release of cortisol induces the 17-hydroxylase enzyme in the fetal membranes to start the conversion of progesterone toestrogen decreases rapidly over the last 48 to 36 h post partum to levels of less than 1 ng/m

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16 Cotyledonary tissue is the main source of placental estrogens (Hoedermarker1990). Characterization of E et al., ilar only r et al., eases gradually from a baseline of 30 to 60 t cher et y of ic acid by atory) ). The PGF2 during the early postpartum period, and the 1 and pooled 17-/ estradiol sulfate (E 2 SO 4 ), reflect simpatterns to estrone sulfate (E 1 SO 4 ). Thus, placental steroid activity is most commdetermined by concentrations of plasma estrone sulfate (E 1 SO 4 ) (Knickerbocke1986). Maternal plasma concentration of E 1 SO 4 incr pg/ml before day 60 to approximately 500 pg/ml by day 100 of pregnancy (Eley eal., 1979). A rapid elevation then occurs until day 150 when estrone sulfate concentrations approach 3000 pg/ml. After 150 days of pregnancy E 1 SO 4 remains constant until approximately day 240 when estrone sulfate increases rapidly (Thatal., 1982). Concentrations of estrogens decline abruptly in association with deliverthe conceptus (calf and placenta). Basal concentrations of P 4 E 1 and E 1 SO 4 are low by 24h after parturition and remains low for approximately 14 day after parturition (Eley etal., 1981). Prostanoids (prostaglandins and thromboxanes) are forms from arachidoncyclooxygenases (COX). At least two different COX enzymes, which are isoenzymes, have been found: COX-1 mainly a constitutive and COX-2 an inducible enzyme COX-2is involved in both physiological (luteolysis, parturition) and pathological (inflammprocesses (Kindahl et al., 2004). The most important product linked to reproduction is PGF 2 The metabolism of PGF 2 is very rapid to its metabolite 15-keto-13,14-dihydroPGF 2 (referred to 15-ketodihydroPGF 2 or PGFM) (Granstrom and Kindahl 1982uterus is the primary source of

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17 carun last 24 h gestation, reaching peak levels of around 5 ng/ml during labor (Hunter, et al.,progesterone 1981). Prolactin acts on the central nervous system to induce maternal behavl seek out and destroy the wide variety of pathogens in their particular habitats within the body and its external and internal surfaces (Janeway, 2005). cles also contribute to the synthesis and metabolism of PGF 2 (Guilbault, et al,. 1984). Utero ovarian venous prostaglandin F 2 levels remained relatively constant at around 500 pg/ml by 48 to 36 h before calving when they increased rapidly especially over the 1977). During the prepartum period levels of PGFM are negatively correlated to P 4 (Eley et al., 1981). A final abrupt increase in PGFM is accomplish by a decline in associated with CL regression just prior parturition (Eley et al,. 1981). Increases in PGFM occurs during the postpartum period (0 to 11 days) after luteolysis and delivery of the calf (Eley et al., 1981). The major increase in concentration of PGFM in plasma during the periparturient period occurs 1 to 4 days postpartum with concentration returning to base level by day 15(Eley et al,. ior (Hafez, 1993). Prolactin concentration begins to rise from a variable baseline of approximately 80ng/ml 2 at weeks prepartum and reach peak values of 200 to 400 ng/mjust prior calving (Eley et al., 1981). The levels of prolactin are maintained elevated up tothe third day post partum, when they return to baseline levels of 80ng/ml (Eley et al., 1981). Uterine Defense Mechanisms The immune system functions to defend the host against infections. Host defense requires different recognition systems and a wide variety of effector mechanisms to

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18 Innate immunity serves as a first line of defense. Once body surfaces (skin, mucosa ), secretions and anatomical barriers are breached, macrophages and neutrophils of thee ncreased protection against subsequent reinfection (Janeway, 2005). al., ndary lymphoid nodules analogous to Peyers patches and bronc, of milk (Mallard et al. 1998). In addition, the onset of lactation imposes tremendous physiological chanisms of the cow which influenerating an immunological innate immunity system provide a first line of defense against many common microorganisms. However, they do not always eliminate the infection. Consequently, thadaptive immune system has evolved to provide a more versatile and specific means of defense, and i The inner layers of the uterus are part of the mucosal immune system with structural and functional similarities, and common lymphocyte trafficking network with the intestinal, bronchial, nasal, ocular, salivary and mammary gland tissue (Ogra et 1999). The most prominent difference between the uterus and the other mucosal surfaces is the lack of organized seco hus-associated lymphoid tissue (BALT) (Head and Billingham, 1986). Furthermorein the majority of domestic species the uterus is exceptional among mucosal tissues because the ovarian steroid hormones have considerable effects on immune events (Lewis, 2004). To the complexity of this specialized immunological system a production stressor factor is added, and the modern dairy cow is unique in her experience of repeated lifetime cycles of pregnancy and parturition followed by lengthy lactations producing high volumes allenges to the homeostatic mech nces immunological responses (Goff and Horst, 1997). Innate Uterine Defense Innate immune mechanisms act immediately, and are followed by early induced responses which can be activated by infection without ge

PAGE 29

19 respom ion, tors (BonDgina by some ciliated columnar epithelium that participates in the process of mucu ogen, mucosa (Senger, 1999). mechanical flushing system for pathogens and antigens that are captu nse (Janeway et al., 2005). The uterus has it own innate immunological systewhich includes anatomical barriers, uterine and cervical secretions and bacterial antagonism. Once this defense mechanism is breached, such as in the case of parturitthe most important line of defense is the innate cell defenses composed of phagocytic cells such as neutrophils, macrophages, effectors cells (basophils, mast cells, and eosinophils) which have the capacity to induce an influx of other immunological fac urant, 1999). Anatomical barriers The uterine environment is protected by anatomical characteristics that act as barriers to the external environment of the cow. These physical barriers consist of the vulva, vaginal vestibule and cervix which are all covered by a mucosal layer that produces and secretes specific and nonspecific immunological factors (Senger, 1999). The cranial vagina as well and the fornix vaginae are characterized by columnar epithelium which is highly secretory under the influence of estrogen. The cranial vais characterized s elimination (Senger, 1999). The caudal vagina is characterized by a stratified squamous epithelium and the dermal epithelium which have an exfoliative process that eliminates microbes adhered to epithelial cells. The secretory function of the vaginavaries according to the endocrine status. During estrus, under the influence of estrthe stratified epithelium becomes thickened, protects the vagina during copulation, and reduces access of microorganisms to the vasculature of the sub Mucus secretion acts as a red by mucoproteins (Senger., 1999).

PAGE 30

20 The two labia of the vulva are in close contact by the action of the constrictor vulvae muscle which minimizes entrance of foreign material to the vulva. The sklabia is part of the integument and has numerous sebaceous and sweat glands which produces antimicrobial factors (Senger, 1999). The cervix isolates the uterus from the external environment by forming an anatomical barrier trough of multiple folds anrings which protrude into the cervical canal (Senger, 1999). During parturiti in of the d three on there is dilation of the cervix which allows expulsion of the es the cervical seal, allowing the interior of the uterus to be in contaal s culating neutrophils and tissue macrophages, that remove the microbes (phagocytosis) and release additional, different mediators (e.g., fetus. This event remov ct with the environment of the cow, and does not return to its normal anatomicform in cows with a normal puerperium until 7 to 10 days post partum (Wehrend et al.2003). Uterine and cervical secretions The internal epithelium of the cervix and the uterus contain cells that secrete mucus, which is composed of glycoproteins (mucins). Mucus traps microorganisms and prevents them from reaching and colonizing the mucosal epithelium. Mucus also containlysozymes that help degrade bacteria, antibodies that prevent microbes from attaching to mucosal cells lactoferrin that binds iron making it unavailable to microbes, and lactoperoxidase that generates toxic superoxide radicals that kill bacteria (Ogra et al.,1999). The complement system is made up of many distinct plasma proteins that react with one another to opsonize pathogens and induce a series of inflammatory responses that help prevent infections (Janeway et al., 2005). The function of complement is to recruit mononuclear phagocytes, initially cir

PAGE 31

21 cytokf in vaginal and uterine secrete and inhibited by progesterone (Hasty et al., 1994). n found in exocrine secretions of the mucosal surfaces of thelands en en s (Lijnen and Collen, 1985). Through the generation of nonspecific, protease plasmthe ines, prostaglandins, leukotrienes, etc.) that enhance the inflammatory process (Janeway et al., 2005). Complement is an important component of the innate immunological reaction in the reproductive tract of the cow, opsonizing and lysing bacteria (Corbeil., 2002). Moreover, its activation seems to be due to the interaction oimmunocomplexes formed by antigens and immunoglobulin, specifically IgG2 (Corbeil., 2002). Increased concentrations of complement C3 are found ions of infected cattle (Kania et al., 2001). Although the exact source of C3 in thuterus and vagina of cattle is still unknown, it has been proposed that increased concentration of C3 in vaginal and uterine secretions result from serum-derived complement (Kania et al., 2001). In rats, C3 production is positively influenced by estrogen Lactoferrin is a ferric protei cow (Dixon and Gibbons, 1979). It is produced and secreted by the exocrine gof the uterus, epithelial cells of the cervix and ampulla of the uterine tube (Inoue et al., 1993). Lactoferrin acts as a powerful bacteriostatic, bactericidal, fungicidal and virucidal agent (Ogra et al., 1999). Another serum protein that is secreted in the reproductive tract is plasminogwhich is converted to plasmin by a series of specific serine proteases called plasminogactivator in, plasminogen activators influence numerous physiopathological processes, including fibrinolysis thrombolysis, and invasiveness, metastasis, and cell migration at sites of inflammation; this precess also involves degradation of the injured tissue and plasminogen activators released by macrophages and granulocytes may contribute to this

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22 process by degrading extracellular proteins (Dano et al., 1995). Two types of plasminogen activators, urokinase (u-PA) and tissue type (t-PA), are present iendometrial tissues and uterine fluids of the cow, and are thought to be involved in tresolution of endometritis in cows (Moraitis et al., 2004). Peroxidase activity has been measured in many mucosal secretions (Ogra et al., 1999), including the uterus. The activity of peroxidase is derived from enzymes synthesized by exocrine glands and secreted onto mucosal surfaces by the glands (Ogra eal., 1999). It produces toxic oxygen-derived products that act as a bactericidal agaddition, leukocytes present in the uterus can also produce large amounts of hydrperoxide (Hansen et al., 1987) which enhance lactoferrin activity (Ogra et al., 1999)addition, nitric oxide is another exocrine product of the uterin n he t ent. In ogen In e mucosa (Lapointe et al., 2000)ive anaerobic and strict anaerobic micro-organisms (Hafez 1993)cus ats et al., 2000). This normal flora of nonpathogenic bacteria compete with pathogenic and produces its bactericidal activity by producing toxic nitrogen oxides on the surface of the mucosa (Janeway et al., 2005). Bacterial antagonism The normal microbial flora of the bovine urogenital tract is made up of a dynamic mixture of aerobic, facultat The normal flora act as an inhibitory flora to help prevent infections by reproductive pathogens (Corbeil and BonDurant, 2001). The normal microbial flora of the reproductive tract is composed of bacteria of the genus Staphylococcus, Streptococand the coliform group (Hafez 1993). Contrary to what it is reported in humans and r(Reid et al. 1985), the number of lactobacilli appears to be lower in cervix and vaginal fluids of the cows (Otero et al., 2000). In contrast, Coagulase-negative Staphylococcus and -haemolytic Streptococcus bacteria are predominant in the vagina of the cow (Otero

PAGE 33

23 microorganisms for nutrients and for attachment sites on the epithelial cells anantimicrobial substances, such as lactic acid (Janeway et al., 2005). Innate cell component The main phagocytic barrier in the uterus is provided d produce by the invasion of neutrophils in rese re the ng neutrophils (BonDurant, 1999). This of cytokines in uterine infections. In addition, the experimental infecte the y ponse to bacteria (Sheldon and Dobson, 2004) that are present on the surface of thendometrium and into the lumen (BonDurant, 1999). Consequently, neutrophils aearliest and most important phagocytic cells to be recruited from the peripheral circulation to the uterine lumen, killing internalized bacteria and contributing to the formation of pus when the phagocytes die (Sheldon and Dobson, 2004). Experimental approaches with bacteria and bacterial components to induce an influx of neutrophils have been reported (Zerbe et al., 2001), However, when a non specific inflammation wasinduced, such as in the case of LPS inflammation, intracellular killing by uterine neutrophils was reduced compared to circulati suggests the importance ion with E. coli and A. pyogenes, the predominant bacteria in cases of bovine uterine infections, resulted in high concentrations of viable neutrophils in uterine secretions. However, the large numbers of neutrophils were not able to eliminatbacterial infection present (Zerbe et al., 2001). Consequently, other unknown factors mainfluence leukocyte activity (Mallard et al., 1998). Macrophages are also important in the uterine immune response. Macrophages sense bacteria or endotoxins through toll-like receptors (TLRs) which are the principal signaling molecules through which mammals sense infection (Beutler et al., 2003). The activation of the macrophages leads to the production of cytokines such as tumor necrosis factor-alpha (TNF ), and interleukins (IL-1, IL-6, IL-8). These cytokines alert other

PAGE 34

24 immune cells such as neutrophils or lymphocytes, by supporting the development of an adaptive immune response (Beutler et al., 2003). In addition, macrop hages induce the tes to inflamed tissue by inducing the expression of selectins, by TNthe d IgE nd st cells, obulins ve arm of the immune response proce and transmigration of leukocy F Selectins cause the rolling of leukocytes on endothelial cells which are attracted to the site of infection by a concentration gradient produced by the above mentioned cytokines, especially IL-8 (Janeway et al., 2005). In addition to neutrophil and macrophage migration mast cells and eosinophils are present on the surface of uterine mucosa. Both eosinophils and mast cells have high affinity receptors that binantibody (Janeway et al., 2005). The eosinophils also release inflammatory mediators aantimicrobial factors such as peroxidase and lytic enzymes (BonDurant 1999). The number of mast cells and eosinophils are known to vary with the stage of the estrous cycle (Likar et al., 1964; Matsuda et al., 1983). Little is known about bovine uterine mabut they secrete some of the usual mast cell mediators, such as histamine, leukotrienes, prostaglandins, heparin, and proteinases, as well as proinflammatory cytokines and Th2 related cytokines. Mast cell and eosinophil mediators increase vascular permeabilitywhich results in the subsequent influx of other immune cells and serum immunoglto the uterine lumen (Corbeil et al., 2005; BonDurant, 1999). Humoral-mediated immunity Antibodies are thought to be the most protecti ss in defense against extracellular pathogens (Corbeil, 2002). The distribution ofimmunoglobulins in external secretions is vastly different from that found in serum. Plasma cells secrete different immunoglobulin after a period of differentiation and isotype switching (Janeway et al., 2005). Different isotypes and allotypes of immunoglobulins are present in different compartments of the body, and the isotype,

PAGE 35

25 quantity may vary depending on the antigen stimulation (Janeway et al., 2005). Immunoglobulin-A (IgA) is the immunoglobulin mainly found on mucosal surfaces (Ogra et al., 2002). However, in the vascular system, secretory IgA (SIgA) is the predominant form, and is principally found as a dimer (Butler, 1972). It is the predominant immunoglobulin in nasal secretions, tears and saliva. In cervico-vaginal secretions, the relative concentration is lower than IgG, but still much higher proportionally to serum (Duncan et al., 1972). The majority of IgA found in externalsecretions is derived from local synthesis by plasma cells rather than selective transport from blood (Duncan et al., 1972). Immunoglobulin A is the major immunoglobulin class in the superficial portion of the reproductive tract and immunoglobulin-G (IgG) is thmajor class in secretions of the uterus, oviduct and follicular fluid (Corbeil et al., 1976; Whitmore and Archbald 1977). Antigenic stimulation of the bovine uterus results in specific antibody response to IgG class, whereas vaginal stimulation leads to an IgA response in vaginal secretions. Two subclasses of IgG are predominantly found in the serum of cattle, IgG1 and IgG2 (Butler, 1973). The rate of transport of IgG1 into vagimucus is more rapid than that of IgG2 (Curtain et al., 1971). In contrast to IgG2, IgG1 is present in higher concentration in the uterus and vagina (Curta e nal in et al., 1971). However, reaches the uterus or vagina are still unknown, because unlike cell in mechanisms by which serum IgG IgA, IgG does not have a secretory piece to mediate transport across epithelial(Corbeil et al., 2005) In addition, the bovine cervix is capable of local antibody production (Corbeil et al., 1976), where IgG predominates. Immunoglobulin-G levelsvaginal mucus exceed those for IgM. Perhaps the lack of detectable IgM in vaginal

PAGE 36

26 mucus can be related to the relative inefficiency of IgM transudation since it is rpredominantly to the vascular space (Wilkie et al., 1972). Cell-Mediated Immunity All lymphocytes are programmed during their development to specific mpathways through the body that enable antigen specific immune response to be concentrated at certain sites (Janeway et al., 2005). Directed migration, or hommucosal tissues is controlled by expression of distinct patterns of adhesion molecules the lymphocyte cell surface which mediate differential recognition and adherence to the endometrium in mucosal sites (Janeway et al., 2005). Intraepithelial lymphocytes are generally present in the stratum compactum of the endometrium, and their number fluctuates with the stage of the estrous cycle (Vander-Wielen and King, 1984). In addition, the main lymphocyte population is formed by CD8 type (Cobb and Watson 1995). The induction of local immune responses in estricted igration ing, to on cies urs in defensive response to the entry into the body of pathogenic agents (IUPS Glossary, the female genital tract of any speis poorly understood (Corbeil et al., 2005). However, during uterine infections with Trichomonas foetus, accumulation of immunocytes, lymphoid nodules, and follicles (some with germinal centers) were detected under the epithelium and adjacent to infected glands (Anderson et al., 1996). The kinetics of isotype-specific antibody responses, mast cell degranulation and clearance of infection demonstrate that immune defense of the uterus is related to increasing antibody levels and decreasing detectable subepithelial mastcells (Corbeil et al., 1974) Fever Pathway During Infections The term fever specifically defines elevation of body core temperature that occ

PAGE 37

27 2002). Functionally, the onset of fever is manifested by an increase in metabolic heat production and cutaneous vasocontriction to reduce heat lo ss from the skin. er is accomplished by the interaction of multiple endogenous mediac rance of S n of al., e pyrogenic and antipyretic properties, depending on the experimental conditions (L The generation of fev tors induced by pyrogens, such as lipopolysaccharides (LPS). Kupffer cells, splenimacrophages, and neutrophils are reported to contribute to the intravascular cleaLPS and produce cytokines (Scapini et al., 2000). Presumably fever is mediated by LPwhich stimulates the Kupffer cells located in the liver (Blatteis, 2006), but to a lesser degree in the spleen. Kupffer cells detect bacterial components such as endotoxins (LPS)and peptidoglycans through the toll-like receptors on the cell surface, specific for LPS(TLR-4) (Beuttler et al., 2003). This stimulation induces the production and secretiocytokines such as tumor necrosis factor (TNF-), and IL-1, IL-6, IL-8 (Beuttler et 2003). Two types of cytokines are responsible for the generation of fever. Pyrogenic cytokines that induce fever include interleukins IL-1, IL-6, IL-8, PGE 2 macrophage-inflammatory protein-1 (MIP-1), and interferon-. The other types of cytokines areendogenous antipyretics which limit the magnitude and duration of fever such as IL-10, arginine vasopressin (AVP), -melanocyte-stimulating hormone (-MSH), and glucocorticoids (Figure 2-6). Although AVP, -MSH, and glucocorticoids are not truecytokines, they still possess endogenous antipyretic properties. Other substances such as tumor necrosis factorhav eon, 2002). Pyrogenic cytokines in the bloodstream are transported to the preoptic-anterior hypothalamic area (POA), which is the primary brain site for thermoregulation The

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28 ventromedial preoptic nucleus (VMPO) is thought to be the fever-producing locus (Boulant, 2000) where cytokines act. Prostaglandin-E (PGE 2 ) is considered t o be the final fever mediator in the POA. The synthesis of PGE 2 in the POA/VMPO is effected through catalysis of arachidonic acid by cyclooxygenase (COX)-2 and microsomal PGE synthase (mPGES)-1 selectively upregulated by the pyrogenic cytokines (Blatteis, 2006). In addition, it is hypothesized that the febrile response to peripheral LPS is not initiated by pyrogenic cytokines released by LPS-stimulated leukocytes generally, but by PGE 2 specifically generated by Kupffer cells activated by LPS (Blatteis, 2006). Infection, Inflammation or trauma Macrophages H, Endo genous Pyrogens (IL-1, IL-6, IL-8, M1P-1, IFN) Endogenous Antipyretics (Il-10, AVP, TNF, -MSglucocorticoids) Hypothalamus + Thermal setpoint echanisms Initiation of effectors m FEVER Fig. 2-6. Pathway of fever development in response to infection, inflammation, or trauma. (Leon, 2002) +/

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29 The interactions of endogenous pyrogens and antipyretics are responsible for magnitude of fever reaction. Interleukin-1 and other inhibitory cytokines stimulate the production of IL-1 receptor agonist which prevents further binding of IL-1, and decreases the effective concentration of IL-1. Interleukin-10 is one of the principal interleukins that down-regulate the pyrogenic process. IL-10 is a product of T helper-2 subset, and isinduced by pyrogenic cytokines. It also inhibits the LPS-induced production of many cytokines implicated in fever, including IL-1, IL-6 and TNF(Leon, 2002).In addthe physiological control of the febrile response may prevent extreme elev the ition ation in body mperature. This regulationa high dose oLPS during sepsis functions to lower the temperature, thus attenuating fever or producing pothermia (Leon, 2002). of Uterine Infections parm cows is usuall contaminated with a wide spectrum of s not consistently associated with clinicay cows 1 to 4 weeks post prtum, species of microorganisms such as reptococcus, Arcanobacteria, Bacillus spp, Staphylococcus idermidis, Staphylococcus aureus, Facteroidespp, Clostridium spp d Proteus can colonize the uterus (Griffin etl., 1974; Olson etl., 1986). This bacterial content is negatively correlated with days postpartum. Within the first 15 days st partum, 90 % of uterine samples have a positive bacteriological culture. However, by 60 days post partum, the percentage of positive bacteriological cultures are reduced to %. (Elliot et al., 1968). Fgure 2-7 representhe percentage of uteri that have positive te seems to be dose related, in which f hy General Review The uterus of postbacteria. However, this i tu y l disease. In the majorit-hemolytic of a St ium pyogenes, Enterobacter ep usobacterium, B s an a a po 10 i t bacteria culture during the first 60 days post partum (Elliott et al., 1968).

PAGE 40

30 In addition, the common contamination of the uterus by these bacteria is not usually associated with clinical signs. Moreover, contamination does not imply infectioas reflected by the adhesion of pathogenic organisms to the mucosa, colonization or penetration into the epithelium, and/or release of bacterial toxins that result in the establishment of uterine disease (Janeway et al., 2005). n, Fig 2 sponse of the cow, ber of pathogenic helm uterine defense mEndometritisPathogenesis Endometritis is defined as an inflammation of the endometrial lining of the uterus. Studies by Griffin et al. (1974) and Elliott et al. (1968) have shown that uterine infections and endometritis are commonly present during the early post partum. In fact, during the 02040608010015 DaDays Postpartum Uthia 1030507090Percentteri wi Bacter y30 Day45 Day60 Day -7. Percentage of uteruses from postpartum cows in which bacteria were recovered in the first 60 days post partum. (Elliott et al., 1968) The development of uterine disease depends on the immune reand the species and number (load or challenge) of bacteria. The numbacteria in the uterus of postpartum cows may be large enough to overwechanisms and cause life-threatening infections, although these are relatively uncommon (Sheldon, and Dobson 2004).

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31 first wn et he h riffin et al. (1974) found a direct correlation between Corynebacterium pyogenes infection and degree of endometritis. However, during the early stages of Corynebacterium pyogenes infections, endometritis usually was classified as mild or moderate, and if the infection persisted for more than a week, the degree of endometritis changed to severe (Griffin, 1974). In most instances, the infection is eliminated within 3 weeks post partum. However, in cows that are unable to clear the infection, their reproductive performance was compromised (Griffin et al, 1974). Consequently, endometritis is a normal process of uterine involution of the uterus include a high proportion of cows that are spontaneously resolving the bacterial uterine infection (Sheldon et al., 2006). Figure 2-8 represents the incidence and degree of endometritis in uterine biopsy samples during the first seven weeks post partum. Histopathological disease definition Endometritis consists for the most part of a diffuse but light infiltration of inflammatory cells with slight desquamation of the superficial epithelium without significant vascular changes and with minimal involvement of the uterine glands. The significance of leukocytes found in the stroma is equivocal in cattle 2 to 3 days after parturition ( Jubb and Kennedy 1992). eek post partum 90 % of cows experience some degree of endometritis (Olsoal., 1986). Inflammation of the uterus (endometrium) and degree of this process is correlated with the type of bacteria cultured (Studer and Morrow, 1978). Bacteria in tuterus such as Coliforms, Streptococus, and Arcanobater are associated highly witendometrial inflammation (Studer and Morrow, 1978). G and classifying a cow as having clinical endometritis less than 21 days post partum will

PAGE 42

32 0%10%40%50%60%70%2128354249Days Postpartumecd cws%) 20%30%1 78 1415 22 29 36 43 Afteo( Figure 2-8. Overall incidence and degree of endometritis in uterine biopsy samples stratum compactum; Moderate (): medium infiltration in stratum compactum and in theand in stratum spongiosum; and no-endometritis (). (Griffin et al., 1974) The best indication of endometritis in all species consists of the accumulatio during the first seven weeks post partum. Mild (): light neutrophils infiltration in upper part of stratum spongiosum; Severe (): dense infiltration in stratum compactum n of e stroma ( Jubb and Kennedy 1992). Changes in the de but dy 1992). plasma cells and lymphocytic foci in th gree of inflammation depends on the duration and severity of inflammationgenerally consists of fibrosis in which leukocytes, lymphocytes and plasma cells predominate. The endometrium can become thickened by inflammatory tissue where endometrial glands may become atrophic, flattened, attenuated, or cystic, due to the periglandular fibrosis ( Jubb and Kenne

PAGE 43

33 arge on reproerized amination in the uterus. Mateus et al. 003) using cows that were within 6 weeks post partum, evaluated the bacteriological Clinical disease definition Studer and Morrow (1978) diagnosed endometritis using uterine biopsy and found a positive correlation with rectal palpation findings and uterine discharge characteristics. Consequently, they suggested that use of rectal palpation can be utilized to identify cows with endometritis (Studer and Morrow, 1978). Sheldon et al. (2006), defined clinical endometritis as those cows with a purulent uterine discharge detectable in the vagina 21 days or more post partum, or mucuopurulent discharge detectable in the vagina after 26 days post partum. This definition was based on the finding from a study conducted by LeBlanc et al. (2002), who reported that presence of purulent vaginal mucus or a cervical diameter >7.5 cm 21 days or more post partum; had a negative effect on reproductive performance. These results agree with those of Oltenacu et al,. (1983), who concluded that the diameter of the cervix estimated by rectal palpation 12 to 26 days post partum (i.e., diameter > 5.5 cm primiparous and 6.0 cmmultiparous) was the best indicator of subsequent poor reproductive performance. However, Oltenacu et al,. (1983) did not find a direct effect of uterine disch ductive performance. Puerperal Metritis Pathogenesis Puerperal metritis has been described as a life-threatening infection, charactby a fetid vulvar discharge that may be associated with clostridial infections (Roberts,1986). The presence of a fetid discharge appears to be an unequivocal sign of uterineinfections which reflects the level of bacterial cont (2

PAGE 44

34 content of the uterus, uterine horn size and fluid content by ultrasonography. In this study (Mate thermore, uteri that contained fetid purul d e tum, e respectively. These results agreed with Hirvonen et al. with a fetid discharge showed higher bacterial growth of E. coli s compared to cows without a purulent discharge. Furthermore, if the cows us et al,. 2003) uterine discharge by vaginoscopy was classified as normal lochia mild endometritis (purulent lochia), and severe endometritis (heavy, fetid purulent lochia)associated with or without systemic symptoms. Results indicated that uterine involution of cows with a fetid discharge was delayed. Fur ent lochia present at examination had a greater horn diameter (i.e,. at 3 to 4 weeks postpartum) and a greater amount of uterine fluid (i.e., at 2 to 6 weeks postpartum) than the normal puerperium group, respectively (Mateus et al,. 2002). No differences in uterine involution were observed between the group classified as normal and the group classified as purulent lochia. In addition, the bacterial cultures showed that Arcanobacterpyogenes, E. coli, Fusobacterium sp. and Bacteroides sp. were more frequently isolatefrom cows with mild endometritis or severe endometritis than in cows without these conditions. In cows with a normal puerperium A. pyogenes was isolated in 74 % of thcases, and gram negative anaerobes only occurred trough the second week post parwhereas cows with mild or severe endometritis, these response were evident until thfourth to 6 week post partum (1999) in which cows and A. pyogene showed systemic signs then Bacteroides sp. and Fusobacterium necrophorum also were isolated. In addition to the previous results (Mateus et al., 2003; Hirvonen et al., 1999), Dohmen et al,. (2000) showed that cows experiencing dystocia or retained fetalmembranes with a fetid discharge showed a high growth rate of E. coli, black pigmented

PAGE 45

35 Ganaerobes and Clostridium spp, from uterine cultures, compared to cows that calved without dystocia or retained fetal membranes and did not have a fetid vulvar discharge. General signs of toxemia can be found in cows with puerperal metritis (Rebhun et al., 1995). Endotoxins or lipopolysaccharides (LPS) are among the most important virulent factors of coliform bacteria. Lipopolysaccharides are somatic antigens of bacomposed of polysaccharides, phospholipids and a small amount of protein (Lohuis et al., 1988). High LPS concentrations in lochia were found to be positively correlated with the presence of a fetid discharge and presence of E. coli bacteria (Dohmen et al., 20However, LPS concentration in the uterus was not correlated with LPS concentrations inblood. In contrast, Mateus et al. (2003) positively correlated the presence of a fetid uterine discharge with LPS concentration in blood. Furthermore, Peter et al. (1990) reported increments of LPS concentrations in blood after intrauterine infusion of endotoxins in postpartum cows and suggested that LPS are absorbed from the uterus. Mechanisms by which LPS are absorbed from the uterus were hypothesized as: direct absorption from the uterus, passive diffusion and/or transmural leakage, or escape through the oviducts and fimbria into the peritoneal cavity (Peter et al., 1990). In addition, Peter et al. (1990) demonstrated that absorption of LPS by the uterus decreaseas days postpartum increased. Intrauterine infusion of LPS in cows 20 days post partudid not show an incremental increase in LPS concentration in blood compared to cows infused at 5 days post partum (Peter et al., 1990). Signs of endotoxemia include depression, respiratory distress, vasomotor disturbance, shock, fever, sometimes followed by hypothermia, disturbance of cteria 00). d m gastrointestinal tract motility and metabolic disturbances (Lohuis et al ., 1988).

PAGE 46

36 Immune cells detect bacterial components such as LPS and peptidoglycan via toll-like receptors that are present in macrophages (TLR-4) (Beuttler et al., 2003) to stimulat e produ nly er (RT ot 92). ttler et ion between level of haptoglobin in blood toglobin ral ction of the cytokines (i.e., TNF, IL-1, IL-6, IL-8). These cytokines act as internal pyrogens to increase core body temperature. Dohmen et al. (2000), found a positive correlation between a fetid uterine discharge, LPS and rectal temperature. Cows with a fetid uterine discharge had higher rectal temperatures (mean = 39.3C). However, fever is not associated always with this type of uterine discharge. Hirvonen et al. (1999) found that only 8 (42%) of 19 cows that were diagnosed with puerperal metritis (acute puerperal metritis, putrid vulvar discharge) between 4 to 11 days postpartum, developed systemic clinical signs of fever (39.5C 41.0C) and poor appetite during the acutephase of the infection. These results agree with those of Pugh et al (1994), in which o42.3% 78 cases of puerperal metritis evaluated within 14 days post partum had fev> 39.4C) at diagnosis. However, in this later study the type of uterine discharge was ndescribed for cows with puerperal metritis. Tissue injury and inflammation induce the release of interleukin-6 (Hirano 19This interleukin is one of the cytokines involved in the development of fever (Beual., 2003), as well as inducing the synthesis of acute phase proteins such as haptoglobin and alpha-1-acid glycoprotein by hepatocytes (MacKay and Lester 1992). Systemic responses that result in fever also increase the levels of acute phase proteins. However, Smith et al. (1998), reported no significant correlat with rectal temperature in cases of puerperal metritis (foul smelling vulvardischarge with a rectal temperature > 102.5). Nevertheless, concentration of hapin cows with puerperal metritis were between 13 to 20mg/dl on the day of puerpe

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37 metri 21 t ucosa, submucosa, musc al. tis diagnosis, which agrees with the ranges (>10 mg/dl) reported by Skinner et al. (1991) in cows with puerperal metritis (fouls smelling vulvar discharge, with or without fever). Williams et al. (2005) compared levels of alpha-1-acid glycoprotein in cowshaving purulent fetid discharge with cows having a purulent but no fetid discharge ator 28 days postpartum. Cows with a fetid uterine discharge had higher concentrations of alpha-1-acid glycoprotein (1.5 mg/dl) than those cows without a fetid discharge (1.03 mg/dl). However, the levels of alpha-1-acid glycoprotein found by Williams et al. (2005)during days 21 or 28 post partum were within the normal levels (1.21.4 mg/dl ) in normal cows within 10 days post partum reported by Sheldon et al. (2001). In contrast, Sheldon et al. (2004), found that cows with one or more events of fever during the firs10 days post partum had higher levels of alpha-1-acid glycoprotein than cows without fever during the same time period. Histopathological disease definition Puerperal metritis is defined as the inflammation of the m ular and the serosal layers of the uterus. It is described as a purulent inflammation, where the sub-serosal connective tissues are edematous and infiltrated with leukocytes, with the same process observed surrounding blood vessels of the myometrum that extendto muscle fibers which undergo granular degeneration. The leukocyte mass on the mucosal surface is associated with extensive hemorrhage, necrosis, and sloughing ( Jubbet al. 1992) induced by bacteria toxins produced by bacteria. The hemorrhage and necrosis, along with bacterial products characterize the clinical findings ( Jubb, et 1992).

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38 The histopathological definition of puerperal metritis is straight forward (Sheldonet al., 2006). However, this is not a common practice because of logistical reasons and the sampling per-se posses a risk to the health of the animal more than the disease its(Etherington et al., 1988). Consequently, as well as in other diseases, a clinical definitionis needed to diagnose uterine infections. These definitions should characterize clinical findings to conclude whether or not the disease is life threatening and weather treatment should be applied. Clinical Disease Definition Highly pathogenic types of bacteria are present in the uterus and along with their toxins are absorbed into the circulation producing symptoms associated with septicemia,endotoxemia, and pyemia (Roberts, 1986). Diagnosis of puerperal metritis has been performed by rectal palpation of the elf uterus (Markusfeld, 1984; Pugh et al., 1994; Risco, noscopy has also been used (Hirvonen et al., 1999) um ity are ia, ss, and metabolic disorders. and Hernandez, 2003). However, vagig Based on clinical experience Rebhun (1995) described puerperal metritis (septic ortoxic metritis) as those cows with a fetid watery uterine discharge from the vulva that varied in color from brown, amber to gray or red, but fluid are low in mucus content and contained purulent material. Cows become ill within the first 7 to 10 days post partand had fever (40.0C to 41.39C), tachycardia, inappetence, decreased production, rumen stasis, and toxemia Dehydration, diarrhea, and depression of varying severalso observed. Extremely severe infection may cause recumbency secondary to toxemweakne

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39 Liters s roduction, and disease incidence. scribed the epidemiology of metritis and estimated the econo 0 t partum was found, that varies from 3 to 45%. In addition, metritits was most commthe as 22 to 49 days postpartum. Late metritis included ulvitis, vulvovaginitis, endometritis, vaginal discharge, metritis or pyometritis diagnosed ature disease definitions Terminology used to classify uterine infections, specially for puerperal metritis habeen vague and inconsistent (Lewis 1997). Many studies have been published that describe the epidemiology of puerperal metritis, its effect on reproduction performance and milk production, and treatment and prevention of this disease. However, differencein disease definition contributed to conflicting results on effect on reproductive performance, milk p Bartlett et al. (1986) de mic impact of metritis in Michigan Holstein-Friesian dairy cows. Metritis was defined according to the thickness of the uterine wall and the fluid content of the uterine cavity in relation to the number of days post partum. The study involved 22 herds andinformation about disease was recovered from a dairy herd health computer network. In over 3773 lactations were studied with an lactational incidence of 18% within 10 to 3days pos only diagnosed between 11 to 20 days post partum. After including the effect on reproductive efficiency, milk production, cost of medication and losses due to culling, total cost estimate was $106.00 for a lactation with metritis. Beaudeau et al. (1995) assessed the effect of health disorders on length of reproductive life in 47 French Holstein commercial dairies during 4 years. Metritis wone of the explanatory variables and was further divided as early metritis and late metritis. Early metritis included vulvitis, vuvlvovaginitis, endometritis, vaginal discharge,metritis or pyometritis diagnosed from v

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40 beyond 50 days post partum. Th e overall incidence for early metritis was 6.4%, however the in g rate. 60 Danish dairy cows.y, n as reported within 14 days postps with cidence ranged form 5.2 % when calculated in cows with less than 90 days postpartum or 8.1 % if calculated in cows with more than 210 days postpartum. Theoverall incidence of late metritis was 5.6 % and ranged from 1.4 % in cows with less than90 day postpartum and 14.1 % in cows with more than 210 days in milk. It was found that cows with late metritis or early abortion had poor survival, thus higher cullinBruun et al. (2002) identified risk factors for metritis in 102,0 Information was recovered from the Danish cattle database. Metritis was not defined. However, diagnoses were made within 1 to 30 days post partum In this studthe incidence risk ranged from 1 to 21 % in 391 herds observed. Callahan and Horstman (1987) reported a retrospective analysis of treatment alternative in dairy cows affected with postpartum metritis in the Purdue University Dairycenter. The criteria for diagnosis of metritis consisted of ballottement of uterine fluid, possible crepitant feel of the uterine content, lack of myometrial tone, retarded involutioand the presence of abnormal discharge. Discharge characteristics ranged from thin, watery and fetid to purulent or mucopurulent. The study was conducted in a 5 year period and 1108 lactations were evaluated. An incidence of 33.8 % w artum. In this study no effect on reproduction performance was found in cowpuerperal metritis. Furthermore, they stated that the early diagnosis of metritis may reduce the impact on the reproductive performance. Chenault et al. (2004) evaluated the efficacy of ceftioufur hydrochloride for the treatment of postpartum metritis. Metritis cases were defined as cows with a rectal temperature 39.5C, with a fetid vaginal discharge that was red or pink to chocolate

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41 brown in color and serous with or without pieces of necrotic tissue, within 1 to 14 days post partum. Given that only metritis cows were reported as an outcome, incidencnot reported. However they concluded that ceftiofur hydrochloride administered at a dosage of 2.2 mg of CE/kg, subcutaneously or intramuscularly once daily for 5 days was efficacious for treatment of acute puerperal metritis in dairy cows. Cobo-Abreu et al. (1979) observed the association between disease, production and es were cullin fined. s related to the calf. Data g in a Holstein dairy herd in Ontario, Canada. This study was conducted duringseven years. Metritis definition, incidence and days in milk to diagnosis were not deCorrea et al. (1993) modeled a path analysis with logistic regression for seven postpartum clinical diseases in cows and also observed the factor were from 7761 lactations from 34 commercial dairy herds close to Cornell University. Metritis was defined as an enlarged uterus found at rectal palpation in cows with or without other clinical signs within 30 days post partum. However a metritis event included cases of endometritis, pyometras and metritis as defined above. A lactational incidence of 7.2 % was observed. It was found that stillbirth increased the odds of developing metritis and retained placenta, cows that twinned had increased odds of developing dystocia and retained placenta. Dystocia was related to an increase in the oddsof retained placenta. Milk fever, dystocia, and ketosis each increased the odds of developing left-displaced abomasum. Postpartum periods with dystocia, retained placenta, or ketosis had increased odds of metritis. Curtis et al. (1985) used path analysis and logistic regression to model direct andindirect relationships among clinical periparturient (within 30 days after calving) diseases. Data were obtained from 1374 lactations of multiparous Holstein cows in 31

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42 commercial herds near the Cornell University area during a period of one year. The definition of metritis was not given. However, an incidence of 7.8 % was reported, within 30 days post partum. Retained placenta, left displaced abomasum, and parturient paredirectly increased risk of complicated ketosis. The study suggested that feeding higher intakes (relative to National Research Council recommendations) o sis f protein and energy in thetreatmtritis tal of 500 illin on 82.9, Reproductive performance did not differ atment last 3 week of the dry period may reduce the incidence of metabolic and reproductive disorders. Drillich et al. (2001) evaluated the efficacy and financial viability of systemic ent of toxic puerperal metritis in dairy cows with ceftiofur. Toxic puerperal mewas defined as the presence of a fetid, reddish-brown vaginal discharge and a rectal temperature 39.5C, within 4 to 6 days postpartum. During the study period, a to1756 calvings were observed and an incidence of 18.5 % was reported. There were no significant differences among the treatment groups regarding clinical efficacy at d 6 after first treatment (group 1 received 600 mg of ceftiofur intramuscularly on 3 consecutive days; group 2 received an intrauterine treatment with antibiotic pills consisting of 2mg of ampicillin and 2500 mg of cloxacillin and an additional 6000 mg IM of ampicon 3 consecutive days and group 3 received the same intrauterine treatment as in group 2, in addition to 600 mg of ceftiofur IM on 3 consecutive days. The cure rates basedrectal temperatures declining to below 39.5 degrees C on d 6 after treatment were 84.8, and 84.6% for groups 1, 2, and 3, respectively. significantly between group 1 and groups 2 and 3 for any of the measures tested. Afinancial analysis with 87 different cost scenarios demonstrated that a systemic tre

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43 of toxic puerperal metritis in cattle with ceftiofur is an effective alternative to the combination of local and systemic treatments. Etherington et al. (1985) used a path analysis to determine the interrelationship between ambient temperature, age at calving, postpartum reproductive events and reproductive performance in dairy cows. Within the reproductive event metritis was included. A cow was considered to have metritis if she exhibited decreased milkproduction, decreased fee dd intake, pyrexia and had foul smelling vaginal discharge. An incideted. e nce of 23% was reported. However days in milk of the diagnosis was not reporIn this study there was an increase in the incidence of retained placenta, in the percentage of cows with abnormal vaginal discharge in the early postpartum period as well as a delay in uterine involution during the winter months. In addition, cows calving during thwinter had prolonged intervals to first estrus, first service and conception compared to cows calving during the summer. Cows calving during the warmest months, on average, were seen in estrus 24 days sooner, received first service 42 days sooner and conceived27 days sooner than cows calving during the coldest months of the year. Erb and Martin (1978), studied age, breed and seasonal patterns in the occurrence of ten diseases on which metritis was one of them. In this study, information of the disease was retrieved from a University central data base. The definition of metritis included endometritis, metritis and pyometra. However, none of these diseases were defined. An incidence of 14 % was observed. No description of the time frame of the diagnoses was made. Using the log-odds method, trends were noted for the youngest cows to be at increased risk of the reproductive diseases such metritis and for the

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44 Guernsey cows to be at increased risk of the uterine diseases. There was a tendencpeaks in disease occurrences in the winter (as o y for pposed to summer) months. e o fers that 76 eeding perfoto ). itis ws, parturition in the spring or summer and being of parity 2 or 3-4 (vs older) increased the Erb et al. (1985) observed the relationship between occurrence of metritis with other diseases. A total of 2960 lactations of Holstein dairy cows were included in thstudy. Metritis was not defined. However, the incidence was reported to be a 9.9 %. Ndescription of the time frame of the diagnoses was reported. It was shown that heiwere older, of lighter weight, or who had lower estimated transmitting ability for milk had more problems, less milk, and poorer survival. Dystocia in heifers had several detrimental consequences including 2.9 to 4 times more retained placenta, metritis, and culling and +7.4 d more to first service. Cystic ovaries were associated directly with 3kg greater milk yield and with a 16.5-d delay in first service. Failure to conceive at first service and mastitis increased risk of culling 5.2 to 10 times. In multiparous cows, milkfever increased risk of reproductive disorders by 1.6 to 4.2 times and indirectly contributed to poor breeding performance and increased culling. Risk of culling was increased 2.1 to 3.7 times directly by mastitis and dystocia and by poor br rmance. Harman et al. (1996) quantified the effect of season of parturition, parity, and diseases on time to conception in 44450 Holstein dairy cows. Metritis was divided inearly (less than 42 days postpartum) and late metritis (more than 42 days post partumHowever no definition of disease was given. The lactational incidences were 2.0% forearly with median day to diagnosis of 18 days after parturition, and 1.1% for late metrwith median day to the diagnosis of 108 days after parturition. For multiparous co

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45 chance of conceiving; 10 diseases or disorders decreased this probability. In primiparousparturition in spring or summer increased the probability of conception, and 6 disorders decre, g y, ted that r creased rtum metritis, the response pattern being less prominent than that for haptority ased it. Disorders that were found to be detrimental in both models were anestrusovulatory dysfunction, other infertility, late metritis, and clinical ketosis. Hirvonen et al. (1999) examined the role of systemic acute phase proteins regardindiagnostic values of Haptoglobin, alpha1acid glycoprotein, and plasma N-acetyl-beta-D-glucosaminidase activity in clinical and bacteriologically defined acute postpartum metritis in dairy cows. Acute metritis was defined as a putrid, reddish-brown, waterfoul smelling vaginal discharge, within 4 to 11 days post partum. No incidence were reported given that puerperal metritis was the principal outcome. Results showed that plasma haptoglobin concentration remained low in most cows with acute postpartum metritis. Only the 3 most severely affected cows exhibited a strong haptoglobin responseThese were later culled due to poor condition and reduced fertility. It was suggesin acute uterine infection a highly increased haptoglobin concentration indicated a pooprognosis for repeat conception. Plasma alpha1-acid glycoprotein concentration inin acute postpa globin. The alpha1-acid glycoprotein concentrations did not correlate with seveof disease, and, consequently, the capacity of alpha1-acid glycoprotein in differentiatinggenital infections was relatively poor. Highest alpha1-acid glycoprotein concentrations were detected in cows with retained placenta and/or dystocia. In addition, plasma N-acetyl-beta-D-glucosaminidase activity levels did not differ between the cows with acute postpartum metritis and healthy control cows.

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46 Lee et al. (1989) described the use of survival analysis to quantify the days open for different diseases during the early post partum period. Metritis was used as an explanatory variable and this disease was further divided into metritis, non systemic andsystemic. However incidences and the definition of metritis and day in milk to diagno sis was n as n d tion cows were measured at wk 1 postpartum. Sire, herd, age, height, season, and BW contributed to peak milk yield. ot provided. It was found that retained placenta, nonsystemic metritis, systemic metritis, ovarian cysts, and lameness were associated with a decrease in conception rate and an increase in median days open. The hazard ratios for conception were .66, .83, .70, .70, and .69 and the increase in median days open 5, 15, 13, 22, and 28 d for the five diseases, respectively. Markusfeld, O (1984) observed the factors associated with retained fetal membranes and postpartum metritis in 2017 Holstein dairy cows. Metritis was definedany purulent foul smelling discharge. The incidence observed was 37.3 % and ranged between 31.2 to 43.8 %. The diagnosis were made within 14 days post partum. In this study risk factors associated with metritis include declining parity, long gestations, induction of parturition, stillbirth, multiple births, low milk yield before drying off, left displacement of the abomasum, ketosis and winter calvings. Markusfeld and Ezra (1993) observed the effect of herd, sire, season, body height, body weight, age at calving, and metritis on performance of first lactation cows. Metritis was defined as described in Markusfeld (1987). Of a total of 621 first lactation heifers, aincidence of 48.6 % was reported, within 5 to 12 days postpartum. This study reportethat short, heavy first lactation cows had an odds ratio of 3.1 of incidence of metritis at calving compared with all others; 648 first lacta

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47 Metrimetritis. e om first AI, independent of milk yield. The relative importance of heigheen e discharge, without fever (>39. meter. n. tis did not affect peak yield. Herd, sire, height, and age contributed to mature equivalent corrected 305-d milk yield. No effect was found for BW, season, or Herd was the only variable contributing to month of peak yield and rate of monthly drop in yield. Interactions between BW, height, and incidence of metritis were significant. Tall, heavy first lactation cows with metritis peaked higher and yielded more than thoswithout metritis. Short, light first lactation cows with metritis yielded less and peaked lower than their healthy counterparts. Metritis did not affect future fertility, but season and the interaction between BW and height did. Tall, heavy first lactation cows had a lower pregnancy rate fr t as a predictor of future milk yield is underestimated. The interaction betwheight and BW may have an antagonistic effect on yield and fertility. Melendez et al,. (2004) evaluated the effect of 2 doses of PGF 2 injected early postpartum on uterine involution, serum concentration of alpha1acid glycoprotein andfertility in Holstein cows with acute puerperal metritis. Acute puerperal metritis wasdiagnosed by per rectum palpation of the uterus at 8 d post partum. Criteria for diagnosiswas an enlarged and flaccid uterus with a foul-smelling uterin 5C). During the study period, 1536 cows calved; an incidence of 15.3% was reported. However only cows diagnosed with retained fetal membranes and metritis were included and those cows with metritis and systemic signs were excluded. In this study postpartum, primiparous, treated cows had smaller uterine diameters and lower uterine scores than controls. Cows with a uterine diameter <5.1 cm at 12 d postpartum were 5.5times more likely to conceive at first service than cows with larger uterine horn diaTreatment significantly reduced the concentrations of serum alpha1-acid glycoprotei

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48 Within primiparous cows, treatment also increased conception at first service by 17%was concluded that 2 doses of PGF It 10 calvings obserigh y al2 8 h apart at 8 d postpartum in primiparous cows with acute puerperal metritis decreased the diameter of uterine horns and serum concentration of alpha1-acid glycoprotein at 12 d postpartum and increased the conception rate at first service. Overton et al. (2003) studied the effect of a prophylactic treatment with estradiolcypionate (ECP) in cows at a high risk to develop metritis. Metritis was defined as any combination of fever 39.7C with a watery and or fetid vulvar discharge within 1 to days postpartum. Metritis were further divided into mild or severe. Metritis was mild when the cow never had a rectal temperature 39.7C and severe when the rectal temperature was 39.7C. An incidence of 10% was observed in 1284 ved. They concluded that prophylactic administration of ECP to dairy cows at hrisk for metritis did not reduce risk for metritis. Pugh et al. (1994) described 78 cases of postpartum metritis in dairy cows. Forttwo and 36 cases of postpartum metritis were recovered from records of the large animal hospital from Aurburn University and Tuskegee University, respectively. The definition was not reported. However, it was stated that the diagnoses were based on the vaginuterine discharge obtained by rectal palpation of the uterus. Older cows and those with hyperthermia were less likely to recover from puerperal metritis. Furthermore, only 42% of the treated cows were with hyperthermia at the moment of the physical exam. Rajala and Grhn (1998) evaluated the effects of dystocia, retained placenta, and metritis on milk yield using repeated, monthly test day milk yields, on 37,776 Finnish Ayrshire dairy cows in 2337 herds, recovered from the national Finnish health recording

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49 system. Metritis was not defined, however diagnosis was further divided in early and latemetritis. The lactational incidence of early metritis ranged from 1.6 to 2.6 % within 28days post partum and the incidence of late metritis, ranged from 1.2 to 1.5 % aftedays postpartum. Dystocia, retained placenta, and early metritis significantly affmilk yield, as indicated by mont r 28 ected hly test day milk yields. Late metritis was not associated with mrst 30 ortion of cows that developed metritis was s n of ceftio. ilk loss. The impact of the diseases differed across parities and also across different levels of milk yield. Using 305-d milk yield as the milk measure, no diseases were associated with reduced milk yield. Risco and Hernandez (2003) compared the administration of ceftiofur hydrochloride and ECP on the prevention of puerperal metritis. In this study the definition of metritis was not stated. However, diagnoses were made within the fidays post partum. Incidences were not reported given that cows with metritis were the experimental units. Results showed that the prop ignificantly different in cows treated with ceftiofur hydrochloride (13%), comparedwith cows treated with ECP (42%) or cows that received no treatment (42%). Uterine involution patterns (i.e. median time to complete retraction of the uterus and mean diameter measure of cervix and uterine horns) were not significantly different between groups. Cows treated with ECP were 0.40 times as likely to conceive as control cows (P=0.05); median time to conception in cows treated with ECP (192 days) was longer, compared to control cows (124 days). It was concluded that systemic administratio ufur hydrochloride is beneficial for prevention of metritis, but its effect on reproductive performance was not significantly different to that of ECP or no treatment

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50 In addition, administration of ECP did not have beneficial effects on metritis prevention and reproductive performance. Schnier et al. (2002) compared the incidence of diseases in 5000 Finland dairy cows kept in cold or loose-housing systems. Information was recovered from the Finishealth data recording system. Metritis included cases of acute and chronic metritis, pyometra, vaginitis and disturbed involution within 0 to 44 days post partum. Hownone of these conditions were defined. The overall incidence for both housing system was 3.3 %. They found that cows in a cold h ever loose-housing system were at lower odds for develof the same loping peral on ups efficacy among antibiotics used to treat cows affected with toxic puerperal metritis. oping late mastitis (15-305 days in milk), and metritis (Friesian breed); odds for ketosis and early mastitis (0-14 days in milk); but at higher odds for deveparturient paresis and metritis (Ayrshire breed). Smith et al. (1998) compared procaine penicillin, intrauterine infusion of oxytetracycline or ceftiofur in dairy cows for the treatment of toxic postpartum metritis.Toxic postpartum metritis was defined as any cow with a rectal temperature >39.2C, a flaccid, non retractable uterus that was located in the abdomen, a cervical diameter >75mm, and a watery, fetid vulvar discharge. Other criteria used to diagnose toxic puermetritis was depressed milk yield (<7.4 kg at the morning milking), within 3 to 20 DIMGiven that only cows with puerperal metritis were used no incidence was reported. Nodifference was observed among groups for milk yield on d 1 and 12 or for temperature d 1 and 5. Serum haptoglobin was elevated to > 10 mg/dl for cows in all groups; however, no difference was observed among groups on d 1 and 5. Because all groshowed a favorable response, this study suggests that there is no difference in treatment

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51 Urton et al. (2005) related cows with high or low feeding behavior during the prepartum transition phase with increased risk of developing metritis after calving. Two metriarge nosed with metritis. A threshold of 75 min otion of as or s tis classification were used. Animals were classified as having metritis if they showed a mucopurulent and foul smelling and fever (rectal temperature > 39.5C) or acutely metritis if they showed a reddish brown, watery, foul smelling vaginal dischand fever. The incidence of metritis ranged between 38% to 27% for metritis and acute metritis respectively, within 3 to 15 days post partum, however the total calvings were not reported. Cows suffering from metritis, exhibit reduced milk yield and reproductive performance. These cows spent on average 22 min/d less time at the feed alley during the transition period than did non-metritic cows. For every 10-min decrease in average daily feeding time, cows were twice as likely to be diag f average daily feeding time was 89% sensitive and 62% specific for detecacute metritis. It was concluded that reduced time at the feeder can be used to identify dairy cows at risk for metritis. Current research definition Given the various definitions used in the literature to describe uterine infections, Sheldon et al. (2006) proposed a series of definitions. In this review, a distinction between puerperal metritis and clinical metritis was made. Puerperal metritis is defined those cows with an abnormally enlarged uterus and a fetid watery red-brown uterine discharge associated with signs of systemic illness (decreased milk yield, dullnessother signs of toxemia) and fever >39.5 C within 21 days post partum. In addition, clinical metritis was defined as those cows that do not appear sick, but had an abnormallyenlarged uterus and a purulent uterine discharge present in the vagina, within 21 day

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52 after pns es and metabolic conditions increased the likelihood s. However, in some cases puerperal metritis was classified as a d ds arturition. The definitions proposed by Sheldon et al. (2006) were based from previous studies related to metritis treatments (Drillich, et al., 2001), pyrexia in postpartum cows (Sheldon et al., 2004), and risk factors for puerperal metritis (Markusfeld, 1984). However, none of the cited studies used to define uterine infectiorelated clinical signs to histopathology and possible pathophyisiology of this disease. Furthermore, they did not include any controlled studies that may relate clinical signs with risk factors to systemic illness or impairment of cow performance. Risk Factors for Puerperal Metritis Puerperal metritis is often associated with retained fetal membranes (RFM), dystocia, stillbirth or twins and usually occurs during the first two weeks post partum (Olson et al., 1986). However, puerperal metritis may also occur in cows without calvingrelated disorders (Olson et al., 1986). Studies using path analysis and risk assessment have consistently indicated that dystocia, retained fetal membran that a cow will develop metriti isease complex without distinguishing the clinical presentation or severity, makingcomparison between studies difficult (Lewis, 1997). Many of these studies used odratios (OR) as a measurement of risk factor, but relative risk (RR) another measurement of association, also has been used. Retain fetal membranes Retained fetal membrane (RFM) is a major predisposing cause of metritis. Thethird stage of parturition involves the expulsion of the fetal membranes, and is completed within 8 hours after parturition. A persistence of the third stage of parturition, that is,

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53 failure to expel the fetal membranes is considered abnormal (Roberts., 1986). Fetal membranes are considered retained when the fetal cotyledonary villi fail to separthe crypts of the maternal caruncles within 12 to 24 hours of parturition (Rober1986) and mechanisms why this process fails has been described (Gunnink 1984; Kimuraet al., 2002). The condition of retained placenta occurs in 4 to 18% of calvings (Markusfeld, 1985; Esslemont and Kossainbati,1996; Erb, et al., 1985). Various risk factors for the ate from ts S. J, rted. Calving problems including dysto be risk factors for RFM. Dohmen et al. (2000), repord is one of the major factor predisposing cattle to 6; Correa et al., 1993). Several studies have related the incideErb development of retained placenta have been repo cia (OR = 4.0) (Correa, et al., 1993; Markusfeld O. 1984; Erb, et al., 1985), stillbirths (Correa, et al., 1993; Markusfeld, 1984), and multiple births (Correa, et al., 1993; Markusfeld, 1984), parturient paresis, low prepartum protein and age of cows (Curtis et al., 1985) have been found to ted that immediately after calving, RFM cows had high concentrations of LPS/endotoxin in lochia and were more often infected with E. coli, Clostridium spp. anGanaerobes (prevalence rates up to 97%) bacteria than cows without periparturient disorders. The principal deleterious effect of retained fetal membranes on the dairy cow isimpaired fertility by delaying involution of the uterus, thereby facilitating the development of uterine infections (Sandals et al., 1979). Retention of fetal membranes metritis (Bartlett et al., 198 nce of metritis with RFM. Sandals et al. (1979) reported an incidence of 54.8 percent of metritis following retained fetal membranes. Erb et al. (l985) reported an important biological causal association between RFM and metritis. It was reported by

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54 et al. (l981) that cows with RFM were almost six times more likely to developed mecompared to cows without RFM. Various studies have associated RFM and metritis OR = 4.4 (Grhn et al., 1990);OR = 6.0 (Correa et al., 1993); OR = 2.5 (Bruun et al., 2002); OR = 5.8 (Erb et al., 1985). Stillbirth, mu tritis ltiple birth and dystocia give 6). The ry ct that e had dystocia using path analyis found Stillbirth, multiple birth and dystocia are events that occur at parturition and are risk factors for metritis and are related to one another. When the first, or the second stages of parturition are prolonged, it becomes difficult or impossible for the dam tobirth without artificial aid and then this condition is termed dystocia (Roberts, 198incidence of dystocia ranges from 6% to 25% (Roberts, 1986; Adamec et al., 2006). Prepartum dietary energy and parturient paresis have direct effects leading to veterinaassisted dystocia (Curtis et al., 1985). However, prepartum energy does not seem to affethe incidence of dystocia (Markusfeld, 1985). Dystocia is greater in pregnanciesterminate early due to uterine disease, fetal death, and twinning (Roberts, 1986). Dystociacan increase the risk of trauma to the uterine wall and thereby increase the odds of metritis (Bruun et al., 2002). In addition, assistance during calving may increase the risk of uterine contamination (Bruun et al., 2002). Erb et al. (1985) differentiated thincidence of metritis by parity and whether on not the cow sis. For primiparous and multiparous cows, dystocia was a risk factor for metrit(OR=3.0; OR = 3.5; respectively). Dohoo et al. (1984), and Curtis et al (1985), alsoa positive association between dystocia and metritis (OR = 2.5) and (OR = 4.9), respectively. In the study reported by Curtis et al. (1985), dystocia were only those calvings assisted by a veterinarian. However, this assistance was not a risk for RFM.

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55 These results are in agreement with studies by Correa et al. ( 1993) who reported that cows requiring assistance at calvin g were 2.1 (OR) times more likely to develop metritis. son, 1996). Martinez et al. (1983) reported that the st ws es been e of en o Stillbirth is the expulsion of a dead fetus at parturition (Roberts, 1986). Calving difficulty has been implicated as the major cause of stillbirth, yet about 50% of stillborn calves are from unassisted births (Philips illbirth rate in U.S. Holsteins is around 10.5% for first lactations, 5.5% for secondlactation and 5.7% for third lactation. Markusfeld (1984), reported that heifers and cowhich gave birth to dead calf had a higher rate of RFM or to develop metritis compared to those which gave birth to live calves (OR = 2.19) These results agree with those of Correa et al. (1993), who reported that cows delivering dead calves were 1.5 (OR) timmore likely to develop metritis than those than did not have stillbirth at parturition. In contrast, Emanuelson et al. (1993) found that stillbirths had a direct effect on the risk of retained placenta but not on metritis. When an uniparous animal aborts two or more or gives birth to fetuses are called twins (Roberts, 1986). The incidence of twinning in dairy cattle has increased dramatically over the past two decades (Nielen et al., 1989; Kinsel at al., 1998). Risk factors for twin calvings such as parity, season, herd and previous twinning have described ((Nielen et al., 1989). The incidence of twinning has been reported to rangfrom 1.04% to 9% (Roberts, 1986; Kinsel et al., 1998). Twin calving increased the risk dystocia (OR = 10.5), RFM (OR = 3.4; Correa et al., 1993) and reduces milk production and increased culling rate than single calvings (Nielen et al., 1989). Twinning oftresults in decreased gestation length and increased dystocia and mortality rates. Markusfeld (1987) found that cows delivering twins were 12 times (OR) more likely t

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56 RFM and 2.3 times (OR) more likely to develop metritis than cows without RFM.. Correa et al. (1993), did not find an effect of twins on the incidence of metritis Parit re likely (OR = 1.6) to develop metritis than seconter cows ted ce ave of y The effect of parity on metritis has been reported previously with conflicting results. Grhn et al., 1990 found no association between parity and metritis. However, Markusfeld (1984; 1987) found an association. As parity increased, the incidence of RFMincreased, but the incidence of metritis decreased (Markusfeld, 1984). In contrast, primiparous cows were more likely (OR = 2.7) than second or greater parity cows to develop metritis (Markusfeld, 1984). This finding is in agreement with Erb and Martin(1978), were first lactation cows compared to other lactation cows were more likely (OR = 1.48) to develop puerperal metritis. Bruum et al. (2002) suggested that there is an u-shaped association between parity and metritis. Primiparous cows are mo d-parity cows because damage to the uterus is more common in heifers given the high incidence of dystocia (Bruum et al., 2002). In contrast, third-parity or greawere more likely (OR = 1.58) to develop metritis compared to second-parity cows relato a delay in uterine involution which increases the risk to develop uterine infections ( Bruum et al., 2002). Tendency for an u-shaped relationship between parity and incidenof metritis was also reported by Rajala and Grhn (1998). However, this relationship was not significant. In addition Smith et al. (1998), found a relationship between parity and dystocia. Primiparous cows were more likely (OR = 2.04) than multiparous cows to hdystocia. However, Pugh et al. (1994) did not find any association between casesmetritis and parity.

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57 Season Season has been reported to be a risk factor for metritis (Sandals et al. 197 9; eld, 1984; Erb and Martin, 1985; Etherington et al., 1985). Markusfeld (1984) foundd a un et t. Etherington et al. (1985), described a higher incides s tis ose Markusf that cows that calved in the summer had a greater risk (RR = 1.64) of RFM anlower risk of developing metritis (RR = 0.64) compared to those that calved in winter. Bruun et al., 2002 found that the odds ratio for metritis in cows calving during the cold season were 1.2 times higher than those cows calving during the warm season. Brual. (2002) stated that during the winter months the general health of cows is lowered making them more prone to infections. In a study that involved 3773 lactations, Bartlettet al., 1986 found that metritis was less common during summer months, although their findings were not statistically significan nce of dystocia during the summer months, contrary to the incidence of RFM which was higher during the winter months. Markusfeld O (1984) reported that cowexperiencing dystocia were more likely to develop endometritis, pyometra and RFM wadirectly associated with metritis. Schnier et al. (2002) compared disease incidence of dairy cows kept in cold or warm loosing house, in Ayrshire and Fresian dairy cows. The odds of contracting metriduring the first 44 days in milk of cows calving in a cool loose house compared to cows calving in a warm loose housing during the indoor period were lower for cows of the Fresian compared to the Ayrshire breed, OR = 0.2 and OR = 1, respectively. In additionGrhn et al. (1990) found that cows calving during January April (OR = 1.6) and September December (OR = 1.7) were more likely to develop early metritis than th

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58 calving d uring the month of May-August. A later report found same results (Grhn et al., 2000) ver, ) artum Health Monitoring and displacement of the abomasum (Curtis et al, 1983). These are costly disorders with estimated economic losses ranging from 200 to 400 dollars per case per lactation (Bartlett Hypocalcemia Uterine inertia and a decrease in uterine involution predispose cows to puerperal metritis (Roberts, 1986). Cows with postpartum hypocalcemia have been found to haveincreased incidence of postpartum metritis compared to cows without this condition (Boseberry and Dobson, 1989). Erb and Martin (1985) found that cattle suffering from hypocalcemia were (OR = 4.2) more likely to have a veterinary assisted dystocia, andmore likely (OR = 2.0) to have RFM. Both dystocia and RFM have been found to be predisposing factors for metritis (Erb et al., 1985; Markusfeld, 1987). Curtis et al. (1983)did not find an association between metritis and milk fever, in agreetment with Markusfeld (1987) who found no association between metritis and milk fever. HoweGrhn et al. (1990) found that cows with parturient paresis were more likely (OR = 1.5to develop puerperal metritis compared to cows without parturient paresis. Postp An important concept of a dairy herd health program is early disease diagnosis treatment of sick cows. A delay in treating a sick cow not only reduces her chances for a full recovery, but results in milk production loss that may impair reproductive performance. Because the early postpartum period of the dairy cow determines productive and reproductive responses during lactation, it is a pivotal time in the production cycle of the cow. During this period, dairy cows are at risk of developingcalving related diseases, such as hypocalcemia, puerperal metritis, ketosis and

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59 et al, 1986). Monitoring the postpartum health of dairy cows allows the opportunity to identify sick cows early and provide appropriate therapy. Furthermore, it can help prevent for ). to ctive A postpartum health muation of rectal tempeian who e re relates the diseases. Diagnostic tests are used to classify or confirm a disease process and to provide appropriate treatment. A test is a device or process designed to detect, or quantify a clinical sign, substance, tissue change, or body response in an animal. Tests are used screening and to identify the proportion of diseased or sick animals correctly (sensitivityAfter a positive result is obtained an in depth diagnostic work-up is performed, in ordercorrectly answer whether or not the animal in question is sick or not (specificity). Sensitivity and specificity are the most important characteristic of a test, however they do not directly tell how useful is the test in detecting a disease. Consequently, predivalues are used to estimate the probability that an animal with a positive test result for a particular disease is truly positive (has the disease; positive predictive value) or is truly negative (does not have the disease; negative predicted value). onitoring program consists of eval rature, attitude, milk production, urine for the presence of ketone bodies and characterization of uterine discharge. To some extent these procedures are objective, however the majority are subjective and related to the experience of the technicperforms them. Attitude Attitude is a subjective parameter that describes the anatomical impression of thpatient (Rosenberger, 1979). Attitude and posture are synonyms, although postumainly to the disposition of the limbs, attitude is used as a behavioral indicator. For assessment of attitude, the evaluator has to assess by external visual inspection the

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60 position or relation of the lips, ears, head, neck, forelimbs and tail, in relation to the bodof the animal (Rebhum, 1995). A healthy cow is one that it is aware of her environment and display y s the common curious behavior of cattle. There are some attitudes or postures that ss s with s the severity of lamenonary as k ed-back posture while walking, her gait remains normal; 3) moderately ed-back posture is evident both while standing and walking, her gait is affected and is uggest a specific diagnosis or a specific system disorder (Rebhum, 1995). Changein the shape of the vertebral column and a tense abdomen are characteristics of an abdominal illness such as peritonitis which can be related to different causes like traumatic reticuloperitonitis, as well as septic metritis (Rosenberger, 1979). Holding the tail up usually indicates a painful process in the anus, rectum or genital tract, accompanied by fractious compressions of the abdominal wall (tenesmus). Animalan extended neck and head usually suffer from pharyngeal and esophageal obstruction or from severe respiratory diseases (Rosenberger, 1979). Certain attitudes displayed by cows are grouped and measured in a scoring system to add objectivity to this subjective approach. One of the most common scoring systemsused in the dairy industry is lameness evaluation in order to asse ess in the cow. This scoring system consists in the observation of the statiwell as the walking attitude of the cow (Sprecher, et al., 1997). The scoring system is based on a five point scale were: 1) normal: the cow stands and walks with a level-bacposture, her gait is normal; 2) mildly lame: the cow stands with a level-back posture but develops an arch lame: arch best described as short striding with one or more limbs; 4) lame: an arched-back posture is always evident and gait is best described as one deliberate step at a time, the cow favors one or more limbs/feet and 5) severely lame: the cow additionally

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61 demonstrates an inability or extreme reluctance to bear weight on one or more of her limbs/feet. Positioning of the cows ears is also a good indicator of a cows attitude. Sick cows usually have ears that droop down due to depression, pain, or fever. Healthy cows on the other hand, appear bright, alert and are curious about their environment. Positioninappearance of the eyes within the socket have also been used to asses the level of dehydration. A scoring system such as 1 (minimal), 2 (mild), 3 (moderate), or 4 (severe) has been proposed to asses dehydrated state (Smith and Risco, 2005). A cow wof 1 usually will have bright eyes that are positioned normally within the eye socket. A cow with a score of 2 will have dull eyes that are slightly sunken (1-2mm) within the eye socket. A cow with a score of 3 will have glazed eyes th g and ith a score at are moderately sunken (2-4 mm) g ) or acute where a cow with a score of 4 will have dry eyes that are severely sunken (>5mm) within the eye socket. However, this method has not been compared with the actual degree of dehydration. Changes in feeding behavior can also be used as an indicator of health. In those farms that have locking stanchions, the attitude of the cow can be observed after feedinto evaluate appetite. A cow that is sick will not eat, conversely a healthy cow aggressively consumes her feed. Smith and Risco (2005) have proposed the following scoring system to evaluate appetite; 1) cows that lock and eat 2) cows that lock appear dull and do not eat and 3) cows that do not lock to eat and appear dull or sick. Cows that fall in categories 2 or 3 should be monitored or examined carefully for illness. Urton et al,. (2005) evaluated if depression in feeding behavior was a good indicator of metritis (mucopurulent, foul smelling discharge and fever > 39.5C

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62 metritis ( reddish brown, watery, foul smelling discharge with fever > 39.5C), in daircows. Both y prepartum and postpartum feeding behavior were observed in healthy cows and in ith Figurs () and 17 Holstein cows without acute metritis () (SE) from 12 d before calving until 19 those that developed metritis or acute metritis postpartum. Cows diagnosed with either metritis or acute metritis spent less time feeding pre and post partum than did non-metritic cows. These cows also spent significantly less time feeding over the post-calvingperiod than did their healthy counterparts. However, only those cows diagnosed wacute metritis showed significantly lower feeding time during the precalving period. Figure 2-9 represents the feeding times for cows with metritis and cows with acute metritis during pre and post partum period. e 2-9. Daily mean feeding time (min/d) of 9 Holstein cows with acute metritid after calving.

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63 Furthermore, the risk of acute metritis related to prepartum feeding behavior was evaluated. It was found that for every 10 minutes in reduction of feeding behavior duringthe prepartum, the odds of developing acute metritis increased by 1.57 when compared tcows without acute metritis. In addition in the final model parity was significant, and wasshown that primiparous cows were at a higher risk Milk Production The evaluation of daily milk production is an objective parameter that can be used to identify sick cows. It has been suggested that an unexpected decrease in milk produmay be reflecting the inappetence of the cow o ction and thus it may be used as a monitoring thod ries apparent. inappetence, possibly resulting from difficult calving or retained placeilk losses that varied from 250 to 800 kg during a parameter to identify sick cows (Smith and Risco, 2005). The application of this mehas been extended with the use of computerized milk-meters which identify and record the production of individual cows on a daily basis. Results in lactation performance have been published in diseases such as ketosis, metritis, or displacement of the abomasumRajala-Schultz et al. (1999) found that milk yield decreased before the diagnosis of clinical ketosis, and the loss of milk continued for at least 2 wk after diagnosis. The effect of metritis, as one diagnosis, did not have any effect on milk yield (Rajala-Schultz and Grhn 1998). Metritis in the cited study (Rajala-Schultz and Grhn 1998) was defined as early 28 days postpartum and late metritis > 28 days postpartum. Once these categowere separated, the importance of the timing of the disease on milk loss becameLate metritis did not have a significant effect on milk yield, but early metritis significantly reduced milk yield. This result could be explained by the possibility that the cases of early metritis were more severe and included systematic symptoms as high temperature and nta. Deluyker et al. (1991) found m

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64 305-day in cows with left displaced abomasums. However, few studies have been conducted to determine the effect of postpartum diseases on daily milk production and its association to postpartum health as a diagnostic tool. Daily milk production can be recorded by milking machine software in which cows are identified if there is a deviation from an expected milk production. Changes in daily n be used to identify those cows that have a drop in milk production, ion r f y and daily milk production ca or are not producing at an expected level (Smith and Risco, 2005). Also, milk productcan be measured as a rate of milk produced per hour between consecutive milking sessions (in kg/h). This parameter may be more accurate than daily milk yield parametesince it reflects the actual milk production changes during milking session intervals. It provides the opportunity to monitor more frequently (2-3 times a day) the performance oan individual cow (Moallem, et al., 2002). Edwards and Tozer (2004) investigated the possibility of predicting an occurrenceof a disease before clinical diagnosis based on changes in daily walking activity (measured with activity meters) and milk production of cows with left displaced abomasum (LDA), ketosis, and digestive problems. Walking activity in cows with these disorders gradually decreased from 8 days to 1 days before clinical diagnosis. In addition, milk yield began to decline 6 days before diagnosis of cows with ketosis, 7 days for cows with an LDA, and 5 days for cows with digestive disorders. Overall activity started to decline before milk production. Figure 2-10 represents the difference in activitmilk yield for cows with an occurrence of ketosis, left displaced abomasum, and general digestive disorders during days 10 to 10 relative to the day of diagnosis (day 0).

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65 Consequently, using both activity and milk production together would be more senin detecting cows with LDA, ketosis and digestive problems. sitive isorders (c), compared with cows without an incidence of a disease in the prebreeding age during day 10 to 10, relative to the day of diagnosis (day 0). Figure 2-10. Difference in activity () and daily milk yield () for cows with an occurrence of ketosis (a), left displaced abomasum LDA (b), and general digestive dst

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66 Rectal Temperature Body temperature affects tissue function and is the resul t of chemical and physiological processes. The cow as a homothermous mammal has the capacity to manditions, through normaa. Fever is defined as o-effector ne system and hyperthermse sufficient heat to bathermomerectal muthe cranial artery of the caudal branch of thact of the rectal ucosa. erature of the al, and 2002). intain a constant temperature range under different environmental cothe inputs and output of heat regulation. An increase in body temperature beyond the l range can be characterized as fever (pyrexia), or hyperthermia controlled elevation of core body temperature by supportive changes in thermactivities. It is commonly regarded as beneficial, that is, having survival value (Kluger, 1986). Fever is the result of communication between the peripheral immuthe brain in response to infection, inflammation and/or trauma, and is clinically characterized by a rise in the body temperature (Cunningham, 2002). In contrast, ia is a rise in core temperature resulting from the inability to lolance the total of endogenous plus exogenous heat loads and can be potentially lethal (Hales et al., 1996). The rectal temperature (RT) is obtained by introducing a glass or a digital ter through the anal canal into the rectum and placed in close contact to the cosa. The blood supply to the rectal mucosa is derived from the caudal mesenteric artery, and by several short middle rectal branches frome urogenital artery (Getty, 1975). Because of the close contmucosa to its blood supply, temperature in blood is transferred to the rectal mConsequently, rectal temperature is a useful indicator of the core temp animal. However, rectal temperature is lower than the core temperature of the animchanges in rectal temperature may lag behind changes in core temperature (Cunningham

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67 Monitoring of re ctal temperature during the first 10 days post partum has been used to idee ). t to artile 39.7C experienced a reduction in RT of 0.6C after treatmthe e at s is ntify cows with fever (Upham, 1996). Normal RT has been reported to rangbetween 38.0C to 39.1C (Rebhun, 1995) or 38.0C to 39.0C (Rosenberger, 1979Cows with RT above the upper limit are defined as febrile or abnormal. However, because of the multiple factors that affect RT, a cut-off value to define fever is difficuldefine (Rebhun, 1995; Rosenberger, 1979). Different cut-off values have been reported to define fever in cows with uterine infections; 39.2C (Smith et al., 1998), 39.3C (Kristula et al., 2001) greater than or equal to 39.4C (Upham, 2001), and 39.5C (Drillich et al., 2001). Sheldon et al. (2004) described a cut off point for defining fever by using the maximum RT between days 1 and 10 post partum: mean of 39.3C, and upper quequal to 39.7C, which was defined as a febrile level. Kristula et al. (2001) havesuggested that cows with RT between 38.8C and 39.4C should be monitored carefullyto determine whether or not they require systemic antibiotic therapy. Furthermore, because cows with a RT of ent, a cut-off value of RT of 39.4C or 39.7C was proposed to be used in decision of whether or not to initiate antibiotic treatment. The protocol for monitoring daily RT in postpartum cows has been readily accepted by dairy producers and veterinarians because it is an objective response that can be used to evaluate health. However, this protocol is time consuming because all postpartum cows are monitored including those cows that calved normally that may ba lower risk to develop puerperal metritis. Therefore, the benefit of evaluating all cow unknown. In a study that evaluated postpartum RT, 48% of cows that calved normally had at least one daily temperature above 39.1 C compared to 93, 83, 100 and 78 % for

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68 cows had at d tia), 4) and ) y that is 82). As a result, the cow must utilizecid that rides calving (Goff and Horst 1997). In addition, ketosis may also occur secondary in cows with RFM, mastitis, metritis and dystocia respectively (Kristula et al, 2001). In addition, cows with an abnormal parturition (RFM, dystocia with or without RFM) rectal temperatures greater than 39.0 C for significantly more days (2.9) than cows thcalved normally (1.9) (Kristula et al, 2001). However, they conclude that two consecutives day with a RT greater than 39.1 C will not be a sufficient indicator to treat the cows with an antibiotic. Ketones Bodies Ketosis is a disorder of carbohydrate and fat metabolism characterized by increaseconcentrations of ketone bodies in blood (ketonemia), urine (ketonuria), milk (ketolacand other body fluids (Geishauser, et al., 1998). The metabolic state of ketonemia has anegative effect on milk production (Rajala-Schultz et al., 1999; Detilleux et al., 199reproduction (Andersson et al., 1991). The major ketone bodies are -hydroxybutyrate (BHBA), acetoacetate ( AcAcand acetone ( Ac) (Andersson, 1988). During early lactation, the amount of energrequired for maintenance of body tissues and milk production exceeds the amount of energy the cow can obtain from dietary sources (Baird, 19 body fat as a source of energy. However there is a limited amount of fatty acan be oxidized to completion by the tricarboxylic acid cycle in the liver or exported from the liver as very low density lipoprotein. When this limit is reached, triglyceaccumulate within the hepatocytes, impair their function, and the acetyl-coenzyme A that is not incorporated into the tricarboxylic acid cycle is converted to acetoacetate and -hydroxybutyrate. The appearance of these ketone bodies in blood, milk, and urine is diagnostic of ketosis and usually becomes clinically evident from 10 d to 3 wk after

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69 with diseases that affect feed intake (Kronfeld, 1982). Risk factors for subclinical ketoinclude metritis, displased abomasum, RFM and hypocalcemia (Curtis et al., 1985; Correa et al., 1993). However a cause-effect relationship has not been clearly defined (Duffield, 2000). Analyses for the presence of ketone bodies in urine and milk are commonly used to diagnose ketosis in cattle. Str sis ips, tablets or powders that contain nitroprussic acid are tively to measure the concentration of ketone bodies in urine or milk (LarsGiven the high incidence of ketosis (29 to 35%) from primary and secondary causes (Emery et al., 1964; Duffield et al., 1998) during the first 2 weeks post partum, monitoring postpartum cows for this condition may be of limited value if applied only to erum BHB Sensitivity (%) Specificity (%) used semiquantita en and Nielsen, et al., 2005). However, these tests vary in sensitivity and specificity (Table 2-1) (Duffield, 2000). Table 2-1 Ketosis threshold, sensitivity and specificity for different ketosis test. Test and body fluid Subclinical Ketosis Threshold (S mol/L) Utrec ht nitroprusside-milk 1400 90 96 Bioketone (urine) 1200 33 100 Utrecht powder (urine) 1200 42 100 Ketostix (urine) 1200 5 100 Ketolact-100 mol/L(Milk) 1200 72 89 Ketolact-200 mol/L(Milk) 1200 45 97 Ketolact-500 mol/L(Milk) 1200 17 100 Ketocheck (Milk) 1400 38 99 Duffield, T. 2000. Subclinical ketosis in lactating dairy cattle. Vet. Clin. North Am. Ketocheck (urine) 1200 28 100 Ketolact-50 mol/L (Milk) 1200 92 55 Ketocheck (Milk) 2000 61 98 Food Anim. Pract.16:231-253.

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70 identify sick cows. In addition, the utility of cow side ketone tests to identify sick animals with a primary disease such as displaced abomasum, mastitis or metritis is not knowEvaluation of Uterine Discharge A major com n. ponent of a postpartum health monitoring program is the evaluation of uterinlity ate uterine health (Studer and Morrow, 1978). Evaluation artum (Uphan, 1996) for early identification t of puerperal metritis to reduce the advers fertilityrsation to evalune infection ied commonlyever, examination of thna with a spec provides a dometritis that rectal examination. A vaginal examination in rectal palpation of the u5 to 50 days p partum resulted in itive culturesthogenic bactcompared to cows ctal palpatio (59% vs 22%spectively) (Mnd Morrow (1978) fous palpation ofrine size was 2002), a cervical diameter at rectal palpation of a >7.4 cm either at 20 to 26 or 27 to 33 days post partum by rectal palpation and presence of a mucopurulent, purulent or foul uterine discharge by vaginoscopy inspection at 27 to 33 days post partum were predictors of reproductive performance. For the palpation-based case definition of clinical e discharge by rectal palpation in order to screen cows that have uterine infections (Griffin et al., 1974; Studer and Morrow, 1978; Roberts, 1986, Upham, 1996). The abito diagnose uterine infections by rectal palpation varies among veterinarians (Lewis, et al., 1997). Typically, evaluation of the uterus by rectal palpation is performed between 25 to 50 days postpartum to evalu of uterine health by rectal palpation has been directed to the first two weeks postp and treatmen (o e effect of metritis on Callahan and Hai tman, 1987). The use of rectal palp te uter s us by veterinarians. How e vagi ulum more accurate diagnosis of en combination with terus 2 ost twice as many cows with pos for pa eria classified as infected using re n alone re iller et al., 1980). Studer a nd gros ute associated weakly with bacterial culture and histology. In a study by LeBlanc et al (

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71 endometritis, ignoring vaginoscopy, the sensitivity and specificity were 17 and 88%, respectively, for non-pregnancy beyond either 120 or 150 DIM. However, between 27 and 33 days pos partum diagnosis of endometritis with vaginoscopy was more sensitive than p n 7.8%. s for et he alpation-based diagnosis (21 vs. 12% sensitivity for classification of non-pregnancy beyond 150 day in milk, respectively). However, during this period, specificity of rectal palpation was higher than vaginoscopy (89 and 94%, respectively). Consequently, because of the changes in size and discharges that the uterus undergoes early post partum, the criteria for assessing uterine health change according to days post partum at examination (Ferguson, 1995). In the study by Callahan and Horstman (1987) ametritis (fluid, crepitus, lack of myometrial tone, and vaginal discharges) incidence of 33% at 1-3 weeks was reported. However, if the first examination had not been performed until 2 weeks later, the incidence of metritis would have bee Based on the review by Sheldon et al. (2006) in which the proposed definitionthe different uterine infections, it can be inferred that the studies previously publishedthat evaluated rectal palpation as a diagnostic tool (Studer and Morrow, 1978; Miller al., 1980; Callahan and Horstman, 1987) refered to endometritis and not puerperal metritis or metritis. Consequently, it remains unclear and more research is needed on ttest characteristics of rectal palpation to evaluate uterine discharge as it relates to puerperal metritis.

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CHAPTER 3 MATERIALS AND METHODS Cows and Herd Management The study was conducted between August 2002 and April 2003 in a commercial dairy farm located in north east Florida (30 18 N, 81 56 W). One thousand lactating cows were milk with a yearly rolling herd milk production average of 9,165Kg. The herdwas milked 3 times per day. The farm was a member of DHIA (Dairy Records Management Systems, Raleigh, NC) and used an on-farm computer based record systemfor maintaining production and health data. Prepartum transition cows that were within 3 weeks of calving were maint ained on dry lo ce s that did not expel the placenta within 24 hrs after calving were considered to have RFM (Risco and Hernandez, 2003). Cows with dystocia delivered by cesarean section or fetotomy were not included in the study. After parturition cows were sent for 2 days to a hospital herd housed in a concrete floor open-sided barn with stanchions that provided free access to a dry lot. At the hospital barn, cows were treated according to standard operation procedures of the farm ts, fed a cationic diet and monitored for signs of calving by farm employees trained to assist with parturition. Calving events such as dystocia and retained fetal membranes(RFM) were recorded by farm personnel. Dystocia was defined and recorded based on afive point scale as follows: (1) no assistance (2) slight problem (assistance for < 15 minutes) (3) needed assistance (assistance for > 15 minutes with moderate difficulty for extraction) (4) considerable force used (5) extreme difficulty or veterinary assistan(DHIA, 1997). Cow 72

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73 which consisted of administration of an oral calcium paste (Balance, Bayer, Shawnee Mission KS) and a single intrauterine infusion of 6 g of oxytetracycline dissolved in 75 ml of sterile water to multiparous cows with rtum, cows were moved to a lactating herd kest bedding, and fed four times a day. Diets for both pr were a total mixed ration formuil chalk ays post partum were examined for cyclic22 22 ) and RFM. At three days post pa pt in an open barn with dry compo e-and postpartum transition cows lated to meet the requirements of lactating dairy cows according to guidelines established by the National Research Council (NRC, 2001). Reproductive management consisted of a voluntary waiting period of 60 days. After that, cows were identified in estrus by visual observation with the aid of taor neck activity meters (WestfaliaSurge, Inc.1880 Country Farm Drive 60563 Naperville IL) Cows not artificially inseminated by 80 d ity, and cows with a corpus luteum (CL) were treated with 25 mg of PGF intramuscularly (IM) (Lutalyse, Pfizer Animal Health, Kalamazoo, MI.). Cows were artificially inseminated at detected estrus. Non-cyclic cows with inactive ovaries were treated with 100 g of GnRH IM (Cystorelin, Meriel Limited, Iselin, NJ.) followed by 25 mg of PGF IM 7 days later and AI at detected estrus. Cows not seen in estrus 14days after PGF treatment were re-examined for cycling status and treated with PGF only if a CL were present. Cows not AI by 120 days post partum were enrolled in the OvSynch program (Pursley et al., 1995). Cows not pregnant > 150 days post partum were also enrolled in the OvSynch program. Study Design This study followed a prospective cohort design. All cows underwent a postpartumhealth monitoring program consisting of daily evaluation of rectal temperature (RTattitude from day 3 to 13 post partum. All cows were examined for clinical endometritis

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74 between 20 to 30 days post partum. Rectal temperature was determined with the use of adigital thermometer (GLA, Agricultural Electronics, San Luis Obispo, CA) between 070to 0900 h immediately after milking. Cows that either appeared sick (depressed, sunken and/or tented eyes) or had a RT 39.4 0 th or perature and incidence of puerp, the d azoo, MI.) IM. Pregnancy diagnosis was determined by trasrectal palpating of s contents 42 to 49 days after artific o C were examined for puerperal metritis. The criterion for diagnosis of puerperal metritis were the presence of a watery, brown-colored, fetid discharge from the vulva (noted after rectal palpation of the uterus), wiwithout a RT 39.4 o C. All information concerning rectal tem eral metritis were stored daily in a separate database belonging to the principal investigator. Cows diagnosed with puerperal metritis were treated daily with ceftiofur hydrochloride (2.2 mg/kg IM. Excenel, Pfizer Animal Health, Kalamazoo, MI) IM for three days. In addition, supportive therapy consisting of anti-inflammatory agentscalcium and energy supplements were administered. Cows that did not respond to three day ceftiofur treatment, based on the persistence of a fetid discharge, received an intrauterine infusion of 3 g of oxytetracycline diluted in 75 ml of sterile water. The criterion used to diagnose clinical endometritis between 20 to 30 days post partum were one of the following condition associated or not with each other: cervicaldiameter greater than 6.0 cm; asymmetry of the uterine horns with fluid content and/or pus present at the vulva following rectal manipulation of the uterus. All cows diagnosewith clinical endometritis were treated with a single injection of 25 mg of PGF 2 (Lutalyse, Pfizer Animal Health, Kalam the uterus and it ial insemination (Zemjanis,1970).

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75 Data Management Data for parity and calving status: (dystocia: calving score 3, RFM, and twins) were recovered from the database, and two groups of cows were established based on calving status. Cows with a normal calving (NC) status were those without calving related problems, and cows with an abnormal calving status (AC) were those with dystocia, RFM with or without dystocia or twins. Cows were classified as having puerperal metritis (M+) or without puerperal metritis (M-) in a two level variable classified as metritis. Cows with and without metritis were also classified in a 3 level variable defined as Mettemp (MT) according to whether or not they had fever (RT 39.4 o C) when puerperal metritis was diagnosed. Cows with puerperal metritis and fever were classified as MT-1, cows with puerperal metritis without fever at the time of diagnosis were classified as MT-2, and cows without puerperal metritis as MT-3. Cows were classified as primiparous or multiparous in a two level variable classified as parity. The different seasons during the study were defined based o n the thermn ril), ence of puerperal metritis and clinical endoemtritis were analyzed using logistic regression PROC GENMOD of SAS 9.1; (SAS, 2003) with a binomial distribution and logit link. al heat index (THI = td (.55 .55RH) (td 58)) (West,1994). This index was calculated using daily ambient temperature (td) and percent relative humidity (RH) recorded at the closest weather station at Macclenny, FL (30 24 N, 82 11 W). Based oa previous report (West, 1994) a cut-off of 76.2 was used to define two seasons. A cool season was defined as those months with an average THI of < 76.2 (October to Apand a warm season were those months with an average THI 76.2 (May to September). Statistical Analyses All outcome variables and the incid

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76 Modeling was performed usilimination starting from the more tervals ain on y rate (%), ctions lso nd ing R) and the survival function estimatesegnancy up to 150 days postpST, ng manual backward e complex model with a third order interaction and the exclusion criteria were determined at P > 0.30. The model fit statistics were performed by comparing the difference in the deviances by the likelihood-ratio statistic test (Agresti, 1986). Maineffects were forced all models. Adjusted odds ratios (AOR) and 95% confidence in(95% CI) were determined. The model used to analyze the incidence of puerperal metritis included the meffects of calving status, parity, and season at calving. A second model was used to analyze the incidence of clinical endometritis and included calving status, parity, seasat calving, and puerperal metritis. A third model was analyzed the incidence of clinical endometritis and the variable puerperal metritis was substituted by the MT variableReproductive performance was evaluated by analyzing first service pregnanc umulative pregnancy rate by 150 days in milk (%) and, number of inseminafor pregnant cows (mean S.E.M). Number of inseminations for pregnant cows was aanalyzed by logistic regression (PROC GENMOD, SAS) with a Poisson distribution alogit link. All models for reproductive performance included the main effects of calvstatus, parity, season at first service, puerperal metritis and clinical endometritis as explanatory variables. Time to pregnancy was analyzed with survival analysis using Coxs proportional hazards regression (PROC PHREG, SAS). Adjusted hazard ratio (Hwas obtain from the proportional hazards regression model and crude median days open for the cumulative pr artum graph were obtained from the Kaplan-Meier analysis (PROC LIFETESAS).

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77 Data for daily RT from days 3 to 13 post partum were used to compare the RT ocows with or without puerperal metritis, and descriptive statistics were determined.Rectal temperature of cows with puerperal metritis 5 days prior and 5 days after diwere compared to the rectal temperature of cows without puerperal metritis (control cows). Control cows, without puerperal metritis, were assigned to cows with puerperal metritis, by controlling for gr f agnosis oup, parity and season, using a random number generator proceas s was There S etry, tell et cows that decreased with increasing lag between measurements was used in the dure. In cows with puerperal metritis, days post partum when puerperal metritis wdiagnosed were reclassified as follow: Day 0 day postpartum when puerperal metritidiagnosed: days -1, -2, -3, 4, 5 before diagnosis, and days 1, 2, 3, 4, 5 after diagnosis. Days post partum of control cows were similarly re-classified and matched to cows withpuerperal metritis in a rate of 3.7 controls cows per each puerperal metritis case. fore, day 0 for control cows (without puerperal metritis) corresponded with the same day post partum when puerperal metritis was diagnosed. This rearrangement of thedata was done to control for the rectal temperature by days post partum of cows with or without puerperal metritis. The analysis of RT between cows with and without puerperal metritis by day post partum was performed using PROC MIXED procedure for repeated measures of SA9.1(SAS, 2003). The model was subjected to 4 covariate structures: compound symmcompound symmetry-heterogeneous, autoregressive order-1 and autoregressive order-1 heterogeneous matrix. The autoregressive order-1 covariance structure was found to havethe smallest Akaikes information criterion and Schwarzs Bayesian criterion (Lital., 2002). Consequently the covariate structure that specified a correlation structure within

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78 mode, ond le l (Ti) + Dayk + Parl + Seasm + Statf + Dmetg (Day T)ki + eijklmfg Yijklmfg = daily rectal temperature Cow(Ti)j = random effect of cow nested in group Parl = fixed effect of parity Statf = fixed effect of metritis (yes, no or MT-1, MT-2, MT-3) (Day T)ki = fixed effect of interaction time and treatment the illustrated in the graphics. l. Two models were analyzed. The first model included the effects of calving statuspuerperal metritis, parity, season at calving and day as main effects as well as the secand third order interactions between the main effects. The second model was analyzed by including the variable Mettemp as a replacement for the variable Metritis. This variab(Mettemp) differentiates the daily rectal temperature of cows with puerperal metritis andfever, and cows with puerperal metritis without fever at the day post partum of puerperametritis diagnosis, and cows without puerperal metritis. For both models, repeated measurements of RT were also analyzed by testing homogeneity of regression curves for day trends. A single polynomial regression for day was fitted for RT, and the differences from fitting individual regressions for the effect of calving status, metritis, parity, season and their interactions were tested. Given that not all puerperal metritis cases werediagnosed on the same day post partum, the actual post partum day at diagnosis was included in the model as a covariable for both models. The mixed model for repeated measures was: Y ijklmfg = + T i + Cow Where: T i = fixed effect of group (normal or abnormal calving status) Day k = fixed effect of time Seas m = fixed effect of season Dmet g = regression coefficient for days in milk to metritis e ijklmfg = random error term Differences among rectal temperature for the different days were determined byuse of PDFF option of SAS. Least squares means ( SEM) of RT were determined and

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CHAPTER 4 RESULTS Final Sample A total of 488 cows calved during the study period and thirty-eight (38) cows were not included in the study because they received antibiotic treatment beyond 3 days postpartum at the hospital barn and did not complete 10 days of health evaluation. Therefore, a total of 450 calvings was evaluated, of which 327 (73%) were classified as normal, and 123 (27%) as abnormal. Ninety four (94) cows were diagnosed with puerperal metritis of which 55 (58.5%) had no fever, and 39 (41.4%) had fever. Incidence of Diseases In the logistic regression multivariate analysis, co ws with normal calving status had a lower incidence of puerperal mal calving status (43/327 (13%) vs. 51/123 (41%), respectiv< 0.01) between parity and season. During the cool season, primiparous cows iparous cows in warm season: 39.4% > 11.0% for primitively; and for primiparous 39.4% > 12.7%, for Cool vs. Ws with abnormal calvintritis was found to be significant for those cows diagnosed with etritis compared to cows with an abnorm ely; P < 0.001). There was a significant interact ion (P had the highest incidence of puerp eral metritis compared to prim season or multiparous cows in either season: Cool parous and multiparous, resp ec arm, respectively (Table 4-1). The overall incidence of clinical endometritis was 24%; Cow gs were more frequently diagnosed with clinical endometritis than those with normal calving status (AOR = 2.8, 95% CI 1.7-4.9, P < 0.001). A higher incidence ( 38.2%) of clinical endome 79

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80 Table 4-1. Incidence and risk factors of puerperal metritis in the first 13 days post partum of lactating dairy cattle. Variable Level Incidence of PRisk of Puerperal Metritis uerperal M etritis % AOR 95% CI P-Value N Metritis Yes 21.1 Calving Status 0.001 Abnormal 41.4 51/123 4.80 2.9 8.00 Season 0.001 Multiparous 11.0 22/200 Referent Referent Warm Primiparous 12.7 6/47 0.84 0.39 3.56 Primiparous Cold 39.4 54/137 Referent Referent Multiparous Warm 18.0 12/66 1.43 0.65 3.18 94 No 79.1 356 Normal 13.1 43/327 Referent Referent Lactation X Cold Primiparous 39.4 54/137 5.05 2.82 9.02 Multiparous 18.1 12/66 Referent Referent Warm 12.7 6/47 0.24 0.09 0.62 Cold 11.0 22/200 Referent Referent puerperal metritis (AOR = 2.2, 95% CI 1.1-3.9, P < 0.005) compared to cows without puerperal metritis. The incidence of clinical endometritis of cows diagnosed with puerperal metritis and fever (38.1%) and puerperal metritis without fever (38.4%) at the day of diagnosis was greater than cows without puerperal metritis (20.2%) (puerperal Metritis with Fever (AOR = 2.2, 95% CI 1.07 4.6, P < 0.02); puerperal Metritis without Fever (AOR = 2.1, 95% CI 1.09 4.19, P < 0.02). Non significant difference was found between fever and no fever puerperal metritis cows on the incidence of clinical endometritis (P < 0.9); Table 4-2.

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81 Table 4-2. Incidence and risk factors of clinical endometritis at 20 to 30 days post partum of lactating dairy cattle. Variable LIncidence of Clinical is Risk of Clinical evel Endometrit Endometritis R P% N AO 95% CI Value Endome tritis Yes 8 No 2 7 ent t 3 .80 90 arous26.0 69/266 1.00 Rferent iparous23.3 43/184 0.63 0.39 1.03 Puerperal Metritis No Metritis 20.2 72/356 eferent Referent 4 90 0.005 5 .10 0 0.02 24.0 10 76.0 34 Calving Status 0.001 Normal 17.7 58/32 Refer Referen Abnormal 40.6 50/12 2 1.70 4. Parity Multip 0.07 e Prim R Metritis 38.2 36/9 2.20 1.10 3. No Fever 38.4 21/5 2 1.09 4.1 Fever38.1 2.20 1.07 4.60 0.02 15/39 Recta l TeatureThe mean ( SEM), medianr a qd thfidenceinterval for RT from day 3 through day 13 post partum are shown in Table 4-3. In both models the effect of day to diagnosis was significant (P < 0.001). The analysis of model one showed a significant interaction between DAY and METRITIS (P < 0.001). Curves of daily rectal temperature during this period described a polynomial of second order for puerperal metritis cows and first order for cows without puerperal metritis. Rectal temperature from cows with puerperal metritis was significantly higher in cows without puerperal metritis 72 h before the diagnosis of puerperal metritis (DAY -3: 38.7C 0.05 > 38.6C 0.03; P < 0.009) and daily comparisons between groups continued to be different until the fourth day after the diagnosis and treatment of puerperal metritis (DAY 4: 38.7C 0.07 > 38.5C 0.04; P < 0.001). mper uppe nd lower uartiles an e 95% con

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82 Table 4-3. LSM (SE), 25 th quartile, median, 75 th quartile, and population 95% confidence intervals rect al temperatures of cows with and without puerperal metritis. SM n796 SD L SE 25th QMedia 5th QLSM 1. No Metritis Cows 38.6 0.0138.3 38.5 37.8 39.3 38.8 Primiparous 38.5 0.0238.2 38.5 38.8 37.5 39.6 lt38.6 38.3 38.8 37.8 39.4 etritis Cows 38.9 38.4 39.1 37.4 40.3 i38.9 38..7 6 Mult.8 38..7 mp-1 (n=55) 38.3 Mettemp-2 38..0 Mu iparou s 0.01 38.5 M 0.03 38.7 Prim parou s 0.04 4 38 39.1 37.7 40. iparous 38 0.05 3 38 39.2 36.7 40.9 Mette 38.7 0.04 38.6 39.0 36.7 40.7 (n=39) 39.2 0.05 5 39 39.4 37.1 41.3 Rectal temperature in cows that were diagnosed with puerperal metritis increased betwee48 h (DA.8C to 24 -1: 38 0.aof puerperal metritis (die 0 0.0 0.09)onteauntil reaching a maximum RT of 3 (day of diagnosis). Twenty-four hd ar and peral d higher RT 72 h before the day of n Y -2: 38 0.07) h (DAY .9C 06) before di gnosis fferenc .13C 8 C, P < and c inued to incr se 9.2C 0.05 on DAY 0 ours before the diagnosis of puerperal metritis, daily increments in RT were founto be significant (difference, 0.28C 0.07 C; P < 0.001). After treatment (DAY 1), RT of cows with puerperal metritis showed a significant reduction (0.33C 0.07 C, P < 0.001), and subsequent daily reductions in RT were not significantly different (Figure 4-1). Curves of RT between cows with puerperal metritis and cows without puerperal metritis tested by homogeneity of regression showed a significantly difference linequadratic effect (P < 0.001). In model 2 there was a significant interaction DAY and METTEMP (P < 0.001). Curves of daily rectal temperature 5 days prior and 5 days after the diagnosis of puermetritis described a polynomial of second order for MT-1 and first order for MT-2 and MT-3. Rectal temperature from MT-1 cows ha

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83 diagnosis of puerperal metritis compared to MT-3 cows (DAY -3: 38.8C 0.10 > 38.6C 0.04; P < 0.04). 38.4 38.6-5-4-3-2-1012345 38.8ectal t 39.0atur 39.2 39.4 Day before and after metritisRer emp e (C) ** ** ** ** ** ** ** Figure 4-1. LSM SEM of daily rectal temperatures of cows 5 days prior to and 5 daysafter the diagnosis of metritis, for cows with puerperal metritis (n=94; ), and cows without puerperal metriti (n=356; ). *P < 0.05 **P < 0.001. This daily difference continued to be significant until 24 h after the diagnosis and treatment of puerperal metritis (DAY 1: 39.0C 0.09 > 38.5C 0.04; P < 0.001). Inthis group (MT-1), the RT started to increase 72 hours (DAY -3: 38.8C 0.10) to 48 h (DAY -2: 39.0C 0.09) before the diagnosis of puerperal metritis (difference 0.200.12 C, P < 0.09), and continued to increase until reaching a maximum of 39.7C 0.09 on the day of diagnosis (Day 0 C ). 4 Twenty four hours before the diagnosis of puerperal metritis, a daily increment inRT of 0.53C 0.11 was found to be significant (P < 0.001). After treatment, cows with puerperal metritis and treated showed a significant reduction in RT in the first 24 h (0.65C 0.11 C, P < 0.001). This reduction continued to be significant between 2

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84 (DAY 1: 39.05C 0.09) to 48 h (DAY 2: 38.7C 0.10) after the diagnosis of puemetritis (difference 0.35C rperal 0.12 C, P < 0.01). Daily rectal temperature from MT-2 cows were higher compared to MT-3 24 h before the diagnosed (DAY -1: 38.8C 0.09 > 38.5C 0.04; P < 0.02), and continued to be different until the fifth day after the diagnosis and treatment of puerperal metritis (DAY 5: 38.7C 0.11 > 38.5C 0.04; P < 0.07). In this group, there was not a statistically significant difference between daily increments of RT before the diagnosis of puerperal metritis. Cows diagnosed with puerperal metritis in the absence of fever had a RT of 38.9C 0.08 on the DAY 0 (day of diagnosis). There was not a significant reduction in the RT after the diagnosis and treatment of puerperal metritis (Figure 4-2). 38.238.439.039.2-5-4-3-2-1012345ctal Teeraturaa*b*c*b*b*b*b*b*b*b*ccc*cFigure 4-2. LSM SEM of daily 38.638.839.840.0Day before and after metritisRempe (a* rectal temperatures of cows 5 days prior to and 5 days after without fever (n=55; 39.439.6C) diagnosis for cows with puerperal metritis and fever (n=39; ), puerperal metritis ) and cows without puerperal metritis (n=356; ). a, b, c differ with a P< 0.05, P< 0.005.

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85 Reproduction There were no detected differences in first-service pregnancy rate (No-Metritis (32%) vs. Metritis (28%); P < 0.3), and accumulated pregnancy rate by 150 days post partum (No-Metritis (59%) vs. Metritis (52%); P < 0.7), or days in postpartum to fiservice or pregnancy for cows with or without metritis. As expected, a seasonal effect was detected in both models for first service pregnancy rate (Cool season (34 %) > thanwarm season (16 %); P < 0.005), accumulated pregnancy rate by 150 days (Cool se(64 %) > Warm season (25 %); P < 0.001). Days to first service as well as days to pregnancy was also influenced by the season effect. No significant differences were found in the number of services for pregnant c rst ason ows between cows with or without puerperal metritis (Mean SE, 1.5 0.05 vs. 1.6 0.11, respectively; P < 0.8). 0%10%20%30%40%50%60%70%80%90%100% 405060708090100110120130140150Days Postpartum Cows Pregnant (%) Cold Season Warm Season uring cold or warm season by 150 days postpartum. Figure 4-3. Proportion of pregnant cows d

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86 0%20%30%50%60%80%90%405060708090100110120130140150Days PostpartumCow Pregnnt (%) 10%40%70%100%sa No Metritis MetritisFigure 4-4. Proportion of pregnant cows with or without puerperal metritis by 150 days postpartum.

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87TABLE 4-4. Logistic regression model of conception rate to first service and pregnancy rate at 150 day postpartum days postpartum by group, presence or not of metritis, parity, season and presence or not of eritis.Variables First Service Conception Rate Pregnancy 150 Days Postpartum ndomet Levels % N AOR 95% CI P % N AOR 95% CI P < < Group 0. 2 0.9 Normal 30 84/284 Referent 58 165/284 Referent Abnormal 35 38/109 1.47 0.87 2.47 56 62/109 1.04 0.63 1.74 Metritis 0. 3 0.7 No 32 98/308 Referent 59 182/308 Referent Yes 28 24/85 0.72 0.39 1.32 52 45/85 0.89 0.50 1.57 Lactation 0.0 6 0.6 Multiparous 28 61/220 Referent 58 127/220 Referent Primiparous 35 61/173 1.56 0.99 2.45 57 100/173 1.10 0.70 1.70 Season 0.0 05 0.001 Cold 34 111/325 Referent 64 210/325 Referent Warm 16 11/68 0.98 0.18 0.72 25 17/68 0.18 0.10 0.33 Endometritis 0. 6 0.7 No 32 92/291 Referent 58 170/291 Referent Yes 29 30/102 0.88 0.52 1.49 55 57/102 0.88 0.54 1.45

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88y Variables Days Postpartum to First Service Days Postpartum to pregnancy TABLE 4-5. Cox proportional model of days postpartum to first service and pregnancy rate at 150 day postpartum days postpartum bgroup, presence or not of metritis, parity, season and presence or not of endometritis. Levels Med ian E Hian H S. azard 95% CI P < Med S.E azard 95% CI P < Group 0.2 0 .9 Normal 69 .0 .0 .0 0 .01 1.7 Referent 126 2.0 Referent Abnormal 78 2.9 .83 0.66 1.05 131 3.3 .01 0.81 1.27 Metritis 0.7 0.0 .0 .0 0 .0 0 .7 No 70 1.7 Referent 127 2.0 Referent Yes 76 3.3 .93 0.72 1.22 149 3.6 .95 0.74 1.23 Lactation 0 .002 0.0 .0 .5 1 .00 .8 Multiparous 73 2.1 Referent 131 2.2 Referent Primiparous 68 2.1 .37 1.21 1.69 124 2.8 .97 0.80 1.18 Season 0.009 0.0 .0 .0 0 .0 0 .001 Cold 69 1.6 Referent 117 1.9 Referent Warm 83 4.0 .70 0.53 0.91 127 3.1 .58 0.45 0.75 Endometritis 0.5 0.0 .0 .0 1 .00 .7 No 70 1.8 Referent 127 2.0 Referent Yes 73 2.7 .10 0.87 1.39 132 3.3 .96 0.76 1.20

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CHDIS APTCUS ER 5SIOIn this study, cows with twins, dystocia, RFM with or without dystocia were sidere h ors fotritis in dairy cows (Markusfeld, 1984; Curtis et al., 1985; Bartlett et al., 6; Coe 199t not t of t to d the il ct of these ders on the incideerpetis. Ie coeith one oore of these disorders as a high risk group to develop puerperal s and retpoo(coith a normal calving). The clinical approach used to identify cows to be tedp 9.4Despite the facat a systec examination of the uterus of all cows was sid,dencpp of (4 o ar wimilar to previous reports (Markusfeld, 1984; Curtis et al., 1985) and within geued sat evaluated all cows for puerperal mis reof e or RT (kusf). etritis used in the present study was based on the ce of a feulvrge withous, only 41.4% hahen puerperal metritis was diagnosed. These results are ement with those of Pugh et al (1994), in which 42.3% of 78 cows evaluated for s 1ys post partum had fever (RT > 39.4C). The diagnosis of puerperal mpre study made by per rectum ption of thrus and vl inspection N confact198effecomegroevRTnocaltheattpremein mein d tor merrea ave an a bnor mal calvi ng. T hes e dis orde rs ha ve b een r epor ted t o be risk t al.,disor 3). I was the ince ntenof pu his sral m tudyetri etermnstea ined, w ndivnsid iduared ws wtritiup alua 3t conving ranitudThe definition of puerperal msentritiagretritithe r m rosp ectiv ely com are the i ncid ence of p uerp eral metr itis t a l w ri sk ws w for uerp eral metr itis wt th as b ased on mati evalu atio n of attitude ( appe ared sick ) or a o C. dereas s in p the inci e of uer era l met ritis 2%) in c ws with an bno mal blishMar tudieeld, s th1984 etrit gard less tid v ar did fev schaer w ith or w t fev er. Of the cows with puerperal 4 dasent etritis was alpa e ute isua 89

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90 of the vulva to observe a uterine discharge. This method of diagnosis of puerperal metritis has been previously described (Markusfeld, 1984; Pugh et al., 1994; Risco, and Hernandez, 2003). In contrast to the diagnosisetritis (Bonnett et al., 1993; Leblanc et al, 2002), no other previously reported method have been proven to be more s rted. s a ns and progesterone during the postpartum period (Lew of clinical endom or less sensitive and or specific to diagnose puerperal metritis. Primiparous cows in the present study had a higher incidence of puerperal metritiduring the cool season. However, no seasonal effect was observed in multiparous cowsAn effect of season and parity on the incidence of postpartum uterine infections have been previously described (Markusfeld, 1984; Bartlett et al., 1986; Grhn et al., 1990; Smith et al., 1998). However, an interaction between these two factors was not repoMarkusfeld (1984) and Bartlett et al (1986) hypothesized that during the winter monthhigh concentration of calvings and a wet and dirty environment may increase the challenge of pathogens to the uterine environment. However, in the present study, both primiparous and multiparous cows were equally exposed to these factors. In the present study, season was determined by the thermal heat index, which is an indirect measurement of heat stress in the cow. Previous reports have shown the relationship between heat stress and uterine involution. Heat stress during late gestation increased concentrations of blood prostaglandi is et al., 1984), and reduced prepartum blood concentrations of estrone sulfate (Collier et al., 1982). These changes in hormonal profiles have been reported to have a positive effect on uterine involution (Lewis et al., 1984; Nakao et al., 1997; Zhang et al., 1999) which may have reduced the incidence of puerperal metritis during the warm season observed in the present study.

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91 The overall incidence of clinical endometritis is in agreement with that obserLeblanc et al. (2002). The relationship between abnormal parturition and chronic uterine infections has been previously reported (Dohmen et al., 2000). Hirvonen et al. (1999) reported that 58 % and 6% of the cows diagnosed with puerperal metritis (putrid smelling, reddish-brown, watery vaginal discharge; rectal temperature ved by 39.5 to 41.0C; withins with clinical for a cut off point for defining fever by using the 4 to 11 days postpartum) had purulent vaginal discharge within 15 to 22 and 32 to 44 days postpartum, respectively. In addition, in the present study, the incidence of clinical endometritis (38.2%) in cows with puerperal metritis treated with ceftiofur is similar to that observed by Drillich et al,. (2001). In that study (Drillich et al., 2001), 44.8% of the cows with puerperal metritis (fetid reddish brown vulvar discharge with a RT 39.5C) treated with ceftiofur hydrochloride developed clinical endometritis within 32 to 34 days post partum. An interesting finding in the present study was that cowpuerperal metritis developed clinical endometritis whether or not they had fever when puerperal metritis was diagnosed. This may suggest that despite treatment of puerperal metritis, cows without fever were just as likely as those with fever to develop endometritis. Normal rectal temperature had been reported to be within the range of 38.0C to39.1C (Rebhun, 1995) 38.0C to 39.0C (Rosenberger, 1979). Cows with a RT above the upper limit are defined as febrile or abnormal. In the present study, the mean RTcows without metritis classified as normal was within the above described ranges, and inagreement with that reported by Kristula et al. (2001). Based on a previous report (Upham, 1996), used in the present study definition of fever was determined to be 39.4C. Sheldon et al. (2004) described

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92 maximn our st f s er. hether or not cows with puerperal metritis had fever, an incremental incread um RT between day 1 and 10 post partum, obtaining a mean of 39.3C, and upper quartile equal to 39.7C, which was defined as a febrile level. In the present study, values of RT between 3 to 13 days post partum for cows without puerperal metritis followed a normal distribution with a mean ( SEM) of 38.5C 0.01C. Based odata, we found that the 95% confidence interval of the RT values within 3 to 13 days popartum were equal to 37.7C 39.3C, after correcting by group, parity, season and presence or not of puerperal metritis. If it were hypothesized that the remaining 5% ovalues will be considered abnormal or febrile, cows with fever will be those with a RT39.4C. Daily incremental increases of RT before diagnosis of puerperal metritis have not been previously reported. Rectal temperature of cows with puerperal metritis started increasing two days before the diagnosis of puerperal metritis. However, the RT value did not reach a febrile level of 39.4C and were similar to those reported in cows with puerperal metritis (Smith et al., 1998). This is related to the fact that a proportion of cowwith puerperal metritis on Day 0 (day of puerperal metritis diagnosis) did not have fevRegardless of w se of daily temperature was significantly different from cows without puerperal metritis. Following treatment of cows with puerperal metritis with ceftiofur, rectal temperature decreased to a level similar to cows without puerperal metritis that were not treated. A similar response in RT reduction has been observed in cows with puerperal metritis treated with ceftiofur (Simith et al., 1998; Chenault et al., 2004; Wagner anApley, 2004).

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93 Results from model two showed two different patterns of RT for the different classifications of puerperal metritis. Cows classified as MT-1 showed a marked increain rectal temperature that was significant 24h before diagnosis, and this high RT wasrapidly reduced after diagnosed and treatment. In contrast, this pattern was not observedin cows classified as MT-2. Physiological control of a febrile response is multifactorial with mechanisms to prevent extreme elevation in body temperature (Leon, 2002). Ouresults showed that RT of cows with puerperal metritis has daily incremental increases ofRT to a point were they became febrile. This may occur from the interaction between the host imm se r une system and bacterial endotoxins which trigger the cascade of events that lead tn ess with higher rate of LPS absorption or concentration than cows with fever. Fever induced by LPS seems to be dose-related in that a high dose of LPS lowers o elevated temperature. Dohmen et al. (2000) correlated the presence of a fetid uterine discharge with high concentration of LPS in the uterus and an elevated rectal temperature. However, comparison of LPS concentrations between the uterus to those iplasma were not correlated. In contrast, Peter et al. (1990) and Mateus et al. (2003) demonstrated that the uterus of cows during the early postpartum period is permeable to Escherichia coli LPS, and that these endotoxins provoked the systemic release of prostaglandin F2 (PGF2 ) and thromboxane A2a. The reason why a proportion of cows did not have fever when puerperal metritiswas diagnosed or why they did not experience an incremental increase in RT beforepuerperal metritis diagnosis is not known. However, because all cows with puerperal metritis without fever were treated on the same day of diagnosis, a lack of disease progression may have occurred. It is also possible that these cows were under a septicemic proc

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94 body of n 87). f cows disease. In addition Harman et al. (1996, a single temperature, attenuates fever and causes hypothermia. In contrast, lower doses LPS seem to stimulate the production of TNF with subsequent production of fever (Leon,2002). Farm personnel involved in the conduction of the reproductive management of the study farm were blinded to the calving or puerperal metritis status of cows. Abnormal calving, puerperal metritis or clinical endometritis did not have any effect on reproductive performance. Typically, rectal examination of postpartum cows had been performed between 25 to 50 days post partum (Studer and Morrow, 1978), recently this procedure had been also done in the first two weeks post partum (Upham, 1996). In thepresent study, the intensity of the post partum health monitoring program resulted in aearlier diagnosis and treatment of puerperal metritis and clinical endometritis. Consequently, it can be assumed that earlier identification and treatment of puerperal metritis, leaded to a decreased adverse effect on fertility (Callahan and Horstman, 19A meta-analysis study on the effect of disease on reproduction (Fourichon et al., 2000) showed less effects of puerperal metritis in studies reporting routine examination ocompared with herds in which the owner reported the ) did not find any effect on the risk of conception for those cows with dystocia, retained placenta, or early metritis during 56 to 120 days postpartum. However, other studies showed a negative effect of abnormal parturition or puerperal metritis on reproductive performance (Borsberry and Dobson, 1989; Grhn and Rajala-Schultz2000). All cows with clinical endometritis in the present study were treated withinjection of 25 mg of PGF 2 In addition, cows were treated with PGF 2 in cases of non observed estrus. It is possible that cows affected with clinical endometritis may have

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95 recovered sufficiently during the 60-day voluntary waiting period after treatment with PGF2a. The use of PGF 2 during the early postpartum period improved fertility in cows which ex perienced an abnormal calving (Risco et al., 1994).

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CHAPTER 6 CONCLUSIONS Occurrence of puerperal metritis was higher in cows experiencing an abnormal calving. Primiparous cows had a greater incidence of puerperal metritis in the cool season for both normal and abnormal calving. In contrast, multiparous cows showed no seasonality in the occurrence of puerperal metritis. A high proportion of cows did not had fever at the time of puerperal diagnosis, suggesting that puerperal metritis may not always be accompanied with a fever. Evaluation of daily RT distinguished both cows with puerperal metritis, and those with or without fever. Prior to diagnosis, sequential increases in RT on two consecutive days prior to the actual diagnosis could serve as a predictor of puerperal metritis and may warrant earlier treatment. Early therapeutic treatment of all cows diagnosed with puerperal metritis resulted in pregnancy rates comparable to cows with normal or abnormal calving status that did not experiencing puerperal metritis. 96

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107 Nakao, T., A. Gamal, T. Osawa, K. Nakada, M. Moriyoshi, and K. Kawata. 1997. Postpartum plasma PGF metabolite profile in cows with dystocia and/or retaiplacenta, and effect of fenprostalene on uterine involution and reproductive perfo ned rmance. J. Vet. Med. Sci. 59: 791-794. National Research Council. 2001. Nutrient Requirements of Dairy Cattle, 7th rev. ed. Nielen, M., Y. H. Schukken, D. T. Scholl, H. J. Wilbrink, and A. Brand, 1989: Twinning 62 Ogra, PMcGhee.1999. Mucosal Immunology, 2nd edn. New York: Academic Press. Olson, etra ve Diseases in Small and Large Animals. W. B. Saunders Co., Philadelphia, PA. Oltenan, type of discharge from genital tract, involution of cervix, and subsequent reproductive performance in Holstein cows. J. Dairy Sci. Ostergad body h herds. J. Dairy Sci. 82:1188-1201. gy. 31:251-254. iol for metritis. J. Am. Vet. Med. Assoc. 15:846-851. Peter, Wxin teri of postpartum dairy cows. Theriogenology. 33: 1011. Philipsroc. Intl. Workshop on Genet. Improvement of Functional Traits in Cattle, Gemloux, Belgium. Interbull. Bull. 12:65-71. Nathanielsz, P. W. 1993. A time to be born: how the fetus signals to the mother that it istime to leave the uterus. Cornell Vet. 83:181-187. Natl. Acad. Sci., Washington, DC. in dairy cattle: a study of risk factors and effects. Theriogenology. 32:845-8. L., J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock, and J. R. J. D., K. N. Bretzlaff, R. G. Mortimer, and L. Ball. 1986. The Metritis-Pyomcomplex in Current Therapy in Theriogenology 2: Diagnosis, Treatment and Prevention of Reproducti cu, P. A., J. H. Britt, R. K. Braun, and R. W. Mellenberger. 1983 Relationships among type of parturitio 66:612-619. ard, S. and Y. T. Grhn. 1999. Effects of diseases on test day milk yield anweight of dairy cows from Danish researc Otero, C., L. Saavedra, C. Silva de Ruiz, O. Wilde, A. R. Holgado, and M. E. Nader-Macas. 2000. Vaginal bacterial microflora modifications during the growth of healthy cows. Letters in Applied Microbiolo Overton, M. W., W. M. Sischo and J. P. Reynolds. 2003. Evaluation of effect of estradcypionate administered prophylactically to postparturient dairy cows at high risk ., T. K. Bosu, and R. O. Gilbert 1990. Absorption of Escherichia coli endoto(lipopolysaccharide) from the u son, J. 1996. Strategies to reduce problems in calving performance and stillbirths by selection and differential use of bulls. P

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BIOGRAPHICAL SKETCH Republ s, until thraised. In 1994 he went to the veterinary school at the recently created Veterinary School at Universidad del Salvador in Argentina. Early in his career he was influenced by several professors with doctorate degrees from North American Universities. With them and especially with Dr. Carlos Eddi (professor of Parasitology), he first encountered the passionate world of research. Mauricios interest in ruminants grew from his interaction with a great clinician (Dr. Enrique Renner, professor of Buiatrics), at that University. After graduation he accepted a 1-year research position in parasitology. Mauricios then took a research position with Dr. Carlos Corbellini at the National Institute of Agricultural Technology where he worked on the dairy industry in projects related to epidemiology and control of mastitis in dairy cows. In 2002 he was given an opportunity to work and learn in the Florida dairy industry, in the United Sates. On August 17, 2002, Mauricio arrived at Mecklenburg Farm where he spent a year and a half on daily dairy activities. There he met Dr. Carlos Risco and Dr. Louis Archbald, and a new chapter of Mauricios life began. In late 2002, Mauricio accepted a residency at the Food Animal and Reproduction Service in the College of Veterinary Medicine at the University of Florida. After 1 year Mauricio Esteban Benzaquen was born in the Federal Capital of the Argentineanic, on November 28, 1975. Mauricio was raised in Escobar, Argentinas flower capital. There he spent time with family, and enjoyed brotherhood, friendship and studiee road of life took him north, still maintaining the principles with which he was 112

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113 of residency (and given his strong interest in research), Mauricio was offered (by Dr. Carlos Risco) the possibility of starting a Master of Science degree program.


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Permanent Link: http://ufdc.ufl.edu/UFE0015643/00001

Material Information

Title: Rectal Temperature, Calving-Related Factors, and the Incidence of Puerperal Metritis in Postpartum Dairy Cows
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015643:00001

Permanent Link: http://ufdc.ufl.edu/UFE0015643/00001

Material Information

Title: Rectal Temperature, Calving-Related Factors, and the Incidence of Puerperal Metritis in Postpartum Dairy Cows
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015643:00001


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RECTAL TEMPERATURE, CALVING-RELATED FACTORS, AND THE
INCIDENCE OF PUERPERAL METRITIS IN POSTPARTUM DAIRY COWS















By

MAURICIO ESTEBAN BENZAQUEN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006
































To my parents, Marta and Alberto who gave me the gift of life, of love and education.















ACKNOWLEDGMENTS

I wish to express my most sincere gratitude to Dr. Carlos A. Risco (my supervisory

committee chair) for seeing in me what even I did not see, and for giving me the

opportunity of a lifetime. It has been an incredible experience that I will never forget: a

mixture of theriogenology, clinical knowledge, and research, blended with friendship. I

thank Dr. Louis F Archbald for his invaluable advice and support, and for having the

right words at the right moment. I thank Dr. William W. Thatcher for his invaluable

advice, for his teaching, and for showing me the fourth dimension of statistical analysis. I

thank Dr. Pedro Melendez for his support, advice and fellowship during these 3 years.

I also thank Marie-Joelle Thatcher, Biological Scientist, for her hard work on my

research and for improving my organizational skills. I thank Dr. Owen Rae and Dr.

Arthur Donovan (of the Food Animal and Reproduction and Medicine Service) for their

support and indulgence during this experience. I gratefully acknowledgeFlorida Dairy

Check-Off for their economic support of my study.

I thank Mr. Ingo Kreig, owner of Mecklenburg Dairy, for his cooperation with the

project; particularly for the use of his cows and facilities. I thank Mr. Roger Rowe, farm

manager at Mecklenburg Dairy for his time and assistance with the project. I thank Dr.

Bronwyn Crane and Dr. Pablo Pinedo, fellow graduate students, for their friendship

andsupport. I thank my fellow residents and interns for the extra effort they gave, which

helpedme to complete this program. I thank God for allowing all of these people to cross

my path.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S .......................................................................... .....................iii

LIST OF TABLES .............. ............................ .............................. vii

LIST OF FIGU RE S ........ ......................................................... ......... ...... ..... viii

A B STR A C T ................................................ ....................................... .. x

CHAPTER

1 IN TR OD U CTION ............................................................................

2 LITER A TU R E R EV IEW ................................................................ ...................... 4

The B ovine U terus ....................................................... .......... .......... .... ....
U terine Involution ......................................................... .. ...... ......... .......... . ..
Endocrine Changes Early Post Partum................................................................ 13
Uterine D defense M echanism s ............................................................ ............... 17
Innate U terine D defense ....................................................................... ............... 18
Anatom ical barriers ............................................ .. .. .... .. ............ 19
U terine and cervical secretions ........................................ ..... ............... 20
Bacterial antagonism ........................................... ........................22
Inn ate cell com p on ent ............................................................ .....................2 3
H um oral-m ediated im m unity ........................................ .......................24
C ell-M ediated Im m unity .............................................................. .....................26
Fever Pathw ay during Infections ......................................................... ............... 26
General Review of Uterine Infections .................................... .......................... ........ 29
E n d o m etritis ................................................................ 3 0
Pathogenesis ......................................... .................... ..........30
H istopathological disease definition ..................................... ............... 31
C clinical disease definition ........................................... ......................... 33
Puerperal M etritis ...................... .................... .............. ........... ..... 33
Pathogenesis ........................................ ................ ................ 33
H istopathological disease definition ..................................... ............... 37
C clinical disease definition ........................................ .......................... 38
Literature disease definitions ........................................ ....................... 39
Current research definition.......................................... 51










Risk Factors for Puerperal M etritis ........................................ ...... ............... 52
R detained fetal m em branes ........................................ .......................... 52
Stillbirth, multiple birth and dystocia.................... ............ .... ........... 54
P a rity ................................................................5 6
S e a so n .......................................................5 7
Hypocalcemia.................................... 58
Postpartum H health M monitoring ............................................... ............................ 58
A ttitu d e ....................................................................................................... 5 9
M ilk P rodu action .......... ................................ ............................ .... .... .... 63
R ectal T em perature ...................... ...... .............. ................ ...........66
K eton e B odies .............................................................6 8
Evaluation of Uterine Discharge ............ ............ ........ ............... 70

3 MATERIALS AND METHODS ............................................................................72

C ow s and H erd M anagem ent............................................................... .....................72
Stu dy D design .....................................................73
D ata M anagem ent .................. ................. ......... .... .. .... .......... .............. 75
Statistical A analysis .............................................75

4 R E S U L T S ..............................................................................7 9

F in al S am p le ........................................... .. ....79..........
Incidence of Diseases ..... ........................... ......... 79
Rectal Tem perature.............................................. 81
R e p ro d u ctio n ......................................... .. ............................................8 5

5 D ISCU SSIO N .................................................................................... .................... 89

6 CON CLU SION ................................................ 96

L IST O F R E FE R E N C E S ................... ............. ......... ..................................... .....................97

BIOGRAPH ICAL SKETCH ............. ................... ........................................ ................... ....... 112


















v
















LIST OF TABLES


Table p

2-1 Ketosis threshold, sensitivity and specificity for different tests ............................69

4-1 Incidence and risk factors for puerperal metritis in the first 13 days postpartum
in lactating dairy cattle ....................... .. .... ................ ........................... 80

4-2 Incidence and risk factors of clinical endometritis at 20 to 30 days postpartum in
lactating dairy cattle .................... .. ........... ........... .... ................. 81

4-3 Mean + (SEM), 25th quartile, median, 75th quartile, and population 95%
confidence intervals rectal temperatures of cows with and without puerperal
m etritis ...................................... ................................... ................ 8 2

4-4 Logistic regression model of conception rate to first service and pregnancy rate
at 150 day postpartum days postpartum by group, presence or not of metritis,
parity, season and presence or not of endometritis. ............................................87

4-5 Cox proportional model of days postpartum to first service and pregnancy rate at
150 day postpartum days postpartum by group, presence or not of metritis,
parity, season and presence or not of endometritis. ............................. ............... 88














LIST OF FIGURES


Figure p

2-1 Changes in lochia volume during the first 20 days postpartum in dairy cows...........7

2-2 Uterine changes in weight, diameter and length during the first 20 days
postpartum in cow s ...................... .. .............. .......................... ... .

2-3 Reduction of the cervical opening during the first 24 h post partum.....................10

2-4 Reduction of the cervical opening during the firstl0 days post partum ................. 10

2-5 Pooled within cows least-squares regressions of plasma progesterone (P4),
estrone sulfate (E1SO4), estrone (El), prolactin (Prl), and 13, 14-dihydro, 15-
keto prostaglandin F2a (PGFM) during the periparturient period ...........................14

2-6 Pathway of fever development in response to infection, inflammation, or trauma..28

2-7 Percentage of uteruses from postpartum cows in which bacteria were recovered
in the first 60 days post partum ......................................................................... 30

2-8 Overall incidence and degree of endometritis in uterine biopsy samples during
the first seven weeks post partum ........................................ ....................... 32

2-9 Daily mean feeding time of Holstein cows with acute metritis and Holstein cows
without acute metritis from 12 d before calving until 19 d after calving ...............62

2-10 Difference in activity and daily milk yield for cows with an occurrence of
ketosis, left displaced abomasum, and general digestive disorders compared to
cows without an incidence of a disease in the prebreeding stage during -10 to 10
days relative to disease diagnosis (day 0). .................................... .................65

4-1 LSM SEM of daily rectal temperatures of cows 5 days before and 5 days after
the diagnosis of metritis in cows with and without puerperal metritis...................83

4-2 LSM SEM of daily rectal temperatures of cows 5 days before and 5 days after
diagnosis for cows with puerperal metritis and fever, puerperal metritis without
fever and cows without puerperal metritis .......... ................. ...... ..............84

4-3 Proportion of pregnant cows during cold or warm season by 150 days
p o stp a rtu m ........................................................................ 8 5









4-4 Proportion of pregnant cows with or without puerperal metritis by 150 days
p o stp a rtu m ........................................................................ 8 6














Abstract of Thesis Presented to the Graduate School of the University of Florida
in Partial Fulfillment of the Requirements for the
Degree of Master of Science

RECTAL TEMPERATURE, CALVING-RELATED FACTORS, AND THE
INCIDENCE OF PUERPERAL METRITIS IN POSTPARTUM DAIRY COWS

By

Mauricio Esteban Benzaquen

August, 2006

Chair: Carlos A. Risco
Major Department: Veterinary Medicine Science.

The objectives of my study were to evaluate the association of calving status, parity

and season on the incidence of puerperal metritis (PM) and clinical endometritis (CE) in

lactating dairy cows; and examine the role of rectal temperature as a predictor for

puerperal metritis, and document the effect of puerperal metritis on subsequent

reproductive performance. This study was a prospective cohort design. Cows were

classified as abnormal calving status (Ac), cows calving with dystocia, RFM with or

without dystocia or twins and cows with a normal calving status (Nc), those without any

calving related problems. Daily rectal temperature (RT) of all cows was taken early in the

morning from days 3 to 13 postpartum, and health examinations were performed by the

on-farm veterinarian. We evaluated a total of 450 calvings. Cows with Nc had a lower

incidence of PM compared to cows with an Ac status (13% vs. 41%, respectively; P <

0.001). During the cool season, primiparous cows had the highest incidence of PM

compared to primiparous cows in warm season or multiparous cows in either season (P <









0.01). Cows with Ac were more frequently diagnosed with CE than those with Nc (AOR

= 2.8, 95% CI 1.7-4.9, P < 0.001). A higher incidence (38.2%) of CE was found in cows

diagnosed with PM (AOR = 2.2, 95% CI 1.1-3.9, P < 0.005). The RT in cows diagnosed

with PM increased between 48 to 24 h before diagnosis of PM and continued to increase

until reaching a maximum RT of 39.20C 0.05 on Day 0 (day of diagnosis). In cows

with PM and fever at diagnosis the RT started to increase between 72 to 48 h before the

diagnosis of PM, and continued to increase until reaching a maximum RT of 39.70C

0.09 on the Day 0 (day of diagnosis). In cows with PM and no fever at diagnosis there

was not a significant daily increment of RT before the diagnosis of PM. Cows without

metritis did not show any variation in RT during the first 13 days postpartum. There were

no detected differences in accumulated pregnancy rate by 150 days post partum (mean=

50%) among normal cows and cows experiencing PM. A season effect was detected

(Cool season [40 %] > than warm season [28 %; P < 0.02]).

Occurrence of PM was higher in cows experiencing an Ac. Primiparous cows had a

greater incidence of PM in the cool season for both normal and abnormal calvings. In

contrast, multiparous cows showed no seasonality in the occurrence of PM. Evaluation of

daily RT was able to distinguished PM with fever and PM without fever. Sequential

increases in RT on two consecutive days prior to the actual diagnosis can serve as a

predictor of PM with fever. Abnormal calving, and PM were risk factors for clinical

endometritis. Pregnancy rates were comparable between cows with normal or abnormal

calving status cows, regardless of the occurrence of PM.














CHAPTER 1
INTRODUCTION

Puerperal metritis has multiple factors contributing to its etiology, severity, and

duration. It occurs during the period from calving to when the anterior pituitary gland

becomes responsive to gonadotrophin releasing hormone (GnRH) approximately 7 to 14

days postpartum (Olson et al., 1986). In puerperal metritis there is inflammation of all

layers of the uterus and it is characterized by the presence of a fetid, watery reddish-

brown vulvar discharge (Lewis, 1997). Sheldom et al. (2006) attempted to standardize the

clinical definition of puerperal metritis. Their definition includes clinical symptoms such

as decreased milk production, dullness, or other signs of toxemia with fever (> 39.50C)

within the first 21 days postpartum.

Factors that predispose cows to puerperal metritis have been reviewed previously

(Curtis et al., 1985; Correa et al., 1993; Markusfeld 1984; Bartlett et al., 1986). However,

in some cases puerperal metritis was classified as a disease complex without

distinguishing clinical severity or presentation, making studies difficult to compare

(Lewis, 1997).

Prevention and early treatment of puerperal metritis is more economical than let

this condition progress to a stage where treatment is not beneficial on cost effective

(Bartlett et al., 1986). Thus the need to identify puerperal metritis early post partum and

provide treatment, monitoring of attitude and fever during the first 10 days postpartum

(Upham, 1996). Rectal temperature is an indicator of the core body temperature, and is

used as a diagnostic method to determine whether the cow has fever. Fever is the result of









a complex communication between the peripheral immune system and the brain in

response to infection, inflammation and/or trauma, and is clinically characterized by a

rise in body temperature (Leon, 2002). Fever can be initiated by bacterial

lipopolysaccharides (LPS) acting directly as exogenous pyrogens; or indirectly by

activating liver macrophages (Steiner et al., 2006) inducing the production of endogenous

pyrogens such as interleukin (IL)-6, IL-1, and TNF-a (Luheshi, 1998).

It has been difficult to establish a minimum rectal temperature to define fever in

postpartum-health monitoring protocols because of the broad range of rectal temperature

described in the literature (Smith et al., 1998; Upham, 1996; Drillich et al., 2001; Zhou et

al., 2001; Sheldon et al., 2004), and the multiple factors that affect rectal temperature

values (Rebhun, 1995; Rosenberger, 1979). Kristula et al. (2001) evaluated postpartum

rectal temperature: 48% of cows that calved normally had at least one daily temperature

above 39.1C compared to 93, 83, 100 and 78% for cows with retained placenta, mastitis,

puerperal metritis and dystocia, respectively. Kristula et al. (2001) concluded that rectal

temperature (per se) is not enough to determine whether antibiotic treatment is needed for

postpartum cows.

Despite the common use of rectal temperature of postpartum cows to evaluate

health, there is a lack of research on the value or significance of rectal temperature and

calving status as tools to diagnose puerperal metritis in dairy cows. Understanding the

factors that predispose cows to puerperal metritis, and understands rectal temperature

responses in cows at risk of developing puerperal metritis should aid in the formulation of

appropriate postpartum health monitoring strategies. In addition, early diagnosis and






3


treatment of puerperal metritis could improve later reproductive performance and

productivity.

Our study objectives were to evaluate the effect of abnormal calving, parity,

season, and rectal temperature on the incidence of puerperal metritis within the first 13

days post partum: and to report the effect (if any) of puerperal metritis and clinical

endometritis on reproductive performance.














CHAPTER 2
LITERATURE REVIEW

The Bovine Uterus

The uterus consists of a tubular structure formed by the cervix, uterine body, and

the uterine horns. The non gravid uterus is located, depending on factors such as age,

breed, and parity of the cow, on the pelvic cavity dorsal to the urinary bladder and ventral

to the rectum. The main function of the uterus is to accept a fertilized ovum which

becomes implanted into the endometrium, and derives nourishment from blood vessels

developed exclusively for this purpose.

The uterus is derived from the embryonic mullerian or paramesonephric ducts, the

tubes will develop into the gonaductal system, giving rise, in the case of the female, to

the oviduct, uterus, cervix and cranial vagina. In mammals the mullerian ducts fuse from

cranial to caudal. The female mullerian ducts start to fuse from the most cranial part,

caudally to form the oviducts, uterus, the cervix and the anterior part of the vagina. The

fusion of the ducts happens in the medial walls, forming the paramesonephric septum.

This partition tends to disappear in time, fusing and forming a single tube. In the cow, the

ducts fuse forming a septum that separates the two horns giving the characterization of a

bipartite type uterus.

Both sides of the uterus are attached to the pelvic and abdominal walls by the broad

ligaments, from were the uterus receives its blood and nerve supply. The middle uterine

artery, a branch of either the internal iliac artery or the external iliac artery, provides the

blood supply to the uterus in the region were the fetus develops. The cranial uterine









artery, a branch of the utero-ovarian artery, supplies blood to the ovary by the ovarian

artery and to the anterior extreme of the uterine horns by the cranial uterine artery. The

utero-ovarian artery runs closely along the surface of the corresponding veins. The artery

terminates by giving rise to a small branch to the tip of the uterine horn and oviduct. All

lymphatics of the uterus are in communication, the cervix drains towards sacral nodes

while the body of the uterus and oviducts drain towards the external iliac nodes, also

there is some drainage towards internal iliac nodes.

The uterus can be divided in three layers. From the lumen to the abdominal cavity

can be divided in endometrium, myometrium and perimetrum or serosa. The inner

portion of the uterus is composed of the mucosa and submucosa. Both compose the

endometrium, characterized by having many endometrial folds. Histologically the

mucosa is characterized by a simple pseudostratified cylindrical cell surface during most

stages of the cycle; few ciliated cells are present, especially in the multiparous cow, less

than 1%. The submucosa is predominantly connective tissue and houses the uterine

glands. The uterine glands develop in the mucosa, projecting to the submucosa where

they coil. Their basic structure is similar to those of a mucosal epithelial surface

(Marinov and Lovell, 1968). The surface of the endometrium of the cow is covered by

non glandular areas denominated caruncles. The caruncles are highly vascularized areas

that give rise to the maternal portion were the cotyledons will attach.

The endometrium is covered by two smooth muscular layers that constitute the

myometrium. In contact with the endometrium a circular smooth muscle layer is present,

it is in this portion where the blood vessels penetrate the submucosa stratum. Up on this

portion, the longitudinal layer of smooth muscle is present, easily recognizable for it









creases, or small ridges, palpable during the early post partum of the cow. The outer layer

of the uterus is the serosa or perimetrum, quite thin and almost transparent covering the

entire uterus and continues dorsally and covers the mesosalpinx. This portion of the

uterus will be in contact with the pelvic and abdominal cavity of the cow.

Uterine Involution

In the pregnant cow, during the time period 1 month before and 1 month after

parturition, several metabolic and endocrine events take place. As the cow enters the

transition period, the dam has to be prepared for the impending parturition, and the uterus

and ovaries must return to a certain stage to be prepared for a new pregnancy (Kindahl, et

al., 2004). Most of these processes are due to or reflected in endocrine changes.

The uterine lumen holds approximately 70 kg at the time of parturition, including

fluids, fetal membranes, and the fetus. Approximately half of this weight contains fluids

and the other half includes the fetus and the fetal membranes (Mortimer et al., 1997).

However, after parturition the uterus of the cow, is a large, flabby sac, nearly a meter

long and 9Kg in weight (Gier and Marion, 1968). The rapid re-organization along with a

fast reduction in the diameter of the organ constitute probably a protective mechanism

against ascending infections (Mortimer et al., 1997).

Involution of the uterus involves loss of intraluminal fluids, reduction in size, and

endometrial repair. During the first two days after calving, the expelled fluids are

serosanguineous, and change in character after caruncular dissolution begins (Olson et

al., 1986; Rasbeck, 1950) describes the elimination of decidual detritus of caruncular

tissue starting 3 to 4 days after parturition and increasing until the 9th day, and gradually

becomes mixed with blood originating from hemorrhages on the surface of the caruncles.

Gier and Marion (1968) found a considerable quantity of blood in the uterus after









parturition, becoming mixed with sloughed caruncular material after day four, which

changed by day 12 post partum to a more lymph like fluid which decreases in quantity by

day 23 postpartum (Figure 2-1) (Gier and Marion, 1968). This discharge is named uterine

lochia, which consist of mucus, tissue, detritus, and blood, that commenced 3 to 4 days

and decreasing until the ninth day post partum (Roberts, 1986). Lochia may assume

different appearances from white, yellow- white or a grey mucopurulent character toward

the latter part of the puerperal period. This discharge is considered a normal process of

uterine involution (Roberts, 1986).


1500



S1000



500



0
1 5 10 15 20
Days postpartum

Figure 2-1. Changes in lochia volume during the first 20 days postpartum in dairy cows
(Gier and Marion, 1968).

During the post partum period, re-organization and involution of the uterus occurs

and a small opening in the cervix remains for elimination of uterine contents (Wehrend et

al., 2003). Reduction in the uterine weight, diameter and length occurs in a decreasing

logarithmic scale (Figure 2-2) (Gier and Marion, 1968). This reduction in size may be

explained partially by peristaltic contractions at intervals of 3 to 4 minutes during the first









day postpartum and continuing through the second day (Gier and Marion, 1968). Bajcsy

et al. (2005) evaluated the contractility of the postpartum uterus by recording and

quantifying the frequency, amplitude and duration of intrauterine pressure changes

between 12 hours to 48 hours after parturition.


5 10 15


Figure 2-2. Uterine changes in A -Weight; m-Diameter and *-Length during the first 20
days postpartum in cows (Gier and Marion, 1968).

In that study (Bajcsy et al., 2005) it was reported that mean frequency of uterine

contractions were 8.9 contractions per hour at 12 h post partum with a range of 6 to 11

contractions every hour. The mean contraction frequency decreased to 1.8 contractions

per hour at 48 h post partum. The largest drop in mean values occurred between 12 and

24 h post partum, and the frequency decreased by 46% of the initial mean value. Results

from amplitude showed an initial individual mean value of 40 mmHg at 12 h. Changes in

mean amplitude showed a similar pattern as frequency, with highest initial mean values at









12 h postpartum (19.6 mmHg) and a reduction of 16% of the starting values by 48 h

postpartum (3.2 mmHg). The most marked drop (42 %) in this response also occurred

between 12 and 24 h post partum. Mean duration at 12 h was 89.8 seconds and varied

between 102.5 at 36 h postpartum to 67.9 seconds at 48 h. In that study (Bajcsy et al.,

2005), the relationship between uterine activity and blood calcium levels also was

investigated. Neither frequency, amplitude and duration showed a significant relationship

to blood Ca2+ levels in cows at any of the four recording times.

Diameter of the cervix at a given time after parturition is influenced by the

involution process of the reproductive tract (Oltenacu et al., 1983). Gier and Marion

(1968) reported that involution of the cervix measured in slaughtered cows. The diameter

of the cervix at day 2 after parturition was about 15 cm; 9 to 11 cm at 10 days, 7 to 8

centimeters by 30 days, and 5 to 6 cm by 60 days. Wehrend et al. (2003) reported results

of cervical involution in vivo by measuring the cervix canal from the time after expulsion

of the calf up to the tenth day post partum. They showed that the cervical folds were in

constant formation and were detected at the third day postpartum. In addition, the

organization of cervical folds started from cranial and continued caudally. After the

expulsion of the calf, a reduction of the opening from 26.9 1.3 cm to 1.9 0.3 cm on

the seventh day post partum was observed (Figure 2-3 and 2-4). Furthermore, up to the

third day post partum, the lumen of the cervix was detected in all cows, disappeared from

the fourth to the seventh day, and re-appeared at the tenth day after parturition. The

authors concluded that this opening was related to allow further elimination of uterine

fluid contents (Wehrend et al., 2003).



































0 2 4 6 8 12
Hours post partum


Figure. 2-3. Reduction
et al., 2003).



30


25


20


J 15-


C 10 -


5-


0 --


16 18 24


of the cervix opening during the first 24 h post partum (Wehrend


0 1 2 3 4 5 6 7 8 9 10
Days post partum


Figure 2-4. Reduction of the cervix opening during the firstl0 days post partum
(Wehrend et al., 2003).









Reduction of the size of the uterus seemed to be produced by early constriction

between 5 to 10 days and a secondary cervix opening relaxation produced at 10 days

postpartum, at the time of final sloughing of the caruncular masses (Gier and Marion,

1968). Morrow et al. (1966), reported that slow regression of the uterus occurred during

the first 4 to 9 days after parturition followed by an accelerated regression during the

period 10 to 14 days after calving. In addition, the maternal portion of the placentome

(caruncle) after the removal of chronic villi, remained as a loose mass of tissue

approximately 70 mm long, 35 mm wide, and 25 mm thick (Gier and Marion, 1968).

Caruncular blood vessels constricted rapidly and were nearly occluded within 2 days

postpartum. However, blood continued to flow from protruding arterioles and contributed

to the luminal fluids for a least 10 days (Gier and Marion, 1968).

Archbald et al. (1972) described the histological involution of the uterus during the

first 60 days post partum. During the first day postpartum, the caruncular epithelium was

degenerated, as well as the intercaruncular epithelium. The myometrium was edematous

with degenerated muscle fibers, and fetal chorionic cells were still present but some were

necrotic. By the fifth day post partum, the caruncular epithelium was regenerated, but

started to be lost given the sloughing of the superficial layer of the caruncles. The

intercaruncular epithelium was regenerated with the exemption of the basilar areas of the

cells. The endometrial glands were degenerated in the basilar area with some ducts

dilated. Vacuolization of the myometrium was almost generalized. However, the nuclei

of the muscle cells were normal and the fetal chorionic cells were necrotic, mineralized

and surrounded by macrophages (Archbald et al., 1972).









At visual examination, most of the necrotic layer is removed by day 10 post partum

and by 15 days post partum all of the caruncular mass that had been involved in the

placentome are sloughed, leaving only stubs of blood vessels extending beyond the

surface of the stratum compactum (Gier and Marion, 1968). By 19 days post partum, the

arterioles within and beyond the stratum compactum disappeared (Gier and Marion,

1968). The histology showed by day 15 post partum that the caruncular epithelium was

absent in some areas, but a cuboidal or flat epithelium was present in other areas. The

intercaruncular epithelium was regenerated with few areas of degeneration in the basilar

area of the cell and the myometrium was shrunken in size, with vacuolation of the muscle

fibers and fetal chorionic cells were not present. By day 19 post partum, the caruncular

epithelium was covered by a columnar to cuboidal epithelium over the entire caruncule.

A cuboidal epithelium was also present over the entire intercaruncular area. Endometrial

glands were normal and non-secretory. The myometrium had few necrotic fibers and

continued to shrink in size. By day 19 postpartum fetal chorionic cells were not present

(Archbald et al., 1972).

The caruncles already reduced their size at 19 days postpartum to 15 to 20 mm in

diameter, and by day 39 post partum, are reduced to smooth knobs of 10 to 15 mm in

diameter composed of circular cones of 8 to 10 mm across the base and 4 to 6 mm across

the crown by 50 to 60 days postpartum (Gier and Marion, 1968).

The final gross involution of the uterus has been reported to be completed in the

previously pregnant horn by day 25 post partum in normal cows, and 30 days post partum

in cows with postpartum disease (Morrow et al., 1966). In contrast, the average interval

for completion of gross uterine involution for multiparious cows was 40 days, and the









interval was affected by parity, season, and stress factors (Marion et al., 1968). The

histological characteristics of the uterus by day 31 to 45 days postpartum showed that the

caruncular and the intercaruncular tissue had epithelium present over the entire structures

(Archbald et al., 1972). The endometrial glands were still normal and non-secretory. The

myometrium was normal, and regained the normal shape and sarcoplasm. By day 60 post

partum the uterus regained it's normal histology. The caruncular area was covered by a

columnar type epithelium, somewhat pseudostratified. Many pigment-bearing histocytes

were present in the stratum compactum and neutrophils were not observed. There were

many plasma cells distributed through the stratum compactum and spongiosum. The

intercaruncular area was covered by a single layer of epithelium and consisted of

cuboidal and columnar cells. Numerous mast cells and histocytes containing hemosiderin

were in the stratum compactum. Numerous mast cells were in the stratum spongiosum,

and the blood vessels of this layer appeared normal. The endometrial glands appeared

normal and non-secretory. The myometrium and the subserosa layer appeared normal,

but were infiltrated by numerous mast cells (Archbald et al., 1972).

Endocrine Changes During Early Post Partum

A balanced, coordinated endocrine system is important for normal reproductive

function. A graphical representation of this complex process is presented in (Figure 2-5).

These changes involve the gonadotrophin releasing hormone (GnRH) from the

hypothalamus, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin

(PRL) from the adenohypophysis, prostaglandin F2a from the uterus, progesterone (P4)

from the corpus luteum, and estrone sulfate (EiSO4), and estrone (Ei), from McDonald

(1980).







14


Cortisol is recognized as a stress hormone and is also responsible for the regulation

of prostaglandin synthesis (Hafez, 1993). In the fetus prior to parturition,

adrenocorticotropin hormone (ACTH) is released from the pituitary gland, stimulating

the adrenal glands to release cortisol. The fetal cortisol is central to the survival of the

neonate, as well as for induction of lactation and parturition in the cow (Nathanielsz

1993).


NG/ML PG/ML
PRL P4 V E ES04 PGF M
200--7 E SO 2000--8000 -4000

P4 PRL PGF M --7000

160- // / 60- -3200
-6000

5000
120 1200-500 2400
-4000 -

o80--3 .' \800\ -3000 -1600

2 *Qo oi
-2000
40- 400- 800
-I '-1000

O I I I I I I i
-13 -11 -9 -7 -5 -3 I 5 7 9

DAYS FROM PARTURITION
Fifure 2-5. Pooled within cows least squares regressions of plasma progesterone (P4),
estrone sulfate (EiSO4), estrone (Ei), prolactin (Prl), and 13, 14-dihydro, 15-keto
prostaglandin F2, (PGFM) during the periparturient period (Eley et al., 1981).

As parturition approaches, the fetal adrenal cortex becomes increasingly sensitive

to adrenocorticotropin (ACTH). Mean fetal levels of corticosteroids are within 5.0 ng/ml

at 20 days to 9.3 ng/ml at 10, and 25 ng/ml 4 days pre partum, and the level progressively

increases to a mean of 74 ng/ml on the day of calving (Hunter, et al., 1977). At 30 days

pre partum, only 50-60% of the corticosteroids fraction is cortisol, whereas in the last 10









days of gestation the proportion of cortisol rise to over 90% (Hunter, et al., 1977).

Dystocia may affect levels of cortisol. Severe dystocia resulted in lower calf rectal

temperature, reduced serum cortisol and increased serum glucose (Bellows and

Lammoglia 2000). Close to parturition the release of cortisol induces the 17a-

hydroxylase enzyme in the fetal membranes to start the conversion of progesterone to

estrogen compounds (Hoedermarker et al., 1990).

Progesterone is secreted by luteal cells of the corpus luteum and by the placenta,

and it secretion is primarily induced by LH (Hafez, 1993). The function of progesterone

is to prepare the endometrium for the implantation of the embryo and for the maintenance

of pregnancy. In contrast to sheep and horses that exhibit placental progesterone

production, the corpus luteum of the cows maintains pregnancy by luteal cells P4

production which fluctuates between 6 to 15 ng/ml through gestation (Knickerbocker et

al., 1986). During the peripartum period there is approximately a 20% reduction of the

levels of progesterone within 4 to 1 week before parturition (Edqvist et al. 1978). A

second phase of reduction can be distinguished with a more abrupt decrease of

progesterone during the last 2-3 days prior to parturition, and this is interpreted as pre-

partal luteolysis (Edqvist et al. 1978). Maternal blood progesterone falls towards term,

decreases rapidly over the last 48 to 36 h post partum to levels of less than 1 ng/ml

(Hunter et al., 1977; Eley et al., 1981), and it is not until day 16 post partum that cows

increase their P4 levels over 1 ng/ml (Eley et al., 1981). The rise in fetal corticosteroids

during the last month of gestation is reflected by the increment of estrogens levels

(Knickerbocker et al., 1986).









Cotyledonary tissue is the main source of placental estrogens (Hoedermarker et al.,

1990). Characterization of El and pooled 17-a/0 estradiol sulfate (E2SO4), reflect similar

patterns to estrone sulfate (EiSO4). Thus, placental steroid activity is most commonly

determined by concentrations of plasma estrone sulfate (EiSO4) (Knickerbocker et al.,

1986).

Maternal plasma concentration of E1SO4 increases gradually from a baseline of 30

to 60 pg/ml before day 60 to approximately 500 pg/ml by day 100 of pregnancy (Eley et

al., 1979). A rapid elevation then occurs until day 150 when estrone sulfate

concentrations approach 3000 pg/ml. After 150 days of pregnancy E1SO4 remains

constant until approximately day 240 when estrone sulfate increases rapidly (Thatcher et

al., 1982). Concentrations of estrogens decline abruptly in association with delivery of

the concepts (calf and placenta). Basal concentrations of P4, El and E1SO4, are low by

24h after parturition and remains low for approximately 14 day after parturition (Eley et

al., 1981).

Prostanoids (prostaglandins and thromboxanes) are forms from arachidonic acid by

cyclooxygenases (COX). At least two different COX enzymes, which are isoenzymes,

have been found: COX-1 mainly a constitutive and COX-2 an inducible enzyme COX-2

is involved in both physiological (luteolysis, parturition) and pathological (inflammatory)

processes (Kindahl et al., 2004). The most important product linked to reproduction is

PGF2,. The metabolism of PGF2, is very rapid to its metabolite 15-keto-13,14-dihydro-

PGF2, (referred to 15-ketodihydro- PGF2 or PGFM) (Granstrom and Kindahl 1982). The

uterus is the primary source of PGF2, during the early postpartum period, and the









caruncles also contribute to the synthesis and metabolism of PGF2a (Guilbault, et al,.

1984).

Utero ovarian venous prostaglandin F2a levels remained relatively constant at

around 500 pg/ml by 48 to 36 h before calving when they increased rapidly especially

over the last 24 h gestation, reaching peak levels of around 5 ng/ml during labor (Hunter,

et al., 1977).

During the prepartum period levels of PGFM are negatively correlated to P4 (Eley

et al., 1981). A final abrupt increase in PGFM is accomplish by a decline in progesterone

associated with CL regression just prior parturition (Eley et al,. 1981). Increases in

PGFM occurs during the postpartum period (0 to 11 days) after luteolysis and delivery of

the calf (Eley et al., 1981).

The major increase in concentration of PGFM in plasma during the periparturient

period occurs 1 to 4 days postpartum with concentration returning to base level by day 15

(Eley et al,. 1981). Prolactin acts on the central nervous system to induce maternal

behavior (Hafez, 1993). Prolactin concentration begins to rise from a variable baseline of

approximately 80ng/ml 2 at weeks prepartum and reach peak values of 200 to 400 ng/ml

just prior calving (Eley et al., 1981). The levels of prolactin are maintained elevated up to

the third day post partum, when they return to baseline levels of 80ng/ml (Eley et al.,

1981).

Uterine Defense Mechanisms

The immune system functions to defend the host against infections. Host defense

requires different recognition systems and a wide variety of effector mechanisms to seek

out and destroy the wide variety of pathogens in their particular habitats within the body

and it's external and internal surfaces (Janeway, 2005).









Innate immunity serves as a first line of defense. Once body surfaces (skin,

mucosa), secretions and anatomical barriers are breached, macrophages and neutrophils

of the innate immunity system provide a first line of defense against many common

microorganisms. However, they do not always eliminate the infection. Consequently, the

adaptive immune system has evolved to provide a more versatile and specific means of

defense, and increased protection against subsequent reinfection (Janeway, 2005).

The inner layers of the uterus are part of the mucosal immune system with

structural and functional similarities, and common lymphocyte trafficking network with

the intestinal, bronchial, nasal, ocular, salivary and mammary gland tissue (Ogra et al.,

1999). The most prominent difference between the uterus and the other mucosal surfaces

is the lack of organized secondary lymphoid nodules analogous to Peyer's patches and

bronchus-associated lymphoid tissue (BALT) (Head and Billingham, 1986). Furthermore,

in the majority of domestic species the uterus is exceptional among mucosal tissues

because the ovarian steroid hormones have considerable effects on immune events

(Lewis, 2004).

To the complexity of this specialized immunological system a production stressor

factor is added, and the modem dairy cow is unique in her experience of repeated lifetime

cycles of pregnancy and parturition followed by lengthy lactations producing high

volumes of milk (Mallard et al. 1998). In addition, the onset of lactation imposes

tremendous physiological challenges to the homeostatic mechanisms of the cow which

influences immunological responses (Goff and Horst, 1997).

Innate Uterine Defense

Innate immune mechanisms act immediately, and are followed by early induced

responses which can be activated by infection without generating an immunological









response (Janeway et al., 2005). The uterus has it own innate immunological system

which includes anatomical barriers, uterine and cervical secretions and bacterial

antagonism. Once this defense mechanism is breached, such as in the case of parturition,

the most important line of defense is the innate cell defenses composed of phagocytic

cells such as neutrophils, macrophages, effectors cells basophilss, mast cells, and

eosinophils) which have the capacity to induce an influx of other immunological factors

(BonDurant, 1999).

Anatomical barriers

The uterine environment is protected by anatomical characteristics that act as

barriers to the external environment of the cow. These physical barriers consist of the

vulva, vaginal vestibule and cervix which are all covered by a mucosal layer that

produces and secretes specific and nonspecific immunological factors (Senger, 1999).

The cranial vagina as well and the fornix vaginae are characterized by columnar

epithelium which is highly secretary under the influence of estrogen. The cranial vagina

is characterized by some ciliated columnar epithelium that participates in the process of

mucus elimination (Senger, 1999). The caudal vagina is characterized by a stratified

squamous epithelium and the dermal epithelium which have an exfoliative process that

eliminates microbes adhered to epithelial cells. The secretary function of the vagina

varies according to the endocrine status. During estrus, under the influence of estrogen,

the stratified epithelium becomes thickened, protects the vagina during copulation, and

reduces access of microorganisms to the vasculature of the submucosa (Senger, 1999).

Mucus secretion acts as a mechanical flushing system for pathogens and antigens that are

captured by mucoproteins (Senger., 1999).









The two labia of the vulva are in close contact by the action of the constrictor

vulvae muscle which minimizes entrance of foreign material to the vulva. The skin of the

labia is part of the integument and has numerous sebaceous and sweat glands which

produces antimicrobial factors (Senger, 1999). The cervix isolates the uterus from the

external environment by forming an anatomical barrier trough of multiple folds and three

rings which protrude into the cervical canal (Senger, 1999).

During parturition there is dilation of the cervix which allows expulsion of the

fetus. This event removes the cervical seal, allowing the interior of the uterus to be in

contact with the environment of the cow, and does not return to it's normal anatomical

form in cows with a normal puerperium until 7 to 10 days post partum (Wehrend et al.,

2003).

Uterine and cervical secretions

The internal epithelium of the cervix and the uterus contain cells that secrete

mucus, which is composed of glycoproteins (mucins). Mucus traps microorganisms and

prevents them from reaching and colonizing the mucosal epithelium. Mucus also contains

lysozymes that help degrade bacteria, antibodies that prevent microbes from attaching to

mucosal cells, lactoferrin that binds iron making it unavailable to microbes, and

lactoperoxidase that generates toxic superoxide radicals that kill bacteria (Ogra et al.,

1999).

The complement system is made up of many distinct plasma proteins that react

with one another to opsonize pathogens and induce a series of inflammatory responses

that help prevent infections (Janeway et al., 2005). The function of complement is to

recruit mononuclear phagocytes, initially circulating neutrophils and tissue macrophages,

that remove the microbes phagocytosiss) and release additional, different mediators (e.g.,









cytokines, prostaglandins, leukotrienes, etc.) that enhance the inflammatory process

(Janeway et al., 2005). Complement is an important component of the innate

immunological reaction in the reproductive tract of the cow, opsonizing and lysing

bacteria (Corbeil., 2002). Moreover, it's activation seems to be due to the interaction of

immunocomplexes formed by antigens and immunoglobulin, specifically IgG2 (Corbeil.,

2002). Increased concentrations of complement C3 are found in vaginal and uterine

secretions of infected cattle (Kania et al., 2001). Although the exact source of C3 in the

uterus and vagina of cattle is still unknown, it has been proposed that increased

concentration of C3 in vaginal and uterine secretions result from serum-derived

complement (Kania et al., 2001). In rats, C3 production is positively influenced by

estrogen and inhibited by progesterone (Hasty et al., 1994).

Lactoferrin is a ferric protein found in exocrine secretions of the mucosal surfaces

of the cow (Dixon and Gibbons, 1979). It is produced and secreted by the exocrine glands

of the uterus, epithelial cells of the cervix and ampulla of the uterine tube (Inoue et al.,

1993). Lactoferrin acts as a powerful bacteriostatic, bactericidal, fungicidal and virucidal

agent (Ogra et al., 1999).

Another serum protein that is secreted in the reproductive tract is plasminogen

which is converted to plasmin by a series of specific serine proteases called plasminogen

activators (Lijnen and Collen, 1985). Through the generation of nonspecific, protease

plasmin, plasminogen activators influence numerous physiopathological processes,

including fibrinolysis thrombolysis, and invasiveness, metastasis, and cell migration at

sites of inflammation; this process also involves degradation of the injured tissue and the

plasminogen activators released by macrophages and granulocytes may contribute to this









process by degrading extracellular proteins (Dano et al., 1995). Two types of

plasminogen activators, urokinase (u-PA) and tissue type (t-PA), are present in

endometrial tissues and uterine fluids of the cow, and are thought to be involved in the

resolution of endometritis in cows (Moraitis et al., 2004).

Peroxidase activity has been measured in many mucosal secretions (Ogra et al.,

1999), including the uterus. The activity of peroxidase is derived from enzymes

synthesized by exocrine glands and secreted onto mucosal surfaces by the glands (Ogra et

al., 1999). It produces toxic oxygen-derived products that act as a bactericidal agent. In

addition, leukocytes present in the uterus can also produce large amounts of hydrogen

peroxide (Hansen et al., 1987) which enhance lactoferrin activity (Ogra et al., 1999). In

addition, nitric oxide is another exocrine product of the uterine mucosa (Lapointe et al.,

2000), and produces it's bactericidal activity by producing toxic nitrogen oxides on the

surface of the mucosa (Janeway et al., 2005).

Bacterial antagonism

The normal microbial flora of the bovine urogenital tract is made up of a dynamic

mixture of aerobic, facultative anaerobic and strict anaerobic micro-organisms (Hafez

1993). The normal flora act as an inhibitory flora to help prevent infections by

reproductive pathogens (Corbeil and BonDurant, 2001). The normal microbial flora of

the reproductive tract is composed of bacteria of the genus Staphylococcus, Streptococcus

and the coliform group (Hafez 1993). Contrary to what it is reported in humans and rats

(Reid et al. 1985), the number of lactobacilli appears to be lower in cervix and vaginal

fluids of the cows (Otero et al., 2000). In contrast, Coagulase-negative Staphylococcus

and a-haemolytic Streptococcus bacteria are predominant in the vagina of the cow (Otero

et al., 2000). This normal flora of nonpathogenic bacteria compete with pathogenic









microorganisms for nutrients and for attachment sites on the epithelial cells and produce

antimicrobial substances, such as lactic acid (Janeway et al., 2005).

Innate cell component

The main phagocytic barrier in the uterus is provided by the invasion of neutrophils

in response to bacteria (Sheldon and Dobson, 2004) that are present on the surface of the

endometrium and into the lumen (BonDurant, 1999). Consequently, neutrophils are the

earliest and most important phagocytic cells to be recruited from the peripheral

circulation to the uterine lumen, killing internalized bacteria and contributing to the

formation of pus when the phagocytes die (Sheldon and Dobson, 2004). Experimental

approaches with bacteria and bacterial components to induce an influx of neutrophils

have been reported (Zerbe et al., 2001), However, when a non specific inflammation was

induced, such as in the case of LPS inflammation, intracellular killing by uterine

neutrophils was reduced compared to circulating neutrophils (BonDurant, 1999). This

suggests the importance of cytokines in uterine infections. In addition, the experimental

infection with E. coli and A. pyogenes, the predominant bacteria in cases of bovine

uterine infections, resulted in high concentrations of viable neutrophils in uterine

secretions. However, the large numbers of neutrophils were not able to eliminate the

bacterial infection present (Zerbe et al., 2001). Consequently, other unknown factors may

influence leukocyte activity (Mallard et al., 1998).

Macrophages are also important in the uterine immune response. Macrophages

sense bacteria or endotoxins through toll-like receptors (TLRs) which are the principal

signaling molecules through which mammals sense infection (Beutler et al., 2003). The

activation of the macrophages leads to the production of cytokines such as tumor necrosis

factor-alpha (TNFa), and interleukins (IL-1, IL-6, IL-8). These cytokines alert other









immune cells such as neutrophils or lymphocytes, by supporting the development of an

adaptive immune response (Beutler et al., 2003). In addition, macrophages induce the

transmigration of leukocytes to inflamed tissue by inducing the expression of selections,

by TNFa. Selectins cause the rolling of leukocytes on endothelial cells which are attracted

to the site of infection by a concentration gradient produced by the above mentioned

cytokines, especially IL-8 (Janeway et al., 2005). In addition to neutrophil and

macrophage migration, mast cells and eosinophils are present on the surface of the

uterine mucosa. Both eosinophils and mast cells have high affinity receptors that bind IgE

antibody (Janeway et al., 2005). The eosinophils also release inflammatory mediators and

antimicrobial factors such as peroxidase and lytic enzymes (BonDurant 1999). The

number of mast cells and eosinophils are known to vary with the stage of the estrous cycle

(Likar et al., 1964; Matsuda et al., 1983). Little is known about bovine uterine mast cells,

but they secrete some of the usual mast cell mediators, such as histamine, leukotrienes,

prostaglandins, heparin, and proteinases, as well as proinflammatory cytokines and Th2

related cytokines. Mast cell and eosinophil mediators increase vascular permeability

which results in the subsequent influx of other immune cells and serum immunoglobulins

to the uterine lumen (Corbeil et al., 2005; BonDurant, 1999).

Humoral-mediated immunity

Antibodies are thought to be the most protective arm of the immune response

process in defense against extracellular pathogens (Corbeil, 2002). The distribution of

immunoglobulins in external secretions is vastly different from that found in serum.

Plasma cells secrete different immunoglobulin after a period of differentiation and

isotype switching (Janeway et al., 2005). Different isotypes and allotypes of

immunoglobulins are present in different compartments of the body, and the isotype, and









quantity may vary depending on the antigen stimulation (Janeway et al., 2005).

Immunoglobulin-A (IgA) is the immunoglobulin mainly found on mucosal surfaces

(Ogra et al., 2002). However, in the vascular system, secretary IgA (SIgA) is the

predominant form, and is principally found as a dimer (Butler, 1972). It is the

predominant immunoglobulin in nasal secretions, tears and saliva. In cervico-vaginal

secretions, the relative concentration is lower than IgG, but still much higher

proportionally to serum (Duncan et al., 1972). The majority of IgA found in external

secretions is derived from local synthesis by plasma cells rather than selective transport

from blood (Duncan et al., 1972). Immunoglobulin A is the major immunoglobulin class

in the superficial portion of the reproductive tract and immunoglobulin-G (IgG) is the

major class in secretions of the uterus, oviduct and follicular fluid (Corbeil et al., 1976;

Whitmore and Archbald 1977). Antigenic stimulation of the bovine uterus results in

specific antibody response to IgG class, whereas vaginal stimulation leads to an IgA

response in vaginal secretions. Two subclasses of IgG are predominantly found in the

serum of cattle, IgG1 and IgG2 (Butler, 1973). The rate of transport of IgG1 into vaginal

mucus is more rapid than that of IgG2 (Curtain et al., 1971). In contrast to IgG2, IgG1 is

present in higher concentration in the uterus and vagina (Curtain et al., 1971). However,

mechanisms by which serum IgG reaches the uterus or vagina are still unknown, because

unlike IgA, IgG does not have a secretary piece to mediate transport across epithelial cell

(Corbeil et al., 2005) In addition, the bovine cervix is capable of local antibody

production (Corbeil et al., 1976), where IgG predominates. Immunoglobulin-G levels in

vaginal mucus exceed those for IgM. Perhaps the lack of detectable IgM in vaginal









mucus can be related to the relative inefficiency of IgM transudation since it is restricted

predominantly to the vascular space (Wilkie et al., 1972).

Cell-Mediated Immunity

All lymphocytes are programmed during their development to specific migration

pathways through the body that enable antigen specific immune response to be

concentrated at certain sites (Janeway et al., 2005). Directed migration, or homing, to

mucosal tissues is controlled by expression of distinct patterns of adhesion molecules on

the lymphocyte cell surface which mediate differential recognition and adherence to the

endometrium in mucosal sites (Janeway et al., 2005). Intraepithelial lymphocytes are

generally present in the stratum compactum of the endometrium, and their number

fluctuates with the stage of the estrous cycle (Vander-Wielen and King, 1984). In

addition, the main lymphocyte population is formed by CD8 type (Cobb and Watson

1995). The induction of local immune responses in the female genital tract of any species

is poorly understood (Corbeil et al., 2005). However, during uterine infections with

Trichomonasfoetus, accumulation of immunocytes, lymphoid nodules, and follicles

(some with germinal centers) were detected under the epithelium and adjacent to infected

glands (Anderson et al., 1996). The kinetics ofisotype-specific antibody responses, mast

cell degranulation and clearance of infection demonstrate that immune defense of the

uterus is related to increasing antibody levels and decreasing detectable subepithelial mast

cells (Corbeil et al., 1974)

Fever Pathway During Infections

The term fever specifically defines elevation of body core temperature that occurs

in defensive response to the entry into the body of pathogenic agents (IUPS Glossary,









2002). Functionally, the onset of fever is manifested by an increase in metabolic heat

production and cutaneous vasocontriction to reduce heat loss from the skin.

The generation of fever is accomplished by the interaction of multiple endogenous

mediators induced by pyrogens, such as lipopolysaccharides (LPS). Kupffer cells, splenic

macrophages, and neutrophils are reported to contribute to the intravascular clearance of

LPS and produce cytokines (Scapini et al., 2000). Presumably fever is mediated by LPS

which stimulates the Kupffer cells located in the liver (Blatteis, 2006), but to a lesser

degree in the spleen. Kupffer cells detect bacterial components such as endotoxins (LPS)

and peptidoglycans through the toll-like receptors on the cell surface, specific for LPS

(TLR-4) (Beuttler et al., 2003). This stimulation induces the production and secretion of

cytokines such as tumor necrosis factor (TNF-a), and IL-1, IL-6, IL-8 (Beuttler et al.,

2003).

Two types of cytokines are responsible for the generation of fever. Pyrogenic

cytokines that induce fever include interleukins IL-1, IL-6, IL-8, PGE2 macrophage-

inflammatory protein-10 (MIP-10), and interferon-y. The other types of cytokines are

endogenous antipyretics which limit the magnitude and duration of fever such as IL-10,

arginine vasopressin (AVP), a-melanocyte-stimulating hormone (a-MSH), and

glucocorticoids (Figure 2-6). Although AVP, a-MSH, and glucocorticoids are not true

cytokines, they still possess endogenous antipyretic properties. Other substances such as

tumor necrosis factor-a have pyrogenic and antipyretic properties, depending on the

experimental conditions (Leon, 2002).

Pyrogenic cytokines in the bloodstream are transported to the preoptic-anterior

hypothalamic area (POA), which is the primary brain site for thermoregulation. The









ventromedial preoptic nucleus (VMPO) is thought to be the fever-producing locus

(Boulant, 2000) where cytokines act. Prostaglandin-E (PGE2) is considered to be the final

fever mediator in the POA. The synthesis of PGE2 in the POA/VMPO is effected through

catalysis of arachidonic acid by cyclooxygenase (COX)-2 and microsomal PGE synthase

(mPGES)-1 selectively upregulated by the pyrogenic cytokines (Blatteis, 2006). In

addition, it is hypothesized that the febrile response to peripheral LPS is not initiated by

pyrogenic cytokines released by LPS-stimulated leukocytes generally, but by PGE2

specifically generated by Kupffer cells activated by LPS (Blatteis, 2006).

Infection, Inflammation or trauma



Macrophages



Endogenous Pyrogens Endogenous
(IL-1, IL-6, IL-8, MIP- Antipyretics (11-10
10, IFNy) AVP, TNFa, a-MSH,
glucocorticoids)




Hypothalamus
T Thermal setpoint



Initiation of effectors mechanisms



FEVER


Fig. 2-6. Pathway of fever development in response to infection, inflammation, or
trauma. (Leon, 2002)









The interactions of endogenous pyrogens and antipyretics are responsible for the

magnitude of fever reaction. Interleukin-1 and other inhibitory cytokines stimulate the

production of IL-1 receptor agonist which prevents further binding of IL-1, and decreases

the effective concentration of IL-1. Interleukin-10 is one of the principal interleukins that

down-regulate the pyrogenic process. IL-10 is a product of T helper-2 subset, and is

induced by pyrogenic cytokines. It also inhibits the LPS-induced production of many

cytokines implicated in fever, including IL-10, IL-6 and TNF-a (Leon, 2002).In addition

the physiological control of the febrile response may prevent extreme elevation in body

temperature. This regulation seems to be dose related, in which a high dose of LPS

during sepsis functions to lower the temperature, thus attenuating fever or producing

hypothermia (Leon, 2002).

General Review of Uterine Infections

The uterus of postpartum cows is usually contaminated with a wide spectrum of

bacteria. However, this is not consistently associated with clinical disease. In the majority

of cows 1 to 4 weeks post partum, species of microorganisms such as a-hemolytic

Streptococcus, Arcanobacterium pyogenes, Enterobacteria, Bacillus spp, Staphylococcus

epidermidis, Staphylococcus aureus, Fusobacterium, Bacteroides spp, Clostridium spp

and Proteus can colonize the uterus (Griffin et al., 1974; Olson et al., 1986). This

bacterial content is negatively correlated with days postpartum. Within the first 15 days

post partum, 90 % of uterine samples have a positive bacteriological culture. However,

by 60 days post partum, the percentage of positive bacteriological cultures are reduced to

10 %. (Elliot et al., 1968). Figure 2-7 represent the percentage of uteri that have positive

bacteria culture during the first 60 days post partum (Elliott et al., 1968).










In addition, the common contamination of the uterus by these bacteria is not

usually associated with clinical signs. Moreover, contamination does not imply infection,

as reflected by the adhesion of pathogenic organisms to the mucosa, colonization or

penetration into the epithelium, and/or release of bacterial toxins that result in the

establishment of uterine disease (Janeway et al., 2005).


100
90 -
80 -
S70-
S60-
50 -
D 40
30 -
S20
10 -
0
0 ---------i-------
15 Day 30 Day 45 Day 60 Day
Days Postpartum
Fig 2-7. Percentage of uteruses from postpartum cows in which bacteria were recovered
in the first 60 days post partum. (Elliott et al., 1968)

The development of uterine disease depends on the immune response of the cow,

and the species and number (load or challenge) of bacteria. The number of pathogenic

bacteria in the uterus of postpartum cows may be large enough to overwhelm uterine

defense mechanisms and cause life-threatening infections, although these are relatively

uncommon (Sheldon, and Dobson 2004).

Endometritis

Pathogenesis

Endometritis is defined as an inflammation of the endometrial lining of the uterus.

Studies by Griffin et al. (1974) and Elliott et al. (1968) have shown that uterine infections

and endometritis are commonly present during the early post partum. In fact, during the









first week post partum 90 % of cows experience some degree of endometritis (Olson et

al., 1986). Inflammation of the uterus (endometrium) and degree of this process is

correlated with the type of bacteria cultured (Studer and Morrow, 1978). Bacteria in the

uterus such as Coliforms, Streptococus, and Arcanobater are associated highly with

endometrial inflammation (Studer and Morrow, 1978). Griffin et al. (1974) found a direct

correlation between Corynebacterium pyogenes infection and degree of endometritis.

However, during the early stages of Corynebacterium pyogenes infections, endometritis

usually was classified as mild or moderate, and if the infection persisted for more than a

week, the degree of endometritis changed to severe (Griffin, 1974). In most instances, the

infection is eliminated within 3 weeks post partum. However, in cows that are unable to

clear the infection, their reproductive performance was compromised (Griffin et al,

1974). Consequently, endometritis is a normal process of uterine involution of the uterus

and classifying a cow as having clinical endometritis less than 21 days post partum will

include a high proportion of cows that are spontaneously resolving the bacterial uterine

infection (Sheldon et al., 2006). Figure 2-8 represents the incidence and degree of

endometritis in uterine biopsy samples during the first seven weeks post partum.

Histopathological disease definition

Endometritis consists for the most part of a diffuse but light infiltration of

inflammatory cells with slight desquamation of the superficial epithelium without

significant vascular changes and with minimal involvement of the uterine glands. The

significance of leukocytes found in the stroma is equivocal in cattle 2 to 3 days after

parturition ( Jubb and Kennedy 1992).










70%

60%

50%

I 40%

-0
a 30% -

< 20% -

10%

0%
1-7 8-14 15- 22- 29- 36- 43-
21 28 35 42 49
Days Postpartum

Figure 2-8. Overall incidence and degree of endometritis in uterine biopsy samples
during the first seven weeks post partum. Mild (e): light neutrophils infiltration in
stratum compactum; Moderate (m): medium infiltration in stratum compactum and in the
upper part of stratum spongiosum; Severe (X): dense infiltration in stratum compactum
and in stratum spongiosum; and no-endometritis (A). (Griffin et al., 1974)

The best indication of endometritis in all species consists of the accumulation of

plasma cells and lymphocytic foci in the stroma ( Jubb and Kennedy 1992). Changes in

the degree of inflammation depends on the duration and severity of inflammation but

generally consists of fibrosis in which leukocytes, lymphocytes and plasma cells

predominate. The endometrium can become thickened by inflammatory tissue where

endometrial glands may become atrophic, flattened, attenuated, or cystic, due to the

periglandular fibrosis ( Jubb and Kennedy 1992).









Clinical disease definition

Studer and Morrow (1978) diagnosed endometritis using uterine biopsy and found

a positive correlation with rectal palpation findings and uterine discharge characteristics.

Consequently, they suggested that use of rectal palpation can be utilized to identify cows

with endometritis (Studer and Morrow, 1978).

Sheldon et al. (2006), defined clinical endometritis as those cows with a purulent

uterine discharge detectable in the vagina 21 days or more post partum, or mucuopurulent

discharge detectable in the vagina after 26 days post partum. This definition was based on

the finding from a study conducted by LeBlanc et al. (2002), who reported that presence

of purulent vaginal mucus or a cervical diameter >7.5 cm 21 days or more post partum;

had a negative effect on reproductive performance. These results agree with those of

Oltenacu et al,. (1983), who concluded that the diameter of the cervix estimated by rectal

palpation 12 to 26 days post partum (i.e., diameter > 5.5 cm primiparous and 6.0 cm

multiparous) was the best indicator of subsequent poor reproductive performance.

However, Oltenacu et al,. (1983) did not find a direct effect of uterine discharge on

reproductive performance.

Puerperal Metritis

Pathogenesis

Puerperal metritis has been described as a life-threatening infection, characterized

by a fetid vulvar discharge that may be associated with clostridial infections (Roberts,

1986). The presence of a fetid discharge appears to be an unequivocal sign of uterine

infections which reflects the level of bacterial contamination in the uterus. Mateus et al.

(2003) using cows that were within 6 weeks post partum, evaluated the bacteriological









content of the uterus, uterine horn size and fluid content by ultrasonography. In this study

(Mateus et al,. 2003), uterine discharge by vaginoscopy was classified as normal lochia

mild endometritis (purulent lochia), and severe endometritis (heavy, fetid purulent lochia)

associated with or without systemic symptoms. Results indicated that uterine involution

of cows with a fetid discharge was delayed. Furthermore, uteri that contained fetid

purulent lochia present at examination had a greater horn diameter (i.e,. at 3 to 4 weeks

postpartum) and a greater amount of uterine fluid (i.e., at 2 to 6 weeks postpartum) than

the normal puerperium group, respectively (Mateus et al,. 2002). No differences in

uterine involution were observed between the group classified as normal and the group

classified as purulent lochia. In addition, the bacterial cultures showed that Arcanobacter

pyogenes, E. coli, Fusobacterium sp. and Bacteroides sp. were more frequently isolated

from cows with mild endometritis or severe endometritis than in cows without these

conditions. In cows with a normal puerperium A. pyogenes was isolated in 74 % of the

cases, and gram negative anaerobes only occurred trough the second week post partum,

whereas cows with mild or severe endometritis, these response were evident until the

fourth to 6 week post partum, respectively. These results agreed with Hirvonen et al.

(1999) in which cows with a fetid discharge showed higher bacterial growth of E. coli

and A. pyogenes compared to cows without a purulent discharge. Furthermore, if the

cows showed systemic signs then Bacteroides sp. and Fusobacterium necrophorum also

were isolated. In addition to the previous results (Mateus et al., 2003; Hirvonen et al.,

1999), Dohmen et al,. (2000) showed that cows experiencing dystocia or retained fetal

membranes with a fetid discharge showed a high growth rate ofE. coli, black pigmented









G- anaerobes and Clostridium spp, from uterine cultures, compared to cows that calved

without dystocia or retained fetal membranes and did not have a fetid vulvar discharge.

General signs of toxemia can be found in cows with puerperal metritis (Rebhun et

al., 1995). Endotoxins or lipopolysaccharides (LPS) are among the most important

virulent factors of coliform bacteria. Lipopolysaccharides are somatic antigens of bacteria

composed of polysaccharides, phospholipids and a small amount of protein (Lohuis et al

., 1988). High LPS concentrations in lochia were found to be positively correlated with

the presence of a fetid discharge and presence of E. coli bacteria (Dohmen et al., 2000).

However, LPS concentration in the uterus was not correlated with LPS concentrations in

blood. In contrast, Mateus et al. (2003) positively correlated the presence of a fetid

uterine discharge with LPS concentration in blood. Furthermore, Peter et al. (1990)

reported increments of LPS concentrations in blood after intrauterine infusion of

endotoxins in postpartum cows and suggested that LPS are absorbed from the uterus.

Mechanisms by which LPS are absorbed from the uterus were hypothesized as: direct

absorption from the uterus, passive diffusion and/or transmural leakage, or escape

through the oviducts and fimbria into the peritoneal cavity (Peter et al., 1990). In

addition, Peter et al. (1990) demonstrated that absorption of LPS by the uterus decreased

as days postpartum increased. Intrauterine infusion of LPS in cows 20 days post partum

did not show an incremental increase in LPS concentration in blood compared to cows

infused at 5 days post partum (Peter et al., 1990).

Signs of endotoxemia include depression, respiratory distress, vasomotor

disturbance, shock, fever, sometimes followed by hypothermia, disturbance of

gastrointestinal tract motility and metabolic disturbances (Lohuis et al ., 1988).









Immune cells detect bacterial components such as LPS and peptidoglycan via toll-

like receptors that are present in macrophages (TLR-4) (Beuttler et al., 2003) to stimulate

production of the cytokines (i.e., TNF-a, IL-1, IL-6, IL-8). These cytokines act as

internal pyrogens to increase core body temperature. Dohmen et al. (2000), found a

positive correlation between a fetid uterine discharge, LPS and rectal temperature. Cows

with a fetid uterine discharge had higher rectal temperatures (mean = 39.30C). However,

fever is not associated always with this type of uterine discharge. Hirvonen et al. (1999)

found that only 8 (42%) of 19 cows that were diagnosed with puerperal metritis (acute

puerperal metritis, putrid vulvar discharge) between 4 to 11 days postpartum, developed

systemic clinical signs of fever (39.50C 41.00C) and poor appetite during the acute

phase of the infection. These results agree with those of Pugh et al (1994), in which only

42.3% 78 cases of puerperal metritis evaluated within 14 days post partum had fever (RT

> 39.40C) at diagnosis. However, in this later study the type of uterine discharge was not

described for cows with puerperal metritis.

Tissue injury and inflammation induce the release of interleukin-6 (Hirano 1992).

This interleukin is one of the cytokines involved in the development of fever (Beuttler et

al., 2003), as well as inducing the synthesis of acute phase proteins such as haptoglobin

and alpha-1-acid glycoprotein by hepatocytes (MacKay and Lester 1992). Systemic

responses that result in fever also increase the levels of acute phase proteins. However,

Smith et al. (1998), reported no significant correlation between level of haptoglobin in

blood with rectal temperature in cases of puerperal metritis (foul smelling vulvar

discharge with a rectal temperature > 102.5). Nevertheless, concentration of haptoglobin

in cows with puerperal metritis were between 13 to 20mg/dl on the day of puerperal









metritis diagnosis, which agrees with the ranges (>10 mg/dl) reported by Skinner et al.

(1991) in cows with puerperal metritis (fouls smelling vulvar discharge, with or without

fever). Williams et al. (2005) compared levels of alpha-i-acid glycoprotein in cows

having purulent fetid discharge with cows having a purulent but no fetid discharge at 21

or 28 days postpartum. Cows with a fetid uterine discharge had higher concentrations of

alpha-1-acid glycoprotein (1.5 mg/dl) than those cows without a fetid discharge (1.03

mg/dl). However, the levels of alpha-1-acid glycoprotein found by Williams et al. (2005)

during days 21 or 28 post partum were within the normal levels (1.2- 1.4 mg/dl ) in

normal cows within 10 days post partum reported by Sheldon et al. (2001). In contrast,

Sheldon et al. (2004), found that cows with one or more events of fever during the first

10 days post partum had higher levels of alpha-1-acid glycoprotein than cows without

fever during the same time period.

Histopathological disease definition

Puerperal metritis is defined as the inflammation of the mucosa, submucosa,

muscular and the serosal layers of the uterus. It is described as a purulent inflammation,

where the sub-serosal connective tissues are edematous and infiltrated with leukocytes,

with the same process observed surrounding blood vessels of the myometrum that extend

to muscle fibers which undergo granular degeneration. The leukocyte mass on the

mucosal surface is associated with extensive hemorrhage, necrosis, and sloughing ( Jubb,

et al. 1992) induced by bacteria toxins produced by bacteria. The hemorrhage and

necrosis, along with bacterial products characterize the clinical findings (Jubb, et al.

1992).









The histopathological definition of puerperal metritis is straight forward (Sheldon

et al., 2006). However, this is not a common practice because of logistical reasons and

the sampling per-se posses a risk to the health of the animal more than the disease itself

(Etherington et al., 1988). Consequently, as well as in other diseases, a clinical definition

is needed to diagnose uterine infections. These definitions should characterize clinical

findings to conclude whether or not the disease is life threatening and weather treatment

should be applied.

Clinical Disease Definition

Highly pathogenic types of bacteria are present in the uterus and along with their

toxins are absorbed into the circulation producing symptoms associated with septicemia,

endotoxemia, and pyemia (Roberts, 1986). Diagnosis of puerperal metritis has been

performed by rectal palpation of the uterus (Markusfeld, 1984; Pugh et al., 1994; Risco,

and Hernandez, 2003). However, vagignoscopy has also been used (Hirvonen et al.,

1999).

Based on clinical experience Rebhun (1995) described puerperal metritis (septic or

toxic metritis) as those cows with a fetid watery uterine discharge from the vulva that

varied in color from brown, amber to gray or red, but fluid are low in mucus content and

contained purulent material. Cows become ill within the first 7 to 10 days post partum

and had fever (40.00C to 41.390C), tachycardia, inappetence, decreased production,

rumen stasis, and toxemia Dehydration, diarrhea, and depression of varying severity are

also observed. Extremely severe infection may cause recumbency secondary to toxemia,

weakness, and metabolic disorders.









Literature disease definitions

Terminology used to classify uterine infections, specially for puerperal metritis has

been vague and inconsistent (Lewis 1997). Many studies have been published that

describe the epidemiology of puerperal metritis, it's effect on reproduction performance

and milk production, and treatment and prevention of this disease. However, differences

in disease definition contributed to conflicting results on effect on reproductive

performance, milk production, and disease incidence.

Bartlett et al. (1986) described the epidemiology of metritis and estimated the

economic impact of metritis in Michigan Holstein-Friesian dairy cows. Metritis was

defined according to the thickness of the uterine wall and the fluid content of the uterine

cavity in relation to the number of days post partum. The study involved 22 herds and

information about disease was recovered from a dairy herd health computer network. In

over 3773 lactations were studied with an lactational incidence of 18% within 10 to 30

days post partum was found, that varies from 3 to 45%. In addition, metritits was most

commonly diagnosed between 11 to 20 days post partum. After including the effect on

reproductive efficiency, milk production, cost of medication and losses due to culling, the

total cost estimate was $106.00 for a lactation with metritis.

Beaudeau et al. (1995) assessed the effect of health disorders on length of

reproductive life in 47 French Holstein commercial dairies during 4 years. Metritis was

one of the explanatory variables and was further divided as early metritis and late

metritis. Early metritis included vulvitis, vuvlvovaginitis, endometritis, vaginal discharge,

metritis or pyometritis diagnosed from 22 to 49 days postpartum. Late metritis included

vulvitis, vulvovaginitis, endometritis, vaginal discharge, metritis or pyometritis diagnosed









beyond 50 days post partum. The overall incidence for early metritis was 6.4%, however

the incidence ranged form 5.2 % when calculated in cows with less than 90 days

postpartum or 8.1 % if calculated in cows with more than 210 days postpartum. The

overall incidence of late metritis was 5.6 % and ranged from 1.4 % in cows with less than

90 day postpartum and 14.1 % in cows with more than 210 days in milk. It was found

that cows with late metritis or early abortion had poor survival, thus higher culling rate.

Bruun et al. (2002) identified risk factors for metritis in 102,060 Danish dairy

cows. Information was recovered from the Danish cattle database. Metritis was not

defined. However, diagnoses were made within 1 to 30 days post partum In this study,

the incidence risk ranged from 1 to 21 % in 391 herds observed.

Callahan and Horstman (1987) reported a retrospective analysis of treatment

alternative in dairy cows affected with postpartum metritis in the Purdue University Dairy

center. The criteria for diagnosis of metritis consisted of ballottement of uterine fluid,

possible crepitant feel of the uterine content, lack of myometrial tone, retarded involution

and the presence of abnormal discharge. Discharge characteristics ranged from thin,

watery and fetid to purulent or mucopurulent. The study was conducted in a 5 year period

and 1108 lactations were evaluated. An incidence of 33.8 % was reported within 14 days

postpartum. In this study no effect on reproduction performance was found in cows with

puerperal metritis. Furthermore, they stated that the early diagnosis of metritis may

reduce the impact on the reproductive performance.

Chenault et al. (2004) evaluated the efficacy of ceftioufur hydrochloride for the

treatment of postpartum metritis. Metritis cases were defined as cows with a rectal

temperature > 39.50C, with a fetid vaginal discharge that was red or pink to chocolate









brown in color and serious with or without pieces of necrotic tissue, within 1 to 14 days

post partum. Given that only metritis cows were reported as an outcome, incidences were

not reported. However they concluded that ceftiofur hydrochloride administered at a

dosage of 2.2 mg of CE/kg, subcutaneously or intramuscularly once daily for 5 days was

efficacious for treatment of acute puerperal metritis in dairy cows.

Cobo-Abreu et al. (1979) observed the association between disease, production and

culling in a Holstein dairy herd in Ontario, Canada. This study was conducted during

seven years. Metritis definition, incidence and days in milk to diagnosis were not defined.

Correa et al. (1993) modeled a path analysis with logistic regression for seven

postpartum clinical diseases in cows and also observed the factors related to the calf.

Data were from 7761 lactations from 34 commercial dairy herds close to Cornell

University. Metritis was defined as an enlarged uterus found at rectal palpation in cows

with or without other clinical signs within 30 days post partum. However a metritis event

included cases of endometritis, pyometras and metritis as defined above. A lactational

incidence of 7.2 % was observed. It was found that stillbirth increased the odds of

developing metritis and retained placenta, cows that twinned had increased odds of

developing dystocia and retained placenta. Dystocia was related to an increase in the odds

of retained placenta. Milk fever, dystocia, and ketosis each increased the odds of

developing left-displaced abomasum. Postpartum periods with dystocia, retained

placenta, or ketosis had increased odds of metritis.

Curtis et al. (1985) used path analysis and logistic regression to model direct and

indirect relationships among clinical periparturient (within 30 days after calving)

diseases. Data were obtained from 1374 lactations of multiparous Holstein cows in 31









commercial herds near the Cornell University area during a period of one year. The

definition of metritis was not given. However, an incidence of 7.8 % was reported, within

30 days post partum. Retained placenta, left displaced abomasum, and parturient paresis

directly increased risk of complicated ketosis. The study suggested that feeding higher

intakes (relative to National Research Council recommendations) of protein and energy

in the last 3 week of the dry period may reduce the incidence of metabolic and

reproductive disorders.

Drillich et al. (2001) evaluated the efficacy and financial viability of systemic

treatment of toxic puerperal metritis in dairy cows with ceftiofur. Toxic puerperal metritis

was defined as the presence of a fetid, reddish-brown vaginal discharge and a rectal

temperature >39.50C, within 4 to 6 days postpartum. During the study period, a total of

1756 calvings were observed and an incidence of 18.5 % was reported. There were no

significant differences among the treatment groups regarding clinical efficacy at d 6 after

first treatment (group 1 received 600 mg of ceftiofur intramuscularly on 3 consecutive

days; group 2 received an intrauterine treatment with antibiotic pills consisting of 2500

mg of ampicillin and 2500 mg of cloxacillin and an additional 6000 mg IM of ampicillin

on 3 consecutive days and group 3 received the same intrauterine treatment as in group 2,

in addition to 600 mg of ceftiofur IM on 3 consecutive days. The cure rates based on

rectal temperatures declining to below 39.5 degrees C on d 6 after treatment were 82.9,

84.8, and 84.6% for groups 1, 2, and 3, respectively. Reproductive performance did not

differ significantly between group 1 and groups 2 and 3 for any of the measures tested. A

financial analysis with 87 different cost scenarios demonstrated that a systemic treatment









of toxic puerperal metritis in cattle with ceftiofur is an effective alternative to the

combination of local and systemic treatments.

Etherington et al. (1985) used a path analysis to determine the interrelationship

between ambient temperature, age at calving, postpartum reproductive events and

reproductive performance in dairy cows. Within the reproductive event metritis was

included. A cow was considered to have metritis if she exhibited decreased milk

production, decreased feedd intake, pyrexia and had foul smelling vaginal discharge. An

incidence of 23% was reported. However days in milk of the diagnosis was not reported.

In this study there was an increase in the incidence of retained placenta, in the percentage

of cows with abnormal vaginal discharge in the early postpartum period as well as a

delay in uterine involution during the winter months. In addition, cows calving during the

winter had prolonged intervals to first estrus, first service and conception compared to

cows calving during the summer. Cows calving during the warmest months, on average,

were seen in estrus 24 days sooner, received first service 42 days sooner and conceived

27 days sooner than cows calving during the coldest months of the year.

Erb and Martin (1978), studied age, breed and seasonal patterns in the occurrence

often diseases on which metritis was one of them. In this study, information of the

disease was retrieved from a University central data base. The definition of metritis

included endometritis, metritis and pyometra. However, none of these diseases were

defined. An incidence of 14 % was observed. No description of the time frame of the

diagnoses was made. Using the log-odds method, trends were noted for the youngest

cows to be at increased risk of the reproductive diseases such metritis and for the









Guernsey cows to be at increased risk of the uterine diseases. There was a tendency for

peaks in disease occurrences in the winter (as opposed to summer) months.

Erb et al. (1985) observed the relationship between occurrence of metritis with

other diseases. A total of 2960 lactations of Holstein dairy cows were included in the

study. Metritis was not defined. However, the incidence was reported to be a 9.9 %. No

description of the time frame of the diagnoses was reported. It was shown that heifers that

were older, of lighter weight, or who had lower estimated transmitting ability for milk

had more problems, less milk, and poorer survival. Dystocia in heifers had several

detrimental consequences including 2.9 to 4 times more retained placenta, metritis, and

culling and +7.4 d more to first service. Cystic ovaries were associated directly with 376

kg greater milk yield and with a 16.5-d delay in first service. Failure to conceive at first

service and mastitis increased risk of culling 5.2 to 10 times. In multiparous cows, milk

fever increased risk of reproductive disorders by 1.6 to 4.2 times and indirectly

contributed to poor breeding performance and increased culling. Risk of culling was

increased 2.1 to 3.7 times directly by mastitis and dystocia and by poor breeding

performance.

Harman et al. (1996) quantified the effect of season of parturition, parity, and

diseases on time to conception in 44450 Holstein dairy cows. Metritis was divided into

early (less than 42 days postpartum) and late metritis (more than 42 days post partum).

However no definition of disease was given. The lactational incidences were 2.0% for

early with median day to diagnosis of 18 days after parturition, and 1.1% for late metritis

with median day to the diagnosis of 108 days after parturition. For multiparous cows,

parturition in the spring or summer and being of parity 2 or 3-4 (vs older) increased the









chance of conceiving; 10 diseases or disorders decreased this probability. In primiparous,

parturition in spring or summer increased the probability of conception, and 6 disorders

decreased it. Disorders that were found to be detrimental in both models were anestrus,

ovulatory dysfunction, other infertility, late metritis, and clinical ketosis.

Hirvonen et al. (1999) examined the role of systemic acute phase proteins regarding

diagnostic values of Haptoglobin, alphal- acid glycoprotein, and plasma N-acetyl-beta-

D-glucosaminidase activity in clinical and bacteriologically defined acute postpartum

metritis in dairy cows. Acute metritis was defined as a putrid, reddish-brown, watery,

foul smelling vaginal discharge, within 4 to 11 days post partum. No incidence were

reported given that puerperal metritis was the principal outcome. Results showed that

plasma haptoglobin concentration remained low in most cows with acute postpartum

metritis. Only the 3 most severely affected cows exhibited a strong haptoglobin response.

These were later culled due to poor condition and reduced fertility. It was suggested that

in acute uterine infection a highly increased haptoglobin concentration indicated a poor

prognosis for repeat conception. Plasma alphal-acid glycoprotein concentration increased

in acute postpartum metritis, the response pattern being less prominent than that for

haptoglobin. The alphal-acid glycoprotein concentrations did not correlate with severity

of disease, and, consequently, the capacity of alphal-acid glycoprotein in differentiating

genital infections was relatively poor. Highest alphal-acid glycoprotein concentrations

were detected in cows with retained placenta and/or dystocia. In addition, plasma N-

acetyl-beta-D-glucosaminidase activity levels did not differ between the cows with acute

postpartum metritis and healthy control cows.









Lee et al. (1989) described the use of survival analysis to quantify the days open for

different diseases during the early post partum period. Metritis was used as an

explanatory variable and this disease was further divided into metritis, non systemic and

systemic. However incidences and the definition of metritis and day in milk to diagnosis

was not provided. It was found that retained placenta, nonsystemic metritis, systemic

metritis, ovarian cysts, and lameness were associated with a decrease in conception rate

and an increase in median days open. The hazard ratios for conception were .66, .83, .70,

.70, and .69 and the increase in median days open 5, 15, 13, 22, and 28 d for the five

diseases, respectively.

Markusfeld, O (1984) observed the factors associated with retained fetal

membranes and postpartum metritis in 2017 Holstein dairy cows. Metritis was defined as

any purulent foul smelling discharge. The incidence observed was 37.3 % and ranged

between 31.2 to 43.8 %. The diagnosis were made within 14 days post partum. In this

study risk factors associated with metritis include declining parity, long gestations,

induction of parturition, stillbirth, multiple births, low milk yield before drying off, left

displacement of the abomasum, ketosis and winter calvings.

Markusfeld and Ezra (1993) observed the effect of herd, sire, season, body height,

body weight, age at calving, and metritis on performance of first lactation cows. Metritis

was defined as described in Markusfeld (1987). Of a total of 621 first lactation heifers, an

incidence of 48.6 % was reported, within 5 to 12 days postpartum. This study reported

that short, heavy first lactation cows had an odds ratio of 3.1 of incidence of metritis at

calving compared with all others; 648 first lactation cows were measured at wk 1

postpartum. Sire, herd, age, height, season, and BW contributed to peak milk yield.









Metritis did not affect peak yield. Herd, sire, height, and age contributed to mature

equivalent corrected 305-d milk yield. No effect was found for BW, season, or metritis.

Herd was the only variable contributing to month of peak yield and rate of monthly drop

in yield. Interactions between BW, height, and incidence of metritis were significant.

Tall, heavy first lactation cows with metritis peaked higher and yielded more than those

without metritis. Short, light first lactation cows with metritis yielded less and peaked

lower than their healthy counterparts. Metritis did not affect future fertility, but season

and the interaction between BW and height did. Tall, heavy first lactation cows had a

lower pregnancy rate from first AI, independent of milk yield. The relative importance of

height as a predictor of future milk yield is underestimated. The interaction between

height and BW may have an antagonistic effect on yield and fertility.

Melendez et al,. (2004) evaluated the effect of 2 doses of PGF2, injected early

postpartum on uterine involution, serum concentration of alphal- acid glycoprotein and

fertility in Holstein cows with acute puerperal metritis. Acute puerperal metritis was

diagnosed by per rectum palpation of the uterus at 8 d post partum. Criteria for diagnosis

was an enlarged and flaccid uterus with a foul-smelling uterine discharge, without fever

(>39.50C). During the study period, 1536 cows calved; an incidence of 15.3% was

reported. However only cows diagnosed with retained fetal membranes and metritis were

included and those cows with metritis and systemic signs were excluded. In this study

postpartum, primiparous, treated cows had smaller uterine diameters and lower uterine

scores than controls. Cows with a uterine diameter <5.1 cm at 12 d postpartum were 5.5

times more likely to conceive at first service than cows with larger uterine horn diameter.

Treatment significantly reduced the concentrations of serum alphal-acid glycoprotein.









Within primiparous cows, treatment also increased conception at first service by 17%. It

was concluded that 2 doses of PGF2, 8 h apart at 8 d postpartum in primiparous cows

with acute puerperal metritis decreased the diameter of uterine horns and serum

concentration of alphal-acid glycoprotein at 12 d postpartum and increased the

conception rate at first service.

Overton et al. (2003) studied the effect of a prophylactic treatment with estradiol

cypionate (ECP) in cows at a high risk to develop metritis. Metritis was defined as any

combination of fever > 39.70C with a watery and or fetid vulvar discharge within 1 to 10

days postpartum. Metritis were further divided into mild or severe. Metritis was mild

when the cow never had a rectal temperature > 39.70C and severe when the rectal

temperature was > 39.70C. An incidence of 10% was observed in 1284 calvings

observed. They concluded that prophylactic administration of ECP to dairy cows at high

risk for metritis did not reduce risk for metritis.

Pugh et al. (1994) described 78 cases of postpartum metritis in dairy cows. Forty

two and 36 cases of postpartum metritis were recovered from records of the large animal

hospital from Aurburn University and Tuskegee University, respectively. The definition

was not reported. However, it was stated that the diagnoses were based on the vaginal-

uterine discharge obtained by rectal palpation of the uterus. Older cows and those with

hyperthermia were less likely to recover from puerperal metritis. Furthermore, only 42%

of the treated cows were with hyperthermia at the moment of the physical exam.

Rajala and Grohn (1998) evaluated the effects of dystocia, retained placenta, and

metritis on milk yield using repeated, monthly test day milk yields, on 37,776 Finnish

Ayrshire dairy cows in 2337 herds, recovered from the national Finnish health recording









system. Metritis was not defined, however diagnosis was further divided in early and late

metritis. The lactational incidence of early metritis ranged from 1.6 to 2.6 % within 28

days post partum and the incidence of late metritis, ranged from 1.2 to 1.5 % after 28

days postpartum. Dystocia, retained placenta, and early metritis significantly affected

milk yield, as indicated by monthly test day milk yields. Late metritis was not associated

with milk loss. The impact of the diseases differed across parities and also across

different levels of milk yield. Using 305-d milk yield as the milk measure, no diseases

were associated with reduced milk yield.

Risco and Hernandez (2003) compared the administration of ceftiofur

hydrochloride and ECP on the prevention of puerperal metritis. In this study the

definition of metritis was not stated. However, diagnoses were made within the first 30

days post partum. Incidences were not reported given that cows with metritis were the

experimental units. Results showed that the proportion of cows that developed metritis

was significantly different in cows treated with ceftiofur hydrochloride (13%), compared

with cows treated with ECP (42%) or cows that received no treatment (42%). Uterine

involution patterns (i.e. median time to complete retraction of the uterus and mean

diameter measure of cervix and uterine horns) were not significantly different between

groups. Cows treated with ECP were 0.40 times as likely to conceive as control cows

(P=0.05); median time to conception in cows treated with ECP (192 days) was longer,

compared to control cows (124 days). It was concluded that systemic administration of

ceftioufur hydrochloride is beneficial for prevention of metritis, but its effect on

reproductive performance was not significantly different to that of ECP or no treatment.









In addition, administration of ECP did not have beneficial effects on metritis prevention

and reproductive performance.

Schnier et al. (2002) compared the incidence of diseases in 5000 Finland dairy

cows kept in cold or loose-housing systems. Information was recovered from the Finish

health data recording system. Metritis included cases of acute and chronic metritis,

pyometra, vaginitis and disturbed involution within 0 to 44 days post partum. However

none of these conditions were defined. The overall incidence for both housing system

was 3.3 %. They found that cows in a cold loose-housing system were at lower odds for

developing late mastitis (15-305 days in milk), and metritis (Friesian breed); of the same

odds for ketosis and early mastitis (0-14 days in milk); but at higher odds for developing

parturient paresis and metritis (Ayrshire breed).

Smith et al. (1998) compared procaine penicillin, intrauterine infusion of

oxytetracycline or ceftiofur in dairy cows for the treatment of toxic postpartum metritis.

Toxic postpartum metritis was defined as any cow with a rectal temperature >39.20C, a

flaccid, non retractable uterus that was located in the abdomen, a cervical diameter >75

mm, and a watery, fetid vulvar discharge. Other criteria used to diagnose toxic puerperal

metritis was depressed milk yield (<7.4 kg at the morning milking), within 3 to 20 DIM.

Given that only cows with puerperal metritis were used no incidence was reported. No

difference was observed among groups for milk yield on d 1 and 12 or for temperature on

d 1 and 5. Serum haptoglobin was elevated to > 10 mg/dl for cows in all groups;

however, no difference was observed among groups on d 1 and 5. Because all groups

showed a favorable response, this study suggests that there is no difference in treatment

efficacy among antibiotics used to treat cows affected with toxic puerperal metritis.









Urton et al. (2005) related cows with high or low feeding behavior during the

prepartum transition phase with increased risk of developing metritis after calving. Two

metritis classification were used. Animals were classified as having metritis if they

showed a mucopurulent and foul smelling and fever (rectal temperature > 39.50C) or

acutely metritis if they showed a reddish brown, watery, foul smelling vaginal discharge

and fever. The incidence of metritis ranged between 38% to 27% for metritis and acute

metritis respectively, within 3 to 15 days post partum, however the total calvings were

not reported. Cows suffering from metritis, exhibit reduced milk yield and reproductive

performance. These cows spent on average 22 min/d less time at the feed alley during the

transition period than did non-metritic cows. For every 10-min decrease in average daily

feeding time, cows were twice as likely to be diagnosed with metritis. A threshold of 75

min of average daily feeding time was 89% sensitive and 62% specific for detection of

acute metritis. It was concluded that reduced time at the feeder can be used to identify

dairy cows at risk for metritis.

Current research definition

Given the various definitions used in the literature to describe uterine infections,

Sheldon et al. (2006) proposed a series of definitions. In this review, a distinction

between puerperal metritis and clinical metritis was made. Puerperal metritis is defined as

those cows with an abnormally enlarged uterus and a fetid watery red-brown uterine

discharge associated with signs of systemic illness (decreased milk yield, dullness or

other signs of toxemia) and fever >39.5 C within 21 days post partum. In addition,

clinical metritis was defined as those cows that do not appear sick, but had an abnormally

enlarged uterus and a purulent uterine discharge present in the vagina, within 21 days









after parturition. The definitions proposed by Sheldon et al. (2006) were based from

previous studies related to metritis treatments (Drillich, et al., 2001), pyrexia in

postpartum cows (Sheldon et al., 2004), and risk factors for puerperal metritis

(Markusfeld, 1984). However, none of the cited studies used to define uterine infections

related clinical signs to histopathology and possible pathophyisiology of this disease.

Furthermore, they did not include any controlled studies that may relate clinical signs

with risk factors to systemic illness or impairment of cow performance.

Risk Factors for Puerperal Metritis

Puerperal metritis is often associated with retained fetal membranes (RFM),

dystocia, stillbirth or twins and usually occurs during the first two weeks post partum

(Olson et al., 1986). However, puerperal metritis may also occur in cows without calving

related disorders (Olson et al., 1986).

Studies using path analysis and risk assessment have consistently indicated that

dystocia, retained fetal membranes, and metabolic conditions increased the likelihood

that a cow will develop metritis. However, in some cases puerperal metritis was classified

as a disease complex without distinguishing the clinical presentation or severity, making

comparison between studies difficult (Lewis, 1997). Many of these studies used odds

ratios (OR) as a measurement of risk factor, but relative risk (RR) another measurement

of association, also has been used.

Retain fetal membranes

Retained fetal membrane (RFM) is a major predisposing cause of metritis. The

third stage of parturition involves the expulsion of the fetal membranes, and is completed

within 8 hours after parturition. A persistence of the third stage of parturition, that is,









failure to expel the fetal membranes is considered abnormal (Roberts., 1986). Fetal

membranes are considered retained when the fetal cotyledonary villi fail to separate from

the crypts of the maternal caruncles within 12 to 24 hours of parturition (Roberts S. J,

1986) and mechanisms why this process fails has been described (Gunnink 1984; Kimura

et al., 2002).

The condition of retained placenta occurs in 4 to 18% of calvings (Markusfeld,

1985; Esslemont and Kossainbati,1996; Erb, et al., 1985). Various risk factors for the

development of retained placenta have been reported. Calving problems including

dystocia (OR = 4.0) (Correa, et al., 1993; Markusfeld 0. 1984; Erb, et al., 1985),

stillbirths (Correa, et al., 1993; Markusfeld, 1984), and multiple births (Correa, et al.,

1993; Markusfeld, 1984), parturient paresis, low prepartum protein and age of cows

(Curtis et al., 1985) have been found to be risk factors for RFM. Dohmen et al. (2000),

reported that immediately after calving, RFM cows had high concentrations of

LPS/endotoxin in lochia and were more often infected with E. coli, Clostridium spp. and

G- anaerobes (prevalence rates up to 97%) bacteria than cows without periparturient

disorders. The principal deleterious effect of retained fetal membranes on the dairy cow is

impaired fertility by delaying involution of the uterus, thereby facilitating the

development of uterine infections (Sandals et al., 1979).

Retention of fetal membranes is one of the major factor predisposing cattle to

metritis (Bartlett et al., 1986; Correa et al., 1993). Several studies have related the

incidence of metritis with RFM. Sandals et al. (1979) reported an incidence of 54.8

percent of metritis following retained fetal membranes. Erb et al. (1985) reported an

important biological causal association between RFM and metritis. It was reported by Erb









et al. (1981) that cows with RFM were almost six times more likely to developed metritis

compared to cows without RFM.

Various studies have associated RFM and metritis OR = 4.4 (Grohn et al., 1990);

OR = 6.0 (Correa et al., 1993); OR = 2.5 (Bruun et al., 2002); OR = 5.8 (Erb et al., 1985).

Stillbirth, multiple birth and dystocia

Stillbirth, multiple birth and dystocia are events that occur at parturition and are

risk factors for metritis and are related to one another. When the first, or the second

stages of parturition are prolonged, it becomes difficult or impossible for the dam to give

birth without artificial aid and then this condition is termed dystocia (Roberts, 1986). The

incidence of dystocia ranges from 6% to 25% (Roberts, 1986; Adamec et al., 2006).

Prepartum dietary energy and parturient paresis have direct effects leading to veterinary

assisted dystocia (Curtis et al., 1985). However, prepartum energy does not seem to affect

the incidence of dystocia (Markusfeld, 1985). Dystocia is greater in pregnancies that

terminate early due to uterine disease, fetal death, and twinning (Roberts, 1986). Dystocia

can increase the risk of trauma to the uterine wall and thereby increase the odds of

metritis (Bruun et al., 2002). In addition, assistance during calving may increase the risk

of uterine contamination (Bruun et al., 2002). Erb et al. (1985) differentiated the

incidence of metritis by parity and whether on not the cow had dystocia using path

analysis. For primiparous and multiparous cows, dystocia was a risk factor for metritis

(OR=3.0; OR = 3.5; respectively). Dohoo et al. (1984), and Curtis et al (1985), also found

a positive association between dystocia and metritis (OR = 2.5) and (OR = 4.9),

respectively. In the study reported by Curtis et al. (1985), dystocia were only those

calvings assisted by a veterinarian. However, this assistance was not a risk for RFM.









These results are in agreement with studies by Correa et al. (1993) who reported that

cows requiring assistance at calving were 2.1 (OR) times more likely to develop metritis.

Stillbirth is the expulsion of a dead fetus at parturition (Roberts, 1986). Calving

difficulty has been implicated as the major cause of stillbirth, yet about 50% of stillborn

calves are from unassisted births (Philipsson, 1996). Martinez et al. (1983) reported that

the stillbirth rate in U.S. Holsteins is around 10.5% for first lactations, 5.5% for second

lactation and 5.7% for third lactation. Markusfeld (1984), reported that heifers and cows

which gave birth to dead calf had a higher rate of RFM or to develop metritis compared

to those which gave birth to live calves (OR = 2.19) These results agree with those of

Correa et al. (1993), who reported that cows delivering dead calves were 1.5 (OR) times

more likely to develop metritis than those than did not have stillbirth at parturition. In

contrast, Emanuelson et al. (1993) found that stillbirths had a direct effect on the risk of

retained placenta but not on metritis.

When an uniparous animal aborts two or more or gives birth to fetuses are called

twins (Roberts, 1986). The incidence of twinning in dairy cattle has increased

dramatically over the past two decades (Nielen et al., 1989; Kinsel at al., 1998). Risk

factors for twin calvings such as parity, season, herd and previous twinning have been

described ((Nielen et al., 1989). The incidence of twinning has been reported to range

from 1.04% to 9% (Roberts, 1986; Kinsel et al., 1998). Twin calving increased the risk of

dystocia (OR = 10.5), RFM (OR = 3.4; Correa et al., 1993) and reduces milk production

and increased culling rate than single calvings (Nielen et al., 1989). Twinning often

results in decreased gestation length and increased dystocia and mortality rates.

Markusfeld (1987) found that cows delivering twins were 12 times (OR) more likely to









RFM and 2.3 times (OR) more likely to develop metritis than cows without RFM..

Correa et al. (1993), did not find an effect of twins on the incidence of metritis

Parity

The effect of parity on metritis has been reported previously with conflicting

results. Grohn et al., 1990 found no association between parity and metritis. However,

Markusfeld (1984; 1987) found an association. As parity increased, the incidence of RFM

increased, but the incidence of metritis decreased (Markusfeld, 1984). In contrast,

primiparous cows were more likely (OR = 2.7) than second or greater parity cows to

develop metritis (Markusfeld, 1984). This finding is in agreement with Erb and Martin

(1978), were first lactation cows compared to other lactation cows were more likely (OR

= 1.48) to develop puerperal metritis.

Bruum et al. (2002) suggested that there is an u-shaped association between parity

and metritis. Primiparous cows are more likely (OR = 1.6) to develop metritis than

second-parity cows because damage to the uterus is more common in heifers given the

high incidence of dystocia (Bruum et al., 2002). In contrast, third-parity or greater cows

were more likely (OR = 1.58) to develop metritis compared to second-parity cows related

to a delay in uterine involution which increases the risk to develop uterine infections (

Bruum et al., 2002). Tendency for an u-shaped relationship between parity and incidence

of metritis was also reported by Rajala and Grohn (1998). However, this relationship was

not significant. In addition Smith et al. (1998), found a relationship between parity and

dystocia. Primiparous cows were more likely (OR = 2.04) than multiparous cows to have

dystocia. However, Pugh et al. (1994) did not find any association between cases of

metritis and parity.









Season

Season has been reported to be a risk factor for metritis (Sandals et al. 1979;

Markusfeld, 1984; Erb and Martin, 1985; Etherington et al., 1985). Markusfeld (1984)

found that cows that calved in the summer had a greater risk (RR = 1.64) of RFM and a

lower risk of developing metritis (RR = 0.64) compared to those that calved in winter.

Bruun et al., 2002 found that the odds ratio for metritis in cows calving during the cold

season were 1.2 times higher than those cows calving during the warm season. Bruun et

al. (2002) stated that during the winter months the general health of cows is lowered

making them more prone to infections. In a study that involved 3773 lactations, Bartlett

et al., 1986 found that metritis was less common during summer months, although their

findings were not statistically significant. Etherington et al. (1985), described a higher

incidence of dystocia during the summer months, contrary to the incidence of RFM

which was higher during the winter months. Markusfeld O (1984) reported that cows

experiencing dystocia were more likely to develop endometritis, pyometra and RFM was

directly associated with metritis.

Schnier et al. (2002) compared disease incidence of dairy cows kept in cold or

warm loosing house, in Ayrshire and Fresian dairy cows. The odds of contracting metritis

during the first 44 days in milk of cows calving in a cool loose house compared to cows

calving in a warm loose housing during the indoor period were lower for cows of the

Fresian compared to the Ayrshire breed, OR = 0.2 and OR = 1, respectively. In addition,

Grohn et al. (1990) found that cows calving during January April (OR = 1.6) and

September December (OR = 1.7) were more likely to develop early metritis than those









calving during the month of May-August. A later report found same results (Grohn et al.,

2000).

Hypocalcemia

Uterine inertia and a decrease in uterine involution predispose cows to puerperal

metritis (Roberts, 1986). Cows with postpartum hypocalcemia have been found to have

increased incidence of postpartum metritis compared to cows without this condition

(Boseberry and Dobson, 1989). Erb and Martin (1985) found that cattle suffering from

hypocalcemia were (OR = 4.2) more likely to have a veterinary assisted dystocia, and

more likely (OR = 2.0) to have RFM. Both dystocia and RFM have been found to be

predisposing factors for metritis (Erb et al., 1985; Markusfeld, 1987). Curtis et al. (1983)

did not find an association between metritis and milk fever, in agreement with

Markusfeld (1987) who found no association between metritis and milk fever. However,

Grohn et al. (1990) found that cows with parturient paresis were more likely (OR = 1.5)

to develop puerperal metritis compared to cows without parturient paresis.

Postpartum Health Monitoring

An important concept of a dairy herd health program is early disease diagnosis and

treatment of sick cows. A delay in treating a sick cow not only reduces her chances for a

full recovery, but results in milk production loss that may impair reproductive

performance. Because the early postpartum period of the dairy cow determines

productive and reproductive responses during lactation, it is a pivotal time in the

production cycle of the cow. During this period, dairy cows are at risk of developing

calving related diseases, such as hypocalcemia, puerperal metritis, ketosis and

displacement of the abomasum (Curtis et al, 1983). These are costly disorders with

estimated economic losses ranging from 200 to 400 dollars per case per lactation (Bartlett









et al, 1986). Monitoring the postpartum health of dairy cows allows the opportunity to

identify sick cows early and provide appropriate therapy. Furthermore, it can help prevent

diseases.

Diagnostic tests are used to classify or confirm a disease process and to provide

appropriate treatment. A test is a device or process designed to detect, or quantify a

clinical sign, substance, tissue change, or body response in an animal. Tests are used for

screening and to identify the proportion of diseased or sick animals correctly (sensitivity).

After a positive result is obtained an in depth diagnostic work-up is performed, in order to

correctly answer whether or not the animal in question is sick or not (specificity).

Sensitivity and specificity are the most important characteristic of a test, however they do

not directly tell how useful is the test in detecting a disease. Consequently, predictive

values are used to estimate the probability that an animal with a positive test result for a

particular disease is truly positive (has the disease; positive predictive value) or is truly

negative (does not have the disease; negative predicted value).

A postpartum health monitoring program consists of evaluation of rectal

temperature, attitude, milk production, urine for the presence of ketone bodies and

characterization of uterine discharge. To some extent these procedures are objective,

however the majority are subjective and related to the experience of the technician who

performs them.

Attitude

Attitude is a subjective parameter that describes the anatomical impression of the

patient (Rosenberger, 1979). Attitude and posture are synonyms, although posture relates

mainly to the disposition of the limbs, attitude is used as a behavioral indicator. For the

assessment of attitude, the evaluator has to assess by external visual inspection the









position or relation of the lips, ears, head, neck, forelimbs and tail, in relation to the body

of the animal (Rebhum, 1995). A healthy cow is one that it is aware of her environment

and displays the common curious behavior of cattle. There are some attitudes or postures

that suggest a specific diagnosis or a specific system disorder (Rebhum, 1995). Changes

in the shape of the vertebral column and a tense abdomen are characteristics of an

abdominal illness such as peritonitis which can be related to different causes like

traumatic reticuloperitonitis, as well as septic metritis (Rosenberger, 1979). Holding the

tail up usually indicates a painful process in the anus, rectum or genital tract,

accompanied by fractious compressions of the abdominal wall (tenesmus). Animals with

an extended neck and head usually suffer from pharyngeal and esophageal obstruction or

from severe respiratory diseases (Rosenberger, 1979).

Certain attitudes displayed by cows are grouped and measured in a scoring system

to add objectivity to this subjective approach. One of the most common scoring systems

used in the dairy industry is lameness evaluation in order to asses the severity of

lameness in the cow. This scoring system consists in the observation of the stationary as

well as the walking attitude of the cow (Sprecher, et al., 1997). The scoring system is

based on a five point scale were: 1) normal: the cow stands and walks with a level-back

posture, her gait is normal; 2) mildly lame: the cow stands with a level-back posture but

develops an arched-back posture while walking, her gait remains normal; 3) moderately

lame: arched-back posture is evident both while standing and walking, her gait is affected

and is best described as short striding with one or more limbs; 4) lame: an arched-back

posture is always evident and gait is best described as one deliberate step at a time, the

cow favors one or more limbs/feet and 5) severely lame: the cow additionally









demonstrates an inability or extreme reluctance to bear weight on one or more of her

limbs/feet.

Positioning of the cow's ears is also a good indicator of a cow's attitude. Sick cows

usually have ears that droop down due to depression, pain, or fever. Healthy cows on the

other hand, appear bright, alert and are curious about their environment. Positioning and

appearance of the eyes within the socket have also been used to asses the level of

dehydration. A scoring system such as 1 (minimal), 2 (mild), 3 (moderate), or 4 (severe)

has been proposed to asses dehydrated state (Smith and Risco, 2005). A cow with a score

of 1 usually will have bright eyes that are positioned normally within the eye socket. A

cow with a score of 2 will have dull eyes that are slightly sunken (1-2mm) within the eye

socket. A cow with a score of 3 will have glazed eyes that are moderately sunken (2-4

mm) where a cow with a score of 4 will have dry eyes that are severely sunken (>5mm)

within the eye socket. However, this method has not been compared with the actual

degree of dehydration.

Changes in feeding behavior can also be used as an indicator of health. In those

farms that have locking stanchions, the attitude of the cow can be observed after feeding

to evaluate appetite. A cow that is sick will not eat, conversely a healthy cow

aggressively consumes her feed. Smith and Risco (2005) have proposed the following

scoring system to evaluate appetite; 1) cows that lock and eat 2) cows that lock appear

dull and do not eat and 3) cows that do not lock to eat and appear dull or sick. Cows that

fall in categories 2 or 3 should be monitored or examined carefully for illness.

Urton et al,. (2005) evaluated if depression in feeding behavior was a good

indicator of metritis (mucopurulent, foul smelling discharge and fever > 39.50C ) or acute








metritis (reddish brown, watery, foul smelling discharge with fever > 39.50C), in dairy

cows. Both prepartum and postpartum feeding behavior were observed in healthy cows

and in those that developed metritis or acute metritis postpartum. Cows diagnosed with

either metritis or acute metritis spent less time feeding pre and post partum than did non-

metritic cows. These cows also spent significantly less time feeding over the post-calving

period than did their healthy counterparts. However, only those cows diagnosed with

acute metritis showed significantly lower feeding time during the precalving period.

Figure 2-9 represents the feeding times for cows with metritis and cows with acute

metritis during pre and post partum period.

120


105








60-


45-


3 0 ,, r
-12 -6 0 6 12 18

Day relative to calving
Figure 2-9. Daily mean feeding time (min/d) of 9 Holstein cows with acute metritis (A)
and 17 Holstein cows without acute metritis (o) (SE) from 12 d before calving until 19
d after calving.









Furthermore, the risk of acute metritis related to prepartum feeding behavior was

evaluated. It was found that for every 10 minutes in reduction of feeding behavior during

the prepartum, the odds of developing acute metritis increased by 1.57 when compared to

cows without acute metritis. In addition in the final model parity was significant, and was

shown that primiparous cows were at a higher risk

Milk Production

The evaluation of daily milk production is an objective parameter that can be used to

identify sick cows. It has been suggested that an unexpected decrease in milk production

may be reflecting the inappetence of the cow, and thus it may be used as a monitoring

parameter to identify sick cows (Smith and Risco, 2005). The application of this method

has been extended with the use of computerized milk-meters which identify and record

the production of individual cows on a daily basis. Results in lactation performance have

been published in diseases such as ketosis, metritis, or displacement of the abomasum.

Rajala-Schultz et al. (1999) found that milk yield decreased before the diagnosis of

clinical ketosis, and the loss of milk continued for at least 2 wk after diagnosis. The effect

of metritis, as one diagnosis, did not have any effect on milk yield (Raj ala-Schultz and

Grohn 1998). Metritis in the cited study (Rajala-Schultz and Grohn 1998) was defined as

early < 28 days postpartum and late metritis > 28 days postpartum. Once these categories

were separated, the importance of the timing of the disease on milk loss became apparent.

Late metritis did not have a significant effect on milk yield, but early metritis

significantly reduced milk yield. This result could be explained by the possibility that the

cases of early metritis were more severe and included systematic symptoms as high

temperature and inappetence, possibly resulting from difficult calving or retained

placenta. Deluyker et al. (1991) found milk losses that varied from 250 to 800 kg during a









305-day in cows with left displaced abomasums. However, few studies have been

conducted to determine the effect of postpartum diseases on daily milk production and its

association to postpartum health as a diagnostic tool.

Daily milk production can be recorded by milking machine software in which cows

are identified if there is a deviation from an expected milk production. Changes in daily

milk production can be used to identify those cows that have a drop in milk production,

or are not producing at an expected level (Smith and Risco, 2005). Also, milk production

can be measured as a rate of milk produced per hour between consecutive milking

sessions (in kg/h). This parameter may be more accurate than daily milk yield parameter

since it reflects the actual milk production changes during milking session intervals. It

provides the opportunity to monitor more frequently (2-3 times a day) the performance of

an individual cow (Moallem, et al., 2002).

Edwards and Tozer (2004) investigated the possibility of predicting an occurrence

of a disease before clinical diagnosis based on changes in daily walking activity

(measured with activity meters) and milk production of cows with left displaced

abomasum (LDA), ketosis, and digestive problems. Walking activity in cows with these

disorders gradually decreased from 8 days to 1 days before clinical diagnosis. In addition,

milk yield began to decline 6 days before diagnosis of cows with ketosis, 7 days for cows

with an LDA, and 5 days for cows with digestive disorders. Overall activity started to

decline before milk production. Figure 2-10 represents the difference in activity and daily

milk yield for cows with an occurrence of ketosis, left displaced abomasum, and general

digestive disorders during days -10 to 10 relative to the day of diagnosis (day 0).












Consequently, using both activity and milk production together would be more sensitive


in detecting cows with LDA, ketosis and digestive problems.


30-





-10 -5 -10

-20-
-30-
-40-
-50-


(a)


I mO


Relative to day of illness


06
I

a.


*5




C3
Pt




(0
ez











C. 2
Sc











T
Q
C3
a



5

i
a)










C. 7
-> --
_ 0


Cu






2




a
reC


o _-U -- -- t *-----,
10 -5 _

-20

-30-

-40
-50
Relative to day of illness


Relative to day of illness


Figure 2-10. Difference in activity (-) and daily milk yield (-) for cows with an
occurrence of ketosis (a), left displaced abomasum LDA (b), and general digestive
disorders (c), compared with cows without an incidence of a disease in the prebreeding
stage during day -10 to 10, relative to the day of diagnosis (day 0).


(b)


I









Rectal Temperature

Body temperature affects tissue function and is the result of chemical and

physiological processes. The cow as a homothermous mammal has the capacity to

maintain a constant temperature range under different environmental conditions, through

the inputs and output of heat regulation. An increase in body temperature beyond the

normal range can be characterized as fever pyrexiaa), or hyperthermia. Fever is defined as

a controlled elevation of core body temperature by supportive changes in thermo-effector

activities. It is commonly regarded as beneficial, that is, having survival value (Kluger,

1986). Fever is the result of communication between the peripheral immune system and

the brain in response to infection, inflammation and/or trauma, and is clinically

characterized by a rise in the body temperature (Cunningham, 2002). In contrast,

hyperthermia is a rise in core temperature resulting from the inability to lose sufficient

heat to balance the total of endogenous plus exogenous heat loads and can be potentially

lethal (Hales et al., 1996).

The rectal temperature (RT) is obtained by introducing a glass or a digital

thermometer through the anal canal into the rectum and placed in close contact to the

rectal mucosa. The blood supply to the rectal mucosa is derived from the cranial artery of

the caudal mesenteric artery, and by several short middle rectal branches from the caudal

branch of the urogenital artery (Getty, 1975). Because of the close contact of the rectal

mucosa to its blood supply, temperature in blood is transferred to the rectal mucosa.

Consequently, rectal temperature is a useful indicator of the core temperature of the

animal. However, rectal temperature is lower than the core temperature of the animal, and

changes in rectal temperature may lag behind changes in core temperature (Cunningham,

2002).









Monitoring of rectal temperature during the first 10 days post partum has been used

to identify cows with fever (Upham, 1996). Normal RT has been reported to range

between 38.0C to 39.10C (Rebhun, 1995) or 38.0C to 39.00C (Rosenberger, 1979).

Cows with RT above the upper limit are defined as febrile or abnormal. However,

because of the multiple factors that affect RT, a cut-off value to define fever is difficult to

define (Rebhun, 1995; Rosenberger, 1979). Different cut-off values have been reported to

define fever in cows with uterine infections; 39.20C (Smith et al., 1998), 39.30C (Kristula

et al., 2001) greater than or equal to 39.40C (Upham, 2001), and 39.50C (Drillich et al.,

2001). Sheldon et al. (2004) described a cut off point for defining fever by using the

maximum RT between days 1 and 10 post partum: mean of 39.30C, and upper quartile

equal to > 39.70C, which was defined as a febrile level. Kristula et al. (2001) have

suggested that cows with RT between 38.80C and 39.40C should be monitored carefully

to determine whether or not they require systemic antibiotic therapy. Furthermore,

because cows with a RT of 39.70C experienced a reduction in RT of 0.60C after

treatment, a cut-off value of RT of 39.40C or 39.70C was proposed to be used in the

decision of whether or not to initiate antibiotic treatment.

The protocol for monitoring daily RT in postpartum cows has been readily

accepted by dairy producers and veterinarians because it is an objective response that can

be used to evaluate health. However, this protocol is time consuming because all

postpartum cows are monitored including those cows that calved normally that may be at

a lower risk to develop puerperal metritis. Therefore, the benefit of evaluating all cows is

unknown. In a study that evaluated postpartum RT, 48% of cows that calved normally

had at least one daily temperature above 39.1 C compared to 93, 83, 100 and 78 % for









cows with RFM, mastitis, metritis and dystocia respectively (Kristula et al, 2001). In

addition, cows with an abnormal parturition (RFM, dystocia with or without RFM) had

rectal temperatures greater than 39.0 C for significantly more days (2.9) than cows that

calved normally (1.9) (Kristula et al, 2001). However, they conclude that two

consecutive day with a RT greater than 39.1 C will not be a sufficient indicator to treat

the cows with an antibiotic.

Ketones Bodies

Ketosis is a disorder of carbohydrate and fat metabolism characterized by increased

concentrations of ketone bodies in blood (ketonemia), urine (ketonuria), milk (ketolactia),

and other body fluids (Geishauser, et al., 1998). The metabolic state of ketonemia has a

negative effect on milk production (Rajala-Schultz et al., 1999; Detilleux et al., 1994) and

reproduction (Andersson et al., 1991).

The major ketone bodies are P-hydroxybutyrate (BHBA), acetoacetate (AcAc),

and acetone ( Ac) (Andersson, 1988). During early lactation, the amount of energy that is

required for maintenance of body tissues and milk production exceeds the amount of

energy the cow can obtain from dietary sources (Baird, 1982). As a result, the cow must

utilize body fat as a source of energy. However there is a limited amount of fatty acid that

can be oxidized to completion by the tricarboxylic acid cycle in the liver or exported

from the liver as very low density lipoprotein. When this limit is reached, triglycerides

accumulate within the hepatocytes, impair their function, and the acetyl-coenzyme A that

is not incorporated into the tricarboxylic acid cycle is converted to acetoacetate and P-

hydroxybutyrate. The appearance of these ketone bodies in blood, milk, and urine is

diagnostic of ketosis and usually becomes clinically evident from 10 d to 3 wk after

calving (Goff and Horst 1997). In addition, ketosis may also occur secondary in cows









with diseases that affect feed intake (Kronfeld, 1982). Risk factors for subclinical ketosis

include metritis, displased abomasum, RFM and hypocalcemia (Curtis et al., 1985;

Correa et al., 1993). However a cause-effect relationship has not been clearly defined

(Duffield, 2000).

Analyses for the presence of ketone bodies in urine and milk are commonly used to

diagnose ketosis in cattle. Strips, tablets or powders that contain nitroprussic acid are

used semiquantitatively to measure the concentration of ketone bodies in urine or milk

(Larsen and Nielsen, et al., 2005). However, these tests vary in sensitivity and specificity

(Table 2-1) (Duffield, 2000).

Table 2-1 Ketosis threshold, sensitivity and specificity for different ketosis test.

Subclinical
Ketosis Threshold .
Test and body fluid Kets Thre d Sensitivity (%) Specificity (%)
(Serum BHB
[tmol/L)
Utrecht nitroprusside-milk 1400 90 96
Bioketone (urine) 1200 33 100
Ketocheck (urine) 1200 28 100
Utrecht powder (urine) 1200 42 100
Ketostix (urine) 1200 5 100
Ketolact-50 atmol/L (Milk) 1200 92 55
Ketolact-100 atmol/L(Milk) 1200 72 89
Ketolact-200 atmol/L(Milk) 1200 45 97
Ketolact-500 tmol/L(Milk) 1200 17 100
Ketocheck (Milk) 1400 38 99
Ketocheck (Milk) 2000 61 98
Duffield, T. 2000. Subclinical ketosis in lactating dairy cattle. Vet. Clin. North Am.
Food Anim. Pract. 16:231-253.

Given the high incidence of ketosis (29 to 35%) from primary and secondary

causes (Emery et al., 1964; Duffield et al., 1998) during the first 2 weeks post partum,

monitoring postpartum cows for this condition may be of limited value if applied only to









identify sick cows. In addition, the utility of cow side ketone tests to identify sick animals

with a primary disease such as displaced abomasum, mastitis or metritis is not known.

Evaluation of Uterine Discharge

A major component of a postpartum health monitoring program is the evaluation of

uterine discharge by rectal palpation in order to screen cows that have uterine infections

(Griffin et al., 1974; Studer and Morrow, 1978; Roberts, 1986, Upham, 1996). The ability

to diagnose uterine infections by rectal palpation varies among veterinarians (Lewis, et

al., 1997). Typically, evaluation of the uterus by rectal palpation is performed between 25

to 50 days postpartum to evaluate uterine health (Studer and Morrow, 1978). Evaluation

of uterine health by rectal palpation has been directed to the first two weeks postpartum

(Uphan, 1996) for early identification and treatment of puerperal metritis to reduce the

adverse effect of metritis on fertility (Callahan and Horstman, 1987).

The use of rectal palpation to evaluate uterine infection is used commonly by

veterinarians. However, examination of the vagina with a speculum provides a more

accurate diagnosis of endometritis that rectal examination. A vaginal examination in

combination with rectal palpation of the uterus 25 to 50 days post partum resulted in

twice as many cows with positive cultures for pathogenic bacteria compared to cows

classified as infected using rectal palpation alone (59% vs 22%, respectively) (Miller et

al., 1980). Studer and Morrow (1978) found gross palpation of uterine size was

associated weakly with bacterial culture and histology. In a study by LeBlanc et al (

2002), a cervical diameter at rectal palpation of a >7.4 cm either at 20 to 26 or 27 to 33

days post partum by rectal palpation and presence of a mucopurulent, purulent or foul

uterine discharge by vaginoscopy inspection at 27 to 33 days post partum were predictors

of reproductive performance. For the palpation-based case definition of clinical









endometritis, ignoring vaginoscopy, the sensitivity and specificity were 17 and 88%,

respectively, for non-pregnancy beyond either 120 or 150 DIM. However, between 27

and 33 days pos partum, diagnosis of endometritis with vaginoscopy was more sensitive

than palpation-based diagnosis (21 vs. 12% sensitivity for classification of non-

pregnancy beyond 150 day in milk, respectively). However, during this period,

specificity of rectal palpation was higher than vaginoscopy (89 and 94%, respectively).

Consequently, because of the changes in size and discharges that the uterus undergoes

early post partum, the criteria for assessing uterine health change according to days post

partum at examination (Ferguson, 1995). In the study by Callahan and Horstman (1987) a

metritis (fluid, crepitus, lack of myometrial tone, and vaginal discharges) incidence of

33% at 1-3 weeks was reported. However, if the first examination had not been

performed until 2 weeks later, the incidence of metritis would have been 7.8%.

Based on the review by Sheldon et al. (2006) in which the proposed definitions for

the different uterine infections, it can be inferred that the studies previously published

that evaluated rectal palpation as a diagnostic tool (Studer and Morrow, 1978; Miller et

al., 1980; Callahan and Horstman, 1987) referred to endometritis and not puerperal

metritis or metritis. Consequently, it remains unclear and more research is needed on the

test characteristics of rectal palpation to evaluate uterine discharge as it relates to

puerperal metritis.














CHAPTER 3
MATERIALS AND METHODS

Cows and Herd Management

The study was conducted between August 2002 and April 2003 in a commercial

dairy farm located in north east Florida (30 18' N, 81 56' W). One thousand lactating

cows were milk with a yearly rolling herd milk production average of 9,165Kg. The herd

was milked 3 times per day. The farm was a member of DHIA (Dairy Records

Management Systems, Raleigh, NC) and used an on-farm computer based record system

for maintaining production and health data.

Prepartum transition cows that were within 3 weeks of calving were maintained on

dry lots, fed a cationic diet and monitored for signs of calving by farm employees trained

to assist with parturition. Calving events such as dystocia and retained fetal membranes

(RFM) were recorded by farm personnel. Dystocia was defined and recorded based on a

five point scale as follows: (1) no assistance (2) slight problem (assistance for < 15

minutes) (3) needed assistance (assistance for > 15 minutes with moderate difficulty for

extraction) (4) considerable force used (5) extreme difficulty or veterinary assistance

(DHIA, 1997). Cows that did not expel the placenta within 24 hrs after calving were

considered to have RFM (Risco and Hernandez, 2003). Cows with dystocia delivered by

cesarean section or fetotomy were not included in the study.

After parturition cows were sent for 2 days to a hospital herd housed in a concrete

floor open-sided barn with stanchions that provided free access to a dry lot. At the

hospital barn, cows were treated according to standard operation procedures of the farm









which consisted of administration of an oral calcium paste (Balance, Bayer, Shawnee

Mission KS) and a single intrauterine infusion of 6 g of oxytetracycline dissolved in 75

ml of sterile water to multiparous cows with RFM. At three days post partum, cows were

moved to a lactating herd kept in an open barn with dry compost bedding, and fed four

times a day. Diets for both pre-and postpartum transition cows were a total mixed ration

formulated to meet the requirements of lactating dairy cows according to guidelines

established by the National Research Council (NRC, 2001).

Reproductive management consisted of a voluntary waiting period of 60 days.

After that, cows were identified in estrus by visual observation with the aid of tail chalk

or neck activity meters (WestfaliaSurge, Inc. 1880 Country Farm Drive 60563 Naperville

IL) Cows not artificially inseminated by 80 days post partum were examined for

cyclicity, and cows with a corpus luteum (CL) were treated with 25 mg of PGF2,

intramuscularly (IM) (Lutalyse, Pfizer Animal Health, Kalamazoo, MI.). Cows were

artificially inseminated at detected estrus. Non-cyclic cows with inactive ovaries were

treated with 100 [g of GnRH IM (Cystorelin, Meriel Limited, Iselin, NJ.) followed by

25 mg of PGF2, IM 7 days later and AI at detected estrus. Cows not seen in estrus 14

days after PGF2, treatment were re-examined for cycling status and treated with PGF2,

only if a CL were present. Cows not AI by 120 days post partum were enrolled in the

OvSynch program (Pursley et al., 1995). Cows not pregnant > 150 days post partum were

also enrolled in the OvSynch program.

Study Design

This study followed a prospective cohort design. All cows underwent a postpartum

health monitoring program consisting of daily evaluation of rectal temperature (RT) and

attitude from day 3 to 13 post partum. All cows were examined for clinical endometritis









between 20 to 30 days post partum. Rectal temperature was determined with the use of a

digital thermometer (GLA, Agricultural Electronics, San Luis Obispo, CA) between 0700

to 0900 h immediately after milking. Cows that either appeared sick (depressed, sunken

and/or tented eyes) or had a RT > 39.4C were examined for puerperal metritis. The

criterion for diagnosis of puerperal metritis were the presence of a watery, brown-

colored, fetid discharge from the vulva (noted after rectal palpation of the uterus), with or

without a RT > 39.4C. All information concerning rectal temperature and incidence of

puerperal metritis were stored daily in a separate database belonging to the principal

investigator. Cows diagnosed with puerperal metritis were treated daily with ceftiofur

hydrochloride (2.2 mg/kg IM. Excenel, Pfizer Animal Health, Kalamazoo, MI) IM for

three days. In addition, supportive therapy consisting of anti-inflammatory agents,

calcium and energy supplements were administered. Cows that did not respond to the

three day ceftiofur treatment, based on the persistence of a fetid discharge, received an

intrauterine infusion of 3 g of oxytetracycline diluted in 75 ml of sterile water.

The criterion used to diagnose clinical endometritis between 20 to 30 days post

partum were one of the following condition associated or not with each other: cervical

diameter greater than 6.0 cm; asymmetry of the uterine horns with fluid content and/or

pus present at the vulva following rectal manipulation of the uterus. All cows diagnosed

with clinical endometritis were treated with a single injection of 25 mg of PGF2,

(Lutalyse, Pfizer Animal Health, Kalamazoo, MI.) IM. Pregnancy diagnosis was

determined by trasrectal palpating of the uterus and its contents 42 to 49 days after

artificial insemination (Zemjanis, 1970).









Data Management

Data for parity and calving status: (dystocia: calving score > 3, RFM, and twins)

were recovered from the database, and two groups of cows were established based on

calving status. Cows with a normal calving (NC) status were those without calving

related problems, and cows with an abnormal calving status (AC) were those with

dystocia, RFM with or without dystocia or twins. Cows were classified as having

puerperal metritis (M+) or without puerperal metritis (M-) in a two level variable

classified as metritis. Cows with and without metritis were also classified in a 3 level

variable defined as Mettemp (MT) according to whether or not they had fever (RT >

39.4C) when puerperal metritis was diagnosed. Cows with puerperal metritis and fever

were classified as MT-1, cows with puerperal metritis without fever at the time of

diagnosis were classified as MT-2, and cows without puerperal metritis as MT-3.

Cows were classified as primiparous or multiparous in a two level variable

classified as parity. The different seasons during the study were defined based on the

thermal heat index (THI = td (.55 .55RH) (td 58)) (West, 1994). This index was

calculated using daily ambient temperature (td) and percent relative humidity (RH)

recorded at the closest weather station at Macclenny, FL (300 24' N, 82 11' W). Based on

a previous report (West, 1994) a cut-off of 76.2 was used to define two seasons. A cool

season was defined as those months with an average THI of < 76.2 (October to April),

and a warm season were those months with an average THI > 76.2 (May to September).

Statistical Analyses

All outcome variables and the incidence of puerperal metritis and clinical

endoemtritis were analyzed using logistic regression PROC GENMOD of SAS 9.1;

(SAS, 2003) with a binomial distribution and logit link.









Modeling was performed using manual backward elimination starting from the

more complex model with a third order interaction and the exclusion criteria were

determined at P > 0.30. The model fit statistics were performed by comparing the

difference in the deviances by the likelihood-ratio statistic test (Agresti, 1986). Main

effects were forced all models. Adjusted odds ratios (AOR) and 95% confidence intervals

(95% CI) were determined.

The model used to analyze the incidence of puerperal metritis included the main

effects of calving status, parity, and season at calving. A second model was used to

analyze the incidence of clinical endometritis and included calving status, parity, season

at calving, and puerperal metritis. A third model was analyzed the incidence of clinical

endometritis and the variable puerperal metritis was substituted by the MT variable.

Reproductive performance was evaluated by analyzing first service pregnancy rate

(%), cumulative pregnancy rate by 150 days in milk (%) and, number of inseminations

for pregnant cows (mean + S.E.M). Number of inseminations for pregnant cows was also

analyzed by logistic regression (PROC GENMOD, SAS) with a Poisson distribution and

logit link. All models for reproductive performance included the main effects of calving

status, parity, season at first service, puerperal metritis and clinical endometritis as

explanatory variables. Time to pregnancy was analyzed with survival analysis using

Cox's proportional hazards regression (PROC PHREG, SAS). Adjusted hazard ratio (HR)

was obtain from the proportional hazards regression model and crude median days open

and the survival function estimates for the cumulative pregnancy up to 150 days

postpartum graph were obtained from the Kaplan-Meier analysis (PROC LIFETEST,

SAS).









Data for daily RT from days 3 to 13 post partum were used to compare the RT of

cows with or without puerperal metritis, and descriptive statistics were determined.

Rectal temperature of cows with puerperal metritis 5 days prior and 5 days after diagnosis

were compared to the rectal temperature of cows without puerperal metritis (control

cows). Control cows, without puerperal metritis, were assigned to cows with puerperal

metritis, by controlling for group, parity and season, using a random number generator

procedure. In cows with puerperal metritis, days post partum when puerperal metritis was

diagnosed were reclassified as follow: Day 0 day postpartum when puerperal metritis was

diagnosed: days -1, -2, -3, 4, 5 before diagnosis, and days 1, 2, 3, 4, 5 after diagnosis.

Days post partum of control cows were similarly re-classified and matched to cows with

puerperal metritis in a rate of 3.7 controls cows per each puerperal metritis case.

Therefore, day 0 for control cows (without puerperal metritis) corresponded with the

same day post partum when puerperal metritis was diagnosed. This rearrangement of the

data was done to control for the rectal temperature by days post partum of cows with or

without puerperal metritis.

The analysis of RT between cows with and without puerperal metritis by day post

partum was performed using PROC MIXED procedure for repeated measures of SAS

9.1(SAS, 2003). The model was subjected to 4 covariate structures: compound symmetry,

compound symmetry-heterogeneous, autoregressive order-1 and autoregressive order-1

heterogeneous matrix. The autoregressive order-1 covariance structure was found to have

the smallest Akaike's information criterion and Schwarz's Bayesian criterion (Littell et

al., 2002). Consequently the covariate structure that specified a correlation structure

within cows that decreased with increasing lag between measurements was used in the









model. Two models were analyzed. The first model included the effects of calving status,

puerperal metritis, parity, season at calving and day as main effects as well as the second

and third order interactions between the main effects. The second model was analyzed by

including the variable Mettemp as a replacement for the variable Metritis. This variable

(Mettemp) differentiates the daily rectal temperature of cows with puerperal metritis and

fever, and cows with puerperal metritis without fever at the day post partum of puerperal

metritis diagnosis, and cows without puerperal metritis. For both models, repeated

measurements of RT were also analyzed by testing homogeneity of regression curves for

day trends. A single polynomial regression for day was fitted for RT, and the differences

from fitting individual regressions for the effect of calving status, metritis, parity, season

and their interactions were tested. Given that not all puerperal metritis cases were

diagnosed on the same day post partum, the actual post partum day at diagnosis was

included in the model as a covariable for both models.

The mixed model for repeated measures was:

Yijklmfg = u + Ti + Cow(Ti) + Dayk + Par + Seasm + Statf + Dmetg (Day x T)ki +
eijklmfg
Where:
Yijklmfg = daily rectal temperature
Ti = fixed effect of group (normal or abnormal calving status)
Cow(Ti)j = random effect of cow nested in group
Dayk = fixed effect of time
Par = fixed effect of parity
Seasm = fixed effect of season
Statf = fixed effect of metritis (yes, no or MT-1, MT-2, MT-3)
Dmetg = regression coefficient for days in milk to metritis
(Day x T)ki = fixed effect of interaction time and treatment
e ijklmfg = random error term

Differences among rectal temperature for the different days were determined by the

use of PDFF option of SAS. Least squares means ( SEM) of RT were determined and

illustrated in the graphics.














CHAPTER 4
RESULTS

Final Sample

A total of 488 cows calved during the study period and thirty-eight (38) cows were

not included in the study because they received antibiotic treatment beyond 3 days

postpartum at the hospital barn and did not complete 10 days of health evaluation.

Therefore, a total of 450 calvings was evaluated, of which 327 (73%) were classified as

normal, and 123 (27%) as abnormal. Ninety four (94) cows were diagnosed with

puerperal metritis of which 55 (58.5%) had no fever, and 39 (41.4%) had fever.

Incidence of Diseases

In the logistic regression multivariate analysis, cows with normal calving status had

a lower incidence of puerperal metritis compared to cows with an abnormal calving status

(43/327 (13%) vs. 51/123 (41%), respectively; P < 0.001). There was a significant

interaction (P < 0.01) between parity and season. During the cool season, primiparous

cows had the highest incidence of puerperal metritis compared to primiparous cows in

warm season or multiparous cows in either season: Cool season: 39.4% > 11.0% for

primiparous and multiparous, respectively; and for primiparous 39.4% > 12.7%, for Cool

vs. Warm, respectively (Table 4-1).

The overall incidence of clinical endometritis was 24%; Cows with abnormal

calvings were more frequently diagnosed with clinical endometritis than those with

normal calving status (AOR = 2.8, 95% CI 1.7-4.9, P < 0.001). A higher incidence (

38.2%) of clinical endometritis was found to be significant for those cows diagnosed with









Table 4-1. Incidence and risk factors of puerperal metritis in the first 13 days post partum
of lactating dairy cattle.

Incidence of
Variable Level Puerperal Risk of Puerperal Metritis
Metritis
% N AOR 95% CI P-Value
Metritis
Yes 21.1 94
No 79.1 356
Calving Status 0.001
Normal 13.1 43/327 Referent Referent
Abnormal 41.4 51/123 4.80 2.9-8.00
Lactation X
Season
Cold
Multiparous 11.0 22/200 Referent Referent
Primiparous 39.4 54/137 5.05 2.82 9.02
Warm
Multiparous 18.1 12/66 Referent Referent
Primiparous 12.7 6/47 0.84 0.39-3.56
Primiparous
Cold 39.4 54/137 Referent Referent
Warm 12.7 6/47 0.24 0.09-0.62
Multiparous
Cold 11.0 22/200 Referent Referent
Warm 18.0 12/66 1.43 0.65-3.18

puerperal metritis (AOR = 2.2, 95% CI 1.1-3.9, P < 0.005) compared to cows without

puerperal metritis. The incidence of clinical endometritis of cows diagnosed with

puerperal metritis and fever (38.1%) and puerperal metritis without fever (38.4%) at the

day of diagnosis was greater than cows without puerperal metritis (20.2%) (puerperal

Metritis with Fever (AOR = 2.2, 95% CI 1.07 4.6, P < 0.02); puerperal Metritis without

Fever (AOR = 2.1, 95% CI 1.09 4.19, P < 0.02). Non significant difference was found

between fever and no fever puerperal metritis cows on the incidence of clinical

endometritis (P < 0.9); Table 4-2.









Table 4-2. Incidence and risk factors of clinical endometritis at 20 to 30 days post partum
of lactating dairy cattle.

SIncidence of Clinical Risk of Clinical
Variable Level
Endometritis Endometritis
P-
% N AOR 95% CI
Value
Endometritis
Yes 24.0 108 -
No 76.0 342 -
Calving Status 0.001
Normal 17.7 58/327 Referent Referent
Abnormal 40.6 50/123 2.80 1.70 4.90
Parity 0.07
Multiparous 26.0 69/266 1.00 Referent
Primiparous 23.3 43/184 0.63 0.39- 1.03
Puerperal
Metritis
No Metritis 20.2 72/356 Referent Referent
Metritis 38.2 36/94 2.20 1.10 3.90 0.005
No Fever 38.4 21/55 2.10 1.09 4.10 0.02
Fever 38.1 15/39 2.20 1.07 4.60 0.02

Rectal Temperature

The mean ( SEM), median, upper and lower quartiles and the 95% confidence

interval for RT from day 3 through day 13 post partum are shown in Table 4-3. In both

models the effect of day to diagnosis was significant (P < 0.001). The analysis of model

one showed a significant interaction between DAY and METRITIS (P < 0.001). Curves

of daily rectal temperature during this period described a polynomial of second order for

puerperal metritis cows and first order for cows without puerperal metritis. Rectal

temperature from cows with puerperal metritis was significantly higher in cows without

puerperal metritis 72 h before the diagnosis of puerperal metritis (DAY -3: 38.70C 0.05

> 38.60C 0.03; P < 0.009) and daily comparisons between groups continued to be

different until the fourth day after the diagnosis and treatment of puerperal metritis (DAY

4: 38.70C 0.07 > 38.50C 0.04; P < 0.001).









Table 4-3. LSM (SE), 25th quartile, median, 75th quartile, and population 95%
confidence intervals rectal temperatures of cows with and without puerperal metritis.

LSM SE 25th Q Median 75th Q LSM 1.96SD
No Metritis Cows 38.6 + 0.01 38.3 38.5 38.8 37.8 39.3
Primiparous 38.5 0.02 38.2 38.5 38.8 37.5 39.6
Multiparous 38.6 + 0.01 38.3 38.5 38.8 37.8 39.4
Metritis Cows 38.9 + 0.03 38.4 38.7 39.1 37.4 40.3
Primiparous 38.9 + 0.04 38.4 38.7 39.1 37.7- 40.6
Multiparous 38.8 0.05 38.3 38.7 39.2 36.7 40.9
Mettemp-1 (n=55) 38.7 + 0.04 38.3 38.6 39.0 36.7- 40.7
Mettemp-2 (n=39) 39.2 0.05 38.5 39.0 39.4 37.1 41.3

Rectal temperature in cows that were diagnosed with puerperal metritis increased

between 48 h (DAY -2: 38.80C 0.07) to 24 h (DAY -1: 38.90C 0.06) before diagnosis

of puerperal metritis (difference 0.130C 0.08 C, P < 0.09), and continued to increase

until reaching a maximum RT of 39.20C 0.05 on DAY 0 (day of diagnosis). Twenty-

four hours before the diagnosis of puerperal metritis, daily increments in RT were found

to be significant (difference, 0.280C 0.07 C; P < 0.001). After treatment (DAY 1), RT

of cows with puerperal metritis showed a significant reduction (0.330C 0.07 C, P <

0.001), and subsequent daily reductions in RT were not significantly different (Figure 4-

1). Curves of RT between cows with puerperal metritis and cows without puerperal

metritis tested by homogeneity of regression showed a significantly difference linear and

quadratic effect (P < 0.001).

In model 2 there was a significant interaction DAY and METTEMP (P < 0.001).

Curves of daily rectal temperature 5 days prior and 5 days after the diagnosis of puerperal

metritis described a polynomial of second order for MT-1 and first order for MT-2 and

MT-3. Rectal temperature from MT-1 cows had higher RT 72 h before the day of










diagnosis of puerperal metritis compared to MT-3 cows (DAY -3: 38.80C 0.10 >

38.60C + 0.04; P < 0.04).



39.4



39.2
I
/ \
0 f** \



7 38.8
S -I

38.6



38.4
-5 -4 -3 -2 -1 0 1 2 3 4 5
Day before and after metritis
Figure 4-1. LSM + SEM of daily rectal temperatures of cows 5 days prior to and 5 days
after the diagnosis of metritis, for cows with puerperal metritis (n=94; i), and cows
without puerperal metriti (n=356; +). *P < 0.05 **P < 0.001.

This daily difference continued to be significant until 24 h after the diagnosis and

treatment of puerperal metritis (DAY 1: 39.00C 0.09 > 38.50C 0.04; P < 0.001). In

this group (MT-1), the RT started to increase 72 hours (DAY -3: 38.80C 0.10) to 48 h

(DAY -2: 39.00C 0.09) before the diagnosis of puerperal metritis (difference 0.200C

0.12 C, P < 0.09), and continued to increase until reaching a maximum of 39.70C 0.09

on the day of diagnosis (Day 0).

Twenty four hours before the diagnosis of puerperal metritis, a daily increment in

RT of 0.530C + 0.11 was found to be significant (P < 0.001). After treatment, cows with

puerperal metritis and treated showed a significant reduction in RT in the first 24 h

(0.650C 0.11 C, P < 0.001). This reduction continued to be significant between 24












(DAY 1: 39.050C 0.09) to 48 h (DAY 2: 38.70C 0.10) after the diagnosis of puerperal


metritis (difference 0.350C 0.12 C, P < 0.01). Daily rectal temperature from MT-2 cows


were higher compared to MT-3 24 h before the diagnosed (DAY -1: 38.80C 0.09 >


38.50C 0.04; P < 0.02), and continued to be different until the fifth day after the


diagnosis and treatment of puerperal metritis (DAY 5: 38.70C 0.11 > 38.50C 0.04; P


< 0.07). In this group, there was not a statistically significant difference between daily


increments of RT before the diagnosis of puerperal metritis. Cows diagnosed with


puerperal metritis in the absence of fever had a RT of 38.90C 0.08 on the DAY 0 (day


of diagnosis). There was not a significant reduction in the RT after the diagnosis and


treatment of puerperal metritis (Figure 4-2).


40.0

a*
39.8

39.6
/ \
/ \
/ \
39.4 /
S1/ \
= 39.2

S39.0 C
I-r

r 38.8

38.6

b* b* b*
38.4 b* b* b* b*

38.2
-5 -4 -3 -2 -1 0 1 2 3 4 5
Day before and after metritis

Figure 4-2. LSM SEM of daily rectal temperatures of cows 5 days prior to and 5 days
after diagnosis, for cows with puerperal metritis and fever (n=39; m), puerperal metritis
without fever (n=55;A) and cows without puerperal metritis (n=356; *).
a, b, c differ with a P< 0.05, P< 0.005.










Reproduction

There were no detected differences in first-service pregnancy rate (No-Metritis

(32%) vs. Metritis (28%); P < 0.3), and accumulated pregnancy rate by 150 days post

partum (No-Metritis (59%) vs. Metritis (52%); P < 0.7), or days in postpartum to first

service or pregnancy for cows with or without metritis. As expected, a seasonal effect

was detected in both models for first service pregnancy rate (Cool season (34 %) > than

warm season (16 %); P < 0.005), accumulated pregnancy rate by 150 days (Cool season

(64 %) > Warm season (25 %); P < 0.001). Days to first service as well as days to

pregnancy was also influenced by the season effect. No significant differences were

found in the number of services for pregnant cows between cows with or without

puerperal metritis (Mean + SE, 1.5 + 0.05 vs. 1.6 + 0.11, respectively; P < 0.8).


100%
90% -
80%
70%
S60%
| 50%
2!
40%
o 30%
20% -- Cold Season
--I Warm Season
10% -
0%
40 50 60 70 80 90 100 110 120 130 140 150
Days Postpartum

Figure 4-3. Proportion of pregnant cows during cold or warm season by 150 days
postpartum.











100% -
90% -
80% -
70%
C 60%
S50%
40% -
o 30%
20% No Metritis
10% --- Metritis
0%
40 50 60 70 80 90 100 110 120 130 140 150
Days Postpartum

Figure 4-4. Proportion of pregnant cows with or without puerperal metritis by 150 days
postpartum.





















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CHAPTER 5
DISCUSSION

In this study, cows with twins, dystocia, RFM with or without dystocia were

considered to have an abnormal calving. These disorders have been reported to be risk

factors for metritis in dairy cows (Markusfeld, 1984; Curtis et al., 1985; Bartlett et al.,

1986; Correa et al., 1993). It was not the intent of this study to determine the individual

effect of these disorders on the incidence of puerperal metritis. Instead, we considered

cows with one or more of these disorders as a high risk group to develop puerperal

metritis and retrospectively compare the incidence of puerperal metritis to a low risk

group (cows with a normal calving). The clinical approach used to identify cows to be

evaluated for puerperal metritis was based on evaluation of attitude (appeared sick) or a

RT > 39.4C. Despite the fact that a systematic examination of the uterus of all cows was

not considered, the incidence of puerperal metritis of (42%) in cows with an abnormal

calving was similar to previous reports (Markusfeld, 1984; Curtis et al., 1985) and within

the range in published studies that evaluated all cows for puerperal metritis regardless of

attitude or RT (Markusfeld, 1984).

The definition of puerperal metritis used in the present study was based on the

presence of a fetid vulvar discharge with or without fever. Of the cows with puerperal

metritis, only 41.4% had fever when puerperal metritis was diagnosed. These results are

in agreement with those of Pugh et al (1994), in which 42.3% of 78 cows evaluated for

metritis 14 days post partum had fever (RT > 39.40C). The diagnosis of puerperal metritis

in the present study was made by per rectum palpation of the uterus and visual inspection









of the vulva to observe a uterine discharge. This method of diagnosis of puerperal

metritis has been previously described (Markusfeld, 1984; Pugh et al., 1994; Risco, and

Hernandez, 2003). In contrast to the diagnosis of clinical endometritis (Bonnett et al.,

1993; Leblanc et al, 2002), no other previously reported method have been proven to be

more or less sensitive and or specific to diagnose puerperal metritis.

Primiparous cows in the present study had a higher incidence of puerperal metritis

during the cool season. However, no seasonal effect was observed in multiparous cows.

An effect of season and parity on the incidence of postpartum uterine infections have

been previously described (Markusfeld, 1984; Bartlett et al., 1986; Grohn et al., 1990;

Smith et al., 1998). However, an interaction between these two factors was not reported.

Markusfeld (1984) and Bartlett et al (1986) hypothesized that during the winter months a

high concentration of calvings and a wet and dirty environment may increase the

challenge of pathogens to the uterine environment. However, in the present study, both

primiparous and multiparous cows were equally exposed to these factors. In the present

study, season was determined by the thermal heat index, which is an indirect

measurement of heat stress in the cow. Previous reports have shown the relationship

between heat stress and uterine involution. Heat stress during late gestation increased

concentrations of blood prostaglandins and progesterone during the postpartum period

(Lewis et al., 1984), and reduced prepartum blood concentrations of estrone sulfate

(Collier et al., 1982). These changes in hormonal profiles have been reported to have a

positive effect on uterine involution (Lewis et al., 1984; Nakao et al., 1997; Zhang et al.,

1999) which may have reduced the incidence of puerperal metritis during the warm

season observed in the present study.