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Effect of Lameness on Ovarian Activity in Post-Partum Holstein Cows


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EFFECT OF LAMENESS ON OVARIAN ACTIVITY IN POST-PARTUM HOLSTEIN COWS By EDUARDO JOSE GARBARINO 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 2004

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Copyright 2004 by Eduardo J Garbarino

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In memory of my father, Eduardo Jos Garbarino

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ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr Jorge Hernandez, Associate Professor of Veterinary Medicine, for his advice and guidance during my 3 years of hard work. I also thank him for his help, support, and friendship, which allowed me to successfully complete my masters program. I thank Dr. Carlos Risco Professor of Veterinary Medicine for his contributions to the project. I thank Dr. Jan P. Shearer, Dairy Extension Veterinarian, for his availability for consultation and interest on the project and Dr. William W. Thatcher, Graduate Research Professor at the Department of Animal Science for his ideas, his contribution in the design and interpretation of the study. I would like to thank Mrs. Coni and Mr. Dale Sauls and the personnel of Condale Dairy Farm for their support and hard work to make this study possible. I also would like to acknowledge Shawn Ward for his hard work and friendship. I thank Julie Oakley and Marie Joelle Thatcher for their help with assay and data handling and analysis. I also need to thank Sally O,Connel, secretary at the Graduate School, for her continuous concern and help for graduate students. I would also thank faculty, residents and staff of the Food Animal Service for their help and patience. I need to acknowledge my friends in Gainesville, Jackie and Mandy, Elisa and Charly for being our hotel, mechanics, electricians, baby sitters, painters and for their constant support and concern but most importantly for their sincere friendship. Thanks to my friends in Argentina, Gerva, Bebe and Paula, Carlitos and Ceci, Chaca, Carlitos, iv

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Luchito and Rosario, Gonzalo and Sole for their support and for more than 15 years of friendship. I would like to thank my mother and sister, for their love and constant support for the past 34 years, and my father, who from heaven, looks after every step I take. Finally I thank my wife Martita, our daughter Sofi and our son Santi, for whom I dont have words to express my gratitude but I am sure that without them this would not be possible. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION..........................................................................................................1 2 LITERATURE REVIEW...............................................................................................4 Anatomy of the Bovine Foot........................................................................................9 Foot......................................................................................................................10 Claws...................................................................................................................11 Suspensory Apparatus and Supporting Structure of the Bovine Digit................12 Horn Formation and Growth...............................................................................13 Microanatomy of the Claw: Structure of the Wall..............................................14 Etiology of Lameness.................................................................................................15 Infectious Diseases of the Digits.........................................................................15 Interdigital phlegmon (Foot-rot, Interdigital necrobacillosis)......................15 Interdigital dermatitis...................................................................................17 Digital dermatitis (DD) (Footwarts, Hairy heel warts, Heel warts).............19 Metabolic Hoof Horn Disease: Claw Horn Disruption.......................................25 Laminitis.......................................................................................................26 Forms of laminitis........................................................................................29 Claw Lesions Associated with Laminitis............................................................30 Hemorrhages of the sole and sole ulcer........................................................30 Softening of the horn of the sole..................................................................30 White line disease.........................................................................................31 Heel erosion..................................................................................................31 Diagnosis of Lameness...............................................................................................31 Lameness and Animal Welfare...................................................................................34 Lameness and Milk Production..................................................................................35 Lameness and Reproductive Performance..................................................................37 Resumption of Ovarian Activity Postpartum.............................................................39 vi

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Physiological Factors Involved in Ovarian Activity Potentially Affected by Energy Balance................................................................................................48 Effects of negative energy balance in LH secretion.....................................48 Metabolic hormones.....................................................................................49 Other factors.................................................................................................53 Lameness and Ovarian Activity..................................................................................57 3 MATERIALS AND METHODS..................................................................................59 Cows and Herd Management......................................................................................59 Study Design...............................................................................................................59 Data Collection...........................................................................................................60 Diagnosis of Lameness...............................................................................................60 Collection of Blood Samples and Detection of Plasma P 4 Concentrations................61 Resumption of Ovarian Cyclicity...............................................................................63 Reproductive and Health Management......................................................................63 Statistical Analyses.....................................................................................................65 4 RESULTS.....................................................................................................................69 5 DISCUSSION...............................................................................................................74 6 CONCLUSION.............................................................................................................80 LIST OF REFERENCES...................................................................................................81 BIOGRAPHICAL SKETCH.............................................................................................97 vii

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LIST OF TABLES Table p age 2-1. Studies reporting incidence of lameness in dairy cows................................................7 2-2. International terminology of digital diseases................................................................9 2-3. Incidence of Digital Dermatitis in US dairy herds by herd size and region 1 ..............22 2-4. Days from calving to 1 st ovulation reported in the literature......................................43 2-5. Incidence rates of delayed cyclicity reported in the literature....................................44 3-1. Protocol for examination of cows postpartum............................................................64 3-2. Definitions of metritis done by farm personnel based on discharge and palpation findings.....................................................................................................................64 3-3. Definition of calving outcomes..................................................................................65 3-4. Criteria for monitoring production health and mastitis using Afimilk system........65 4-1. Frequency distribution of cows classified as lame or non-lame using a modification of the locomotion scoring system developed by Sprecher, 1997.............................71 4-2. Descriptive statistics and unadjusted odds ratios for risk of delayed ovarian cyclicity in post partum Holstein cows...................................................................................72 4-3. Final logistic regression model for risk of delayed ovarian cyclicity in post partum Holstein cows...........................................................................................................73 4-4. Attributable proportion of cows that experienced delayed resumption of ovarian cyclicity....................................................................................................................73 viii

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ix LIST OF FIGURES Figure page 1. Normal ovarian cyclicity.............................................................................................67 2. Normal ovarian cyclicity for cows treated with PGF2...............................................67 3. Delayed resumption of ovarian cyclicity....................................................................67 4. Extended luteal phase.................................................................................................68

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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 EFFECT OF LAMENESS ON OVARIAN ACTIVITY IN POSTPARTUM HOLSTEIN COWS By Eduardo Jos Garbarino May, 2004 Chair: Jorge Hernandez Major Department: Veterinary Medical Sciences An observational cohort study was conducted to examine the relationship between lameness and delayed ovarian cyclicity in post partum Holstein cows. We used 253 cows from a 600-cow dairy herd that calved during a 12-month period. Cows were classified into one of six categories of lameness during the first 35 days post partum using a locomotion scoring system. Cows were blood sampled weekly for detection of plasma P 4 concentrations during the first 60 days post partum. Cows with a delayed resumption of ovarian cyclicity were those with consistent P 4 concentrations < 1 ng/mL during the first 60 days post partum. The null hypothesis that risk of delayed cyclicity is the same in cows classified as non-lame, moderately lame, or lame (after adjusting for potential modifying or confounding effects of loss of body condition and other variables related with delayed cyclicity) was tested using logistic regression. Results of the study support the hypothesis that lameness has a detrimental effect on ovarian activity in Holstein cows x

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during the early post partum period. Cows classified as lame were 3.5 times at higher risk of delayed cyclicity, compared to cows classified as non-lame (OR = 3.5; 95% CI = 1.0 12.2; P = 0.04). Attributable proportion analysis indicated that delayed ovarian cyclicity in lame cows would be reduced by 71% if lameness had been prevented. In addition, cows classified as moderately lame were 2.1 times at higher risk of delayed cyclicity, compared to non-lame cows (OR = 2.1; 95% CI = 0.7 6.1; P = 0.15). xi

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CHAPTER 1 INTRODUCTION Lameness is one of the top three health problems that cause premature culling of dairy cows in the United States. The National Animal Health Monitoring System Dairy 2002 Study (NAHMS 2002) reported that lameness was the reason for culling 16% of dairy cows sent to slaughter. Overall, 10% of cows were reported affected with lameness in the previous 12 months. The economic importance of lameness is attributed to cost of treatment and control methods (Shearer and Elliot, 1998; Shearer et al., 1998; Hernandez et al., 1999, 2000; Moore et al., 2001), impaired reproductive performance (Lucey et al., 1986; Lee et al., 1989; Sprecher et al., 1997; Hernandez et al., 2001; Melendez et al., 2003), decreased milk yield (Warnick et al., 2001; Green et al., 2002; Hernandez et al., 2002), increased risk of culling (Sprecher et al., 1997; Collick et al., 1989), and decreased carcass value of culled cows (Van Arendonk et al., 1984). In addition, because of the pain, discomfort, and high incidence of lameness in dairy cows, this disorder should be considered an animal welfare issue. Delayed ovarian cyclicity in the preservice postpartum period is a common ovarian dysfunction in dairy cows (Opsomer et al., 1998). Late resumption of ovarian activity post partum has a detrimental effect on reproductive performance in dairy cows (Thatcher and Wilcox, 1973; Stevenson and Call, 1983; Lucy et al., 1992). Cows ovulating earlier post partum have fewer services per conception and a shorter calving-to-conception interval (Lucy et al., 1992). Minimizing the interval from calving 1

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2 to first ovulation provides ample time for completion of multiple ovarian cycles before to insemination, which in turn improves conception rates (Butler and Smith, 1989). Losses in body condition, puerperal disturbances, and ketosis have been identified as risk factors significantly associated with delayed ovarian cyclicity in dairy cows (Opsomer et al., 2000). While previous studies have shown that lameness has a detrimental effect on reproductive performance (i.e., a prolonged calving-to-conception interval) (Lucey et al., 1986; Collick et al., 1989; Sprecher et al., 1997; Hernandez et al., 2001), the relationship between lameness and ovarian activity in dairy cows has not been investigated using objective research methods. Results of previous studies in Florida suggest that as cows experience increasing positive energy status, there is increased ovarian follicular activity leading to return to ovulation (Staples et al., 1990; Lucy et al., 1992). As energy status becomes more positive for cows early post partum, diameter of the largest follicle increases, the number of double ovulations increases, and time for detection of the first corpus luteum decreases (Lucy et al., 1991). These changes in follicle size and numbers and the number of ovulations, are thought to be caused by increases in luteinizing hormone, follicle-stimulating hormone, insulin, BST, insulin-like growth factor-1, and possibly other yet-to-be determined compounds that are activated by an improved energy status (Beam and Butler, 1998). Clinical observations by veterinarians and dairy farmers in Florida suggest that lameness has a detrimental effect on ovarian activity in lactating dairy cows. We hypothesized that as lame cows experience a more pronounced loss in body condition (hence a prolonged state of negative energy balance) during the early postpartum period,

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3 lame cows are at higher risk of delayed ovarian cyclicity than non-lame cows. Under field conditions, evidence of corpus luteum function can be determined by monitoring plasma P 4 concentrations weekly during lactation, before and after diagnosis of lameness in dairy cows. The objective of this study was to examine the relationship between lameness and delayed resumption of ovarian cyclicity in Holstein cows during the first 60 days post partum. Knowledge of the epidemiologic aspects of diseases and lesions that cause lameness is essential to further develop control and prevention methods for lameness in dairy cows. Risk of lameness during lactation, severity and duration of lameness, and the relationship between lameness and ovarian activity in lame cows have not been investigated under US dairy farming conditions. Our prospective research study will allow us to better characterize lameness in dairy cows under commercial farming conditions, and examine the relationship between lameness and ovarian activity. An understanding of the reasons for individual-cow differences in lost revenues will aid producers in making management decisions at the cow and herd levels to manage lameness, reproductive performance, and animal welfare.

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CHAPTER 2 LITERATURE REVIEW Lameness is one of the top three health problems that cause premature culling of dairy cows in the United States. Several studies have reported that the incidence of lameness in dairy cattle operations varies from 7% to 54.6% (Harris et al., 1988; Clarkson et al., 1996; Collick et al., 1989; Barkema et al., 1994; Green et al., 2002; Deluyker et al., 1991), with the highest proportion of cases occurring in the first 100 days in milk (Collick et al., 1989; Barkema et al., 1994; Green et al., 2002). The economic importance of lameness is reportedly attributable to cost of treatment and control methods (Moore et al., 2001; Hernandez et al., 1999, 2000; Shearer et al., 1998), impaired reproductive performance (Lee et al., 1989; Tranter and Morris, 1991; Sprecher et al., 1997; Hernandez et al., 2001; Melendez et al., 2003), decreased milk yield (Tranter and Morris, 1991; Coulon et al., 1996; Warnick et al., 2001; Hernandez et al., 2002), increased risk of culling (Sprecher et al., 1997; Collick et al., 1989), and decreased carcass value of culled cows (Van Arendonk et al., 1984). In addition, because of the pain, discomfort, and high incidence of lameness in dairy cows, this disorder should be considered an animal-welfare issue. It is difficult to describe the incidence and prevalence of the problem of lameness in dairy cows, because of the wide variation that exists in published reports. This variation is due to differences in geographic locations and production systems (pasture-based vs. confinement) of study herds, that may alter the frequency distribution of lameness. In addition, the method of gathering data varies among studies (some 4

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5 collecting data from veterinary practices, and others gathering data directly from the farmer, or by researchers). Whitaker (1983) reported an average incidence of lameness of 25% (range = 2% to 55%) in 21,000 dairy cows in 185 herds in England and Wales. In the study by Whitaker (1983), data were collected by a survey of the number of treatments for lameness either by the farmer or veterinary surgeon. Another study conducted in 37 dairy farms in the United Kingdom and Wales, Clarkson (1996) reported an annual incidence of lameness of 54.6% (new cases/100 cows) with a range from 10.7% to 170.1%. This same study (Clarkson et al., 1996) reported the prevalence of lameness to be 20.6%. Diagnosis of lameness was performed either by the farmer, student, hoof trimmer, or a veterinarian. All treatments were recorded and were used to calculate the prevalence and incidence of lameness. Collick (1989) performed a study in 17 dairies in the UK, and reported an incidence of lameness of 17% during a 6month period, with a range of 8% to 28%, with 65% of cases occurring in the first 100 days in milk. In this case, diagnosis of lameness was made by the attending veterinarian. A study conducted in 80 dairies in France (Faye and Lescourret, 1989), the reported annual incidence of lameness was 29.5%. They stated that incidence of lameness was higher than reported incidence rates of mastitis, and that lameness was the most common disease reported in dairies. A study conducted in 101 dairy farms in Sweden (Manske et al., 2002a), found a prevalence of lameness of 5%. In this study (Manske et al., 2002a) lameness examination of cows was done by researchers who looked at every cow on each farm, with the purpose of identifying claw lesions in lame and non-lame cows. In other parts of the world with more extensive production system such as New Zealand, Australia, and Argentina, the incidence/prevalence of lameness differs from

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6 those reported in Europe. Dewes (1978) reported an incidence of lameness of 14%, and more than 85% of the cases of lameness were detected before 90 days in milk. This study (Dewes, 1978) was conducted using data provided by four dairy producers from Waikato, New Zealand. In another study in New Zealand by Tranter and Morris (1991) in three dairy herds, the annual incidence of lameness was 16% with a range from 2% to 38%. In this study the mean days to onset of lameness was 92 (mean SD). Harris (1988) conducted a study in 73 dairies in Southern Australia and reported and annual incidence of lameness of 7% with a range of 0% to 31%. In this study, diagnosis of lameness was done by the farmer. In a study done in Argentina (Rutter, 1994) involving 4580 dairy cows from 25 farms, the incidence of lameness was 23.4% with the highest incidence occurring in first lactation heifers (45%). In most of these studies, sole ulcers and white line disease were the most predominant lesions observed. When we examined the incidence and prevalence of lameness studies in the United States, we also found important variations. Reported incidences of lameness were 4.4%, 5.1% and 9.5% (Bartlett et al., 1987; Weigler et al., 1990; Kaneene et al., 1990). In these studies, lameness was diagnosed by the farmers. This motivated Wells (1993a), to design a study on prevalence of lameness, and to compare the diagnosis of lameness performed by researchers and farmers. In both seasons spring and summer, the observed prevalence reported by researchers were 3 times higher than that by the farmers. This study (Wells et al., 1993a) involved 17 dairy farms from Minnesota and Wisconsin and reported a prevalence of lameness of 14% in the summer and 17% in the spring. In another study conducted in 13 dairy farms in Ohio, researchers observed that the incidence of hemorrhages and discoloration of the sole in first calf heifers (from 60 days

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7 pre partum until 100 days post partum) was 62% during this period. In another study by Sprecher (1997) using a locomotion scoring system, the incidence of lame cows (Locomotion Score 4) was 24.5%, the incidence of moderately lame plus lame cows (LS 3) was 49.1% at the time of the first service. Table 2-1. Studies reporting incidence of lameness in dairy cows Author, year Country Incidence of lameness (%) Rutter, 1994 Argentina 23 Harris et al., 1988 Australia 7 (0 to 31) Faye and Lescourret, 1989 France 29 Dewes, 1978 New Zealand 14 Tranter et al., 1991 New Zealand 16 (2 to 38) Manske et al., 2002a Sweden 5 Whitaker et al., 1983 United Kingdom 25 (2 to 55) Collick et al., 1989 United Kingdom 17 (8 to 28) Clarkson et al., 1996 United Kingdom 54 Bartlett et al., 1987 United States 4 Weigler et al., 1990 United States 5 Kaneene et al., 1990 United States 9 Wells et al., 1993 United States 13.7 summer & 16.7 winter Sprecher et al., 1997 United States 49 Warnick et al., 2001 United States 46 Almost all reports describing lameness in dairy cows considered claw diseases as the most common causes of lameness. Some of the papers listed above (Tranter and Morris, 1991; Deluyker et al., 1991; Murray, 1996) reported claw diseases as responsible for more than 90% of the cases of lameness. When we examined the most common claw disorder, reports are not so much in agreement. Differences maybe due to, different housing types, feeding strategies, environmental challenges and managements systems.

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8 Studies from the United Kingdom (Clarkson et al., 1996; Murray et al., 1996) agreed that sole ulcers, white line disease, laminitis and subsolar abscess are the most common diseases affecting the bovine claw. In another study conducted on 101 dairy farms in Sweden (Manske et al., 2002), sole ulcers and white line lesions were the most common lesions found in lame cows. In New Zealand and Australia (Tranter and Morris, 1991; Dewes, 1978; Harris et al., 1988) where cows are kept in pasture all year around, traumatic pododermatitis (sole bruising), worn soles and interdigital dermatitis the most common diseases of the claw affecting dairy cows. In a study conducted in Argentina (Rutter, 1994), digital dermatitis (39.4%) and interdigital dermatitis (26.3%) were the most common diseases affecting the bovine foot. In the United States, all studies identified claw lesions as the most common lesions affecting dairy cows (Deluyker et al., 1991; Smilie et al., 1996; Hernandez, 2002). These studies, however, were not designed to establish the prevalence of the different diseases of the bovine foot. With an average incidence of 30 cases per 100 cows per year, a case fatality rate of 2%, involuntary culling of 20% of cases; an average increase of 28 days open, treatment costs including veterinary fees, drugs and farmer labor of $23 per case. The total cost of lameness per 100 cows per year is estimated to be about $9000 (Guard, 1996). This paper is probably underestimating the cost of lameness as it is not taking into account other factors that are affected by lameness. Lameness has been shown to affect milk production and this was not included in the estimation, as well as the decrease of the carcass value of cows sent to slaughter and the increased probability of a cow being culled if experiencing lameness. In another report from the UK, Kossaibati and Esslemont (1997) reported that the average total cost per affected cow of a case of sole

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9 ulcer was (approx $600). This estimation included the cost of treatment, herdsmans time, discarded milk, reduced milk yield, veterinarians time, increased risk of culling and longer calving interval. Increased risk of culling and a longer calving interval accounted for more than 60% of the cost. Unfortunately, the authors did not explain how they estimated milk loss and increased risk of culling. Table 2-2. International terminology of digital diseases International terminology English Common terms Dermatitis interdigitalis Interdigital dermatitis Superficial Foot-Rot Phlegmona interdigitalis Interdigital phlegmon Foot-Rot, Foul in the foot Erosio ungulae Heel erosion Underrun heel, Slurry heel Hyperplasia interdigitalis Interdigital hyperplasia Corn, Interdigital fibroma Dermatitis digitalis Digital dermatitis Hairy foot warts, Hairy heels Pododermatitis aseptica difusa Diffuse aseptic pododermatitis Laminitis Pododermatitis circumscripta Circumscript pododermatitis Sole ulcer Pododermatitis septica traumatica Traumatic septic pododermatitis Subsolar, toe and white line abscesses Fissura ungulae Hoof wall cracks Sand cracks: longitudinal and transverse Pododermatitis locale Localizedpododermatitis Bruises Ungulae deformans Overgrown hooves Long toes To compare results from different studies it is important to standardize the terminology regarding claw lesions. In order to clarify the rest of this manuscript we will follow the terminology proposed by Weaver (1994) (Table 2-2). Anatomy of the Bovine Foot Before we start describing the different pathologies affecting the bovine foot, it is important to review its anatomy and microanatomy.

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10 Foot The foot includes the entire limb below the fetlock joint. It is comprised of two digits each of which has a horn-covered claw. It should be noted that in cattle the term claw is preferable to hoof. The front aspect of the foot is referred to as the dorsal side. The back side of the front foot is referred to as the palmar aspect whereas in the rear foot is referred to as plantar aspect. When referring to an area nearest the longitudinal axis (i.e., toward the center) it is designated as axial, whereas items farther away (away from the center) are designated as abaxial. Each digit of the foot has 4 bones: phalange 1 (P1), phalange 2 (P2), phalange 3 (P3), and navicular bone; and 2 joints: proximal interphalangeal (PIP) and distal interphalangeal (DIP). The proximal end of P1 articulates with the metacarpus (in the front leg) or metatarsus (in the rear leg) in the fetlock joint, whereas the distal (away from the center of the body) end of P1 articulates with the proximal end of P2. This articulation between P1 and P2 is referred to as the proximal interphalangeal joint (PIP). The distal end of P2 articulates with the proximal end of P3. This joint is referred to as the distal interphalangeal joint (DIP). P3 is completely enclosed within the claw horn capsule. Its solar surface is concave or arch shaped and marked on the back edge by a bump known as flexor tuberosity. The flexor tuberosity is the site of attachment of the deep flexor tendon. This tuberosity has an important role in the pathogenesis of sole ulcers as it becomes involved in the process of compression of the corium subsequent to laminitis and the displacement of P3 (Toussaint Raven, 1989). The navicular bone (also referred to as the distal sesamoid bone) is attached to P3 by three small ligaments and also to P2 by collateral ligaments. Between the navicular

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11 bone and the deep flexor tendon is the navicular bursa. The navicular bursa contains joint-fluid which permits movement of the deep flexor tendon over the surface of the navicular bone during extension and flexion of the claw. P3, the DIP joint, navicular bone and navicular bursa all lie within the claw capsule. Claws The purpose of the claw horn capsule is to protect the underlying sensitive tissues of the corium and dissipate the concussion forces that occur when the digits impact the ground. It consists of the wall which can be divided into the axial (inside) and the abaxial (outside). The abaxial wall is further subdivided into the dorsal (or front) and lateral (abaxial side) aspects. The wall is demarcated from the heel on the abaxial side of the claw by the abaxial groove. The wall consists of two types of horn: perioplic and coronary. Perioplic horn is the softer horn lying just below the coronet at the skin-horn junction (corresponding to the human cuticule). At the back of the foot the periople gradually widens and eventually becomes the horn of the heel. Coronary horn, the hardest horn within the claw capsule makes up the bulk of the horn of the wall. The wall has faint ridges or rugae, which run horizontally and parallel to each other. Toward the heel these ridges diverge reflecting a more rapid rate of growth in the heel region due to faster rates of wear. In mature Holstein cattle the length of the dorsal wall should be a minimum of 3 inches in length from just below the top of the hairless portion of the wall to the weight-bearing surface. Ideal heel height is 1.5 inches (Toussaint Raven, 1989; Blowey, 1993). The sole is produced by the solar corium and merges imperceptibly with the horn of the heel at the heel-sole junction. The sole is connected to the wall by means of the white line. White line horn is produced by laminar corium. It courses forward from the

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12 area of the heel on the abaxial side of the claw, around the tip of the toe and about 1/3 of the way back on the axial side of the claws weight bearing surface. Where the white line leaves the weight bearing surface it courses upward on the axial side of the claw. This white line is a unique and important structure. It is the softest horn within the claw capsule. This permits it to provide a flexible junction between the harder horn of the wall and the softer horn of the sole. On the other hand, because of its softer nature it also represents a weak spot on the weight-bearing surface that is vulnerable to damage. Suspensory Apparatus and Supporting Structure of the Bovine Digit Cattle (and all animals with claws or hooves) are suspended in their feet, that is, they stand in their feet, not on them. In other words, the bone within the claw (also known as P3) is suspended within the claw horn capsule by the laminar corium and a series of collagen fibers bundles that stretch from the insertion zone on the surface of P3 to the basement membrane of the epidermis (the line of demarcation between dermis and epidermis). The interface between dermal and epidermal components is the interdigitating dermal and epidermal laminae. The result is that P3 hangs within the claw capsule and weight is transferred as tension onto the wall of the claw capsule. The suspensory system in cattle differs significantly from that in horses. First, the laminar corium is much less extensive in cattle as compared to horses. Secondly, there are no secondary laminae in the laminar corium of cattle. Therefore, capabilities with respect to mechanical load carried on the claws of cattle vary significantly. In the horse load bearing is primarily on the wall. Cattle, on the other hand, simply cannot handle the same amount of mechanical load on the walls of their claws. Instead, weight-bearing in cattle requires displacement of load to the wall, and support structures within the sole and heel.

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13 The primary structures within the supportive apparatus of the bovine claw are the solar corium and associated connective tissue, and the digital cushion, which consists of loose connective tissue and varying amounts of adipose (fat) tissue. The digital cushions are arranged in a series of three parallel cylinders similar to the design used in the cushion of a running sole. In the cows foot these cushions act like shock absorbers within the claw protecting the corium and permitting limited movement of P3 in the region of the heel. Horn Formation and Growth The horn-producing germinal layer of the epidermis and its supporting structure, the corium, consist of four different regions, each producing a structurally different type of horn (Budras et al., 1996). Perioplic horn, overlying the perioplic corium, is found just below the skin-horn junction and extends to the back of the claw to include the heel horn (Budras et al., 1996). Horn of the wall is produced in the area of the coronary corium and it is situated between the perioplic corium and the sensitive laminae. The area overlying the laminar corium produces the horn of the white line, also known as laminar horn. The solar horn overlies the solar corium and is situated between the laminar horn of the white line and the perioplic horn of the heel (Budras et al., 1996; van Amstel and Shearer, 2001). Horn production and growth are supported by the corium, which corresponds to the dermis. The corium consists of a rich vascular network that terminates in dermal papillae, also called vascular peg (Greenough, 1997). A vascular peg consists of a main arteriole and a venule, which are connected at the tip. Between the arteriole and the venule is an extensive capillary network, and there are also several vascular shunts between such arterioles and venules. These shunts may open under certain circumstances,

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14 cutting of the blood supply to the tip of the vascular peg, which adversely affect horn cell formation. The epidermal layer overlying the vascular pegs produces horn cells in the form of tubules (tubular horn) (Budras et al., 1996; Greenough, 1997). Intertubular horn is produced between the papillae and interconnects the tubular horn. There are approximately 80 vascular pegs or dermal papillae per square millimeter of coronary corium surface (Greenough, 1997), which means that the wall consists of tightly packed tubular horn that is cemented together by intertubular horn. The perioplic corium of the heel horn and the solar corium has fewer vascular pegs per square millimeter. Because tubular horn supplies structural strength to the horn capsule, it follows that the horn of the wall is structurally the strongest, followed by the sole and the heel. Keratin filaments produced by horn cells enhances the rigidity and strength of horn cells as they progress to the exterior. Laminar horn is immature, nontubular, so it is soft and flexible and has a high turnover rate. Horn cells, whether tubular or nontubular, are cemented by a substance known as membrane-cementing substance (Budras et al., 1998). This substance, a lipoprotein, is permeable and holds water, giving the horn its flexibility (Budras et al., 1998). Horn quality is dependant on internal and external and factors. Internal factors relates to blood and nutrient supply, whereas external factors relates to environment where the claw is found. Any compromise in blood flow has a negative effect on horn production. Microanatomy of the Claw: Structure of the Wall The structure of the claw consists of modified skin that is a continuation of the epidermis of the coronary band. The claw has the same basic structures as the skin. It has

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15 an epidermis (horny wall), a dermis (corium or quick), and a subcutis (fibroelastic heel pad and coronary and digital cushion). The epidermis itself is divided into basement membrane, germinal epithelium (stratum germinativum), stratum spinosum (layers of horn undergoing keratinization) and stratum corneum (the layer of cornified epithelium). The basement membrane is the junction between the epidermis and corium. The stratum germinativum is the germinative layer responsible for horn growth. The stratum corneum is the cornified epithelium forming the claw horn. Cells are arranged into tubular and intertubular horn. The mechanical strength of the bovine claw is a function of the keratinization of cells in the germinal layers of the epidermis (Hendry et al., 1994). Etiology of Lameness Lameness in cattle can be caused by a variety of reasons. The purpose of the next section is to describe the different causes of lameness in cattle. Infectious Diseases of the Digits Several systemic diseases can be associated with digital lesions potentially leading, as a result of localized pain, to stiffness and lameness. They include Foot-and-Mouth Disease (FMD), Bovine Virus Diarrhea (BVD), Bovine Malignant Catarrh, Bluetongue and Vesicular Stomatitis. This review of lameness is focused on major specific infections of the digits: Interdigital Phlegmon (Foot-Rot), Interdigital Dermatitis and Digital Dermatitis. Interdigital phlegmon (Foot-rot, Interdigital necrobacillosis) Interdigital Phlegmon is characterized by fissuring, caseous necrosis of the subcutis in the interdigital space and diffuse digital swelling. Pain, moderate to severe lameness, and pyrexia are also common signs of this disease. A characteristic fetid odor

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16 is usually present because of the presence of Fusobacterium necrophorum which if not treated early a common sequela is septic arthritis (Berry, 2001). Although the pathogenesis of foot rot is not understood completely, bacteria gain entry through abraided skin on the lower part of the foot. Hard surfaces contribute to foot injury, and continuous wetting likely favors abrasions by softening the interdigital skin (Radostits et al., 2000). The greatest economic impact of bovine foot rot is in dairy operations, where milk production may be affected (Hernandez et al., 2002). In this study, cows affected with foot rot produced 10% less milk than normal cows. Also this disease can affect feedlots where antimicrobial treatments require withdrawal times that could delay marketing of products (Radostits et al., 2000). Although spontaneous recovery may occur, lameness may persist for several weeks when infections are left untreated, and complications may cause more severe problems that could eventually lead to death or euthanasia of the animal (Radostits et al., 2000). Treatment of foot rot can be accomplished with a variety of antimicrobials (Cook et al., 1995; Morck, 1998; Berry, 2001). A recent study looked at the efficacy of Ceftiofur Sodium and Hydrochloride formulation for the treatment of foot rot (Kausche et al., 2003). This was a multilocation study conducted on 11 farms in the US to compare the efficacy of Ceftiofur at a dose of 1.1 mg/kg once a day for 3 consecutive days with a placebo group. Results of this study indicated that cure rate for Ceftiofur was 62.2% versus 14% for the placebo group (P < 0.003). These same authors did another study to compare the efficacy of Ceftiofur versus Oxytetracycline at 10 mg/kg. Results of this study indicated that Ceftiofur and Oxytetracycline were comparable in efficacy, with Ceftiofur having excellent injection-site tolerance and a short or no milk discard or

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17 pre-slaughter withdrawal (Kausche et al., 2003). Treatment can also be accomplished by the use of Sulfadimethoxine orally (25 g/lb followed by 12.5 g/lb SID for no more than 5 days) or intravenously (55 mg/kg followed by 27.5 mg/kg SID for 2 days after remission of clinical signs). Good results also can be obtained with Penicillin G intramuscularly for 3 days (Bergsten, 1997). Prevention and control of foot rot can be accomplisheded by the use of foot baths with 5% to 10% copper sulfate or zinc sulfate (Rebhun, 1982). Formaldehyde solutions of 3 to 5% in water have been reported to be effective in the prevention of foot-rot (Bergesten, 1997). Caution should be emphasized when using formaldehyde due to potential hazards for handlers as well as contamination of the environment. Other measures recommended to prevent foot rot are to maintain clean passageways to reduce the exposure of the feet to feces, maintaining a dry environment and avoiding rough floor surfaces that can traumatize the interdigital skin and allow the entry of bacteria (Blowey, 1994). Efforts to produce vaccines against Fusobacterium necrophorum have failed because of the weak immune response to the bacterium (Smith, 1992). There are vaccines available in the US market but there are no peer-reviewed studies to support their use. Interdigital dermatitis Interdigital dermatitis occurs as an acute or chronic inflammation of the interdigital skin that does not usually cause lameness (Blowey, 1994; Guard, 1995). The inflammation does not extend to the subcutaneous tissues and in this respect differs from foot-rot, where infection extends to the dermis, leading to fissure formation, infection of deeper structures, and cellulitis of the pastern and fetlock (Blowey, 1994). Some authors implicate F.necrophorum, Dichelobcater nodosus and Bacetroides sp as the causative agents of interdigital dermatitis (Toussaint Raven, 1989, Peterse, 1982).

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18 Interdigital dermatitis occurs in dairy cattle, especially in wet environments. It is usually an incidental finding when trimming feet because it rarely causes lameness. A study conducted in 17 Danish dairy herds reported that interdigital dermatitis occurred in 4.5% and 7.6% of first and 2+ lactation cows, respectively (Enevoldsen et al., 1991). In this study, severity of disease increased with parity and risk increased with stage of lactation. In a Dutch study on 86 dairy farms, researchers reported that the prevalence of interdigital dermatitis and heel horn erosion was 24% (range = 3 to 92%) (Manske et al., 2002c). Clinical signs of interdigital dermatitis include hyperemia of the interdigital skin, including the palmar and plantar areas, superficial erosion and ulceration followed by hyperemia with serous or grayish exudates. More aggressive forms interfere with the horn formation in the bulbs, where fissures, hemorrhages and necrosis can arise. The subcutaneous tissue is inflamed secondarily. Swelling and hyperkeratosis may develop in a more chronic stage. Chronic interdigital irritation may cause slight to severe interdigital hyperplasia (Bergsten, 1997). The most common complication of interdigital dermatitis is heel horn erosion. Results of a study conducted by Enevoldsen (1991) support the hypothesis that severe heel erosion and interdigital dermatitis are two manifestations of the same disease with Dichelobacter nodosus as an important component and are closely related. In this study (Enevoldsen, 1991) the incidence of heel erosions in 1 st and 2+ lactation cows was 43% and 69%, respectively. This study (Enevoldsen, 1991) also reported unhygienic and moist conditions as important risk factors for interdigital dermatitis.

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19 Prevention and treatment is usually accomplished by the use of 5 to 10% copper sulfate footbath or zinc sulfate (10 to 20%), or formalin (3 to 5%) footbaths. Care must be taken to ensure that the footbath remains clean. Interdigital dermatitis can persist in dairies that practice regular footbaths (Guard, 1995). This same report suggested that the causative organisms may survive within deep heel cracks that are not permeated by footbath solutions; hence, heel cracks must be trimmed during hoof trimming to allow for exposure to footbath solutions. Claw trimming causes a mechanical cleansing of affected tissues and an exposure to air that might be beneficial for the curing of dermatitis lesions (Manske et al., 2002b). As reported in this same study, every third hoof affected with a severe dermatitis and concurrent heel-horn erosion had recovered 1 month after trimming. Digital dermatitis (DD) (Footwarts, Hairy heel warts, Heel warts) Digital dermatitis is an important cause of lameness in dairy cattle. It was first reported by Cheli and Mortellaro in 1974 (Mortellaro, 1994) as a mysterious cause of epidemic lameness affecting up to 70% of adult cattle in the Po Valley of Italy. Since then, the disease has been reported in other countries such as the Netherlands, France, England, Czechoslovakia, Germany, and Ireland (Bassett et al., 1990; Brizzi, 1993). In the United States, Rebhun (1980) first reported the disease as outbreaks of lameness in New York dairy herds. Clinically, digital dermatitis typically appears within dairy herds as outbreaks of lameness. It is a superficial skin disease of the bovine digit with variable presentation (i.e., depending upon the stage of the lesion), from painful, moist, strawberry-like lesions to raised, hairy, wart-like lesions (Read and Walker, 1998). These lesions (i.e., usually located on the rear of the foot between the bulbs of the heel) have been referred to by

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20 several names, including: hairy footwarts, strawberry (or raspberry) heelwarts, and papillomatous digital dermatitis. Early lesions produce matting of the hairs, which stand erect in thick, light brown exudates, which have a characteristic pungent odor (Blowey et al., 1994). A study conducted in California described the lesions as being distinctly demarcated, circumscribed, spherical to oval, 0.5 cm to 6 cm across, partially or completely alopecic, moist, painful-to-touch, prone-to-bleed plaques of flat or raised proliferative tissue. Lesion surfaces vary in appearance from being extensively red and granular (31%), often with patches of yellow or gray filiform papillae (41%) to extensively gray, brown or black with papillary outgrowth of the epithelium (28%) (Read and Walker, 1994). In spite of many studies and specific research, the exact etiology of DD is still unknown. Researchers still believe that DD is a multifactorial disease, even though in some cases high morbidity, apparent contagiousness and response to antimicrobial treatments suggest that an infectious agent is primarily involved (Mortellaro, 1994). In one study the incidence of DD was higher in heifers a few months after they entered the milking herd and may be due to lack of immunity (Read et al., 1992). Initial studies (Rebhun et al., 1980; Cheli and Mortellaro, 1986; Peterse, 1982) were unable to identify any viral pathogens, and results of bacteriology were inconsistent. Peterse (1982) was able occasionally to isolate Dichelobacter nodosus from some typical lesions. Bassett (1990) was unsuccessful in isolating a microorganism from DD lesions and also failed to replicate the disease after inoculation of heifers with homogenate from fresh lesions. Read (1992) examined histological lesions and were able to demonstrate the presence of large numbers of spirochetes invading the stratum spinosum and dermal papillae (Blowey

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21 et al., 1994). In a more recent study, Walker (1995) isolated two different spirochetes from cows with DD lesions in California dairy herds. These spirochetes were further categorized based on morphology (intracytoplasmatic tubules), antigenicity and enzymatic activity in the genus Treponema. This finding was supported later by another study (Demirkan et al., 1999) where serological evidence suggested that spirochetes are involved in the pathogenesis of DD. Also, this study (Demirkan et al., 1999) supports the hypothesis that Borrelia burgdorferi may be involved in the pathogenesis of DD, as first proposed by Blowey (1994). To date, there is still no isolation of the bovine spirochete. In a study conducted in California dairy herds (Read and Walker, 1994), the prevalence of DD was approximately 90%. Between-herd morbidity varied from 0.5 to 12% per month. Within-herd morbidity was generally greater during spring and summer months. Most lesions occur on the plantar interdigital cleft of the rear foot and less common sites for lesions are the palmar interdigital ridge of a front foot or a dorsal aspect of any foot (Mortellaro, 1994; Read and Walker, 1998). Another study conducted by the National Animal Health Monitoring System involving 83% of US dairy cows in 20 states observed the incidence of digital dermatitis and risk factors (Wells et al., 1999). This study reported, that within the last 12 months of the study, 43.5% of the US dairy herds had cows that showed clinical signs of DD with variation by herd size and region (Table 2-3). The study by Wells (1999) reported that the average percent of cows affected was 18.9%. A high percentage of digital dermatitis-affected cattle were also reported lame (81.9% of affected cows and 85.9% of bred heifers). This study also looked at risk factors associated with digital dermatitis. Interesting results from this study identified several

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22 factors that contribute to herd incidences > 5%. The percent of cows not born on the dairy operation was strongly associated with high digital-dermatitis incidence, and there was evidence for a dose-response relationship. Table 2-3. Incidence of Digital Dermatitis in US dairy herds by herd size and region Variable Level Herds with digital dermatitis in the previous 12 months (%) Herd Size < 100 cows 36.4 100 to 199 cows 61.9 >200 or more cows 80.3 Total 43.5 Region Northwest 56.1 Southwest 70.3 North Midwest 35.4 South Midwest 45.5 Notheast 53.1 Southeast 20.8 Wells et al., 1999 Rodriguez-Lainz (1996) showed a strong association between introduction of heifers and digital-dermatitis prevalence in southern California dairy herds. These results were in agreement with results from Argez Rodrguez (1997) who reported that purchased animals were 3.4 times more likely to be affected than animals born on the farm. Farm factors such as type of concrete flooring with concrete abrasive floors or slippery floors were associated with > 5% incidence of DD. Rodriguez-Lainz (1996) reported an association between the incidence of digital-dermatitis and corral moisture in southern California dairy operations with dirt dry lot corrals. Some biosecurity factors identified were hoof trimmers that trim cows on other farms. Herds in which the primary hoof trimmer also trimmed cows hooves on other operations were 2.8 times more likely to have > 5% incidence of digital dermatitis compared to herds where the primary hoof trimmer did not trim hooves on other operations or where cows hooves were not

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23 trimmed. Also, herds in which hoof-trimming equipment was not washed between cows were 1.9 times more likely to have > 5% incidence of digital dermatitis than those where the equipment was washed or where no hooves were trimmed (Wells et al., 1999). Blowey (1988) reported different treatments when describing one of the first outbreaks of the disease in the UK. Treatment of DD started by the application of parenteral injections of penicillin, streptomycin, tetracyclines, cephalexin, and sulphonamides, but none of these treatments proved effective and most cases recovered on their own. The most effective treatment in this report appeared to be deep scraping of the lesion with a hoof knife, followed by topical oxytetracycline/gentian violet aerosol spray. This treatment led to a reduction of lameness in 6 to 12 hours and complete resolution in 2 days. In this study, footbaths with 5% formalin or 2.5% copper sulfate were used to try to control the outbreak with poor results. Sheldon (1994) further supported treatment with oxytetracycline spray (4 g/L), although this was not a controlled study. Further research conducted by Britt (1996) confirmed the efficacy of Oxytetracycline (100 mg/mL) as a treatment for digital dermatitis applied as a spray. Manske et al., (2002c) reported that Oxytetracycline applied as a bandage was significantly more effective than hoof trimming alone of the affected foot (P < 0.001). This study was done to try to find an alternative treatment to antibiotics and Glutaraldehyde bandage was tested as the alternative nonantibiotic treatment. Results of this study did not support the use of this product. Treatment of DD with topical oxytetracycline does not require any milk withdrawal as shown by Britt (1999) who was unable to demonstrate antibiotic residues in milk using standard routines after cows were treated topically with this antibiotic. The most common treatments for digital dermatitis

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24 involve the use of topical antibiotics (Blowey, 1988; Blowey, 1994; Guard, 1995; Berry et al., 1996; Britt et al., 1996; Britt et al., 1998; Read et al., 1998; Berry et al., 1999a; Berry et al., 1999b, Hernandez et al., 1999; Shearer et al., 2000; Manske et al., 2002c). Some of the antibiotics used in these studies were Oxytetracycline, Tetracycline, Lincomycin/Spectinomycin, and Erythromycin. There also are reports of non-antibiotic products being effective in the treatment of DD (Shearer and Hernandez, 2000; Hernandez et al., 1999). Hernandez (1999) used a topical spray with four different non-antibiotic products in the lame cow with DD. Treatment with an Oxytetracycline solution, or a soluble copper, peroxide compound and a cationic agent appeared to be effective for the treatment of DD, compared to a 5% copper sulfate (CuSO 4 ) solution, or acidified copper solution, or hydrogen peroxide-peroxyacetic acid (HPPA) solution. In another study (Shearer et al., 2000) non-antibiotic compounds were used to treat DD in 78 cows affected with lameness. In this study (Shearer et al., 2000), a previously tested product (Hernandez et al., 1999) was modified to improve its handling and storage characteristics. There were four treatment groups in this study: cows in group A were treated with oxytetracycline solution, cows in group B were treated with the original formulation also containing soluble copper, peroxide compound and a cationic agent, cows in group C were treated with a modified formulation with reduced soluble copper, peroxide compound but increased levels of cationic agent and cows in group D were treated with a modified formulation containing levels of soluble copper and cationic agent similar to the original formulation but reduced concentrations of peroxide compounds. Results of this study (Shearer et al., 2000) indicated that the modified non-antibiotic formulation used on cows in group C appeared

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25 to be the most effective treatment of papillomatous digital dermatitis compared to the other formulations as the proportion of cows with signs of pain was significantly lower in this group of cows (group C). Also this study reported an unexpected low efficacy of the oxytetracycline treatment suggesting the possible development of resistance in dairy cows affected with DD. This study supports the use of non-antibiotic products as an efficacious tool for the treatment of DD, thus minimizing the potential risk for residues in milk and meat. Control measures of biosecurity, as stated in a previous study (Wells et al., 1999), includes examination of animals entering the herd, cleaning hoof trimming equipment between cows, and to have a farm-set of trimming tools available for the hoof trimmer to avoid contamination incoming from other farms. Footbaths can be used as a part of control measures of DD on infected herds (formalin 5%; copper sulfate 2.5%; oxytetracycline 1 to 4 g/L; zinc sulfate 20%) (Blowey et al., 1988; Brizzi, 1993; Mortellaro, 1994; Guard, 1995). If no preventive herd measures are taken, a relapse may be expected within 5 to 7 weeks after a successful single topical treatment of DD (Berry et al., 1999a) (Guard and Williams, 1995). Although the efficacy of footbaths remains controversial, individual treatment of affected cows combined with the use of footbaths for the herd represent the most effective method of prevention (Mortellaro, 1994). Metabolic Hoof Horn Disease: Claw Horn Disruption Hoof horn of low quality is a frequent cause of lameness in cattle. Studies in the UK reported that claw disorders accounted for 70 to 90% of diagnosed cases of lameness in dairy cattle (Whitaker et al., 1983; Clarkson et al., 1996; Murray et al., 1996). These researchers identified subclinical laminitis related disorders, such as sole ulcers and white line disease, as the most important conditions affecting dairy cattle in the UK. Laminitis

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26 was regarded by some authors (Bradley et al., 1989; Greenough and Vermunt, 1991) as an important predisposing factor in lameness caused by claw disorders such as sole ulcers, white line disease, and abscesses in the subsole. Subclinical laminitis has been identified as the underlying cause of abnormalities of hoof horn formation which results in claw disorders (Hoblet et al., 2001). To better understand the pathophysiology of lameness, it is important to review the anatomy of the bovine foot. Laminitis Laminitis, or pododermatitis aseptica diffusa, is an aseptic inflammation of the dermal layers of the claw (Nielsson, 1963) characterized by defective claw horn production with thrombosis and hemorrhages in the digital corium (Mortensen, 1994). It was originally described as consisting of three clinical forms (acute, subacute, chronic), but Peterse (1979) later described a fourth form of laminitis, subclinical laminitis. Because the disease process finally affects horn formation at the cellular level, regardless of whether an initial primary inflammatory response had occurred, the term claw horn disruption has been proposed (Logue et al., 1998). Although many different terms have been proposed for this disorder, I will refer to it as laminitis for the purpose of this discussion. Laminitis has a multifactorial etiology and is thought to be associated with several, largely interdependent factors such as genetic predisposition, claw size, body weight, architecture of limb angles, claw hardness, pigmentation of the claw and the quality of the surface over which the animal walks (Mortensen, 1994; Nordlund and Garret, 1994). Nutritional management has been identified as a key component in the development of laminitis, particularly the feeding of increased fermentable carbohydrates which result in an acidotic state. There seems to be no doubt that the disease is related to

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27 high energy intake, frequency and quantity of consumption. Factors such as body condition, body weight, and feet and leg structure, can unnaturally increase the weight load and stress on feet and exacerbate the internal mechanical damage that is associated with laminitis (Nocek, 1997). Nocek (1997) has described the mechanisms causing development of laminitis in detail on a recent review of the topic. The mechanistic phases of laminitic development were described as alternating stages of disturbances relating to metabolic and subsequent mechanical degradation of the internal foot structure (Mortensen, 1994; Nordlund and Garret, 1994). The process can be segmented into various phases (Nocek, 1997). Phase 1. The initial activation phase of laminitis, phase 1, is associated with a systemic metabolic insult. This phase is a result of ruminal acidosis, and subsequently an altered systemic pH. The reduction in systemic pH activates a vasoactive mechanism that increases digital pulse and total blood flow. Depending upon the insult that initiates the process, endotoxins, histamine and lactate can be released, which create increased vascular constriction and dilation and, in turn, cause the development of several unphysiological arteriovenous (AV) shunts, further increasing blood pressure. The increased blood pressure causes seepage of serum through vessel walls, which ultimately are damaged. Damaged vessels then exude serum, which results in edema, internal hemorrhaging of the solar corium from thrombosis, and ultimately expansion of the corium, causing severe pain. Phase 2. As a result of the initial insult, there is mechanical damage, phase 2, which is associated with the vascular system. Once vascular edema has occurred, ischemia results in hypoxemia of the local internal digital tissue causing tissue hypoxia

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28 which results in fewer nutrients and less oxygen reaching the epidermal cells. Ischemia itself can trigger a further increase in AV shunting. Trauma and stress can increase AV shunting. As a result of previous events, increased blood pressure further increases vascular seepage in the lower part of the digit as well as edema and ischemia. This cycle continues as long as the initial insult continues. Phase 3. In phase 3, as a result of the mechanical damage associated with microvasculature and fewer nutrients provided to the epidermal cells, the stratum germinativum in the epidermis breaks down. These events ultimately cause corium degeneration and breakdown of the laminar region associated with the dermal-epidermal junction. Phase 4. Ultimately, in phase 4, local mechanical damage occurs. A situation develops in which the epidermal junction is broken down which results in the separation of the strata germinativum and corium. This separation results in a breakdown between the dorsal and lateral laminar supports of the hoof tissue. Ultimately, the laminar layer separates, and P3 takes on a different configuration in relationship to its position in the corium and dorsal wall. As the bone shifts in position it causes a compression of the soft tissue between the bone and sole which is extremely susceptible to damage. The compression of this soft tissue results in hemorrhage, thrombosis, and further enhancement of edema and ischemia which result in a necrotic area within the solar region of the foot. Small areas of scar tissue accumulate because of the necrotic process. Once this process is triggered, continued potential for tissue degeneration persists because cellular debris is incorporated into the cellular matrix and the production and integrity of new horn tissue layers are hindered. Ultimately, a variety of processes can

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29 occur as a result of the incorporation of scar tissue intervention, which includes double sole phenomenon, sole hemorrhages (red blood patches), bruises, white line lesions, and sole ulcers (Vermunt, 1994). Forms of laminitis Acute and subacute laminitis. In the acute and subacute stage of the disease, an aseptic inflammation of the corium coincides with a systemically sick animal. At this stage, the claw horn shows few, if any, visible changes. Vessel seepege, edema of capillary beds, and AV shunting are all initiated. Vascular congestion is present. The major clinical sign in addition to pain includes swelling and temperatures that are slightly warmer than normal above the coronary band in the soft tissue (Nocek, 1997). These forms of laminitis are prone to recurrence at varying intervals and often progress to the chronic form (Vermunt, 1994). Chronic laminitis. Chronic laminitis has no systemic symptoms and changes are localized to the claw. A disturbed horn growth pattern and an alteration in the shape of the claw with an elongated flattened and broadened sole are characteristic (Vermunt, 1994; Greenough, 1997). Internally P3 has separated from the dorsal aspect of the wall. Continued ischemia results in destruction of the capillary beds and development of AV shunts. Cellular destruction results in separation of the dermal-epidermal junction, and internal foot destruction (Nocek, 1997). Grooves and ridges caused by irregular episodes of horn growth can be seen in the claw wall. This deformation of the claw shape often predisposes to sole ulcers, white line disease or subsolar abscesses (Greenough and Vermunt, 1991; Mortensen, 1994). Subclinical laminitis. Subclinical laminitis was first described in 1979 by Peterse and later by others (Bradley et al., 1989; Greenough and Vermunt, 1991). Lameness is

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30 usually not observed with this form of laminitis, but changes in the hooves can lead to chronic laminitis. The horn becomes softer, discolored, and waxy in appearance. It often stains yellow and hemorrhages can be seen in the weight-bearing surface of the claw, in particular the white zone, apex of the sole and the axial side of the sole-bulb junction (Bradley et al., 1989; Greenough and Vermunt, 1991). Internally, ischemia, hypoxia and epidermal damage are key aspects associated with this stage (Nocek, 1997). The concept of subclinical lameness is now universally accepted (Mortensen, 1994). Claw Lesions Associated with Laminitis Hemorrhages of the sole and sole ulcer Hemorrhages in the sole are the major and characteristic indication of past laminitic insults. The hemorrhages can take the form of a slight pink tinge, a pronounced brush stroke of red coloration, or a dark solid red stain. Hemorrhages of the sole are considered part of the same pathologic process as sole ulcers and are represented by a continuum that ranges, from barely perceptible hemorrhages to severe ulceration of the sole with exposed corium (Leach et al., 1997). Sole hemorrhages have a particularly high incidence in first lactation heifers managed in confinement in the interval from 60 to 100 days after calving (Greenough and Vermunt, 1991; Smilie et al., 1996). The most common spot for sole hemorrhages and sole ulcers is the so-called typical spot of the lateral claw of the hind limb under the flexor tuberosity (axial prominence) of P3 (Smilie et al., 1996). Softening of the horn of the sole Although objective evidence is not available, it has been proposed that sole horn produced after an episode of laminitis is softer than normal (Mortensen, 1994).

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31 White line disease There are a number of names for the lesions in the White Line (WL): WL disease, WL abscess, WL separation, WL fissure, WL lesions, widening of the WL, WL hemorrhages (Bergsten, 2000; Blowey, 1993; Kempson and Logue, 1993; Leach, 1997). These names describe clinical signs or morphological changes in the WL. Different names are descriptions of different stages of development and degree of diseases whose pathogenesis has a common origin (Mlling, 2002). This condition is also considered to be associated with laminitis (Mortensen, 1994). Heel erosion Heel erosion has been described as one of the infectious diseases affecting the bovine foot in this manuscript, but there are some authors that also relate this disease with laminitis (Mortensen, 1994). Diagnosis of Lameness Several locomotion scoring systems have been developed to standardize gait analysis in cattle. Manson and Leaver (1988) devised a system of locomotion scoring which is a subjective assessment based on observation of cows walking away from the observer on a level concrete surface (Table 2-4). Using this scoring system, cows classified with a score of 3 or higher were counted as lame for calculation of prevalence of lameness. Two problems with this system were its subjectivity and complexity (Ward, 1998). Collick (1989) used this system and factored percentage reduction of weight bearing for the affected foot, foot structure affected and duration of lameness, which made this system more complicated.

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32 Table 2-4. Locomotion scoring developed by Manson and Leaver (1988) Score Description 1.0 Minimal abduction/adduction, no unevenness of gait, no tenderness 1.5 Slight abduction/adduction, no unevenness or tenderness 2.0 Abduction/adduction present, uneven gait, perhaps tender 2.5 Abduction/adduction present, uneven gait, tenderness of feet 3.0 Slight lameness, not affecting behavior 3.5 Obvious lameness, some difficulty in turning, not affecting behavior 4.0 Obvious lameness, difficulty in turning, behavior pattern affected 4.5 Some difficulty in rising, difficulty in walking, behavior affected 5.0 Extreme difficulty rising, difficulty walking, adverse effect on behavior Wells (1993) described a similar but different locomotion scoring system (Table 2-5) arguing that simplicity was necessary when using this type of scoring. This system was used to estimate the prevalence of lameness in 17 dairies in Wisconsin and Minnesota and cows with a score of 2 or higher were classified as lame Table 2-5. Locomotion scoring used described by Wells (1993) Score Gait abnormality Description 0 None Gait abnormality not visible at walk; not reluctant to walk. 1 Mild Mild variation from normal gait at walk; includes intermittent mild gait asymmetry or mild bilateral or quadrilateral restriction in free movement. 2 Moderate Moderate and consistent gait asymmetry or symmetric gait abnormality, but able to walk 3 Sever Marked gait asymmetry or severe symmetric abnormality 4 Nonambulatory Recumbent This system was used to estimate the prevalence of lameness in 17 dairies in Wisconsin and Minnesota and cows with a score of 2 or higher were classified as lame. In 1997, Boelling (1998) used the locomotion system developed by Manson and Leaver (1988) and described it using a 9 point-scale, from 1 (perfect walk) to 9 (near inability to walk). A score of 1 to 4 was considered sound locomotion, while a score of 5 or higher was regarded as clinical lameness. They used this to estimate the heritability of locomotion, which they found to be 0.06 to 0.11. Sprecher (1997) based on an

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33 observation of Morrow in 1966, where an arched-back posture was associated with acute and chronic laminitis, developed a simple locomotion scoring system. He developed this method of classification of lameness to try to predict future reproductive performance and culling risk of cows classified within different scores. This locomotion scoring system was based on gait and back posture (Table 2-6). If used consistently, all these scoring systems are useful to screen cows and identify early lesions associated with lameness. Early intervention prevents the more serious stages of a claw disorder (Toussaint Raven, 1989). Table 2-6. Locomotion scoring system developed by Sprecher et al., 1997 Locomotion score Clinical description Assessment criteria 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 3 Moderately lame An 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 5 Severely lame The cow additionally demonstrates an inability or extreme reluctance to bear weight on one or more of her limbs/feet If used consistently, all these scoring systems are useful to screen cows and identify early lesions associated with lameness. Early intervention prevents the more serious stages of a claw disorder (Toussaint Raven, 1989). On the basis of differences in anatomic location and morphologic characteristics, it is possible to make a clinical diagnosis of interdigital phlegmon, digital dermatitis, or claw lesions in cows affected with lameness. Lame cows with interdigital phlegmon will be cows characterized by fissuring, caseous necrosis of the subcutis in the interdigital

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34 space, and swelling of the entire foot above the dewclaws and separation of the digits. Pain and moderate to severe lameness are often seen with this disease. A characteristic fetid odor is usually present (Berry, 2001). Lame cows with digital dermatitis will have demarcated lesions, circumscribed, spherical to oval, 0.5cm to 6cm across, partially or completely alopecic, moist, painful-to-touch, prone-to-bleed plaques of flat or raised proliferative tissue on the interdigital cleft, heels, or dewclaw (Read and Walker, 1994). Lame cows with claw lesions will be cows that have white line lesions, abscess, or sole ulcers and will be treated by use of corrective foot trimming techniques (Shearer and van Amstel, 2001). Lameness and Animal Welfare Because of the pain, discomfort, and high incidence of lameness in dairy cows, lameness is an animal welfare issue. Disturbed claw health is an unequivocal source of suffering for cows, because the disorder is usually long term and painful (Alban, 1995). Some countries are already setting acceptable levels of clinical lameness. As an example, the Dutch Advisory Board for Animal Affairs (RDA) has considered the actual levels of clinical and subclinical claw disorders (30% cow cases per year) in The Netherlands as unacceptable from an animal welfare point of view (Somers et al., 2003). In other countries as well, the prevalence of lameness has also stated as not acceptable and have give rise to a growing concern about animal welfare. Welfare can be defined as the state of animals regarding their attempts to cope with their environment (Broom, 1988). A lame cow is less able to cope with her environment, as pain might seriously affect walking and other movements (Hassall et al., 1993). The secondary effects of a reduced ability to walk may impact important physiological activities such as reduction in the time feeding (Hassall et al., 1993;

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35 Galindo et al., 2002), also affecting their behavior as suggested by Peeler and Esslemont (1994), were lame cows experienced similar inhibitions as cows on poor footing and would less likely be observed in estrus. These welfare factors are important to consider when evaluating the effects of lameness disease on production. In most US dairies, incidence of lameness is underestimated because only cows affected with severe signs of lameness are detected and treated; cows with mild or moderate signs of lameness are often not diagnosed. In a study by Wells (1993) the prevalence of lameness diagnosed by farmers in 17 dairies was compared to that by researchers in the same farms. The prevalence of lameness reported by researchers was three times higher than that by farmers. The underestimation of prevalence of lameness keeps farmers and the industry unaware of the importance of this disease. Lameness and Milk Production Several studies have been carried out around the world to test the effect of lameness on milk production. Results of these studies are conflicting. Some authors reported a decrease in milk yield after diagnosis of lameness (Whitaker et al., 1983; Tranter and Morris, 1991; Rajala-Schultz et al., 1999; Warnick et al., 2001), a decrease in milk yield before and after treatment (Lucey et al., 1986; Green et al., 2002) or no change in milk yield (Cobo-Abreu et al., 1979). Another study (Barkema et al., 1994) reported an increase in milk yield from 100 to 270 DIM during the same lactation in lame cows with sole ulcers. Green (2002) reported an increase in 100-day cumulative milk volume in the previous lactation for cows with any cause of lameness. Culling bias may in part account for these results because cows with both lameness and low production would be expected to be culled more often than cows with lameness and high production. Argez-Rodrguez (1997) did a retrospective study a in Mexico to examine the effects of digital dermatitis in

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36 milk production. The authors (Argez-Rodrguez et al., 1997) reported a statistically non-significant difference between milk production of lame cows due to DD and healthy cows, with cows experiencing DD producing less milk. In this study lame cows due to DD were compared with healthy cows and lame cows for other reasons were included as healthy which can mask the effects of DD on milk production. Three studies have examined the relationship between lameness and milk yield in US dairy herds. In a study conducted on a 500-cow dairy in California (Deluyker et al., 1991), cows diagnosed as lame during the first 49 days postpartum coincided with higher milk yield. The positive association of lameness and high milk yield during early lactation found in this study suggested that high yield was a risk factor for lameness. Although diseases or foot lesions associated with lameness were not investigated, white line and sole lesions were the most common lesions in this study. In another study conducted on two dairy herds in New York (Warnick et al., 2001), lame cows with claw lesions or interdigital phlegmon produced less milk than healthy cows. Lameness was more common in early lactation and more likely to occur in older cows. Finally, in a study conducted in Florida, lame cows with interdigital phlegmon produced 10% less milk, compared to non-lame cows (Hernandez et al., 2002). The authors (Hernandez et al., 2002) estimated that such a decrease in milk yield of 1,885 lb/cow represented a loss of $301/cow (assuming a milk value of $16.00/100 lb). In that study, most lame cows with interdigital phlegmon were affected during early lactation (within 100 days postpartum), when cows reach peak yields and 60% were culled during lactation. These findings may suggest that lame cows affected with interdigital phlegmon during early

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37 lactation may be sufficiently compromised to adversely affect a cows ability to achieve its own milk yield potential during the current lactation (Hernandez et al., 2002). Lameness and Reproductive Performance A relationship between lameness and reproductive performance has been established in several studies in the United States and in other parts of the world. In 1985, Weaver postulated lameness as a possible cause of reduced fertility because animals spend more time lying down, are less willing to demonstrate standing heat, and are less able to compete for available feed. Some of these suggestions were later confirmed by different studies. Varner (1994) looking at pedometer readings, reported that cows in estrus move and interact with other cows significantly more than the rest of the herd. Britt (1986) found that excellent footing greatly increases the duration of estrus activity in dairy cows. Peeler and Esslemont (1994) reported that lame cows experience similar inhibitions as cows on poor footing and would less likely be observed in estrus. These findings may in part explain results of studies indicating that lameness has a detrimental effect on reproductive performance. In a study conducted on five dairy farms in the UK, Lucey (1986) found that lame cows affected with sole ulcer and white line disease between 36 and 70 days after calving were associated with longer calving to conception intervals (17 and 30 days, respectively). Collick (1989) did a larger study on 17 dairy farms in the UK involving 427 cases of lameness. The authors reported that lameness happening before 120 days after calving was associated with significantly increased intervals from calving to conception. The largest increase in the intervals from calving to conception were associated with sole ulceration (40 days, P < 0.01). In a retrospective study of digital dermatitis in a commercial dairy in Mexico Argez-Rodrguez (1997) reported that healthy cows conceived 93 days after calving (median),

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38 compared to affected cows with digital dermatitis which conceived 113 days after calving (P < 0.01). Three previous studies have examined the relationship between lameness and reproductive performance in US dairy herds. In one study conducted in five dairy herds in Pennsylvania, cows affected with lameness had a 28-day-longer calving to conception interval, compared to healthy cows (Lee et al., 1989). In another study, a scoring system was developed to identify lameness and predict future reproductive performance in dairy cows, lame cows were 15.6 times more likely to require an interval greater than the mean for days open compared to healthy cows (Sprecher et al., 1997). In a study conducted on a 500-cow dairy in Florida (Hernandez et al., 2001), claw lesions were the most important cause of lameness and impaired reproductive performance in dairy cows, as indicated by a higher incidence of affected cows, a greater time from calving to conception (median, 140 days), and a higher number of services required per conception (median, 5), compared to non-lame cows (100 days and 3 services, respectively). In this study herd, cows were synchronized and time-inseminated; thus the authors were not able to assess the calving to first breeding interval nor to draw any conclusion that lameness has an effect on estrus behavior. Conversely, the significantly higher number of services required per conception and the longer time from calving to conception in lame cows with claw lesions, compared to healthy cows, may suggest that lameness has an effect on conception. Melendez (2003) examined the association between lameness, ovarian cysts, and fertility on a 3000-cow dairy in Florida. Results of this study showed that cows that became lame within the first 30 days postpartum were associated with higher incidence

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39 of ovarian cysts, a lower likelihood of pregnancy, and a lower fertility than non-lame cows. Although all these studies found a significant association between lameness and reproductive performance, the relationship between lameness and ovarian activity was not investigated. Resumption of Ovarian Activity Postpartum Early resumption of ovarian cyclicity postpartum is important for high reproductive efficiency in dairy cows. Delays in the commencement of ovarian cyclicity and estrous expression are associated with reduced conception rates, pregnancy rates and an increased interval from calving to conception (Thatcher and Wilcox, 1973; Stevenson and Call, 1983; Lucy et al., 1992; Senatore et al., 1996). The study done by Thatcher and Wilcox, (1973) reported that cows exhibiting 0 or 1 heat postpartum required significantly more services per conception than cows exhibiting 2 to 4 heats. These authors reported that non-return rates improved as frequency of postpartum heats increased (0 and 1 heat, 37%; 2 to 4 heats 44%; P < 0.05). In a study conducted to identify the influence of early estrus, ovulation and insemination on fertility in postpartum dairy cows, Stevenson and Call (1983) reported that the interval to first detected heat had a significant influence on first service intervals and days open. When estrus was not expressed before 60 days postpartum, average days to first service were 18 days longer and days open were 19 days longer than in cows that expressed heat before 60 days postpartum (P < 0.05). These authors concluded that more expressed heats early postpartum were associated with positive effects on fertility. A study conducted by Lucy (1992) who monitored the interval to first ovulation to clarify the importance of milk production, dry-matter intake and energy balance in the interval to first ovulation, reported that cows having first ovulation before 42 days tended

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40 to have a shorter interval from calving to detected estrus, required fewer services per conception and had a shorter interval from calving to conception compared with cows having first ovulation after 42 days postpartum. More recently, Darwash (1997) reported that the interval to postpartum commencement of luteal activity was correlated favorably with measures of fertility such that for every day delay in the interval to commencement of ovarian activity, there was an average delay of 0.24 and 0.41 (P < 0.001) days in the interval to first service and conception respectively. These studies pointed out the importance of early resumption of ovarian cyclicity postpartum as a factor contributing to high reproductive efficiency on dairy cows. Following calving, the reproductive strategy of the cow is transformed from delivering and nourishing a healthy calf to reestablishing pregnancy. The dormancy of ovarian follicular development that prevailed during late pregnancy must now be replaced by a sequence of events culminating with behavioral estrus, ovulation of healthy follicles and normal luteal function. These are the requirements for successful reproductive performance in any type of cattle production system (Rhodes et al., 2003). After regression of the corpus luteum of pregnancy, there is a variable anovulatory period before first ovulation takes place (Savio et al., 1990a). This period is characterized by an absence of estrus behaviour and lack of progesterone secretion by the ovary and a return to basal concentrations of estradiol during the first week postpartum (Echternkamp and Hansel, 1973; Stevenson and Britt, 1979; Webb et al., 1980; Humphrey et al., 1983; Peters, 1984; Savio et al., 1990a; Rhodes et al., 2002). Following parturition, a wave of follicular development occurs in 5 to 7 days regardless of negative energy balance and in response to an elevation in plasma FSH concentrations (Beam and

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41 Butler, 1997). As reported by Savio (1990a), ovarian follicular turnover starts early after calving and is similar to that observed during normal estrous cycles. In this study Savio (1990a) reported that the postpartum interval to the detection of the first dominant follicle was 11.6 8.9 days and the interval to first ovulation was 27.4 23 days. Beam and Butler (1997) reported that the emergence of the first follicular wave postpartum occurred after a peak in mean peripheral FSH levels and rather synchronously with the clearance of gestational estradiol from blood. Subsequent, mean levels of FSH increased in the first 5 days postpartum and decreased from 5 to 11 days. This is in accordance with previous work (Butler et al., 1983; Price and Webb, 1988) that indicated that removal of estradiol negative feedback inhibition of FSH release would account for the increase in mean plasma FSH observed between days 2 and 9 postpartum. After returning to basal levels, estradiol concentrations fluctuate or remain low until 2 to 3 days before estrus when they peak (Echternkamp and Hansel, 1973; Stevenson and Britt, 1979). Estradiol levels declined from the 1 st day postpartum until day 7, when estradiol levels slowly increase with the concurrent development of a dominant ovarian follicle (Beam and Butler, 1997). Plasma estradiol concentration is related with the degree of the peak of LH release in response to GnRH as reported by Zolman (1974); Kesler (1977); and Fernandes (1978). These studies agreed with that of Moss (1985) who reported that pituitary responsiveness to GnRH is not restored until approximately 8 to 10 days post-partum. As described by Savio (1990a), follicular growth is accompanied by episodic LH secretion of variable amplitude and frequency. The use of ultrasound techniques allowed Savio (1990a) to relate the stage of follicular development and the pulsatile secretion of LH. Within a 6-hour period, 2 to3 LH pulses occurred when concentrations of estradiol were low (< 5

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42 pg/mL), and the frequency of LH pulses increased to 6 when estradiol concentrations increased (> 10 pg/mL) coinciding with a dominant follicle. Stevenson and Britt (1979) reported that the interval from calving to first postpartum ovulation was associated inversely with the number of episodic LH surges and magnitude of the largest LH surge. This high frequency mode of pulsatile LH secretion has been identified as necessary for the final phase of maturation of ovarian follicles and thus induction of estrus and ovulation (Webb et al., 1980; Humphrey et al., 1983; Randel, 1990). Canfield and Butler (1990a) reported a high correlation between the interval from parturition to the highest LH pulse frequencies and first ovulation, emphasizes the importance of achieving this pattern of secretion for the stimulation of first ovulation, support this. These series of events can lead to three different outcomes of follicular development as described by Beam and Butler (1997): a. Ovulation of the first dominant follicle; b. Non-ovulation of the first dominant follicle followed by turnover and a new follicular wave; c. The dominant follicle fails to ovulate and becomes cystic. The development of non-ovulatory dominant or cystic follicles prolongs the interval from calving to first ovulation. In dairy cattle, the interval from calving to first ovulation has been reported to be between 17 and 34 days (Table 2-7) The variation observed between some of these studies may be due to differences in study populations such as animal breeds, production systems, level of production, feeding systems, or it might be as concluded by Opsomer (2000) that the first postpartum ovulation in modern high-yielding dairy cows tends to occur later than it did a decade ago. Results of these studies are difficult to compare since mean days to first ovulation were calculated only

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43 for normal cows in some of these studies, which excluded cows that had follicular cysts or those who did not ovulate early postpartum; while in other studies, all cows were in the calculations. Table 2-7. Days from calving to 1 st ovulation reported in the literature Author, year Days to 1 st postpartum ovulation (d) Schams (1978) 17 Stevenson (1979) 18 Webb (1980) 17 Butler (1981) 36 Stevenson (1983) 19 Fonseca (1983) 20 Meisterling (1987) 33 Short luteal phase 25 Normal luteal phase Butler (1989) 30 Canfield (1990) 19 Harrison (1990) 29 Staples (1990) 22 Savio (1990) 22 Spicer (1990) 24 Canfield (1990) 29 Etherington (1991) 24 Nakao (1992) 30 Zurek (1995) 24 Darwash (1997) 22 Opsomer (2000) 32 Reist (2000) 34 If ovulation occurs then plasma progesterone concentrations increase to greater than 1ng/mL within 2 to 3 days after ovulation (Schams et al., 1978; Stevenson and Britt, 1979). An elevation of plasma Progesterone concentration above 1 ng/mL has been used as an indication of resumption of postpartum ovarian cyclicity in several studies (Butler et al., 1981; Harrison et al., 1990; Staples et al., 1990; Canfield et al., 1991; Zurek et al., 1995; Beam and Butler, 1997; Beam et al., 1998) The duration of the first postpartum estrous cycle can be variable, being shorter than normal or normal, and it can occur with or without estrus behavior. Stevenson and

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44 Britt (1979) reported 32% of first estrous cycles were shorter (16 d vs. 20 d) than subsequent estrous cycles. These observations have been supported by different studies (Schams et al., 1978; Webb et al., 1980; Stevenson and Call, 1983; Fonseca et al., 1983; Savio et al., 1990b; Staples et al., 1990; Senatore et al., 1996). Among dairy cows, those that have not ovulated by 60 days post partum have been defined as having a delayed resumption of ovarian cyclicity (Humboldt and Thibier, 1980; Stevenson and Call, 1983; Staples et al., 1990; Moreira et al., 2001). Incidence rates of delayed cyclicity reported in the literature are summarized in Table 2-8. Table 2-8. Incidence rates of delayed cyclicity reported in the literature Author, year Delayed cyclicity Milk/Plasma Incidence of delayed cyclicity (%) Humboldt (1980) P4 1.5 ng/mL 60d Plasma 29 Bartlett (1987) Palpation 70d 23 Miesterling (1987) P4 4 ng/mL 65d Milk 21 Archbald (1990) Presence of CL and P4 > 1ng/mL 1 st month PP Plasma 30 Nakao (1990) P4 < 1 ng/mL 50d Milk 25 Staples (1990) P4 < 1 ng/mL 63d Plasma 28 Etherington (1991) P4 2 ng/mL 50d Milk 33 Lamming (1998) P4 > 3 ng/mL 45d Milk 11 Opsomer (2000) P4 < 15 ng/mL 50d Milk 21 Moreira (2001) P4 1 ng/mL 63d Plasma 23 Several factors can affect the interval between calving and first postpartum ovulation, the incidence rate of delayed cyclicity, and the reproductive performance of affected cows. Some of theses factors cited in the literature are energy balance, season, parity, and periparturient diseases (Butler et al., 1981; Fonseca et al., 1983; Canfield et al., 1990b; Lucy et al., 1992; Senatore et al., 1996; Beam and Butler, 1997; Darwash at al., 1997; Jonsson et al., 1997; Opsomer et al., 2000; Moreira et al., 2001).

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45 Several studies have found associations relating energy balance to ovarian activity postpartum, and some of these studies have proposed a casual path to this association. However, the effects of energy balance affecting postpartum ovarian activity are not completely elucidated. Milk production and dry matter intake increase after calving but at different rates with the maximum feed intake occurring some weeks after maximum milk production. The result of this delay is negative energy balance that persists for 4 to 12 weeks of lactation (Butler at al., 1981), when most dairy cows must mobilize body reserves to support milk production (Bauman and Currie, 1980). Negative energy balance is usually maximal during the first 3 weeks of lactation (Canfield et al., 1990b). Butler (1981) reported that first ovulation occurred 10 days after the negative energy balance nadir was reached, and energy balance was still negative at ovulation, but was returning towards zero. The authors (Canfield et al., 1990b) concluded that there was an inverse relationship between the interval from parturition to first normal ovulation and the average energy balance during the first 20 days postpartum. These results were further supported by other studies (Staples et al., 1990; Canfield and Butler, 1990a; Lucy et al., 1992; Senatore et al., 1996). The greater the average energy deficit incurred, the longer the delay to ovulation. Canfield and Butler (1990b) showed a high correlation between days to negative energy balance nadir and days to first ovulation. This relationship suggested that first ovulation does not occur in an individual animal until energy balance progresses beyond its most negative value and is returning toward balance. In this study, 1 st ovulation also occurred 10 and 13 days following negative energy balance nadir in non-lactating and lactating cows respectively. It was proposed from this study that energy status could be acting to

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46 slow an increasing LH pulse frequency until the cow has begun to return to a positive energy balance. Cows that reached the energy balance nadir earlier postpartum (4 d vs. 14 d) ovulated earlier (14 d vs. 27 d) and had higher concentrations of insulin. These results, together with the finding that the pulse frequency of LH was not different for both groups, made the authors propose that insulin levels may act permissively on the ovary to enhance follicular responsiveness. These same authors (Canfield and Butler, 1990a) examined the effect of energy balance and changes in plasma concentrations of glucose, insulin, non-esterified fatty acids (NEFA), and ketones on pulsatile LH secretion in early postpartum period. They reported a direct relationship between postpartum energy balance and first ovulation, and between negative energy balance nadir and changes in pulsatile LH secretion, suggesting that as negative energy balance reaches its nadir and starts returning towards balance, LH secretion is disinhibited and first ovulation occurs. The relationship between energy balance and first ovulation was further supported by different studies. A study by Staples (1990) examined the relationship between ovarian activity and energy status during the early postpartum period. In this study, cows were classified according to the time of first ovulation as early responders (resume ovarian activity within 40 days postpartum), late responders (resume ovarian activity between 40 and 60 days postpartum), and no responders (resumption of ovarian activity after 63 days PP). These authors concluded that the early and late responders cows were in less negative energy balance than non-responders and were able to restore ovarian activity during the first 63 days postpartum. On the other hand, the non-responder cows did not have the capability to consume as much dietary energy, produced less milk and were more dependent on energy from body reserves to produce milk. As a consequence,

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47 metabolic status inhibited the initiation of postpartum ovarian activity in cows during the first 63 days postpartum. In addition, an increased loss of body weight during the first 2 weeks of lactation coincided with decreased ovarian activity; early responder cows losing the least weight and non-responder cows losing the most weight. Lucy (1992) examined the influence of diet composition, dry-matter intake, milk production, and energy balance on time to post-partum ovulation. Results of this study confirmed those of Staples (1990) where the interval to first ovulation was shorter in cows that consumed more dry-matter and produced more milk. In contrast, low milk producing cows consumed less dry-matter and were more likely to be classified as late responders. These two studies suggested that the fact that higher producing cows ovulating earlier than low producing cows did not contradict the effects of energy balance on the interval to first ovulation, as higher producing cows can be in a less negative energy balance and thus ovulate earlier. It was also reported that cows ovulating earlier (before day 42 postpartum) had a superior reproductive performance. Also results from the study done by Lucy (1992) are in agreement with those of Fonseca (1983) where Jersey cows producing more milk ovulated earlier than lower producing herdmates. These two studies concluded that the data do not support linear relationships between days to first estrus, days to first insemination, and days open with increasing milk yield as suggested by others. They proposed that this contradiction occurred because lowest producing cows in commercial herds are culled before they have the opportunity to express their reproductive potential. As stated by Staples (1990) and supported by several studies (Fonseca et al., 1983; Harrison et al., 1990; Lucy et al., 1992), if energy status of the cow is more important than milk yield in determining return to estrus, then

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48 milk yield alone may be either positively or negatively correlated with days to first ovulation. Although an association between energy balance and postpartum ovarian activity has been clearly established, the causal path remains unclear. Detrimental effects of negative energy balance on ovarian activity could be due to effects on any of the components of the reproductive endocrine axis. Physiological Factors Involved in Ovarian Activity Potentially Affected by Energy Balance Effects of negative energy balance in LH secretion It has been already mentioned the importance of a high frequency mode of pulsatile LH secretion in the final phase of maturation and ovulation of ovarian follicles. Although pituitary content of gonadotropins increase rapidly after calving and are capable of supporting ovulation by day 8 to 10 postpartum (Kesler et al., 1977; Fernandes et al., 1978; Moss et al., 1985), however, pulsatile LH secretion capable of inducing ovulation generally occurs near 1 st ovulation which usually takes place at 17 to 34 days (see table 2.4). There is strong evidence that secretion of LH is impaired in cows not recovering from negative energy balance. Peters (1985) reported that very few lactating cows ovulated in response to pulsatile administration of LHRH when delivered at a time when animals were in a negative energy balance. Canfield (1988) compared LH secretion at 2 weeks postpartum and again at the energy balance nadir (during the return towards EB), and reported an increase in LH frequency and a decrease in pulse amplitude. These authors concluded that energy balance plays an important role in the control of first ovulation by suppressing LH pulse frequency following calving. Canfield and Butler (1990a) demonstrated a direct relationship between postpartum energy balance and 1 st ovulation, and between negative

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49 energy balance nadir and changes in pulsatile LH secretion. Therefore, it was concluded that as negative energy balance reaches it nadir and starts returning towards balance, LH secretion is disinhibited and 1 st ovulation occurs. Canfield and Butler (1991) reported that dairy cows in a negative energy balance had similar LH patterns but ovulated later than cows in positive energy balance. In another study, increased energy balance was associated with increase pulse amplitude of LH secretion (Lucy et al., 1991). Schillo (1992), in a review of the topic proposed that, the reduction on the LH pulse frequency observed during negative energy balance, represents one of the most important means by which energy balance impairs reproductive activity in cattle. The above results are consistent with findings of Beam and Butler (1997) which reported that follicles emerging after the negative energy balance nadir, rather than before, exhibited greater growth and diameter, enhanced estradiol production, and were more likely to ovulate. Metabolic hormones There is evidence that metabolic hormones such as growth hormone, insulin, IGF-I, and leptin have important roles in the control of ovarian follicular development and are likely to be important mediators of the effects of dietary intake and energy balance on cow fertility. Insulin like growth factor-I. IGF-I and insulin are effectors of follicle cell function in vitro with stimulation of steroidogenesis and cell proliferation in granulosa and thecal cells (Monniaux et al., 1992; Spicer et al., 1993; Magoffin et al., 1993; Spicer et al., 1996). Insulin-like growth factor-1 (IGF-I) is decreased in postpartum cows when experiencing negative energy balance (Spicer et al., 1990; Beam and Butler, 1999) Cows in poor body condition or cows not recovering body condition during lactation have also been identified as having low blood IGF-I concentrations. Beam and Butler (1997)

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50 reported that levels of plasma IGF-I averaged approximately 40% higher during the first 2 weeks postpartum in cows ovulating during the first follicular wave postpartum than in cows not ovulating. Plasma IGF-I in cows ovulating the 1 st follicular wave was higher at day 1 postpartum, before the establishment of follicular dominance and subsequent increases in peripheral estradiol. Therefore, the authors suggested that higher IGF-I in ovulatory cows did not result from greater dominant follicle estradiol production, but preceded and possibly contributed to differences in follicular function (Beam and Butler, 1997). Results of this study also suggest that low concentrations of circulating IGF-I are related to low steroidogenic output of dominant ovarian follicles early post partum. Results of another study (Cohick et al., 1996) also suggested that changes in systemic levels of IGF-I and IGFBP affect their concentrations in follicular fluid and follicular development. Finally, Beam and Butler (1999) proposed that during the negative energy balance period, the ability of follicles to produce sufficient estradiol for ovulation seems to depend on the availability of insulin and IGF-I in serum and the changing energy balance profile. Insulin. There is significant evidence that dietary restriction and negative energy balance reduce circulating concentrations of insulin (Vizcarra et al., 1998; Mackey et al., 2000). In vitro studies (Stewart et al., 1995) showed that insulin at physiological levels affected proliferation of bovine thecal cells, and acted synergistically with luteinizing hormone in stimulating steroidogenesis. Spicer (2001) reported that insulin by itself was a more effective stimulator of aromatase activity than FSH in vitro. Beam and Butler (1997) reported a greater insulin:GH ratio during the first week postpartum in cows ovulating during the first follicular wave than those that did not, suggesting that levels of

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51 insulin and GH during the very early stages of follicular recruitment may be important to later follicular function. Gong (2002) showed that dairy cows fed a diet that increased circulating concentrations of insulin during the first days 50 postpartum had shorter postpartum anestrus intervals, and an increased conception rate to first service independent of any effects on LH or FSH and without affecting milk yield or energy balance. The authors proposed that the increase in insulin concentrations promoted differentiation and maturation of dominant follicles during early lactation, thereby increasing the chance of these dominant follicles of ovulating in response to the LH surge. These results suggest that insulin may have a direct effect at the ovarian level. Thyroid hormones (T 3 and T 4 ). Reist (2003) examining associations between postpartum reproductive function and metabolic status in high yielding cows, reported that cows with higher plasma levels of thyroid hormones (T 3 and T 4 ), were associated with early start of ovarian cycle; proposing that Thyroid hormones can also play an important role in the resumption of ovarian activity postpartum. These results are supported by previous findings of Spicer (2001) who provided evidence for a role of T 3 and T 4 in regulating steroidogenesis of bovine follicles. The author proposed that T 3 and T 4 may have a minor positive impact on FSH-induced progesterone production by bovine granulosa cells, and a major positive impact on LH-induced androstenedione production by bovine thecal cells, both of which would result in a net increase in estrogen production by the follicle, however, T 3 and T 4 have little or no direct effect on aromatase activity. This evidence supports a role of Thyroid hormones as part of a multihormonal complex regulating ovarian activity in cattle.

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52 Leptin. Leptin is a protein hormone secreted by adipocytes (Bradley et al., 2000) and acts on the central nervous system to reduce voluntary feed intake (Schwartz et al., 2000). Block (2001) reported that postparturient cows undergoing negative energy balance had significantly lower plasma concentrations of leptin compared with postparturient cows in positive energy balance. In addition, at the first week of lactation the plasma concentration of leptin was correlated positively with plasma concentrations of glucose and insulin and negatively correlated with plasma concentrations of GH and NEFA. The author concluded that these correlations could represent a co-regulation by energy balance and these factors in mediating the effect of energy balance on leptin synthesis. Leptin has diverse effects on the neuroendocrine axis in addition to appetite and body weight regulation (Ahima and Flier, 2000). Leptin stimulated gonadotropin release and inhibited insulin-like growth factor-mediated release of estradiol in ovarian follicular cells in rat ovarian granulosa cells (Zachow and Magoffin, 1997). Another study (Chehab et al., 1996) reported that correction of the sterility defect in homozygous (ob/ob) obese female mice could be accomplished by repeated administration of human recombinant leptin, resulting in ovulation, pregnancy and parturition. Williams (2002) reported that short term fasting of growing prepubertal heifers causes marked reductions in circulating leptin, concomitant with declines in LH pulse frequency, and serum concentrations of insulin and IGF-I. In this same study results could not be repeated for mature cows under the same short term fasting. Altogether, these results may indicate that leptin signals the adequacy of energy stores for reproduction, by interacting with different target organs in the hypothalamic-pituitary-gonadal axis in cattle and other species. It has been proposed

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53 that the effects of leptin might be mediated in part by NPY, which in turn has been shown to regulate gonadotrophin release by inhibiting LH secretion in ewes (McShane et al., 1992). NPY is a potent inhibitor of LH release and unlike leptin, is a potent stimulator of food intake (Houseknecht et al., 1998) Concentrations of NPY increase in cerebrospinal fluid during undernutrition and can negatively modulate the secretion of LH when centrally infused in cattle (Gazal et al., 1998). These results may stimulate future research to explore the role and potential interactions between hormones, neuropeptides and resumption of ovarian cyclicity under negative energy balance in postpartum dairy cattle. It has been well established in cattle that ovarian function is controlled primarily by an integrated GnRH-gonadotrophin-ovarian axis. Recent work has shown that factors classically thought to be mainly involved in the regulation of metabolic processes, such as GH, insulin and IGF-I, thyroid hormones, leptin,and neuropeptide-Y may play an important role in the control of ovarian activity in the postpartum dairy cow (Spicer at al., 1995; Reist et al., 2003; Williams et al., 2002). Metabolic hormones can act either directly to control gonadotrophin independent stages of follicle development (Gong et al., 1996), or in synergy with gonadotrophins to modulate follicular recruitment and final development and maturation of preovulatory follicles (Spicer et al., 1995; Armstrong et al., 2002). These effects could represent at least part of the mechanism underlying well documented but not completely understood nutritional influence on reproductive function in cattle. Other factors Season. The effect of season has been reported as having an effect on resumption of ovarian activity postpartum. In a study conducted in North Carolina, Fonseca (1983)

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54 reported that cows calving in the winter had 6.5 more days to first ovulation compared with cows calving in the fall. Peters (1984) reported that cows calving in the spring underwent longer periods between calving and first ovulation than autumn calvers. Savio (1990a) reported that the postpartum interval to detection of the first dominant follicle was shorter in autumn than in the spring. When only normal dominant follicles were considered, the cows that calved in autumn tended to have a shorter, and less variable, intervals from calving to first ovulation. In Australia, cows calving in summer had significantly longer intervals from calving to first postpartum ovulation than those calving in winter (23d vs. 18d) (Jonsson et al., 1997). In addition, cows losing more bodyweight had longer intervals from calving to first ovulation. In Belgium, cows calving in the winter (stable housing) were more prone to delayed ovarian function compared to cows calving in the spring (pasture housing) (Opsomer et al., 2000). None of these studies proposed a path as to how season affects ovarian cyclicity. In Switzerland, Reist (2003) examined the relationship between reproductive function and metabolic and endocrine status in dairy cows. Results of this study showed a significant effect of season in resumption of ovarian activity. Cows calving in the fall were more likely to start ovarian cyclicity earlier postpartum than cows calving in the spring. Parity. The data on the effect of parity on the interval to first ovulation is contradictory and does not support an effect of parity on resumption of ovarian cyclicity. Stevenson and Britt (1979) reported that interval to first ovulation tended to be longer for pluriparous than for primiparous cows (18.7 vs 16.3 days), but the interval to first estrus was not different between parity groups (26.1 vs 27.7 days). Fonseca (1983) reported that in cows between 33 and 60 months of age, younger cows had their first ovulation earlier

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55 than older cows. In this study, there were very few observations in older cows. In contrast, Lucy (1992) reported that ovarian activity was delayed in primiparous cows compared with multiparous cows. However, primiparous cows were similar to multiparous cows with respect to first service, first service conception rate, services per conception, and days open. Moreira (2001) reported that the incidence of anestrus at 63 days was greater in primiparus than in multiparous cows. Periparturient diseases. The effects of periparturient diseases in resumption of ovarian cyclicity in dairy cows have been reported. Fonseca (1983) reported that cows with abnormalities after calving had 8.8 more days to first ovulation than cows without abnormalities after calving. In this study the main abnormalities at parturition were retained placenta and milk fever in Holstein cows and Jersey cows, respectively. During the postpartum period, uterine infection was the most frequent clinical abnormality in Holsteins, while ovarian cysts ranked first (followed by injury or disease and uterine infection) in Jerseys. Opsomer (2000) conducted a study to identify risk factors associated with postpartum ovarian dysfunction in dairy cows. In this study, cows suffering from clinical diseases such as mastitis, severe lameness, or pneumonia during the first month of lactation, were 5 times more at risk of developing delayed resumption of ovarian activity than healthy cows. In addition, cows developing clinical symptoms of ketosis with a positive prussiate test were 11 times more at risk of delayed resumption of ovarian cyclicity than normal cows. Cows with abnormal calvings and abnormal vaginal discharges were 3.6 and 4.5 times more likely to develop delayed ovulation compared to cows with normal vaginal discharges respectively. Similar results were reported by

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56 Etherington (1991) where cows with retained placenta were associated with longer intervals to first ovulation. Loss of body condition early in the postpartum period was another factor increasing the risk of delayed cyclicity. Cows losing more in body condition were 19 and 11 times more at risk of delayed cyclicity at 30 days and 2 months after calving. Cows with normal progesterone profiles lost on average 0.26 points during the first month after calving and 0.29 points during the first 2 months after calving, while cows with delayed ovarian function lost 0.39 and 0.49 points, respectively (Opsomer et al., 2000). These results were in agreement with those of Moreira (2001), where an effect of body condition at 63 days was associated with the frequency of cows classified as anestrus. As body condition increases the incidence of anestrus decreases. In another study conducted by Mateus (2003) a relationship between endotoxin concentrations in blood from cows with endometritis and a prolonged anestrus period was observed. This result is in agreement with a previous study from Peter (1990) where intrauterine infusions of endotoxins reduced the preovulatory LH surge in cows. This could have been mediated by high cortisol levels and resulted in ovulation failure. Ketosis is another periparturient disorder related to delayed resumption of ovarian activity. Reist (2000) examined the relationship between ketone body concentration and first ovulation in dairy cows. Cows classified as late responders (cows with a first ovulation between 31 and 87 days postpartum) had higher blood and milk ketone bodies concentrations compared to early responders (cows with first ovulation within 30 days postpartum), with no significant differences in body condition scores between groups.

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57 Lameness and Ovarian Activity While several studies have shown a relationship between lameness and reproductive performance, the relationship between lameness and ovarian activity in dairy cows has not been investigated using objective research methods. To our knowledge, there is only one study that looked at the relationship between lameness and ovarian activity in dairy cows. This study was conducted in 335 dairy cows on six high producing dairy herds in Belgium. Cows diagnosed with clinical mastitis, severe lameness, or pneumonia by farmers were at higher risk of delayed cyclicity, compared to cows classified as clinically healthy (Opsomer et al., 2000); however, the actual number of cows affected with clinical mastitis, severe lameness, or pneumonia was not reported. In Florida, clinical observations by veterinarians and dairy farmers suggest that cycle cows affected with lameness experience anestrus, and the duration of anestrus is associated with severity, duration, and diseases or lesions associated with lameness (eg, interdigital phlegmon, papillomatous digital dermatitis, claw lesions). We hypothesized that as lame cows experience a more pronounced loss in body condition (hence a prolonged state of negative energy balance) during the early post partum period, lame cows are at higher risk of delayed ovarian cyclicity than non-lame cows. Lame cows eat less and go into a negative energy state. In order to meet energy deficit, body reserves are mobilized resulting in body weight loss. The output of energy in lame cows (body maintenance and milk yield) may exceed the energy input in the form of feed. It is possible that cows that become lame are in a progressive negative energy state, go into anestrus and experience long delays to restore ovulation. Results of previous studies suggest that as cows experience increasing positive energy status, there is increased ovarian follicle activity leading to early return to

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58 ovulation (Butler et al., 1981, Staples et al., 1990; Lucy et al., 1991). As energy status becomes more positive for cows in early postpartum, diameter of the largest follicle increases, the number of double ovulations increases, and time for detection of the first corpus luteum decreases. (Lucy et al., 1991).These changes in follicle size and numbers and the number of ovulations are thought to be aroused by increases in luteinizing hormone (LH), follicle stimulating hormone (FSH), insulin, BST, insulin-like growth hormone-1 (IGF-1), and possibly other yet-to-be determined compounds as activated by improving energy status (Beam and Butler, 1998). Therefore, lameness may have an effect on feed intake and energy status leading to changes in concentrations of reproductive hormones. Under field conditions, evidence of corpus luteum function can be determined by monitoring plasma progesterone (P 4 ) concentrations weekly during lactation, before and after diagnosis of lameness in dairy cows.

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CHAPTER 3 MATERIALS AND METHODS Cows and Herd Management Cows in this study were from a high-producing dairy herd (rolling herd average milk production, approx 12,000 kg) of approximately 600 Holstein cows located in Florida. Cows were milked and fed a TMR ration three times per day. Cows were housed in lots equipped with sprinklers, fans and shade cloth over the feed bunks to reduce the effects of heat stress. This herd was selected for study on the basis of a history of lameness, quality of veterinary records, and willingness of the owner to participate in the study. Study Design This study was designed as an observational cohort study. Sample size calculations were made on the basis of our estimate of the number of cows affected with delayed ovarian cyclicity increasing from 10% in non-lame cows to 30% in lame cows (type I error = 0.05; type II error = 0.20). A total of 253 (45%) of 563 Holstein cows identified with an even numbered ear-tag that calved from June 1, 2002 until May 31, 2003 was used in the study. Cows with an even numbered ear-tag were enrolled in the study as they calved (instead of cows randomly selected) to overcome logistical identification procedures and to reduce disruption of routine veterinary medical and management procedures at the study farm. Cows were classified into one of six categories of lameness during the first 35 days post partum using a modification of the locomotion scoring developed by Sprecher et al., 1997. Blood samples were obtained for weekly for 59

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60 detection of plasma progesterone (P 4 ) concentrations during the first 60 days post partum. Risk of delayed cyclicity was compared between cows classified as non-lame, moderately lame, or lame. Data Collection Using farm records, the following data were collected for each cow: lactation number, calving date, calving season (winter months: Jan to Apr and Oct to Dec; summer months: May to Sep), dystocia (yes, no), retained placenta (yes, no), metritis (yes, no), mastitis (yes, no), ketosis (yes, no), body condition score at calving (Edmonson et al., 1989), change in body condition score in the first 50 days post partum, use of PGF 2 (Lutalyse, Pharmacia, Kalamazoo, MI) prior to resumption of ovarian activity (yes, no), and 305-day mature equivalent milk yield. From Dairy Herd Improvement Association (DHIA) records, projected 305 day ME milk yield data were collected based upon production at 60 days post partum. Levels of milk yield were defined as low (5,530 to 10,619 kg), medium (10,620 to 12,978 kg) and high (12,979 to 15,137 kg) on the basis of the frequency of distribution (first, second and third, and fourth quartiles, respectively). Diagnosis of Lameness During the first 35 days post partum, study cows were examined weekly (Tuesday) for diagnosis of lameness using a locomotion scoring system described by Sprecher (1997) with modifications (Table 1). Cows were observed and scored by the same veterinarian (EJG) as they walked-out of the wash pen to the holding area prior to milking. Cows with a locomotion score of 4 or 5 were further examined on a tilt table for diagnosis and treatment of lameness, noting lesions observed and date of occurrence. Lame cows with claw lesions had white line lesions or sole ulcers and were treated by corrective foot trimming techniques (Shearer and van Amstel, 2001). Lame cows with

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61 subacute laminitis were those with yellow and red discoloration of the sole and white line, and in most cases they had thin soles and were sensitive at examination with hoof testers (Mortensen, 1994; Toussaint Raven, 1989). Lame cows with interdigital dermatitis were those with inflammation confined to the epidermis and in some cases hyperkeratosis, which creates a roughened appearance to the interdigital skin (Blowey, 1994); a fetid serous exudate could be present, and there was mild sensitivity to pressure. This condition was frequently accompanied by cracks in the heel, heel horn erosion, with potential under-running of the heel horn (Berry, 2001). Collection of Blood Samples and Detection of Plasma P 4 Concentrations Cows were blood-sampled and scored for body condition (Edmonson et al., 1989) weekly (Thursday) for detection of plasma progesterone concentrations during the first 60 days post partum. Cows were blood-sampled via coccygeal venipuncture using vacutainer collection tubes containing K 3 EDTA (Becton, Dickinson, and Company, Franklin Lakes, NJ). Blood samples were refrigerated until and during transportation to a laboratory at the University of Florida where they were centrifuged for 20 min at 3000 RPM at room temperature for plasma harvest. Plasma samples were frozen at 20 C until tested for P 4 concentrations using the Coat-A-Count Progesterone Kit (DPC Diagnostics Products Corporation) radioimmunoassay. The Coat-A-Count Progesterone procedure is a solid-phase radioimmunoassay where 125 I-labeled progesterone competes for a fixed time with the progesterone content of the cows plasma sample. Because antibody is bound to the wall of the polypropylene tube, simply decanting the supernatant is enough to terminate the competition and to isolate the antibody-bound fraction of the radiolabeled progesterone. A standard curve dilution was prepared using coated tubes and non-coated tubes were used for total counts and non-specific binding. A 100 l volume of

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62 increasing concentrations of progesterone calibrators, (0, 0.1, 0.25, 0.5, 2, 5, 10, 20, and 40 ng/mL) were placed in the tubes. Plasma samples (100 l) were added to coated tubes and 1 mL of 125 I-labeled progesterone (25000 cpm) to all tubes. Every 6 th plasma sample was evaluated in duplicate. After an incubation period of 3 hours, the supernatant was discarded and tubes were dried for 15 minutes. They were then placed in a gamma counter. Calculation of the progesterone concentration in the plasma sample was made by computerized data process using a spline fitting curve. Accuracy of assay procedures was determined by measuring known quantities of exogenous progesterone (0.625, 1.25, 2.5, and 5.0 ng/mL) in plasma in seven different assays. Recovery of added (x) versus measured (Y) P 4 concentrations was described by linear regression (Y= 0.57 + 0.93x; R 2 = 0.89). The regression intercept value (0.57 ng/mL) represented original P 4 concentrations measured in a plasma pool prior to addition of exogenous masses. Parallelism of logit plots between the displacement curves for different volumes of a plasma pool containing 8 ng/mL of P 4 (i.e., 25, 50 and 100 l) and standard P 4 amounts (i.e., 0.1, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 ng/mL) was tested for homogeneity using regression analysis (Wilcox et al., 1990). The linear regression curves for plasma and P 4 standards were parallel (Yp= 0.60 1.44x, R 2 = 0.99; Ys = 0.21 1.61x, R 2 = 0.99; where Yp and Ys = ln B/F, x = log 10 of assay volume or log 10 standard P 4 concentrations, respectively). The slopes were not different. Coefficients of variation were calculated from a reference sample (luteal phase) and duplicate samples obtained from all assays. Duplicate plasma concentrations of P 4 were categorized into high ( 3.0 ng/mL; n = 359), medium (1.0 and <3.0 ng/mL; n = 128),

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63 and low ( 0.3 and <1.0 ng/mL; n = 52), and the coefficients of variation were 12.4%, 12.4%, and 14.2%, respectively. Inter-and intraassay coefficients of variation for the luteal phase reference sample were 8.9% and 8.34%, respectively. Resumption of Ovarian Cyclicity Cows with evidence of normal ovarian cyclicity during the first 60 days post partum were those with: i) weekly plasma P 4 concentrations > 1 ng/mL for 2 or 3 consecutive samples followed by a decline in P 4 ; or ii) if P 4 concentration > 1 ng/mL was followed by a marked decrease after PGF 2 injection and this followed by an increase in P 4 concentration. Cows with a delayed resumption of ovarian cyclicity were those with consistently low P 4 concentrations 1 ng/mL during the first 60 days post partum (Staples et al., 1990). For the purpose of this study, cows with P4 values above 1 ng/mL for 4 or more consecutive samples were classified as cows with extended luteal phases (Opsomer et al., 1999). First luteal phase was defined as the first rise in P 4 above 1 ng/mL. Reproductive and Health Management All cows were subjected to a pre-synchronization program. After days 30 to 35 postpartum cows received an injection of prostaglandin F2 and then again at 44 to 49 days. Cows observed in heat after the second injection of prostaglandin F2 were inseminated, and those that did not demonstrate behavioral signs of estrus were enrolled in a timed insemination program. This program involved the use of GnRH on Day 0, 7 days later prostaglandin F2, a second GnRH on Day 9, and timed inseminated 16 to 18 hours later. All cows were examined for pregnancy at 42 to 49 days post insemination by palpation of the uterus and its contents by the attending veterinarian.

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64 Farm personnel examined cows for the detection of health problems several times post partum following a pre-established protocol (Table 3-1). Cows were grouped by days in milk and a group of fresh cows (less than 30 DIM) were kept together. Cows were examined after each milking. On Day one cows were checked for retained fetal membranes (fetal membranes visible at the vulva for more than 24hs after calving), udder edema (by observation and palpation of the udder), and mastitis (by stripping all four quarters). On Day 4, cows were checked for production (using Afimilk computerized system), udder edema, metritis (Table 3-2) and rectal temperatures were taken. On day 7 cows were checked for Ketonuria (using Ketostixs), metritis, body temperature, and using a stethoscope, rumen movements, and displaced abomasum (DA) (tympanic sound on the left side at simultaneous auscultation-percussion of the left paralumbar fossa). This 7-day check was repeated at day 10 and 15. In all checks, daily milk yield was monitored for every cow for deviations in production. Table 3-1. Protocol for examination of cows postpartum Check day Health checks 1 Retained fetal membranes (RFM), udder edema, mastitis 4 Temp, udder edema, metritis, production, mastitis, manure consistency 7 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency 10 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency 15 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency Table 3-2. Definitions of metritis done by farm personnel based on discharge and palpation findings Metritis code Definition U1 Normal size and abnormal discharge. U2 Abnormal size and abnormal discharge (pus discharge) U3 Abnormal size and abnormal discharge (watery and foul smelling) U4 Abnormal size, abnormal discharge and the cow looks sick

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65 Cows with U1 and some with U2 were treated with an injection of prostaglandin F2. Some cows with U2 and all with U3 were treated with an intrauterine infusion of tetracycline (100 mg/mL) solution. Cows with U4 were treated with systemic antibiotics. Other health codes recorded were calving outcomes (See table 3-3) Table 3-3. Definition of calving outcomes Calving code Definition Pull 1 Easy pull, 1 person. Pull 2 Difficult pull, 1 person Pull 3 Easy for 2 persons Pull 4 Difficult pull for 2 persons Pull 5 Extreme pull. Cows with pulls 4 and 5 were started with systemic antibiotics after calving. All cows in the herd were monitored daily for deviations in milk (Afimilk) production, and milk conductivity for detection of mastitis following a pre-established criteria. (Table 3.4). All health events and treatments were recorded on a cow-side computer program (Visi-Cow) used by farm personnel. Table 3-4. Criteria for monitoring production health and mastitis using Afimilk system Population % Decrease in daily milk production (%) Increase in daily conductivity (%) Increase daily milk production (%) 1 to 15 DIM ------< 10 1 to 40 DIM 15 12 --41 to 100 DIM 20 15 --> 100 DIM 25 18 --Statistical Analyses The null hypothesis that risk of delayed ovarian cyclicity is the same in cows classified as non-lame, moderately lame, or lame was tested using logistic regression. In the analysis, non-lame cows were those with a score of 3 for one week only, or scores of 2. Cows classified as moderately lame were those with a score of 3 on two consecutive

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66 weeks. Lame cows were those classified at least once with a locomotion score of 4 or 5. Additional independent variables (lactation number, calving season, milk yield, dystocia, retained placenta, metritis, ketosis, body condition score, use of PGF 2 ) were included in the analysis to address possible modifying or confounding effects of these factors on risk of delayed ovarian cyclicity. Stepwise forward regression was used, and a variable had to be significant at the 0.20 level before it could enter the model. A variable remained in the model when its significance level was < 0.10. Variables for lactation number and calving season were forced into the model. In the final model, adjusted odds ratios (OR) and 95% confidence intervals (CI) were reported. The OR was used as an epidemiologic measure of association between a variable (i.e., lameness) and the outcome of interest (i.e., delayed ovarian cyclicity). In each variable, the reference category had an OR = 1. An assessed OR > 1.0 indicates that the probability of delayed ovarian cyclicity increased, compared with cows in the reference category. The attributable proportion was estimated as (OR 1)/OR, and interpreted to represent the proportion of lame cows that experienced delayed ovarian cyclicity because of lameness (Martin et al., 1987). The null hypothesis that number of days post partum to first luteal phase did not differ among groups of cows classified as non-lame, moderately lame, or lame was tested by use of the Kruskal-Wallis nonparametric test (because the dependent variable failed to meet assumptions of parametric testing), and multiple ANOVA for the dependent variable of days to first luteal phase (ranked data) while simultaneously adjusting for variables related to ovarian cyclicity (i.e., lactation number, calving season, ketosis, milk yield). Significance was set at P 0.05.

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67 012345678714212835424956637077Days postpartumP4 ng/ml Figure 1. Normal ovarian cyclicity 012345671421283542495663707784Days postpartumP4 ng/ml Figure 2. Normal ovarian cyclicity for cows treated with PGF2 00.20.40.60.8171421283542495663707784Days postpartumP4 ng/ml Figure 3. Delayed resumption of ovarian cyclicity

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68 0123456714212835424956637077849198105Days postpartumP4 ng/ml Figure 4. Extended luteal phase

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CHAPTER 4 RESULTS All 253 cows enrolled in the study were followed-up successfully during the 60-day study period. Two hundred and thirty-eight (94%) cows met the criteria for ovarian delayed cyclicity used in this study. A visual examination of plasma P 4 concentration patterns revealed that 15 cows (6%) experienced an extended luteal phase. A total of 101 of 238 (42%) cows were classified as moderately lame (locomotion score = 3) and 41 (17%) as lame (score = 4) (Table 4-1). The mean number of days post partum when cows were classified as lame was 15 days (1 34 days). The most common lesions observed were subacute laminitis (26/41 = 63%), and claw lesions such as sole ulcers and white line disease (9/41 = 22%). The overall incidence of delayed ovarian cyclicity was 11%. The incidence of delayed ovarian cyclicity was higher in cows classified as moderately lame (14/101; 14%) or lame (7/41; 17%), compared to non-lame cows (6/96; 6%). In the univariable analysis, cows classified as moderately lame were 2.4 times at higher risk of delayed ovarian cyclicity compared to non-lame cows (OR = 2.4; 95% CI = 0.9 6.7; P = 0.07) (Table 4-2). Cows classified as lame were 3.1 times at higher risk of delayed ovarian cyclicity compared to non-lame cows (OR = 3.1; 95% CI = 0.9 9.9; P = 0.05). In the multivariable analysis, lameness, lactation number, season, ketosis and milk yield were retained in the final modeling process (Table 4-3). Addition of two-way interaction terms did not contribute to the final model for risk of delayed ovarian cyclicity, and these terms were removed from the model. Cows classified as moderately 69

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70 lame were 2.1 times at higher risk of delayed ovarian cyclicity, compared to non-lame cows (OR = 2.1; 95% CI = 0.7 6.1; P = 0.15). Cows classified as lame were 3.5 times at higher risk of delayed ovarian cyclicity compared to non-lame cows (OR = 3.5; 95% CI = 1.0 12.2; P = 0.04). The attributable proportions of cows that experienced delayed ovarian cyclicity associated with moderate lameness and lameness were 0.52 and 0.71, respectively (Table 4-4). Overall, the time interval (median) from calving to first luteal activity in the study population was 31 days. This time period was more prolonged in cows classified as lame (median = 36; range = 17 to 97) or moderately lame (median = 32; range = 4 to 146), compared with non-lame cows (median = 29; range = 2-172) (P 0.05).

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71 Table 4-1. Frequency distribution of cows classified as lame or non-lame using a modification of the locomotion scoring system developed by Sprecher, 1997 Locomotion score Clinical description Assessment criteria Cows n = 238 No. of cows (%) 0 1 2 3 4 5 Normal Barely lame Mildly lame Moderately lame Lame Severely lame The cow stands and walks with a level-back posture. Gait is normal. The cow stands with a level-back posture but develops an arched back posture while walking. Gait remains normal. An arched-back posture is evident both while standing and walking. Normal gait. An arched-back posture is evident both while standing and walking. Gait is affected and best described as short strides with one or more limbs. 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. In addition to criteria in LS4, the cow demonstrates an inability or extreme reluctance to bear weight on one or more of her limbs/feet. 3 (1) 17 (7) 76 (32) 101 (42) 41 (17) 0 (0)

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72 Table 4-2. Descriptive statistics and unadjusted odds ratios for risk of delayed ovarian cyclicity in post-partum Holstein cows Variable Delayed cyclicity Yes n = 27 Delayed cyclicity No n = 211 OR 95% CI P Lameness group Locomotion score 2 3 4 Lactation number 1 2 Season Winter Summer Milk yield Low Medium High Dystocia No Yes Retained placenta No Yes Metritis No Yes Mastitis No Yes Ketosis No Yes BCS at calving < 2.75 2.75 3.5 > 3.5 BCS change (0.75) No Yes Use of PGF 2 No Yes No. of cows (%) 6 (22) 14 (52) 7 (26) 10 (37) 17 (63) 18 (67) 9 (33) 9 (33) 16 (59) 2 (8) 20 (74) 3 (11) 23 (85) 4 (15) 16 (59) 11 (41) 25 (93) 2 (7) 18 (67) 9 (33) 3 (11) 20 (74) 4 (15) 20 (74) 7 (26) 17 (63) 10 (37) No. of cows (%) 90 (43) 87 (41) 34 (16) 77 (36) 134 (64) 123 (58) 88 (42) 50 (24) 102 (48) 56 (27) 163 (77) 17 (8) 181 (86) 30 (14) 132 (63) 79 (37) 174 (82) 37 (18) 177 (84) 34 (16) 34 (16) 151 (71) 26 (12) 173 (82) 38 (18) 125 (59) 86 (41) 1.0 2.4 3.1 1.0 0.9 1.0 0.7 1.0 1.0 0.2 1.0 1.4 1.0 1.0 1.0 1.1 1.0 0.3 1.0 2.6 0.6 1.0 1.1 1.0 1.5 1.0 0.8 Reference 0.9 6.7 0.9 9.9 Reference 0.4 2.2 Reference 0.3 1.6 0.4 2.5 Reference 0.05 0.9 Reference 0.3 5.3 Reference 0.3 3.2 Reference 0.5 2.5 Reference 0.09 1.6 Reference 1.0 6.2 0.1 2.3 Reference 0.3 3.6 Reference 0.6 4.0 Reference 0.3 1.9 NA 0.07 0.05 NA 0.95 NA 0.40 0.91 NA 0.04 NA 0.58 NA 0.93 NA 0.73 NA 0.19 NA 0.03 0.52 NA 0.79 NA 0.32 NA 0.70

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73 Table 4-3. Final logistic regression model for risk of delayed ovarian cyclicity in postpartum Holstein cows Variable Adjusted odds ratio 95% confidence interval P value Lameness group Locomotion score 2 1.0 Reference NA 3 2.1 0.7 6.1 0.15 4 3.5 1.0 12.2 0.04 Lactation number 1 1.0 Reference NA 2 1.2 0.5 2.3 0.65 Season Winter 1.0 Reference NA Summer 0.9 0.3 2.3 0.90 Ketosis No 1.0 Reference NA Yes 2.7 1.0 7.0 0.03 Milk yield Low 0.9 0.3 2.5 0.98 Medium 1.0 Reference NA High 0.2 0.05 0.9 0.04 NA = Not applicable Table 4-4. Attributable proportion of cows that experienced delayed resumption of ovarian cyclicity Locomotion score N OR Attributable proportion 2 6/96 1 NA 3 14/101 2.1 0.52 4 7/41 3.5 0.71

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CHAPTER 5 DISCUSSION The results of the study reported here support the hypothesis that lameness has a detrimental effect on ovarian activity in Holstein cows during the early post partum period. Cows classified as lame were 3.5 times at higher risk of delayed cyclicity, compared to non-lame cows. Attributable proportion analysis indicated that delayed ovarian cyclicity in lame cows would be reduced by 71% if lameness had been prevented. In addition, cows classified as moderately lame were 2.1 times at higher risk of delayed ovarian cyclicity compared to non-lame cows (OR = 2.1; 95% CI = 0.7 6.1; P = 0.15). Even though this association was not statistically significant, the OR and the position of the confidence interval (Szklo and Nieto, 2000) suggest that cows classified as moderately lame were at high risk of delayed ovarian cyclicity. This observation is further supported by the fact that the interval from calving to first luteal phase was more prolonged in both lame cows (median = 36 days) or moderately lame cows (32 days) compared with non-lame cows (29 days) (P 0.05). Thus preventive measures (such as examination of cows feet and, if necessary, use of corrective foot trimming techniques) should be targeted at the group of moderately lame cows since as they represented 42% of the study population. We examined a second logistic regression model which included the 15 cows that experienced an extended luteal phase (in addition to the 238 cows that met the criteria for ovarian cyclicity used in this study), and the effect of lameness on delayed cyclicity did not disappear. Cows classified as moderately lame and lame were 2 (OR = 2.0; 95% CI = 0.7, 5.9; P = 0.17) and 3 times (OR = 3.0; 95% CI = 0.9, 10.3; P = 74

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75 0.07) at higher risk of delayed cyclicity, respectively, compared to non-lame cows. To our knowledge, only one previous study has examined the relationship between lameness and ovarian activity. In a study conducted in 335 dairy cows on six high producing dairy herds in Belgium, cows diagnosed with clinical mastitis, severe lameness, or pneumonia by farmers were at higher risk of delayed ovarian cyclicity compared to cows classified as clinically healthy (Opsomer et al., 2000). However, the actual number of cows affected with clinical mastitis, severe lameness, or pneumonia was not reported. The incidence of cows classified as moderately lame and lame during the first 35 days post partum was 42% and 17%, respectively. In a previous study involving 66 dairy cows on a farm in Michigan (Sprecher et al., 1997), a locomotion scoring system similar to that in our study was used for diagnosis of lameness. In the Michigan study, 27 (49%) cows and 14 (24%) cows were classified as moderately lame and lame, respectively. Results from that study are difficult to compare with results of the present study because of differences in the scoring system. After testing the locomotion scoring system (Sprecher et al., 1997) weekly for two months in the study herd, a new category was added to include cows that were observed with an arched-back posture that was evident both while standing and walking, but their gait seemed normal (score = 2, mildly lame); 76 (32%) cows were included in this category. In our analysis, this group of cows was classified as non-lame. Assuming that this group of cows was misclassified as non-lame, the incidence of cows classified as moderately lame (score = 3) would have been higher (76 + 101 = 177 cows or 74%). However, study results support our clinical observations and locomotion scoring system for diagnosis of lameness. The possibility that cows classified as mildly lame

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76 were misclassified is unlikely since the incidence of delayed ovarian cyclicity was lower in mildly lame cows (3/76 or 4%) compared to moderately lame cows (14/101 or 14%). If mildly lame cows were misclassified, there would have been an expected an incidence of delayed ovarian cyclicity similar to that observed in cows classified as moderately lame. Although we established an association between lameness and delayed ovarian cyclicity, we failed to identify loss of body condition (or a modifying effect of lameness and loss of body condition) as a significant risk factor associated with delayed ovarian cyclicity. The risk of delayed ovarian cyclicity was 1.5 times higher in cows that had a change in BCS 0.75 in the first 50 days post partum compared to cows with a change in BCS < 0.75. Therefore, this association was not significant (OR = 1.5; 95% CI = 0.6 4.0; P = 0.32). The observed incidence of delayed ovarian cyclicity in the study population was low (11%) compared to other studies (23 to 29%), (Humboldt and Thibier, 1980; Bartlett et al., 1987; Staples et al., 1990), creating a sample size limitation. In the previous study conducted in 335 dairy cows in Belgium, cows losing more body condition during the first and second month after calving were at higher risk of delayed ovarian cyclicity (Opsomer et al., 2000). In our study, ketosis was, by itself, a risk factor for delayed resumption of ovarian cyclicity. This result is in agreement with a study conducted in 84 dairy cows on 8 farms in Switzerland (Reist et al., 2000) where blood serum and milk ketone body concentrations during the first 6 weeks post partum were higher in cows classified as late responders (i.e., cows started post partum ovarian cyclicity after 30 days) than in early responders, with no significant differences in body condition scores between groups. It is possible that lameness and ketosis may additionally interact with each other to affect the

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77 risk of delayed ovarian cyclicity, but the small sample size was too small in the present study did not allow detection of such an interaction. Lameness can depress dry matter intake (Hassall et al., 1993; Galindo and Broom, 2002) and result in negative energy balance. It has been reported that negative energy balance contributes to increased ketone body formation and delays the onset of ovarian activity (Reist et al., 2000). A negative energy balance post partum not only contributes to increased ketogenesis, but also delays the onset of ovarian cyclicity, especially if energy deficiency is prolonged (Butler and Smith, 1989; Staples et al., 1990; Lucy et al., 1992). Furthermore, results of the study reported here revealed that the risk of delayed ovarian cyclicity was lower in high milk producing cows, compared to medium or low producing cows. The results of previous studies suggested that low producing cows have reduced inferior dry matter intake, a more negative energy balance, and are less likely to restore ovarian activity during the first 63 days post partum compared to high producing cows (Staples et al., 1990; Lucy et al., 1992). Although is clear that lameness has an effect on resumption of ovarian cyclicity in postpartum cows, we could not establish the cause of this effect. We hypothesized that energy balance would be responsible for the delayed in resumption of ovarian cyclicity, but our study could not support such pathway. Even though there are no studies reporting a pathway to explain the effect of lameness on ovarian activity, we cannot ignore other proposed mechanisms by which lameness may affect the hypothalamus-pituitary-ovarian axis. Dobson (2000) proposed that activation of the hypothalamus-pituitary-adrenal axis by stressors reduces the pulasatility of GnRH/LH by actions at both the hypothalamus

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78 and pituitary gland, depriving the ovarian follicle of adequate LH support. This will lead to reduced estradiol production by slower growing follicles. A combination of a reduced GnRH/LH pulsatility with a reduced production of estradiol, contributes to the delay and reduced magnitude of the LH surge and a delayed or absence of ovulation (Dobson et al., 1999; Dobson et al., 2000). Phogat (1997; 1999) provided evidence that the effect of increased concentrations of ACTH either exogenously, or after transport, reduced the amounts of LH released after challenges with small doses of GnRH, providing support for additional effects at the pituitary level. Dobson (2000) proposed that in situations such as during chronic stress of severe lameness or fever, the pulse GnRH/LH frequency will be so slow that initial follicular growth will occur but will be unable to continue into the later stages that depend on faster pulse frequencies. Thus the animal fails to maintain an estrus cycle developing anestrous. Another hypothesis is that the effect of lameness on cyclicity could be driven by endotoxins released after an event of ruminal acidosis (Nocek, 1997). As shown by Peter (1990) increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at ovarian level resulting in failure of a preovulatory LH surge. This may lead to anovulation or delayed ovarian cyclicity. Supporting the effects of endotoxins, Bataglia (1997) found that the suppressive effects of endotoxins on the reproductive axis can be mediated centrally through an inhibition of GnRH and thus LH pulsatile secretion. Ruminal acidosis has been identified as risk factor for lameness (Nocek, 1997; Hoblet et al., 2001). So if lame cows are suffering from acidosis with endotoxin released by gram-negative bacteria, endotoxins can mediate the effect of

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79 lameness in delayed resumption of ovarian cyclicity. At this point these hypotheses are only speculative and need further research to be proved.

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CHAPTER 6 CONCLUSION Analysis of results of the study reported here support the hypothesis that lameness has a detrimental effect on ovarian activity in Holstein cows during the early post partum period. The locomotion scoring system used in this study is a useful management tool that veterinarians and dairy farmers can adopt for early detection of lameness in dairy cows. The use of corrective foot trimming techniques in moderately lame cows may help reduce the risk of delayed ovarian cyclicity associated with the more severe forms of lameness (i.e., score = 4 and 5) in dairy cows. 80

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BIOGRAPHICAL SKETCH Eduardo Jose Garbarino is the son of Marta Pisarenko and Eduardo Jose Garbarino. He was born in Buenos Aires, Argentina, on July the 8 th of 1970. He lived in Buenos Aires during his entire childhood and finished his high school studies in 1989. He started veterinary medicine in 1994 at Universidad del Salvador, College of Veterinary Medicine, in Argentina. In 1998, he was honored with the Presidents Award (from the President of the Argentinean Nation) for the most qualified graduate from all Veterinary Medicine Schools in Argentina. He completed the degree of Medico Veterinario (MV) in March 1999 (with Honors Diploma). After graduation, he obtained a scholarship from the Argentinean government to work at the National Institute of Agricultural Research, in the areas of mastitis and subclinical ketosis in dairy cows. In the year 2000, he obtained a 1-year scholarship from the Secretary of Science and Techniques of the Argentinean Nation (Secretara de Ciencia y Tcnica de la Nacin Argentina) to work on research in the dairy project of the Agricultural Experimental Station of the National Institute of Agricultural Research (INTA), comparing two different milk-production systems. From December of 2000 to April 2001, he did and externship at a 2300-cow dairy farm in Torren, Mexico. In May of 2002, he started a 3-year residency program at the University of Florida, College of Veterinary Medicine, in the Food Animal Reproduction and Medicine Service. In 2002, he enrolled in the graduate program at the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, to obtain the degree of Master of Science. 97


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Physical Description: Mixed Material
Copyright Date: 2008

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EFFECT OF LAMENESS ON OVARIAN ACTIVITY IN POST-PARTUM HOLSTEIN
COWS













By

EDUARDO JOSE GARBARINO


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


2004

































Copyright 2004

by

Eduardo J Garbarino





























In memory of my father, Eduardo Jose Garbarino
















ACKNOWLEDGMENTS

I would like to express my sincere gratitude to Dr Jorge Hernandez, Associate

Professor of Veterinary Medicine, for his advice and guidance during my 3 years of hard

work. I also thank him for his help, support, and friendship, which allowed me to

successfully complete my masters program.

I thank Dr. Carlos Risco Professor of Veterinary Medicine for his contributions to

the proj ect. I thank Dr. Jan P. Shearer, Dairy Extension Veterinarian, for his availability

for consultation and interest on the proj ect and Dr. William W. Thatcher, Graduate

Research Professor at the Department of Animal Science for his ideas, his contribution in

the design and interpretation of the study.

I would like to thank Mrs. Coni and Mr. Dale Sauls and the personnel of Condale

Dairy Farm for their support and hard work to make this study possible. I also would like

to acknowledge Shawn Ward for his hard work and friendship.

I thank Julie Oakley and Marie Joelle Thatcher for their help with assay and data

handling and analysis. I also need to thank Sally O,Connel, secretary at the Graduate

School, for her continuous concern and help for graduate students. I would also thank

faculty, residents and staff of the Food Animal Service for their help and patience.

I need to acknowledge my friends in Gainesville, Jackie and Mandy, Elisa and

Charly for being our hotel, mechanics, electricians, baby sitters, painters and for their

constant support and concern but most importantly for their sincere friendship. Thanks to

my friends in Argentina, Gerva, Bebe and Paula, Carlitos and Ceci, Chaca, Carlitos,









Luchito and Rosario, Gonzalo and Sole for their support and for more than 15 years of

friendship.

I would like to thank my mother and sister, for their love and constant support for

the past 34 years, and my father, who from heaven, looks after every step I take. Finally I

thank my wife Martita, our daughter Sofl and our son Santi, for whom I don't have words

to express my gratitude but I am sure that without them this would not be possible.





















TABLE OF CONTENTS


page


ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ................. ................. viii............


LIST OF FIGURES .............. .................... ix


AB S TRAC T ......_ ................. ............_........x


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


2 LITERATURE REVIEW .............. ...............4.....


Anatomy of the Bovine Foot ................. ...............9........... ...
Foot ................. ...............10.................
Claws ................. ..... ... ...... ......... .... .... ... .... .... .......1

Suspensory Apparatus and Supporting Structure of the Bovine Digit ................ 12
Horn Formation and Growth ................ ........... ..... ....... ....... ........ 1
Microanatomy of the Claw: Structure of the Wall ................ ................. ... 14
Etiology of Lameness ................... ......_._ ...............15......
Infectious Diseases of the Digits .................. ...... ....._ ... ....... ...........1
Interdigital phlegmon (F oot-rot, Interdigital necrob acill osi s) ........._.._..........15
Interdigital dermatitis ........._...... .... .... ._.. ... ......._..... .............1
Digital dermatitis (DD) (Footwarts, Hairy heel warts, Heel warts)............. 19
Metabolic Hoof Horn Disease: Claw Horn Disruption .........._.... ......._.......25
Laminiti s........._._ ......_ _. ...............26...
Forms of laminitis .............. .... ...............29.
Claw Lesions Associated with Laminitis .................... ............... 3
Hemorrhages of the sole and sole ulcer. .........._.._. ......_. .................30
Softening of the horn of the sole ................. ...............30......__ ..
White line disease............... ...............31
Heel erosion............... ...............3 1

Diagnosis of Lameness ................. .............. 1......... ....
Lameness and Animal Welfare............... ...............34
Lameness and Milk Production .............. ...............35....
Lameness and Reproductive Performance............... ..............3
Resumption of Ovarian Activity Postpartum .............. ...............39....












Physiological Factors Involved in Ovarian Activity Potentially Affected by
Energy Balance ................. .... ........ ..... ........ .............4
Effects of negative energy balance in LH secretion ................. .................4 8
Metabolic hormones ................. ...............49.................
Other factors ................. ...............53.................
Lameness and Ovarian Activity............... ...............57
3 MATERIALS AND METHODS............... ...............59


Cows and Herd Management............... ...............5
Study Design............... ...............59.
Data Collection .............. ...............60....

Diagnosis of Lameness ................. ............ ..... ... ...... ....... ...... .........6
Collection of Blood Samples and Detection of Plasma P4 COncentrations ................61
Resumption of Ovarian Cyclicity .............. ...............63....
Reproductive and Health Management .............. ...............63....
Statistical Analyses ................. ...............65.................
4 RE SULT S .............. ...............69....


5 DI SCUS SSION ................. ...............74................


6 CONCLU SION ................. ...............8.. 0..............


LI ST OF REFERENCE S ................. ...............8.. 1......... ....


BIOGRAPHICAL SKETCH .............. ...............97....

















LIST OF TABLES


Table page

2-1. Studies reporting incidence of lameness in dairy cows ................. ............ .........7

2-2. International terminology of digital diseases............... ...............9

2-3. Incidence of Digital Dermatitis in US dairy herds by herd size and region ........._....22

2-4. Days from calving to 1st ovulation reported in the literature ............... ................. 43

2-5. Incidence rates of delayed cyclicity reported in the literature. ................. ...............44

3-1. Protocol for examination of cows postpartum ................. ...............64.............

3-2. Definitions of metritis done by farm personnel based on discharge and palpation
findings ................. ...............64.................

3-3. Definition of calving outcomes. ............. ...............65.....

3-4. Criteria for monitoring production health and mastitis using Afimilk@ system........65

4-1. Frequency distribution of cows classified as lame or non-lame using a modification
of the locomotion scoring system developed by Sprecher, 1997. ............................71

4-2. Descriptive statistics and unadjusted odds ratios for risk of delayed ovarian cyclicity
in post partum Holstein cows ................ ...............72......_... ...

4-3. Final logistic regression model for risk of delayed ovarian cyclicity in post partum
Hol stein cows .............. ...............73....

4-4. Attributable proportion of cows that experienced delayed resumption of ovarian
cy clicity. ............. ...............73.....


















LIST OF FIGURES

Figure pg

1. Normal ovarian cyclicity............... ...............6

2. Normal ovarian cyclicity for cows treated with PGF2 .............. .....................6

3. Delayed resumption of ovarian cyclicity .............. ...............67....

4. Extended luteal phase .............. ...............68....
















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

EFFECT OF LAMENESS ON OVARIAN ACTIVITY INT POSTPARTUM HOLSTEIN
COWS

By

Eduardo Jose Garbarino

May, 2004

Chair: Jorge Hernandez
Major Department: Veterinary Medical Sciences

An observational cohort study was conducted to examine the relationship between

lameness and delayed ovarian cyclicity in post partum Holstein cows. We used 253 cows

from a 600-cow dairy herd that calved during a 12-month period. Cows were classified

into one of six categories of lameness during the first 35 days post partum using a

locomotion scoring system. Cows were blood sampled weekly for detection of plasma P4

concentrations during the first 60 days post partum. Cows with a delayed resumption of

ovarian cyclicity were those with consistent P4 COncentrations < 1 ng/mL during the first

60 days post partum. The null hypothesis that risk of delayed cyclicity is the same in

cows classified as non-lame, moderately lame, or lame (after adjusting for potential

modifying or confounding effects of loss of body condition and other variables related

with delayed cyclicity) was tested using logistic regression. Results of the study support

the hypothesis that lameness has a detrimental effect on ovarian activity in Holstein cows










during the early post partum period. Cows classified as lame were 3.5 times at higher risk

of delayed cyclicity, compared to cows classified as non-lame

(OR = 3.5; 95% CI = 1.0 12.2; P = 0.04). Attributable proportion analysis indicated that

delayed ovarian cyclicity in lame cows would be reduced by 71% if lameness had been

prevented. In addition, cows classified as moderately lame were 2.1 times at higher risk

of delayed cyclicity, compared to non-lame cows

(OR = 2. 1; 95% CI = 0.7 6. 1; P = 0. 15).















CHAPTER 1
INTRODUCTION

Lameness is one of the top three health problems that cause premature culling of

dairy cows in the United States. The National Animal Health Monitoring System Dairy

2002 Study (NAHMS 2002) reported that lameness was the reason for culling 16% of

dairy cows sent to slaughter. Overall, 10% of cows were reported affected with lameness

in the previous 12 months. The economic importance of lameness is attributed to cost of

treatment and control methods (Shearer and Elliot, 1998; Shearer et al., 1998; Hernandez

et al., 1999, 2000; Moore et al., 2001), impaired reproductive performance (Lucey et al.,

1986; Lee et al., 1989; Sprecher et al., 1997; Hernandez et al., 2001; Melendez et al.,

2003), decreased milk yield (Warnick et al., 2001; Green et al., 2002; Hernandez et al.,

2002), increased risk of culling (Sprecher et al., 1997; Collick et al., 1989), and decreased

carcass value of culled cows (Van Arendonk et al., 1984). In addition, because of the

pain, discomfort, and high incidence of lameness in dairy cows, this disorder should be

considered an animal welfare issue.


Delayed ovarian cyclicity in the preservice postpartum period is a common

ovarian dysfunction in dairy cows (Opsomer et al., 1998). Late resumption of ovarian

activity post partum has a detrimental effect on reproductive performance in dairy cows

(Thatcher and Wilcox, 1973; Stevenson and Call, 1983; Lucy et al., 1992). Cows

ovulating earlier post partum have fewer services per conception and a shorter

calving-to-conception interval (Lucy et al., 1992). Minimizing the interval from calving









to first ovulation provides ample time for completion of multiple ovarian cycles before to

insemination, which in turn improves conception rates (Butler and Smith, 1989). Losses

in body condition, puerperal disturbances, and ketosis have been identified as risk factors

significantly associated with delayed ovarian cyclicity in dairy cows (Opsomer et al.,

2000).

While previous studies have shown that lameness has a detrimental effect on

reproductive performance (i.e., a prolonged calving-to-conception interval) (Lucey et al.,

1986; Collick et al., 1989; Sprecher et al., 1997; Hernandez et al., 2001), the relationship

between lameness and ovarian activity in dairy cows has not been investigated using

objective research methods. Results of previous studies in Florida suggest that as cows

experience increasing positive energy status, there is increased ovarian follicular activity

leading to return to ovulation (Staples et al., 1990; Lucy et al., 1992). As energy status

becomes more positive for cows early post partum, diameter of the largest follicle

increases, the number of double ovulations increases, and time for detection of the first

corpus luteum decreases (Lucy et al., 1991). These changes in follicle size and numbers

and the number of ovulations, are thought to be caused by increases in luteinizing

hormone, follicle-stimulating hormone, insulin, BST, insulin-like growth factor-1, and

possibly other yet-to-be determined compounds that are activated by an improved energy

status (Beam and Butler, 1998).

Clinical observations by veterinarians and dairy farmers in Florida suggest that

lameness has a detrimental effect on ovarian activity in lactating dairy cows. We

hypothesized that as lame cows experience a more pronounced loss in body condition

(hence a prolonged state of negative energy balance) during the early postpartum period,









lame cows are at higher risk of delayed ovarian cyclicity than non-lame cows. Under

field conditions, evidence of corpus luteum function can be determined by monitoring

plasma P4 COncentrations weekly during lactation, before and after diagnosis of lameness

in dairy cows. The obj ective of this study was to examine the relationship between

lameness and delayed resumption of ovarian cyclicity in Holstein cows during the first 60

days post partum.

Knowledge of the epidemiologic aspects of diseases and lesions that cause

lameness is essential to further develop control and prevention methods for lameness in

dairy cows. Risk of lameness during lactation, severity and duration of lameness, and the

relationship between lameness and ovarian activity in lame cows have not been

investigated under US dairy farming conditions. Our prospective research study will

allow us to better characterize lameness in dairy cows under commercial farming

conditions, and examine the relationship between lameness and ovarian activity. An

understanding of the reasons for individual-cow differences in lost revenues will aid

producers in making management decisions at the cow and herd levels to manage

lameness, reproductive performance, and animal welfare.















CHAPTER 2
LITERATURE REVIEW

Lameness is one of the top three health problems that cause premature culling of

dairy cows in the United States. Several studies have reported that the incidence of

lameness in dairy cattle operations varies from 7% to 54.6% (Harris et al., 1988;

Clarkson et al., 1996; Collick et al., 1989; Barkema et al., 1994; Green et al., 2002;

Deluyker et al., 1991), with the highest proportion of cases occurring in the first 100 days

in milk (Collick et al., 1989; Barkema et al., 1994; Green et al., 2002).

The economic importance of lameness is reportedly attributable to cost of

treatment and control methods (Moore et al., 2001; Hernandez et al., 1999, 2000; Shearer

et al., 1998), impaired reproductive performance (Lee et al., 1989; Tranter and Morris,

1991; Sprecher et al., 1997; Hernandez et al., 2001; Melendez et al., 2003), decreased

milk yield (Tranter and Morris, 1991; Coulon et al., 1996; Warnick et al., 2001;

Hernandez et al., 2002), increased risk of culling (Sprecher et al., 1997; Collick et al.,

1989), and decreased carcass value of culled cows (Van Arendonk et al., 1984). In

addition, because of the pain, discomfort, and high incidence of lameness in dairy cows,

this disorder should be considered an animal-welfare issue.

It is difficult to describe the incidence and prevalence of the problem of lameness

in dairy cows, because of the wide variation that exists in published reports. This

variation is due to differences in geographic locations and production systems

(pasture-based vs. confinement) of study herds, that may alter the frequency distribution

of lameness. In addition, the method of gathering data varies among studies (some









collecting data from veterinary practices, and others gathering data directly from the

farmer, or by researchers). Whitaker (1983) reported an average incidence of lameness of

25% (range = 2% to 55%) in 21,000 dairy cows in 185 herds in England and Wales. In

the study by Whitaker (1983), data were collected by a survey of the number of

treatments for lameness either by the farmer or veterinary surgeon. Another study

conducted in 37 dairy farms in the United Kingdom and Wales, Clarkson (1996) reported

an annual incidence of lameness of 54.6% (new cases/100 cows) with a range from

10.7% to 170.1%. This same study (Clarkson et al., 1996) reported the prevalence of

lameness to be 20.6%. Diagnosis of lameness was performed either by the farmer,

student, hoof trimmer, or a veterinarian. All treatments were recorded and were used to

calculate the prevalence and incidence of lameness. Collick (1989) performed a study in

17 dairies in the UK, and reported an incidence of lameness of 17% during a 6- month

period, with a range of 8% to 28%, with 65% of cases occurring in the first 100 days in

milk. In this case, diagnosis of lameness was made by the attending veterinarian.

A study conducted in 80 dairies in France (Faye and Lescourret, 1989), the

reported annual incidence of lameness was 29.5%. They stated that incidence of lameness

was higher than reported incidence rates of mastitis, and that lameness was the most

common disease reported in dairies. A study conducted in 101 dairy farms in Sweden

(Manske et al., 2002a), found a prevalence of lameness of 5%. In this study (Manske et

al., 2002a) lameness examination of cows was done by researchers who looked at every

cow on each farm, with the purpose of identifying claw lesions in lame and non-lame

cows. In other parts of the world with more extensive production system such as New

Zealand, Australia, and Argentina, the incidence/prevalence of lameness differs from









those reported in Europe. Dewes (1978) reported an incidence of lameness of 14%, and

more than 85% of the cases of lameness were detected before 90 days in milk. This study

(Dewes, 1978) was conducted using data provided by four dairy producers from Waikato,

New Zealand. In another study in New Zealand by Tranter and Morris (1991) in three

dairy herds, the annual incidence of lameness was 16% with a range from 2% to 38%. In

this study the mean days to onset of lameness was 92154 (mean +SD). Harris (1988)

conducted a study in 73 dairies in Southern Australia and reported and annual incidence

of lameness of 7% with a range of 0% to 31%. In this study, diagnosis of lameness was

done by the farmer. In a study done in Argentina (Rutter, 1994) involving 4580 dairy

cows from 25 farms, the incidence of lameness was 23.4% with the highest incidence

occurring in first lactation heifers (45%). In most of these studies, sole ulcers and white

line disease were the most predominant lesions observed.

When we examined the incidence and prevalence of lameness studies in the

United States, we also found important variations. Reported incidences of lameness were

4.4%, 5.1% and 9.5% (Bartlett et al., 1987; Weigler et al., 1990; Kaneene et al., 1990). In

these studies, lameness was diagnosed by the farmers. This motivated Wells (1993a), to

design a study on prevalence of lameness, and to compare the diagnosis of lameness

performed by researchers and farmers. In both seasons spring and summer, the observed

prevalence reported by researchers were 3 times higher than that by the farmers. This

study (Wells et al., 1993a) involved 17 dairy farms from Minnesota and Wisconsin and

reported a prevalence of lameness of 14% in the summer and 17% in the spring. In

another study conducted in 13 dairy farms in Ohio, researchers observed that the

incidence of hemorrhages and discoloration of the sole in first calf heifers (from 60 days










pre partum until 100 days post partum) was 62% during this period. In another study by

Sprecher (1997) using a locomotion scoring system, the incidence of lame cows

(Locomotion Score > 4) was 24.5%, the incidence of moderately lame plus lame cows

(LS > 3) was 49. 1% at the time of the first service.

Table 2-1. Studies reporting incidence of lameness in dairy cows
Author, year Country Incidence of lameness (%)

Rutter, 1994 Argentina 23
Harris et al., 1988 Australia 7 (0 to 31)

Faye and Lescourret, 1989 France 29
Dewes, 1978 New Zealand 14
Tranter et al., 1991 New Zealand 16 (2 to 38)
Manske et al., 2002a Sweden 5
Whitaker et al., 1983 United Kingdom 25 (2 to 55)
Collick et al., 1989 United Kingdom 17 (8 to 28)
Clarkson et al., 1996 United Kingdom 54
Bartlett et al., 1987 United States 4

Weigler et al., 1990 United States 5
Kaneene et al., 1990 United States 9
Wells et al., 1993 United States 13.7 summer & 16.7 winter

Sprecher et al., 1997 United States 49
Warnick et al., 2001 United States 46



Almost all reports describing lameness in dairy cows considered claw diseases as

the most common causes of lameness. Some of the papers listed above (Tranter and

Morris, 1991; Deluyker et al., 1991; Murray, 1996) reported claw diseases as responsible

for more than 90% of the cases of lameness. When we examined the most common claw

disorder, reports are not so much in agreement. Differences maybe due to, different

housing types, feeding strategies, environmental challenges and managements systems.









Studies from the United Kingdom (Clarkson et al., 1996; Murray et al., 1996) agreed that

sole ulcers, white line disease, laminitis and subsolar abscess are the most common

diseases affecting the bovine claw. In another study conducted on 101 dairy farms in

Sweden (Manske et al., 2002), sole ulcers and white line lesions were the most common

lesions found in lame cows. In New Zealand and Australia (Tranter and Morris, 1991;

Dewes, 1978; Harris et al., 1988) where cows are kept in pasture all year around,

traumatic pododermatitis (sole bruising), worn soles and interdigital dermatitis the most

common diseases of the claw affecting dairy cows. In a study conducted in Argentina

(Rutter, 1994), digital dermatitis (39.4%) and interdigital dermatitis (26.3%) were the

most common diseases affecting the bovine foot. In the United States, all studies

identified claw lesions as the most common lesions affecting dairy cows (Deluyker et al.,

1991; Smilie et al., 1996; Hernandez, 2002). These studies, however, were not designed

to establish the prevalence of the different diseases of the bovine foot.

With an average incidence of 30 cases per 100 cows per year, a case fatality rate

of 2%, involuntary culling of 20% of cases; an average increase of 28 days open,

treatment costs including veterinary fees, drugs and farmer labor of $23 per case. The

total cost of lameness per 100 cows per year is estimated to be about $9000 (Guard,

1996). This paper is probably underestimating the cost of lameness as it is not taking into

account other factors that are affected by lameness. Lameness has been shown to affect

milk production and this was not included in the estimation, as well as the decrease of the

carcass value of cows sent to slaughter and the increased probability of a cow being

culled if experiencing lameness. In another report from the UK, Kossaibati and

Esslemont (1997) reported that the average total cost per affected cow of a case of sole









ulcer was 424 (approx $600). This estimation included the cost of treatment, herdsman' s

time, discarded milk, reduced milk yield, veterinarian's time, increased risk of culling

and longer calving interval. Increased risk of culling and a longer calving interval

accounted for more than 60% of the cost. Unfortunately, the authors did not explain how

they estimated milk loss and increased risk of culling.

Table 2-2. International terminology of digital diseases
International terminology English Common terms
Dermatitis interdigitalis Interdigital dermatitis Superficial Foot-Rot
Phlegmona interdigitalis Interdigital phlegmon Foot-Rot, Foul in the foot
Erosio ungulae Heel erosion Underrun heel, Slurry heel
Hyperplasia interdigitalis Interdigital hyperplasia Corn, Interdigital fibroma
.Hairy foot warts, Hairy
Dermatitis digitalis Digital dermatitis el

Diffuse aseptic
Pododermatitis aseptica difusa Laminitis
pododermatiti s
Circumscript
Pododermatitis circumscripta Sole ulcer
pododermatiti s
Pododermatitis septica Traumatic septic Subsolar, toe and white
traumatica pododermatitis line abscesses
Sand cracks: longitudinal
Fissura ungulae Hoof wall cracks
and transverse
Pododermatitis locale Localizedpododermatiti s Bruises
Ungulae deformans Overgrown hooves Long toes



To compare results from different studies it is important to standardize the

terminology regarding claw lesions. In order to clarify the rest of this manuscript we will

follow the terminology proposed by Weaver (1994) (Table 2-2).

Anatomy of the Bovine Foot

Before we start describing the different pathologies affecting the bovine foot, it is

important to review it' s anatomy and microanatomy.









Foot

The foot includes the entire limb below the fetlock j oint. It is comprised of two

digits each of which has a horn-covered claw. It should be noted that in cattle the term

"claw" is preferable to hoof. The front aspect of the foot is referred to as the dorsal side.

The back side of the front foot is referred to as the palmar aspect whereas in the rear foot

is referred to as plantar aspect. When referring to an area nearest the longitudinal axis

(i.e., toward the center) it is designated as axial, whereas items farther away (away from

the center) are designated as abaxial.

Each digit of the foot has 4 bones: phalange 1 (Pl), phalange 2 (P2), phalange 3

(P3), and navicular bone; and 2 joints: proximal interphalangeal (PIP) and distal

interphalangeal (DIP). The proximal end of Pl articulates with the metacarpus (in the

front leg) or metatarsus (in the rear leg) in the fetlock j oint, whereas the distal (away from

the center of the body) end of Pl articulates with the proximal end of P2. This

articulation between Pl and P2 is referred to as the proximal interphalangeal joint (PIP).

The distal end of P2 articulates with the proximal end of P3. This j oint is referred to as

the distal interphalangeal joint (DIP).

P3 is completely enclosed within the claw horn capsule. Its solar surface is

concave or arch shaped and marked on the back edge by a bump known as flexor

tuberosity. The flexor tuberosity is the site of attachment of the deep flexor tendon. This

tuberosity has an important role in the pathogenesis of sole ulcers as it becomes involved

in the process of compression of the corium subsequent to laminitis and the displacement

of P3 (Toussaint Raven, 1989).

The navicular bone (also referred to as the distal sesamoid bone) is attached to P3

by three small ligaments and also to P2 by collateral ligaments. Between the navicular









bone and the deep flexor tendon is the navicular bursa. The navicular bursa contains

j oint-fluid which permits movement of the deep flexor tendon over the surface of the

navicular bone during extension and flexion of the claw. P3, the DIP j oint, navicular bone

and navicular bursa all lie within the claw capsule.

Claws

The purpose of the claw hom capsule is to protect the underlying sensitive tissues

of the corium and dissipate the concussion forces that occur when the digits impact the

ground. It consists of the wall which can be divided into the axial (inside) and the abaxial

(outside). The abaxial wall is further subdivided into the dorsal (or front) and lateral

abaxiall side) aspects. The wall is demarcated from the heel on the abaxial side of the

claw by the abaxial groove. The wall consists of two types of horn: perioplic and

coronary. Perioplic hom is the softer horn lying just below the coronet at the skin-hom

junction (corresponding to the human cuticule). At the back of the foot the periople

gradually widens and eventually becomes the horn of the heel. Coronary horn, the hardest

horn within the claw capsule makes up the bulk of the horn of the wall. The wall has faint

ridges or rugae, which run horizontally and parallel to each other. Toward the heel these

ridges diverge reflecting a more rapid rate of growth in the heel region due to faster rates

of wear. In mature Holstein cattle the length of the dorsal wall should be a minimum of 3

inches in length from just below the top of the hairless portion of the wall to the

weight-bearing surface. Ideal heel height is 1.5 inches (Toussaint Raven, 1989; Blowey,

1993).

The sole is produced by the solar corium and merges imperceptibly with the hom

of the heel at the heel-sole junction. The sole is connected to the wall by means of the

white line. White line horn is produced by laminar corium. It courses forward from the









area of the heel on the abaxial side of the claw, around the tip of the toe and about 1/3 of

the way back on the axial side of the claw' s weight bearing surface. Where the white line

leaves the weight bearing surface it courses upward on the axial side of the claw. This

white line is a unique and important structure. It is the softest horn within the claw

capsule. This permits it to provide a flexible junction between the harder horn of the wall

and the softer horn of the sole. On the other hand, because of its softer nature it also

represents a weak spot on the weight-bearing surface that is vulnerable to damage.

Suspensory Apparatus and Supporting Structure of the Bovine Digit

Cattle (and all animals with claws or hooves) are suspended in their feet, that is,

they stand in their feet, not on them. In other words, the bone within the claw (also

known as P3) is suspended within the claw horn capsule by the laminar corium and a

series of collagen fibers bundles that stretch from the insertion zone on the surface of P3

to the basement membrane of the epidermis (the line of demarcation between dermis and

epidermis). The interface between dermal and epidermal components is the

interdigitating dermal and epidermal laminae. The result is that P3 hangs within the claw

capsule and weight is transferred as tension onto the wall of the claw capsule.

The suspensory system in cattle differs significantly from that in horses. First, the

laminar corium is much less extensive in cattle as compared to horses. Secondly, there

are no secondary laminae in the laminar corium of cattle. Therefore, capabilities with

respect to mechanical load carried on the claws of cattle vary significantly. In the horse

load bearing is primarily on the wall. Cattle, on the other hand, simply cannot handle the

same amount of mechanical load on the walls of their claws. Instead, weight-bearing in

cattle requires displacement of load to the wall, and support structures within the sole and

heel .










The primary structures within the supportive apparatus of the bovine claw are the

solar corium and associated connective tissue, and the digital cushion, which consists of

loose connective tissue and varying amounts of adipose (fat) tissue. The digital cushions

are arranged in a series of three parallel cylinders similar to the design used in the

cushion of a running sole. In the cows foot these cushions act like shock absorbers within

the claw protecting the corium and permitting limited movement of P3 in the region of

the heel.

Horn Formation and Growth

The horn-producing germinal layer of the epidermis and its supporting structure,

the corium, consist of four different regions, each producing a structurally different type

of horn (Budras et al., 1996). Perioplic horn, overlying the perioplic corium, is found just

below the skin-hom junction and extends to the back of the claw to include the heel horn

(Budras et al., 1996). Horn of the wall is produced in the area of the coronary corium and

it is situated between the perioplic corium and the sensitive laminae. The area overlying

the laminar corium produces the horn of the white line, also known as laminar horn. The

solar horn overlies the solar corium and is situated between the laminar hom of the white

line and the perioplic hom of the heel (Budras et al., 1996; van Amstel and Shearer,

2001).

Horn production and growth are supported by the corium, which corresponds to

the dermis. The corium consists of a rich vascular network that terminates in dermal

papillae, also called vascular peg (Greenough, 1997). A vascular peg consists of a main

arteriole and a venule, which are connected at the tip. Between the arteriole and the

venule is an extensive capillary network, and there are also several vascular shunts

between such arterioles and venules. These shunts may open under certain circumstances,










cutting of the blood supply to the tip of the vascular peg, which adversely affect horn cell

formation. The epidermal layer overlying the vascular pegs produces horn cells in the

form of tubules (tubular horn) (Budras et al., 1996; Greenough, 1997). Intertubular horn

is produced between the papillae and interconnects the tubular horn. There are

approximately 80 vascular pegs or dermal papillae per square millimeter of coronary

corium surface (Greenough, 1997), which means that the wall consists of tightly packed

tubular homn that is cemented together by intertubular horn. The perioplic corium of the

heel horn and the solar corium has fewer vascular pegs per square millimeter. Because

tubular homn supplies structural strength to the homn capsule, it follows that the horn of the

wall is structurally the strongest, followed by the sole and the heel. Keratin filaments

produced by horn cells enhances the rigidity and strength of homn cells as they progress to

the exterior.

Laminar horn is immature, nontubular, so it is soft and flexible and has a high

turnover rate. Homn cells, whether tubular or nontubular, are cemented by a substance

known as membrane-cementing substance (Budras et al., 1998). This substance, a

lipoprotein, is permeable and holds water, giving the horn its flexibility (Budras et al.,

1998).

Horn quality is dependant on internal and external and factors. Internal factors

relates to blood and nutrient supply, whereas external factors relates to environment

where the claw is found. Any compromise in blood flow has a negative effect on horn

production.

Microanatomy of the Claw: Structure of the Wall

The structure of the claw consists of modified skin that is a continuation of the

epidermis of the coronary band. The claw has the same basic structures as the skin. It has










an epidermis (horny wall), a dermis coriumm or quick), and a subcutis (Hibroelastic heel

pad and coronary and digital cushion). The epidermis itself is divided into basement

membrane, germinal epithelium (stratum germinativum), stratum spinosum (layers of

horn undergoing keratinization) and stratum corneum (the layer of comified epithelium).

The basement membrane is the junction between the epidermis and corium. The stratum

germinativum is the germinative layer responsible for horn growth. The stratum corneum

is the cornified epithelium forming the claw hom. Cells are arranged into tubular and

intertubular horn. The mechanical strength of the bovine claw is a function of the

keratinization of cells in the germinal layers of the epidermis (Hendry et al., 1994).

Etiology of Lameness

Lameness in cattle can be caused by a variety of reasons. The purpose of the next

section is to describe the different causes of lameness in cattle.

Infectious Diseases of the Digits

Several systemic diseases can be associated with digital lesions potentially

leading, as a result of localized pain, to stiffness and lameness. They include

Foot-and-Mouth Disease (FMD), Bovine Virus Diarrhea (BVD), Bovine Malignant

Catarrh, Bluetongue and Vesicular Stomatitis. This review of lameness is focused on

maj or specific infections of the digits: Interdigital Phlegmon (Foot-Rot), Interdigital

Dermatitis and Digital Dermatitis.

Interdigital phlegmon (Foot-rot, Interdigital necrobacillosis)

Interdigital Phlegmon is characterized by fissuring, caseous necrosis of the

subcutis in the interdigital space and diffuse digital swelling. Pain, moderate to severe

lameness, and pyrexia are also common signs of this disease. A characteristic fetid odor









is usually present because of the presence of Fusobacterium necrophorum which if not

treated early a common sequela is septic arthritis (Berry, 2001).

Although the pathogenesis of foot rot is not understood completely, bacteria gain

entry through abraided skin on the lower part of the foot. Hard surfaces contribute to foot

injury, and continuous wetting likely favors abrasions by softening the interdigital skin

(Radostits et al., 2000). The greatest economic impact of bovine foot rot is in dairy

operations, where milk production may be affected (Hernandez et al., 2002). In this

study, cows affected with foot rot produced 10% less milk than normal cows. Also this

disease can affect feedlots where antimicrobial treatments require withdrawal times that

could delay marketing of products (Radostits et al., 2000). Although spontaneous

recovery may occur, lameness may persist for several weeks when infections are left

untreated, and complications may cause more severe problems that could eventually lead

to death or euthanasia of the animal (Radostits et al., 2000).

Treatment of foot rot can be accomplished with a variety of antimicrobials (Cook

et al., 1995; Morck, 1998; Berry, 2001). A recent study looked at the efficacy of Ceftiofur

Sodium and Hydrochloride formulation for the treatment of foot rot (Kausche et al.,

2003). This was a multilocation study conducted on 11 farms in the US to compare the

efficacy of Ceftiofur at a dose of 1.1 mg/kg once a day for 3 consecutive days with a

placebo group. Results of this study indicated that cure rate for Ceftiofur was 62.2%

versus 14% for the placebo group (P < 0.003). These same authors did another study to

compare the efficacy of Ceftiofur versus Oxytetracycline at 10 mg/kg. Results of this

study indicated that Ceftiofur and Oxytetracycline were comparable in efficacy, with

Ceftiofur having excellent inj ection-site tolerance and a short or no milk discard or









pre-slaughter withdrawal (Kausche et al., 2003). Treatment can also be accomplished by

the use of Sulfadimethoxine orally (25 g/1b followed by 12.5 g/1b SID for no more than 5

days) or intravenously (55 mg/kg followed by 27.5 mg/kg SID for 2 days after remission

of clinical signs). Good results also can be obtained with Penicillin G intramuscularly for

3 days (Bergsten, 1997).

Prevention and control of foot rot can be accomplisheded by the use of foot baths

with 5% to 10% copper sulfate or zinc sulfate (Rebhun, 1982). Formaldehyde solutions

of 3 to 5% in water have been reported to be effective in the prevention of foot-rot

(Bergesten, 1997). Caution should be emphasized when using formaldehyde due to

potential hazards for handlers as well as contamination of the environment. Other

measures recommended to prevent foot rot are to maintain clean passageways to reduce

the exposure of the feet to feces, maintaining a dry environment and avoiding rough floor

surfaces that can traumatize the interdigital skin and allow the entry of bacteria (Blowey,

1994). Efforts to produce vaccines against Fusobacterium necrophorum have failed

because of the weak immune response to the bacterium (Smith, 1992). There are vaccines

available in the US market but there are no peer-reviewed studies to support their use.

Interdigital dermatitis

Interdigital dermatitis occurs as an acute or chronic inflammation of the

interdigital skin that does not usually cause lameness (Blowey, 1994; Guard, 1995). The

inflammation does not extend to the subcutaneous tissues and in this respect differs from

foot-rot, where infection extends to the dermis, leading to fissure formation, infection of

deeper structures, and cellulitis of the pastern and fetlock (Blowey, 1994). Some authors

implicate F.necrophorum, Dichelobcater nodosus and Bacetroides sp as the causative

agents of interdigital dermatitis (Toussaint Raven, 1989, Peterse, 1982).









Interdigital dermatitis occurs in dairy cattle, especially in wet environments. It is

usually an incidental finding when trimming feet because it rarely causes lameness. A

study conducted in 17 Danish dairy herds reported that interdigital dermatitis occurred in

4.5% and 7.6% of first and 2+ lactation cows, respectively (Enevoldsen et al., 1991). In

this study, severity of disease increased with parity and risk increased with stage of

lactation. In a Dutch study on 86 dairy farms, researchers reported that the prevalence of

interdigital dermatitis and heel horn erosion was 24% (range = 3 to 92%) (Manske et al.,

2002c).

Clinical signs of interdigital dermatitis include hyperemia of the interdigital skin,

including the palmar and plantar areas, superficial erosion and ulceration followed by

hyperemia with serious or grayish exudates. More aggressive forms interfere with the

horn formation in the bulbs, where fissures, hemorrhages and necrosis can arise. The

subcutaneous tissue is inflamed secondarily. Swelling and hyperkeratosis may develop in

a more chronic stage. Chronic interdigital irritation may cause slight to severe interdigital

hyperplasia (Bergsten, 1997). The most common complication of interdigital dermatitis is

heel horn erosion. Results of a study conducted by Enevoldsen (1991) support the

hypothesis that severe heel erosion and interdigital dermatitis are two manifestations of

the same disease with Dichelobacter nodosus as an important component and are closely

related. In this study (Enevoldsen, 1991) the incidence of heel erosions in 1st and 2+

lactation cows was 43% and 69%, respectively. This study (Enevoldsen, 1991) also

reported unhygienic and moist conditions as important risk factors for interdigital

dermatitis.









Prevention and treatment is usually accomplished by the use of 5 to 10% copper

sulfate footbath or zinc sulfate (10 to 20%), or formalin (3 to 5%) footbaths. Care must be

taken to ensure that the footbath remains clean. Interdigital dermatitis can persist in

dairies that practice regular footbaths (Guard, 1995). This same report suggested that the

causative organisms may survive within deep heel cracks that are not permeated by

footbath solutions; hence, heel cracks must be trimmed during hoof trimming to allow for

exposure to footbath solutions. Claw trimming causes a mechanical cleansing of affected

tissues and an exposure to air that might be beneficial for the curing of dermatitis lesions

(Manske et al., 2002b). As reported in this same study, every third hoof affected with a

severe dermatitis and concurrent heel-horn erosion had recovered 1 month after

trimming.

Digital dermatitis (DD) (Footwarts, Hairy heel warts, Heel warts)

Digital dermatitis is an important cause of lameness in dairy cattle. It was first

reported by Cheli and Mortellaro in 1974 (Mortellaro, 1994) as a mysterious cause of

epidemic lameness affecting up to 70% of adult cattle in the Po Valley of Italy. Since

then, the disease has been reported in other countries such as the Netherlands, France,

England, Czechoslovakia, Germany, and Ireland (Bassett et al., 1990; Brizzi, 1993). In

the United States, Rebhun (1980) first reported the disease as outbreaks of lameness in

New York dairy herds.

Clinically, digital dermatitis typically appears within dairy herds as outbreaks of

lameness. It is a superficial skin disease of the bovine digit with variable presentation

(i.e., depending upon the stage of the lesion), from painful, moist, strawberry-like lesions

to raised, hairy, wart-like lesions (Read and Walker, 1998). These lesions (i.e., usually

located on the rear of the foot between the bulbs of the heel) have been referred to by









several names, including: hairy footwarts, strawberry (or raspberry) heelwarts, and

papillomatous digital dermatitis. Early lesions produce matting of the hairs, which stand

erect in thick, light brown exudates, which have a characteristic pungent odor (Blowey et

al., 1994). A study conducted in California described the lesions as being distinctly

demarcated, circumscribed, spherical to oval, 0.5 cm to 6 cm across, partially or

completely alopecic, moist, painful-to-touch, prone-to-bleed plaques of flat or raised

proliferative tissue. Lesion surfaces vary in appearance from being extensively red and

granular (3 1%), often with patches of yellow or gray filiform papillae (41%) to

extensively gray, brown or black with papillary outgrowth of the epithelium (28%) (Read

and Walker, 1994).

In spite of many studies and specific research, the exact etiology of DD is still

unknown. Researchers still believe that DD is a multifactorial disease, even though in

some cases high morbidity, apparent contagiousness and response to antimicrobial

treatments suggest that an infectious agent is primarily involved (Mortellaro, 1994). In

one study the incidence of DD was higher in heifers a few months after they entered the

milking herd and may be due to lack of immunity (Read et al., 1992). Initial studies

(Rebhun et al., 1980; Cheli and Mortellaro, 1986; Peterse, 1982) were unable to identify

any viral pathogens, and results of bacteriology were inconsistent. Peterse (1982) was

able occasionally to isolate Dichelobacter nodosus from some typical lesions. Bassett

(1990) was unsuccessful in isolating a microorganism from DD lesions and also failed to

replicate the disease after inoculation of heifers with homogenate from fresh lesions.

Read (1992) examined histological lesions and were able to demonstrate the presence of

large numbers of spirochetes invading the stratum spinosum and dermal papillae (Blowey









et al., 1994). In a more recent study, Walker (1995) isolated two different spirochetes

from cows with DD lesions in California dairy herds. These spirochetes were further

categorized based on morphology (intracytoplasmatic tubules), antigenicity and

enzymatic activity in the genus Treponema. This finding was supported later by another

study (Demirkan et al., 1999) where serological evidence suggested that spirochetes are

involved in the pathogenesis of DD. Also, this study (Demirkan et al., 1999) supports the

hypothesis that Borrelia burgdorferi may be involved in the pathogenesis of DD, as first

proposed by Blowey (1994). To date, there is still no isolation of the bovine spirochete.

In a study conducted in California dairy herds (Read and Walker, 1994), the

prevalence of DD was approximately 90%. Between-herd morbidity varied from 0.5 to

12% per month. Within-herd morbidity was generally greater during spring and summer

months. Most lesions occur on the plantar interdigital cleft of the rear foot and less

common sites for lesions are the palmar interdigital ridge of a front foot or a dorsal aspect

of any foot (Mortellaro, 1994; Read and Walker, 1998). Another study conducted by the

National Animal Health Monitoring System involving 83% of US dairy cows in 20 states

observed the incidence of digital dermatitis and risk factors (Wells et al., 1999). This

study reported, that within the last 12 months of the study, 43.5% of the US dairy herds

had cows that showed clinical signs of DD with variation by herd size and region (Table

2-3 ).

The study by Wells (1999) reported that the average percent of cows affected was

18.9%. A high percentage of digital dermatitis-affected cattle were also reported lame

(81.9% of affected cows and 85.9% of bred heifers). This study also looked at risk factors

associated with digital dermatitis. Interesting results from this study identified several










factors that contribute to herd incidences > 5%. The percent of cows not born on the dairy

operation was strongly associated with high digital-dermatitis incidence, and there was

evidence for a dose-response relationship.

Table 2-3. Incidence of Digital Dermatitis in US dairy herds by herd size and region
.aibe ee Herds with digital dermatitis in the
previous 12 months (%)
Herd Size < 100 cows 36.4
100 to 199 cows 61.9
>200 or more cows 80.3
Total 43.5

Region Northwest 56.1
Southwest 70.3
North Midwest 35.4
South Midwest 45.5
Notheast 53.1
Southeast 20.8
Wells et al., 1999

Rodriguez-Lainz (1996) showed a strong association between introduction of

heifers and digital-dermatitis prevalence in southern California dairy herds. These results

were in agreement with results from Argaez Rodriguez (1997) who reported that

purchased animals were 3.4 times more likely to be affected than animals born on the

farm. Farm factors such as type of concrete flooring with concrete abrasive floors or

slippery floors were associated with > 5% incidence of DD. Rodriguez-Lainz (1996)

reported an association between the incidence of digital-dermatitis and corral moisture in

southern California dairy operations with dirt dry lot corrals. Some biosecurity factors

identified were hoof trimmers that trim cows on other farms. Herds in which the primary

hoof trimmer also trimmed cows hooves on other operations were 2.8 times more likely

to have > 5% incidence of digital dermatitis compared to herds where the primary hoof

trimmer did not trim hooves on other operations or where cows hooves were not









trimmed. Also, herds in which hoof-trimming equipment was not washed between cows

were 1.9 times more likely to have > 5% incidence of digital dermatitis than those where

the equipment was washed or where no hooves were trimmed (Wells et al., 1999).

Blowey (1988) reported different treatments when describing one of the first

outbreaks of the disease in the UK. Treatment of DD started by the application of

parenteral injections of penicillin, streptomycin, tetracyclines, cephalexin, and

sulphonamides, but none of these treatments proved effective and most cases recovered

on their own. The most effective treatment in this report appeared to be deep scraping of

the lesion with a hoof knife, followed by topical oxytetracycline/genti an violet aerosol

spray. This treatment led to a reduction of lameness in 6 to 12 hours and complete

resolution in 2 days. In this study, footbaths with 5% formalin or 2.5% copper sulfate

were used to try to control the outbreak with poor results. Sheldon (1994) further

supported treatment with oxytetracycline spray (4 g/L), although this was not a controlled

study. Further research conducted by Britt (1996) confirmed the efficacy of

Oxytetracycline (100 mg/mL) as a treatment for digital dermatitis applied as a spray.

Manske et al., (2002c) reported that Oxytetracycline applied as a bandage was

significantly more effective than hoof trimming alone of the affected foot (P < 0.001).

This study was done to try to Eind an alternative treatment to antibiotics and

Glutaraldehyde bandage was tested as the alternative non- antibiotic treatment. Results of

this study did not support the use of this product. Treatment of DD with topical

oxytetracycline does not require any milk withdrawal as shown by Britt (1999) who was

unable to demonstrate antibiotic residues in milk using standard routines after cows were

treated topically with this antibiotic. The most common treatments for digital dermatitis









involve the use of topical antibiotics (Blowey, 1988; Blowey, 1994; Guard, 1995; Berry

et al., 1996; Britt et al., 1996; Britt et al., 1998; Read et al., 1998; Berry et al., 1999a;

Berry et al., 1999b, Hernandez et al., 1999; Shearer et al., 2000; Manske et al., 2002c).

Some of the antibiotics used in these studies were Oxytetracycline, Tetracycline,

Li ncomy cin/S p ecti nomy ci n, and Erythromy ci n.

There also are reports of non-antibiotic products being effective in the treatment

of DD (Shearer and Hernandez, 2000; Hernandez et al., 1999). Hernandez (1999) used a

topical spray with four different non-antibiotic products in the lame cow with DD.

Treatment with an Oxytetracycline solution, or a soluble copper, peroxide compound and

a cationic agent appeared to be effective for the treatment of DD, compared to a 5%

copper sulfate (CuSO4) Solution, or acidified copper solution, or hydrogen

peroxide-peroxyacetic acid (HPPA) solution. In another study (Shearer et al., 2000)

non-antibiotic compounds were used to treat DD in 78 cows affected with lameness. In

this study (Shearer et al., 2000), a previously tested product (Hernandez et al., 1999) was

modified to improve its handling and storage characteristics. There were four treatment

groups in this study: cows in group A were treated with oxytetracycline solution, cows in

group B were treated with the original formulation also containing soluble copper,

peroxide compound and a cationic agent, cows in group C were treated with a modified

formulation with reduced soluble copper, peroxide compound but increased levels of

cationic agent and cows in group D were treated with a modified formulation containing

levels of soluble copper and cationic agent similar to the original formulation but reduced

concentrations of peroxide compounds. Results of this study (Shearer et al., 2000)

indicated that the modified non-antibiotic formulation used on cows in group C appeared









to be the most effective treatment of papillomatous digital dermatitis compared to the

other formulations as the proportion of cows with signs of pain was significantly lower in

this group of cows (group C). Also this study reported an unexpected low efficacy of the

oxytetracycline treatment suggesting the possible development of resistance in dairy

cows affected with DD. This study supports the use of non-antibiotic products as an

efficacious tool for the treatment of DD, thus minimizing the potential risk for residues in

milk and meat.

Control measures of biosecurity, as stated in a previous study (Wells et al., 1999),

includes examination of animals entering the herd, cleaning hoof trimming equipment

between cows, and to have a farm-set of trimming tools available for the hoof trimmer to

avoid contamination incoming from other farms. Footbaths can be used as a part of

control measures of DD on infected herds (formalin 5%; copper sulfate 2.5%;

oxytetracycline 1 to 4 g/L; zinc sulfate 20%) (Blowey et al., 1988; Brizzi, 1993;

Mortellaro, 1994; Guard, 1995). If no preventive herd measures are taken, a relapse may

be expected within 5 to 7 weeks after a successful single topical treatment of DD (Berry

et al., 1999a) (Guard and Williams, 1995). Although the efficacy of footbaths remains

controversial, individual treatment of affected cows combined with the use of footbaths

for the herd represent the most effective method of prevention (Mortellaro, 1994).

Metabolic Hoof Horn Disease: Claw Horn Disruption

Hoof horn of low quality is a frequent cause of lameness in cattle. Studies in the

UJK reported that claw disorders accounted for 70 to 90% of diagnosed cases of lameness

in dairy cattle (Whitaker et al., 1983; Clarkson et al., 1996; Murray et al., 1996). These

researchers identified subclinical laminitis related disorders, such as sole ulcers and white

line disease, as the most important conditions affecting dairy cattle in the UK. Laminitis










was regarded by some authors (Bradley et al., 1989; Greenough and Vermunt, 1991) as

an important predisposing factor in lameness caused by claw disorders such as sole

ulcers, white line disease, and abscesses in the subsole. Subclinical laminitis has been

identified as the underlying cause of abnormalities of hoof horn formation which results

in claw disorders (Hoblet et al., 2001). To better understand the pathophysiology of

lameness, it is important to review the anatomy of the bovine foot.

Laminitis

Laminitis, or pododermatitis a~septica diffuse, is an aseptic inflammation of the

dermal layers of the claw (Nielsson, 1963) characterized by defective claw horn

production with thrombosis and hemorrhages in the digital corium (Mortensen, 1994). It

was originally described as consisting of three clinical forms (acute, subacute, chronic),

but Peterse (1979) later described a fourth form of laminitis, "subclinical laminitis".

Because the disease process finally affects horn formation at the cellular level, regardless

of whether an initial primary inflammatory response had occurred, the term claw horn

disruption has been proposed (Logue et al., 1998). Although many different terms have

been proposed for this disorder, I will refer to it as laminitis for the purpose of this

discussion.

Laminitis has a multifactorial etiology and is thought to be associated with

several, largely interdependent factors such as genetic predisposition, claw size, body

weight, architecture of limb angles, claw hardness, pigmentation of the claw and the

quality of the surface over which the animal walks (Mortensen, 1994; Nordlund and

Garret, 1994). Nutritional management has been identified as a key component in the

development of laminitis, particularly the feeding of increased fermentable carbohydrates

which result in an acidotic state. There seems to be no doubt that the disease is related to










high energy intake, frequency and quantity of consumption. Factors such as body

condition, body weight, and feet and leg structure, can unnaturally increase the weight

load and stress on feet and exacerbate the internal mechanical damage that is associated

with laminitis (Nocek, 1997).

Nocek (1997) has described the mechanisms causing development of laminitis in

detail on a recent review of the topic. The mechanistic phases of laminitic development

were described as alternating stages of disturbances relating to metabolic and subsequent

mechanical degradation of the internal foot structure (Mortensen, 1994; Nordlund and

Garret, 1994). The process can be segmented into various phases (Nocek, 1997).

Phase 1. The initial activation phase of laminitis, phase 1, is associated with a

systemic metabolic insult. This phase is a result of ruminal acidosis, and subsequently an

altered systemic pH. The reduction in systemic pH activates a vasoactive mechanism that

increases digital pulse and total blood flow. Depending upon the insult that initiates the

process, endotoxins, histamine and lactate can be released, which create increased

vascular constriction and dilation and, in turn, cause the development of several

unphysiological arteriovenous (AV) shunts, further increasing blood pressure. The

increased blood pressure causes seepage of serum through vessel walls, which ultimately

are damaged. Damaged vessels then exude serum, which results in edema, internal

hemorrhaging of the solar corium from thrombosis, and ultimately expansion of the

corium, causing severe pain.

Phase 2. As a result of the initial insult, there is mechanical damage, phase 2,

which is associated with the vascular system. Once vascular edema has occurred,

ischemia results in hypoxemia of the local internal digital tissue causing tissue hypoxia










which results in fewer nutrients and less oxygen reaching the epidermal cells. Ischemia

itself can trigger a further increase in AV shunting. Trauma and stress can increase AV

shunting. As a result of previous events, increased blood pressure further increases

vascular seepage in the lower part of the digit as well as edema and ischemia. This cycle

continues as long as the initial insult continues.

Phase 3. In phase 3, as a result of the mechanical damage associated with

microvasculature and fewer nutrients provided to the epidermal cells, the stratum

germinativum in the epidermis breaks down. These events ultimately cause corium

degeneration and breakdown of the laminar region associated with the dermal-epidermal

junction.

Phase 4. Ultimately, in phase 4, local mechanical damage occurs. A situation

develops in which the epidermal junction is broken down which results in the separation

of the strata germinativum and corium. This separation results in a breakdown between

the dorsal and lateral laminar supports of the hoof tissue. Ultimately, the laminar layer

separates, and P3 takes on a different configuration in relationship to its position in the

corium and dorsal wall. As the bone shifts in position it causes a compression of the soft

tissue between the bone and sole which is extremely susceptible to damage. The

compression of this soft tissue results in hemorrhage, thrombosis, and further

enhancement of edema and ischemia which result in a necrotic area within the solar

region of the foot. Small areas of scar tissue accumulate because of the necrotic process.

Once this process is triggered, continued potential for tissue degeneration persists

because cellular debris is incorporated into the cellular matrix and the production and

integrity of new horn tissue layers are hindered. Ultimately, a variety of processes can









occur as a result of the incorporation of scar tissue intervention, which includes double

sole phenomenon, sole hemorrhages (red blood patches), bruises, white line lesions, and

sole ulcers (Vermunt, 1994).

Forms of laminitis

Acute and subacute laminitis. In the acute and subacute stage of the disease, an

aseptic inflammation of the corium coincides with a systemically sick animal. At this

stage, the claw horn shows few, if any, visible changes. Vessel seepege, edema of

capillary beds, and AV shunting are all initiated. Vascular congestion is present. The

maj or clinical sign in addition to pain includes swelling and temperatures that are slightly

warmer than normal above the coronary band in the soft tissue (Nocek, 1997). These

forms of laminitis are prone to recurrence at varying intervals and often progress to the

chronic form (Vermunt, 1994).

Chronic laminitis. Chronic laminitis has no systemic symptoms and changes are

localized to the claw. A disturbed horn growth pattern and an alteration in the shape of

the claw with an elongated flattened and broadened sole are characteristic (Vermunt,

1994; Greenough, 1997). Internally P3 has separated from the dorsal aspect of the wall.

Continued ischemia results in destruction of the capillary beds and development of AV

shunts. Cellular destruction results in separation of the dermal-epidermal junction, and

internal foot destruction (Nocek, 1997). Grooves and ridges caused by irregular episodes

of horn growth can be seen in the claw wall. This deformation of the claw shape often

predisposes to sole ulcers, white line disease or subsolar abscesses (Greenough and

Vermunt, 1991; Mortensen, 1994).

Subclinical laminitis. Subclinical laminitis was first described in 1979 by Peterse

and later by others (Bradley et al., 1989; Greenough and Vermunt, 1991). Lameness is









usually not observed with this form of laminitis, but changes in the hooves can lead to

chronic laminitis. The horn becomes softer, discolored, and waxy in appearance. It often

stains yellow and hemorrhages can be seen in the weight-bearing surface of the claw, in

particular the white zone, apex of the sole and the axial side of the sole-bulb junction

(Bradley et al., 1989; Greenough and Vermunt, 1991). Internally, ischemia, hypoxia and

epidermal damage are key aspects associated with this stage (Nocek, 1997). The concept

of subclinical lameness is now universally accepted (Mortensen, 1994).

Claw Lesions Associated with Laminitis

Hemorrhages of the sole and sole ulcer

Hemorrhages in the sole are the maj or and characteristic indication of past

laminitic insults. The hemorrhages can take the form of a slight pink tinge, a pronounced

brush stroke of red coloration, or a dark solid red stain. Hemorrhages of the sole are

considered part of the same pathologic process as sole ulcers and are represented by a

continuum that ranges, from barely perceptible hemorrhages to severe ulceration of the

sole with exposed corium (Leach et al., 1997). Sole hemorrhages have a particularly high

incidence in first lactation heifers managed in confinement in the interval from 60 to 100

days after calving (Greenough and Vermunt, 1991; Smilie et al., 1996). The most

common spot for sole hemorrhages and sole ulcers is the so-called typical spot of the

lateral claw of the hind limb under the flexor tuberosity (axial prominence) of P3 (Smilie

et al., 1996).

Softening of the horn of the sole

Although obj ective evidence is not available, it has been proposed that sole horn

produced after an episode of laminitis is softer than normal (Mortensen, 1994).









White line disease

There are a number of names for the lesions in the White Line (WL): WL disease,

WL abscess, WL separation, WL fissure, WL lesions, widening of the WL, WL

hemorrhages (Bergsten, 2000; Blowey, 1993; Kempson and Logue, 1993; Leach, 1997).

These names describe clinical signs or morphological changes in the WL. Different

names are descriptions of different stages of development and degree of diseases whose

pathogenesis has a common origin (Miilling, 2002). This condition is also considered to

be associated with laminitis (Mortensen, 1994).

Heel erosion

Heel erosion has been described as one of the infectious diseases affecting the

bovine foot in this manuscript, but there are some authors that also relate this disease with

laminitis (Mortensen, 1994).

Diagnosis of Lameness

Several locomotion scoring systems have been developed to standardize gait

analysis in cattle. Manson and Leaver (1988) devised a system of locomotion scoring

which is a subj ective assessment based on observation of cows walking away from the

observer on a level concrete surface (Table 2-4). Using this scoring system, cows

classified with a score of 3 or higher were counted as lame for calculation of prevalence

of lameness. Two problems with this system were its subj activity and complexity (Ward,

1998). Collick (1989) used this system and factored percentage reduction of weight

bearing for the affected foot, foot structure affected and duration of lameness, which

made this system more complicated.









Table 2-4. Locomotion scoring developed by Manson and Leaver (1988)
Score Description
1.0 Minimal abduction/adduction, no unevenness of gait, no tenderness
1.5 Slight abduction/adduction, no unevenness or tenderness
2.0 Abduction/adduction present, uneven gait, perhaps tender
2.5 Abduction/adduction present, uneven gait, tenderness of feet
3.0 Slight lameness, not affecting behavior
3.5 Obvious lameness, some difficulty in turning, not affecting behavior
4.0 Obvious lameness, difficulty in turning, behavior pattern affected
4.5 Some difficulty in rising, difficulty in walking, behavior affected
5.0 Extreme difficulty rising, difficulty walking, adverse effect on behavior


Wells (1993) described a similar but different locomotion scoring system (Table

2-5) arguing that simplicity was necessary when using this type of scoring. This system

was used to estimate the prevalence of lameness in 17 dairies in Wisconsin and

Minnesota and cows with a score of 2 or higher were classified as lame

Table 2-5. Locomotion scoring used described by Wells (1993)
Score Gait abnormality Description
0 None Gait abnormality not visible at walk; not reluctant to walk.
1 Mild Mild variation from normal gait at walk; includes
intermittent mild gait asymmetry or mild bilateral or
quadrilateral restriction in free movement.
2 Moderate Moderate and consistent gait asymmetry or symmetric gait
abnormality, but able to walk
3 Sever Marked gait asymmetry or severe symmetric abnormality
4 Nonambulatory Recumbent


This system was used to estimate the prevalence of lameness in 17 dairies in

Wisconsin and Minnesota and cows with a score of 2 or higher were classified as lame.

In 1997, Boelling (1998) used the locomotion system developed by Manson and Leaver

(1988) and described it using a 9 point-scale, from 1 (perfect walk) to 9 (near inability to

walk). A score of 1 to 4 was considered sound locomotion, while a score of 5 or higher

was regarded as clinical lameness. They used this to estimate the heritability of

locomotion, which they found to be 0.06 to 0. 11. Sprecher (1997) based on an









observation of Morrow in 1966, where an arched-back posture was associated with acute

and chronic laminitis, developed a simple locomotion scoring system. He developed this

method of classification of lameness to try to predict future reproductive performance and

culling risk of cows classified within different scores. This locomotion scoring system

was based on gait and back posture (Table 2-6).

If used consistently, all these scoring systems are useful to screen cows and

identify early lesions associated with lameness. Early intervention prevents the more

serious stages of a claw disorder (Toussaint Raven, 1989).

Table 2-6. Locomotion scoring system developed by Sprecher et al., 1997
Locomotion Clinical
Assessment criteria
score description
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
3 Moderately An arched-back posture is evident both while standing and
lame 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
5 Severely The cow additionally demonstrates an inability or extreme
lame reluctance to bear weight on one or more of her limbs/feet


If used consistently, all these scoring systems are useful to screen cows and identify

early lesions associated with lameness. Early intervention prevents the more serious

stages of a claw disorder (Toussaint Raven, 1989).

On the basis of differences in anatomic location and morphologic characteristics,

it is possible to make a clinical diagnosis of interdigital phlegmon, digital dermatitis, or

claw lesions in cows affected with lameness. Lame cows with interdigital phlegmon will

be cows characterized by Hissuring, caseous necrosis of the subcutis in the interdigital










space, and swelling of the entire foot above the dewclaws and separation of the digits.

Pain and moderate to severe lameness are often seen with this disease. A characteristic

fetid odor is usually present (Berry, 2001). Lame cows with digital dermatitis will have

demarcated lesions, circumscribed, spherical to oval, 0.5cm to 6cm across, partially or

completely alopecic, moist, painful-to-touch, prone-to-bleed plaques of flat or raised

proliferative tissue on the interdigital cleft, heels, or dewclaw (Read and Walker, 1994).

Lame cows with claw lesions will be cows that have white line lesions, abscess, or sole

ulcers and will be treated by use of corrective foot trimming techniques (Shearer and van

Amstel, 2001).

Lameness and Animal Welfare

Because of the pain, discomfort, and high incidence of lameness in dairy cows,

lameness is an animal welfare issue. Disturbed claw health is an unequivocal source of

suffering for cows, because the disorder is usually long term and painful (Alban, 1995).

Some countries are already setting acceptable levels of clinical lameness. As an example,

the Dutch Advisory Board for Animal Affairs (RDA) has considered the actual levels of

clinical and subclinical claw disorders (30% cow cases per year) in The Netherlands as

unacceptable from an animal welfare point of view (Somers et al., 2003). In other

countries as well, the prevalence of lameness has also stated as not acceptable and have

give rise to a growing concern about animal welfare.

Welfare can be defined as the state of animals regarding their attempts to cope

with their environment (Broom, 1988). A lame cow is less able to cope with her

environment, as pain might seriously affect walking and other movements (Hassall et al.,

1993). The secondary effects of a reduced ability to walk may impact important

physiological activities such as reduction in the time feeding (Hassall et al., 1993;









Galindo et al., 2002), also affecting their behavior as suggested by Peeler and Esslemont

(1994), were lame cows experienced similar inhibitions as cows on poor footing and

would less likely be observed in estrus. These welfare factors are important to consider

when evaluating the effects of lameness disease on production.

In most US dairies, incidence of lameness is underestimated because only cows

affected with severe signs of lameness are detected and treated; cows with mild or

moderate signs of lameness are often not diagnosed. In a study by Wells (1993) the

prevalence of lameness diagnosed by farmers in 17 dairies was compared to that by

researchers in the same farms. The prevalence of lameness reported by researchers was

three times higher than that by farmers. The underestimation of prevalence of lameness

keeps farmers and the industry unaware of the importance of this disease.

Lameness and Milk Production

Several studies have been carried out around the world to test the effect of

lameness on milk production. Results of these studies are conflicting. Some authors

reported a decrease in milk yield after diagnosis of lameness (Whitaker et al., 1983;

Tranter and Morris, 1991; Rajala-Schultz et al., 1999; Warnick et al., 2001), a decrease in

milk yield before and after treatment (Lucey et al., 1986; Green et al., 2002) or no change

in milk yield (Cobo-Abreu et al., 1979). Another study (Barkema et al., 1994) reported an

increase in milk yield from 100 to 270 DIM during the same lactation in lame cows with

sole ulcers. Green (2002) reported an increase in 100-day cumulative milk volume in the

previous lactation for cows with any cause of lameness. Culling bias may in part account

for these results because cows with both lameness and low production would be expected

to be culled more often than cows with lameness and high production. Argaez-Rodriguez

(1997) did a retrospective study a in Mexico to examine the effects of digital dermatitis in









milk production. The authors (Argaez-Rodriguez et al., 1997) reported a statistically non-

significant difference between milk production of lame cows due to DD and healthy

cows, with cows experiencing DD producing less milk. In this study lame cows due to

DD were compared with healthy cows and lame cows for other reasons were included as

healthy which can mask the effects of DD on milk production.

Three studies have examined the relationship between lameness and milk yield in

US dairy herds. In a study conducted on a 500-cow dairy in California (Deluyker et al.,

1991), cows diagnosed as lame during the first 49 days postpartum coincided with higher

milk yield. The positive association of lameness and high milk yield during early

lactation found in this study suggested that high yield was a risk factor for lameness.

Although diseases or foot lesions associated with lameness were not investigated, white

line and sole lesions were the most common lesions in this study. In another study

conducted on two dairy herds in New York (Warnick et al., 2001), lame cows with claw

lesions or interdigital phlegmon produced less milk than healthy cows. Lameness was

more common in early lactation and more likely to occur in older cows. Finally, in a

study conducted in Florida, lame cows with interdigital phlegmon produced 10% less

milk, compared to non-lame cows (Hernandez et al., 2002). The authors (Hernandez et

al., 2002) estimated that such a decrease in milk yield of 1,885 lb/cow represented a loss

of $301/cow (assuming a milk value of $16.00/100 lb). In that study, most lame cows

with interdigital phlegmon were affected during early lactation (within 100 days

postpartum), when cows reach peak yields and 60% were culled during lactation. These

findings may suggest that lame cows affected with interdigital phlegmon during early









lactation may be sufficiently compromised to adversely affect a cow's ability to achieve

its own milk yield potential during the current lactation (Hernandez et al., 2002).

Lameness and Reproductive Performance

A relationship between lameness and reproductive performance has been

established in several studies in the United States and in other parts of the world. In 1985,

Weaver postulated lameness as a possible cause of reduced fertility because animals

spend more time lying down, are less willing to demonstrate standing heat, and are less

able to compete for available feed. Some of these suggestions were later confirmed by

different studies. Varner (1994) looking at pedometer readings, reported that cows in

estrus move and interact with other cows significantly more than the rest of the herd.

Britt (1986) found that excellent footing greatly increases the duration of estrus

activity in dairy cows. Peeler and Esslemont (1994) reported that lame cows experience

similar inhibitions as cows on poor footing and would less likely be observed in estrus.

These Eindings may in part explain results of studies indicating that lameness has a

detrimental effect on reproductive performance. In a study conducted on Hyve dairy farms

in the UK, Lucey (1986) found that lame cows affected with sole ulcer and white line

disease between 36 and 70 days after calving were associated with longer calving to

conception intervals (17 and 30 days, respectively). Collick (1989) did a larger study on

17 dairy farms in the UK involving 427 cases of lameness. The authors reported that

lameness happening before 120 days after calving was associated with significantly

increased intervals from calving to conception. The largest increase in the intervals from

calving to conception were associated with sole ulceration (40 days, P < 0.01). In a

retrospective study of digital dermatitis in a commercial dairy in Mexico Argaez-

Rodriguez (1997) reported that healthy cows conceived 93 days after calving (median),










compared to affected cows with digital dermatitis which conceived 113 days after calving

(P < 0.0 1).

Three previous studies have examined the relationship between lameness and

reproductive performance in US dairy herds. In one study conducted in five dairy herds

in Pennsylvania, cows affected with lameness had a 28-day-longer calving to conception

interval, compared to healthy cows (Lee et al., 1989). In another study, a scoring system

was developed to identify lameness and predict future reproductive performance in dairy

cows, lame cows were 15.6 times more likely to require an interval greater than the mean

for days open compared to healthy cows (Sprecher et al., 1997). In a study conducted on

a 500-cow dairy in Florida (Hernandez et al., 2001), claw lesions were the most

important cause of lameness and impaired reproductive performance in dairy cows, as

indicated by a higher incidence of affected cows, a greater time from calving to

conception (median, 140 days), and a higher number of services required per conception

(median, 5), compared to non-lame cows (100 days and 3 services, respectively). In this

study herd, cows were synchronized and time-inseminated; thus the authors were not able

to assess the calving to first breeding interval nor to draw any conclusion that lameness

has an effect on estrus behavior. Conversely, the significantly higher number of services

required per conception and the longer time from calving to conception in lame cows

with claw lesions, compared to healthy cows, may suggest that lameness has an effect on

conception.

Melendez (2003) examined the association between lameness, ovarian cysts, and

fertility on a 3000-cow dairy in Florida. Results of this study showed that cows that

became lame within the first 30 days postpartum were associated with higher incidence









of ovarian cysts, a lower likelihood of pregnancy, and a lower fertility than non-lame

cows. Although all these studies found a significant association between lameness and

reproductive performance, the relationship between lameness and ovarian activity was

not investigated.

Resumption of Ovarian Activity Postpartum

Early resumption of ovarian cyclicity postpartum is important for high

reproductive efficiency in dairy cows. Delays in the commencement of ovarian cyclicity

and estrous expression are associated with reduced conception rates, pregnancy rates and

an increased interval from calving to conception (Thatcher and Wilcox, 1973; Stevenson

and Call, 1983; Lucy et al., 1992; Senatore et al., 1996). The study done by Thatcher and

Wilcox, (1973) reported that cows exhibiting 0 or 1 heat postpartum required

significantly more services per conception than cows exhibiting 2 to 4 heats. These

authors reported that non-return rates improved as frequency of postpartum heats

increased (0 and 1 heat, 37%; 2 to 4 heats 44%; P < 0.05). In a study conducted to

identify the influence of early estrus, ovulation and insemination on fertility in

postpartum dairy cows, Stevenson and Call (1983) reported that the interval to first

detected heat had a significant influence on first service intervals and days open. When

estrus was not expressed before 60 days postpartum, average days to first service were 18

days longer and days open were 19 days longer than in cows that expressed heat before

60 days postpartum (P < 0.05). These authors concluded that more expressed heats early

postpartum were associated with positive effects on fertility.

A study conducted by Lucy (1992) who monitored the interval to first ovulation to

clarify the importance of milk production, dry-matter intake and energy balance in the

interval to first ovulation, reported that cows having first ovulation before 42 days tended









to have a shorter interval from calving to detected estrus, required fewer services per

conception and had a shorter interval from calving to conception compared with cows

having first ovulation after 42 days postpartum. More recently, Darwash (1997) reported

that the interval to postpartum commencement of luteal activity was correlated favorably

with measures of fertility such that for every day delay in the interval to commencement

of ovarian activity, there was an average delay of 0.24 and 0.41 (P < 0.001) days in the

interval to first service and conception respectively. These studies pointed out the

importance of early resumption of ovarian cyclicity postpartum as a factor contributing to

high reproductive efficiency on dairy cows.

Following calving, the reproductive strategy of the cow is transformed from

delivering and nourishing a healthy calf to reestablishing pregnancy. The dormancy of

ovarian follicular development that prevailed during late pregnancy must now be

replaced by a sequence of events culminating with behavioral estrus, ovulation of healthy

follicles and normal luteal function. These are the requirements for successful

reproductive performance in any type of cattle production system (Rhodes et al., 2003).

After regression of the corpus luteum of pregnancy, there is a variable

anovulatory period before first ovulation takes place (Savio et al., 1990a). This period is

characterized by an absence of estrus behaviour and lack of progesterone secretion by the

ovary and a return to basal concentrations of estradiol during the first week postpartum

(Echternkamp and Hansel, 1973; Stevenson and Britt, 1979; Webb et al., 1980;

Humphrey et al., 1983; Peters, 1984; Savio et al., 1990a; Rhodes et al., 2002). Following

parturition, a wave of follicular development occurs in 5 to 7 days regardless of negative

energy balance and in response to an elevation in plasma FSH concentrations (Beam and









Butler, 1997). As reported by Savio (1990a), ovarian follicular turnover starts early after

calving and is similar to that observed during normal estrous cycles. In this study Savio

(1990a) reported that the postpartum interval to the detection of the first dominant follicle

was 11.6 & 8.9 days and the interval to first ovulation was 27.4 & 23 days. Beam and

Butler (1997) reported that the emergence of the first follicular wave postpartum occurred

after a peak in mean peripheral FSH levels and rather synchronously with the clearance

of gestational estradiol from blood. Subsequent, mean levels of FSH increased in the first

5 days postpartum and decreased from 5 to 11 days. This is in accordance with previous

work (Butler et al., 1983; Price and Webb, 1988) that indicated that removal of estradiol

negative feedback inhibition of FSH release would account for the increase in mean

plasma FSH observed between days 2 and 9 postpartum. After returning to basal levels,

estradiol concentrations fluctuate or remain low until 2 to 3 days before estrus when they

peak (Echtemnkamp and Hansel, 1973; Stevenson and Britt, 1979). Estradiol levels

declined from the 1st day postpartum until day 7, when estradiol levels slowly increase

with the concurrent development of a dominant ovarian follicle (Beam and Butler, 1997).

Plasma estradiol concentration is related with the degree of the peak of LH release in

response to GnRH as reported by Zolman (1974); Kesler (1977); and Fernandes (1978).

These studies agreed with that of Moss (1985) who reported that pituitary responsiveness

to GnRH is not restored until approximately 8 to 10 days post-partum. As described by

Savio (1990a), follicular growth is accompanied by episodic LH secretion of variable

amplitude and frequency. The use of ultrasound techniques allowed Savio (1990a) to

relate the stage of follicular development and the pulsatile secretion of LH. Within a

6-hour period, 2 to3 LH pulses occurred when concentrations of estradiol were low (< 5









pg/mL), and the frequency of LH pulses increased to 6 when estradiol concentrations

increased (> 10 pg/mL) coinciding with a dominant follicle. Stevenson and Britt (1979)

reported that the interval from calving to first postpartum ovulation was associated

inversely with the number of episodic LH surges and magnitude of the largest LH surge.

This high frequency mode of pulsatile LH secretion has been identified as necessary for

the final phase of maturation of ovarian follicles and thus induction of estrus and

ovulation (Webb et al., 1980; Humphrey et al., 1983; Randel, 1990). Canfield and Butler

(1990a) reported a high correlation between the interval from parturition to the highest

LH pulse frequencies and first ovulation, emphasizes the importance of achieving this

pattern of secretion for the stimulation of first ovulation, support this.

These series of events can lead to three different outcomes of follicular

development as described by Beam and Butler (1997):

a. Ovulation of the first dominant follicle;
b. Non-ovulation of the first dominant follicle followed by turnover and a new
follicular wave;
c. The dominant follicle fails to ovulate and becomes cystic.


The development of non-ovulatory dominant or cystic follicles prolongs the

interval from calving to first ovulation. In dairy cattle, the interval from calving to first

ovulation has been reported to be between 17 and 34 days (Table 2-7) The variation

observed between some of these studies may be due to differences in study populations

such as animal breeds, production systems, level of production, feeding systems, or it

might be as concluded by Opsomer (2000) that the first postpartum ovulation in modern

high-yielding dairy cows tends to occur later than it did a decade ago. Results of these

studies are difficult to compare since mean days to first ovulation were calculated only









for normal cows in some of these studies, which excluded cows that had follicular cysts

or those who did not ovulate early postpartum; while in other studies, all cows were in

the calculations.

Table 2-7. Days from calving to 1st ovulation reported in the literature
Author, year Days to 1st postpartum ovulation (d)
Schams (1978) 17
Stevenson (1979) 18
Webb (1980) 17
Butler (1981) 36
Stevenson (1983) 19
Fonseca (1983) 20
Meisterling (1987) 33 Short luteal phase 25 Normal luteal phase
Butler (1989) 30
Canfield (1990) 19
Harrison (1990) 29
Staples (1990) 22
Savio (1990) 22
Spicer (1990) 24
Canfield (1990) 29
Etherington (1991) 24
Nakao (1992) 30
Zurek (1995) 24
Darwash (1997) 22
Opsomer (2000) 32
Reist (2000) 34


If ovulation occurs then plasma progesterone concentrations increase to greater

than Ing/mL within 2 to 3 days after ovulation (Schams et al., 1978; Stevenson and Britt,

1979). An elevation of plasma Progesterone concentration above 1 ng/mL has been used

as an indication of resumption of postpartum ovarian cyclicity in several studies (Butler

et al., 1981; Harrison et al., 1990; Staples et al., 1990; Canfield et al., 1991; Zurek et al.,

1995; Beam and Butler, 1997; Beam et al., 1998)

The duration of the first postpartum estrous cycle can be variable, being shorter

than normal or normal, and it can occur with or without estrus behavior. Stevenson and









Britt (1979) reported 32% of first estrous cycles were shorter (16 d vs. 20 d) than

subsequent estrous cycles. These observations have been supported by different studies

(Schams et al., 1978; Webb et al., 1980; Stevenson and Call, 1983; Fonseca et al., 1983;

Savio et al., 1990b; Staples et al., 1990; Senatore et al., 1996).

Among dairy cows, those that have not ovulated by 60 days post partum have been

defined as having a delayed resumption of ovarian cyclicity (Humboldt and Thibier,

1980; Stevenson and Call, 1983; Staples et al., 1990; Moreira et al., 2001). Incidence

rates of delayed cyclicity reported in the literature are summarized in Table 2-8.

Table 2-8. Incidence rates of delayed cyclicity reported in the literature
Incidence of delayed
Author, year Delayed cyclicity Milk/Plasma yit )
Humboldt (1980) P4 > 1.5 ng/mL 60d Plasma 29
Bartlett (1987) Palpation 70d 23
Miesterling (1987) P4 > 4 ng/mL 65d Milk 21
Presence of CL and P4 >
Archbald (1990) stPlasma 30
Ing/mL 1~ month PP
Nakao (1990) P4 < 1 ng/mL 50d Milk 25
Staples (1990) P4 < 1 ng/mL 63d Plasma 28
Etherington (1991) P4 > 2ng/mL 50d Milk 33
Lamming (1998) P4 > 3 ng/mL 45d Milk 11
Opsomer (2000) P4 < 15 ng/mL 50d Milk 21
Moreira (2001) P4 < 1 ng/mL 63d Plasma 23


Several factors can affect the interval between calving and first postpartum

ovulation, the incidence rate of delayed cyclicity, and the reproductive performance of

affected cows. Some of theses factors cited in the literature are energy balance, season,

parity, and periparturient diseases (Butler et al., 1981; Fonseca et al., 1983; Canfield et

al., 1990b; Lucy et al., 1992; Senatore et al., 1996; Beam and Butler, 1997; Darwash at

al., 1997; Jonsson et al., 1997; Opsomer et al., 2000; Moreira et al., 2001).









Several studies have found associations relating energy balance to ovarian activity

postpartum, and some of these studies have proposed a casual path to this association.

However, the effects of energy balance affecting postpartum ovarian activity are not

completely elucidated. Milk production and dry matter intake increase after calving but at

different rates with the maximum feed intake occurring some weeks after maximum milk

production. The result of this delay is negative energy balance that persists for 4 to 12

weeks of lactation (Butler at al., 1981), when most dairy cows must mobilize body

reserves to support milk production (Bauman and Currie, 1980). Negative energy balance

is usually maximal during the first 3 weeks of lactation (Canfield et al., 1990b). Butler

(1981) reported that first ovulation occurred 10 days after the negative energy balance

nadir was reached, and energy balance was still negative at ovulation, but was returning

towards zero. The authors (Canfield et al., 1990b) concluded that there was an inverse

relationship between the interval from parturition to first normal ovulation and the

average energy balance during the first 20 days postpartum. These results were further

supported by other studies (Staples et al., 1990; Canfield and Butler, 1990a; Lucy et al.,

1992; Senatore et al., 1996).

The greater the average energy deficit incurred, the longer the delay to ovulation.

Canfield and Butler (1990b) showed a high correlation between days to negative energy

balance nadir and days to first ovulation. This relationship suggested that first ovulation

does not occur in an individual animal until energy balance progresses beyond its most

negative value and is returning toward balance. In this study, 1st ovulation also occurred

10 and 13 days following negative energy balance nadir in non-lactating and lactating

cows respectively. It was proposed from this study that energy status could be acting to









slow an increasing LH pulse frequency until the cow has begun to return to a positive

energy balance. Cows that reached the energy balance nadir earlier postpartum (4 d vs. 14

d) ovulated earlier (14 d vs. 27 d) and had higher concentrations of insulin. These results,

together with the finding that the pulse frequency of LH was not different for both

groups, made the authors propose that insulin levels may act permissively on the ovary to

enhance follicular responsiveness. These same authors (Canfield and Butler, 1990a)

examined the effect of energy balance and changes in plasma concentrations of glucose,

insulin, non-esterified fatty acids (NEFA), and ketones on pulsatile LH secretion in early

postpartum period. They reported a direct relationship between postpartum energy

balance and first ovulation, and between negative energy balance nadir and changes in

pulsatile LH secretion, suggesting that as negative energy balance reaches it's nadir and

starts returning towards balance, LH secretion is disinhibited and first ovulation occurs.

The relationship between energy balance and first ovulation was further supported

by different studies. A study by Staples (1990) examined the relationship between

ovarian activity and energy status during the early postpartum period. In this study, cows

were classified according to the time of first ovulation as early responders (resume

ovarian activity within 40 days postpartum), late responders (resume ovarian activity

between 40 and 60 days postpartum), and no responders (resumption of ovarian activity

after 63 days PP). These authors concluded that the early and late responders cows were

in less negative energy balance than non-responders and were able to restore ovarian

activity during the first 63 days postpartum. On the other hand, the non-responder cows

did not have the capability to consume as much dietary energy, produced less milk and

were more dependent on energy from body reserves to produce milk. As a consequence,









metabolic status inhibited the initiation of postpartum ovarian activity in cows during the

first 63 days postpartum. In addition, an increased loss of body weight during the first 2

weeks of lactation coincided with decreased ovarian activity; early responder cows losing

the least weight and non-responder cows losing the most weight.

Lucy (1992) examined the influence of diet composition, dry-matter intake, milk

production, and energy balance on time to post-partum ovulation. Results of this study

confirmed those of Staples (1990) where the interval to first ovulation was shorter in

cows that consumed more dry-matter and produced more milk. In contrast, low milk

producing cows consumed less dry-matter and were more likely to be classified as late

responders. These two studies suggested that the fact that higher producing cows

ovulating earlier than low producing cows did not contradict the effects of energy balance

on the interval to first ovulation, as higher producing cows can be in a less negative

energy balance and thus ovulate earlier. It was also reported that cows ovulating earlier

(before day 42 postpartum) had a superior reproductive performance. Also results from

the study done by Lucy (1992) are in agreement with those of Fonseca (1983) where

Jersey cows producing more milk ovulated earlier than lower producing herdmates.

These two studies concluded that the data do not support linear relationships

between days to first estrus, days to first insemination, and days open with increasing

milk yield as suggested by others. They proposed that this contradiction occurred because

lowest producing cows in commercial herds are culled before they have the opportunity

to express their reproductive potential. As stated by Staples (1990) and supported by

several studies (Fonseca et al., 1983; Harrison et al., 1990; Lucy et al., 1992), if energy

status of the cow is more important than milk yield in determining return to estrus, then









milk yield alone may be either positively or negatively correlated with days to first

ovulation. Although an association between energy balance and postpartum ovarian

activity has been clearly established, the causal path remains unclear. Detrimental effects

of negative energy balance on ovarian activity could be due to effects on any of the

components of the reproductive endocrine axis.

Physiological Factors Involved in Ovarian Activity Potentially Affected by Energy
Balance

Effects of negative energy balance in LH secretion

It has been already mentioned the importance of a high frequency mode of

pulsatile LH secretion in the final phase of maturation and ovulation of ovarian follicles.

Although pituitary content of gonadotropins increase rapidly after calving and are

capable of supporting ovulation by day 8 to 10 postpartum (Kesler et al., 1977; Fernandes

et al., 1978; Moss et al., 1985), however, pulsatile LH secretion capable of inducing

ovulation generally occurs near 1st ovulation which usually takes place at 17 to 34 days

(see table 2.4). There is strong evidence that secretion of LH is impaired in cows not

recovering from negative energy balance. Peters (1985) reported that very few lactating

cows ovulated in response to pulsatile administration of LHRH when delivered at a time

when animals were in a negative energy balance.

Canfield (1988) compared LH secretion at 2 weeks postpartum and again at the

energy balance nadir (during the return towards EB), and reported an increase in LH

frequency and a decrease in pulse amplitude. These authors concluded that energy

balance plays an important role in the control of first ovulation by suppressing LH pulse

frequency following calving. Canfield and Butler (1990a) demonstrated a direct

relationship between postpartum energy balance and 1st ovulation, and between negative










energy balance nadir and changes in pulsatile LH secretion. Therefore, it was concluded

that as negative energy balance reaches it nadir and starts returning towards balance, LH

secretion is disinhibited and 1st ovulation occurs. Canfield and Butler (1991) reported that

dairy cows in a negative energy balance had similar LH patterns but ovulated later than

cows in positive energy balance. In another study, increased energy balance was

associated with increase pulse amplitude of LH secretion (Lucy et al., 1991). Schillo

(1992), in a review of the topic proposed that, the reduction on the LH pulse frequency

observed during negative energy balance, represents one of the most important means by

which energy balance impairs reproductive activity in cattle. The above results are

consistent with Eindings of Beam and Butler (1997) which reported that follicles

emerging after the negative energy balance nadir, rather than before, exhibited greater

growth and diameter, enhanced estradiol production, and were more likely to ovulate.

Metabolic hormones

There is evidence that metabolic hormones such as growth hormone, insulin,

IGF-I, and leptin have important roles in the control of ovarian follicular development

and are likely to be important mediators of the effects of dietary intake and energy

balance on cow fertility.

Insulin -like growth factor-I. IGF-I and insulin are effectors of follicle cell

function in vitro with stimulation of steroidogenesis and cell proliferation in granulosa

and thecal cells (Monniaux et al., 1992; Spicer et al., 1993; Magoffin et al., 1993; Spicer

et al., 1996). Insulin-like growth factor-1 (IGF-I) is decreased in postpartum cows when

experiencing negative energy balance (Spicer et al., 1990; Beam and Butler, 1999) Cows

in poor body condition or cows not recovering body condition during lactation have also

been identified as having low blood IGF-I concentrations. Beam and Butler (1997)









reported that levels of plasma IGF-I averaged approximately 40% higher during the first

2 weeks postpartum in cows ovulating during the first follicular wave postpartum than in

cows not ovulating. Plasma IGF-I in cows ovulating the 1st follicular wave was higher at

day 1 postpartum, before the establishment of follicular dominance and subsequent

increases in peripheral estradiol. Therefore, the authors suggested that higher IGF-I in

ovulatory cows did not result from greater dominant follicle estradiol production, but

preceded and possibly contributed to differences in follicular function (Beam and Butler,

1997). Results of this study also suggest that low concentrations of circulating IGF-I are

related to low steroidogenic output of dominant ovarian follicles early post partum.

Results of another study (Cohick et al., 1996) also suggested that changes in systemic

levels of IGF-I and IGFBP affect their concentrations in follicular fluid and follicular

development. Finally, Beam and Butler (1999) proposed that during the negative energy

balance period, the ability of follicles to produce sufficient estradiol for ovulation seems

to depend on the availability of insulin and IGF-I in serum and the changing energy

balance profile.

Insulin. There is significant evidence that dietary restriction and negative energy

balance reduce circulating concentrations of insulin (Vizcarra et al., 1998; Mackey et al.,

2000). In vitro studies (Stewart et al., 1995) showed that insulin at physiological levels

affected proliferation of bovine thecal cells, and acted synergistically with luteinizing

hormone in stimulating steroidogenesis. Spicer (2001) reported that insulin by itself was

a more effective stimulator of aromatase activity than FSH in vitro. Beam and Butler

(1997) reported a greater insulin:GH ratio during the first week postpartum in cows

ovulating during the first follicular wave than those that did not, suggesting that levels of









insulin and GH during the very early stages of follicular recruitment may be important to

later follicular function. Gong (2002) showed that dairy cows fed a diet that increased

circulating concentrations of insulin during the first days 50 postpartum had shorter

postpartum anestrus intervals, and an increased conception rate to first service

independent of any effects on LH or FSH and without affecting milk yield or energy

balance. The authors proposed that the increase in insulin concentrations promoted

differentiation and maturation of dominant follicles during early lactation, thereby

increasing the chance of these dominant follicles of ovulating in response to the LH

surge. These results suggest that insulin may have a direct effect at the ovarian level.

Thyroid hormones (T3 and T4). Reist (2003) examining associations between

postpartum reproductive function and metabolic status in high yielding cows, reported

that cows with higher plasma levels of thyroid hormones (T3 and T4), WeTO aSsociated

with early start of ovarian cycle; proposing that Thyroid hormones can also play an

important role in the resumption of ovarian activity postpartum. These results are

supported by previous findings of Spicer (2001) who provided evidence for a role of T3

and T4 in regulating steroidogenesis of bovine follicles. The author proposed that T3 and

T4 may have a minor positive impact on FSH-induced progesterone production by bovine

granulosa cells, and a maj or positive impact on LH-induced androstenedione production

by bovine thecal cells, both of which would result in a net increase in estrogen production

by the follicle, however, T3 and T4 have little or no direct effect on aromatase activity.

This evidence supports a role of Thyroid hormones as part of a multihormonal complex

regulating ovarian activity in cattle.










Leptin. Leptin is a protein hormone secreted by adipocytes (Bradley et al., 2000)

and acts on the central nervous system to reduce voluntary feed intake (Schwartz et al.,

2000). Block (2001) reported that postparturient cows undergoing negative energy

balance had significantly lower plasma concentrations ofleptin compared with

postparturient cows in positive energy balance. In addition, at the first week of lactation

the plasma concentration of leptin was correlated positively with plasma concentrations

of glucose and insulin and negatively correlated with plasma concentrations of GH and

NEFA. The author concluded that these correlations could represent a co-regulation by

energy balance and these factors in mediating the effect of energy balance on leptin

synthesis.

Leptin has diverse effects on the neuroendocrine axis in addition to appetite and

body weight regulation (Ahima and Flier, 2000). Leptin stimulated gonadotropin release

and inhibited insulin-like growth factor-mediated release of estradiol in ovarian follicular

cells in rat ovarian granulosa cells (Zachow and Magoffin, 1997). Another study (Chehab

et al., 1996) reported that correction of the sterility defect in homozygous (ob/ob) obese

female mice could be accomplished by repeated administration of human recombinant

leptin, resulting in ovulation, pregnancy and parturition. Williams (2002) reported that

short term fasting of growing prepubertal heifers causes marked reductions in circulating

leptin, concomitant with declines in LH pulse frequency, and serum concentrations of

insulin and IGF-I. In this same study results could not be repeated for mature cows under

the same short term fasting. Altogether, these results may indicate that leptin signals the

adequacy of energy stores for reproduction, by interacting with different target organs in

the hypothalamic-pituitary-gonadal axis in cattle and other species. It has been proposed









that the effects of leptin might be mediated in part by NPY, which in turn has been shown

to regulate gonadotrophin release by inhibiting LH secretion in ewes (McShane et al.,

1992).

NPY is a potent inhibitor of LH release and unlike leptin, is a potent stimulator of

food intake (Houseknecht et al., 1998) Concentrations ofNPY increase in cerebrospinal

fluid during undernutrition and can negatively modulate the secretion of LH when

centrally infused in cattle (Gazal et al., 1998). These results may stimulate future research

to explore the role and potential interactions between hormones, neuropeptides and

resumption of ovarian cyclicity under negative energy balance in postpartum dairy cattle.

It has been well established in cattle that ovarian function is controlled primarily

by an integrated GnRH-gonadotrophin-ovari an axi s. Recent work has shown that factors

classically thought to be mainly involved in the regulation of metabolic processes, such

as GH, insulin and IGF-I, thyroid hormones, leptin,and neuropeptide-Y may play an

important role in the control of ovarian activity in the postpartum dairy cow (Spicer at al.,

1995; Reist et al., 2003; Williams et al., 2002). Metabolic hormones can act either

directly to control gonadotrophin -independent stages of follicle development (Gong et

al., 1996), or in synergy with gonadotrophins to modulate follicular recruitment and final

development and maturation of preovulatory follicles (Spicer et al., 1995; Armstrong et

al., 2002). These effects could represent at least part of the mechanism underlying well

documented but not completely understood nutritional influence on reproductive function

in cattle.

Other factors

Season. The effect of season has been reported as having an effect on resumption

of ovarian activity postpartum. In a study conducted in North Carolina, Fonseca (1983)










reported that cows calving in the winter had 6.5 more days to first ovulation compared

with cows calving in the fall. Peters (1984) reported that cows calving in the spring

underwent longer periods between calving and first ovulation than autumn calvers. Savio

(1990a) reported that the postpartum interval to detection of the first dominant follicle

was shorter in autumn than in the spring. When only normal dominant follicles were

considered, the cows that calved in autumn tended to have a shorter, and less variable,

intervals from calving to first ovulation. In Australia, cows calving in summer had

significantly longer intervals from calving to first postpartum ovulation than those

calving in winter (23d vs. 18d) (Jonsson et al., 1997). In addition, cows losing more

bodyweight had longer intervals from calving to first ovulation. In Belgium, cows calving

in the winter (stable housing) were more prone to delayed ovarian function compared to

cows calving in the spring (pasture housing) (Opsomer et al., 2000). None of these

studies proposed a path as to how season affects ovarian cyclicity. In Switzerland, Reist

(2003) examined the relationship between reproductive function and metabolic and

endocrine status in dairy cows. Results of this study showed a significant effect of season

in resumption of ovarian activity. Cows calving in the fall were more likely to start

ovarian cyclicity earlier postpartum than cows calving in the spring.

Parity. The data on the effect of parity on the interval to first ovulation is

contradictory and does not support an effect of parity on resumption of ovarian cyclicity.

Stevenson and Britt (1979) reported that interval to first ovulation tended to be longer for

pluriparous than for primiparous cows (18.7 vs 16.3 days), but the interval to first estrus

was not different between parity groups (26.1 vs 27.7 days). Fonseca (1983) reported that

in cows between 33 and 60 months of age, younger cows had their first ovulation earlier









than older cows. In this study, there were very few observations in older cows. In

contrast, Lucy (1992) reported that ovarian activity was delayed in primiparous cows

compared with multiparous cows. However, primiparous cows were similar to

multiparous cows with respect to first service, first service conception rate, services per

conception, and days open. Moreira (2001) reported that the incidence of anestrus at 63

days was greater in primiparus than in multiparous cows.

Periparturient diseases. The effects of periparturient diseases in resumption of

ovarian cyclicity in dairy cows have been reported. Fonseca (1983) reported that cows

with abnormalities after calving had 8.8 more days to first ovulation than cows without

abnormalities after calving. In this study the main abnormalities at parturition were

retained placenta and milk fever in Holstein cows and Jersey cows, respectively. During

the postpartum period, uterine infection was the most frequent clinical abnormality in

Holsteins, while ovarian cysts ranked first (followed by injury or disease and uterine

infection) in Jerseys. Opsomer (2000) conducted a study to identify risk factors

associated with postpartum ovarian dysfunction in dairy cows. In this study, cows

suffering from clinical diseases such as mastitis, severe lameness, or pneumonia during

the first month of lactation, were 5 times more at risk of developing delayed resumption

of ovarian activity than healthy cows. In addition, cows developing clinical symptoms of

ketosis with a positive prussiate test were 1 1 times more at risk of delayed resumption of

ovarian cyclicity than normal cows. Cows with abnormal calvings and abnormal vaginal

discharges were 3.6 and 4.5 times more likely to develop delayed ovulation compared to

cows with normal vaginal discharges respectively. Similar results were reported by









Etherington (1991) where cows with retained placenta were associated with longer

intervals to first ovulation.

Loss of body condition early in the postpartum period was another factor

increasing the risk of delayed cyclicity. Cows losing more in body condition were 19 and

11 times more at risk of delayed cyclicity at 30 days and 2 months after calving. Cows

with normal progesterone profiles lost on average 0.26 points during the first month after

calving and 0.29 points during the first 2 months after calving, while cows with delayed

ovarian function lost 0.39 and 0.49 points, respectively (Opsomer et al., 2000). These

results were in agreement with those of Moreira (2001), where an effect of body

condition at 63 days was associated with the frequency of cows classified as anestrus. As

body condition increases the incidence of anestrus decreases. In another study conducted

by Mateus (2003) a relationship between endotoxin concentrations in blood from cows

with endometritis and a prolonged anestrus period was observed. This result is in

agreement with a previous study from Peter (1990) where intrauterine infusions of

endotoxins reduced the preovulatory LH surge in cows. This could have been mediated

by high cortisol levels and resulted in ovulation failure. Ketosis is another periparturient

disorder related to delayed resumption of ovarian activity. Reist (2000) examined the

relationship between ketone body concentration and first ovulation in dairy cows. Cows

classified as late responders (cows with a first ovulation between 31 and 87 days

postpartum) had higher blood and milk ketone bodies concentrations compared to early

responders (cows with first ovulation within 30 days postpartum), with no significant

differences in body condition scores between groups.









Lameness and Ovarian Activity

While several studies have shown a relationship between lameness and

reproductive performance, the relationship between lameness and ovarian activity in

dairy cows has not been investigated using obj ective research methods. To our

knowledge, there is only one study that looked at the relationship between lameness and

ovarian activity in dairy cows. This study was conducted in 335 dairy cows on six high

producing dairy herds in Belgium. Cows diagnosed with clinical mastitis, severe

lameness, or pneumonia by farmers were at higher risk of delayed cyclicity, compared to

cows classified as clinically healthy (Opsomer et al., 2000); however, the actual number

of cows affected with clinical mastitis, severe lameness, or pneumonia was not reported.

In Florida, clinical observations by veterinarians and dairy farmers suggest that

cycle cows affected with lameness experience anestrus, and the duration of anestrus is

associated with severity, duration, and diseases or lesions associated with lameness (eg,

interdigital phlegmon, papillomatous digital dermatitis, claw lesions). We hypothesized

that as lame cows experience a more pronounced loss in body condition (hence a

prolonged state of negative energy balance) during the early post partum period, lame

cows are at higher risk of delayed ovarian cyclicity than non-lame cows. Lame cows eat

less and go into a negative energy state. In order to meet energy deficit, body reserves are

mobilized resulting in body weight loss. The output of energy in lame cows (body

maintenance and milk yield) may exceed the energy input in the form of feed. It is

possible that cows that become lame are in a progressive negative energy state, go into

anestrus and experience long delays to restore ovulation.

Results of previous studies suggest that as cows experience increasing positive

energy status, there is increased ovarian follicle activity leading to early return to









ovulation (Butler et al., 1981, Staples et al., 1990; Lucy et al., 1991). As energy status

becomes more positive for cows in early postpartum, diameter of the largest follicle

increases, the number of double ovulations increases, and time for detection of the first

corpus luteum decreases. (Lucy et al., 1991).These changes in follicle size and numbers

and the number of ovulations are thought to be aroused by increases in luteinizing

hormone (LH), follicle stimulating hormone (FSH), insulin, BST, insulin-like growth

hormone-1 (IGF-1), and possibly other yet-to-be determined compounds as activated by

improving energy status (Beam and Butler, 1998). Therefore, lameness may have an

effect on feed intake and energy status leading to changes in concentrations of

reproductive hormones. Under field conditions, evidence of corpus luteum function can

be determined by monitoring plasma progesterone (P4) COncentrations weekly during

lactation, before and after diagnosis of lameness in dairy cows.















CHAPTER 3
MATERIALS AND METHODS

Cows and Herd Management

Cows in this study were from a high-producing dairy herd (rolling herd average

milk production, approx 12,000 kg) of approximately 600 Holstein cows located in

Florida. Cows were milked and fed a TMR ration three times per day. Cows were housed

in lots equipped with sprinklers, fans and shade cloth over the feed bunks to reduce the

effects of heat stress. This herd was selected for study on the basis of a history of

lameness, quality of veterinary records, and willingness of the owner to participate in the

study .

Study Design

This study was designed as an observational cohort study. Sample size calculations

were made on the basis of our estimate of the number of cows affected with delayed

ovarian cyclicity increasing from 10% in non-lame cows to 30% in lame cows (type I

error = 0.05; type II error = 0.20). A total of 253 (45%) of 563 Holstein cows identified

with an even numbered ear-tag that calved from June 1, 2002 until May 31, 2003 was

used in the study. Cows with an even numbered ear-tag were enrolled in the study as they

calved (instead of cows randomly selected) to overcome logistical identification

procedures and to reduce disruption of routine veterinary medical and management

procedures at the study farm. Cows were classified into one of six categories of lameness

during the first 3 5 days post partum using a modification of the locomotion scoring

developed by Sprecher et al., 1997. Blood samples were obtained for weekly for










detection of plasma progesterone (P4) COncentrations during the first 60 days post partum.

Risk of delayed cyclicity was compared between cows classified as non-lame, moderately

lame, or lame.

Data Collection

Using farm records, the following data were collected for each cow: lactation

number, calving date, calving season (winter months: Jan to Apr and Oct to Dec; summer

months: May to Sep), dystocia (yes, no), retained placenta (yes, no), metritis (yes, no),

mastitis (yes, no), ketosis (yes, no), body condition score at calving (Edmonson et al.,

1989), change in body condition score in the first 50 days post partum, use of PGF2a

(Lutalyse, Pharmacia, Kalamazoo, MI) prior to resumption of ovarian activity (yes, no),

and 305-day mature equivalent milk yield. From Dairy Herd Improvement Association

(DHIA) records, proj ected 305 day ME milk yield data were collected based upon

production at 60 days post partum. Levels of milk yield were defined as low (5,530 to

10,619 kg), medium (10,620 to 12,978 kg) and high (12,979 to 15,137 kg) on the basis of

the frequency of distribution (first, second and third, and fourth quartiles, respectively).

Diagnosis of Lameness

During the first 35 days post partum, study cows were examined weekly (Tuesday)

for diagnosis of lameness using a locomotion scoring system described by Sprecher

(1997) with modifications (Table 1). Cows were observed and scored by the same

veterinarian (EJG) as they walked-out of the wash pen to the holding area prior to

milking. Cows with a locomotion score of 4 or 5 were further examined on a tilt table for

diagnosis and treatment of lameness, noting lesions observed and date of occurrence.

Lame cows with claw lesions had white line lesions or sole ulcers and were treated by

corrective foot trimming techniques (Shearer and van Amstel, 2001). Lame cows with









subacute laminitis were those with yellow and red discoloration of the sole and white

line, and in most cases they had thin soles and were sensitive at examination with hoof

testers (Mortensen, 1994; Toussaint Raven, 1989). Lame cows with interdigital dermatitis

were those with inflammation confined to the epidermis and in some cases

hyperkeratosis, which creates a roughened appearance to the interdigital skin (Blowey,

1994); a fetid serious exudate could be present, and there was mild sensitivity to pressure.

This condition was frequently accompanied by cracks in the heel, heel horn erosion, with

potential under-running of the heel horn (Berry, 2001).

Collection of Blood Samples and Detection of Plasma P4 COncentrations

Cows were blood-sampled and scored for body condition (Edmonson et al., 1989)

weekly (Thursday) for detection of plasma progesterone concentrations during the first

60 days post partum. Cows were blood-sampled via coccygeal venipuncture using

vacutainer collection tubes containing K3 EDTA (Becton, Dickinson, and Company,

Franklin Lakes, NJ). Blood samples were refrigerated until and during transportation to a

laboratory at the University of Florida where they were centrifuged for 20 min at 3000

RPM at room temperature for plasma harvest. Plasma samples were frozen at 20 C until

tested for P4 COncentrations using the Coat-A-Count@ Progesterone Kit (DPC@

Diagnostics Products Corporation) radioimmunoassay. The Coat-A-Count@ Progesterone

procedure is a solid-phase radioimmunoassay where 125I-labeled progesterone competes

for a fixed time with the progesterone content of the cow' s plasma sample. Because

antibody is bound to the wall of the polypropylene tube, simply decanting the supernatant

is enough to terminate the competition and to isolate the antibody-bound fraction of the

radiolabeled progesterone. A standard curve dilution was prepared using coated tubes and

non-coated tubes were used for total counts and non-specific binding. A 100 Cl~ volume of









increasing concentrations of progesterone calibrators, (0, 0.1i, 0.25, 0.5, 2, 5, 10, 20, and

40 ng/mL) were placed in the tubes. Plasma samples (100 Cl) were added to coated tubes

and 1 mL of 125I-labeled progesterone (25000 cpm) to all tubes. Every 6th plasma sample

was evaluated in duplicate. After an incubation period of 3 hours, the supernatant was

discarded and tubes were dried for 15 minutes. They were then placed in a gamma

counter. Calculation of the progesterone concentration in the plasma sample was made by

computerized data process using a spline fitting curve.

Accuracy of assay procedures was determined by measuring known quantities of

exogenous progesterone (0.625, 1.25, 2.5, and 5.0 ng/mL) in plasma in seven different

assays. Recovery of added (x) versus measured (Y) P4 COncentrations was described by

linear regression (Y= 0.57 + 0.93x; R2 = 0.89). The regression intercept value (0.57

ng/mL) represented original P4 COncentrations measured in a plasma pool prior to

addition of exogenous masses.


Parallelism of logit plots between the displacement curves for different volumes of

a plasma pool containing 8 ng/mL of P4 (i.e., 25, 50 and 100 Cl~) and standard P4 amOunts

(i.e., 0.1, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0 and 20.0 ng/mL) was tested for homogeneity using

regression analysis (Wilcox et al., 1990). The linear regression curves for plasma and P4

standards were parallel (Yp= 0.60 1.44x, R2 = 0.99; Ys = 0.21 1.61x, R2 = 0.99; where

Yp and Ys = In B/F, x = loglo of assay volume or loglo standard P4 COncentrations,

respectively). The slopes were not different.

Coefficients of variation were calculated from a reference sample (luteal phase) and

duplicate samples obtained from all assays. Duplicate plasma concentrations of P4 were

categorized into high (2 3.0 ng/mL; n = 359), medium ( 1.0 and <3.0 ng/mL; n = 128),









and low (> 0.3 and <1.0 ng/mL; n = 52), and the coefficients of variation were 12.4%,

12.4%, and 14.2%, respectively. Inter-and intraassay coefficients of variation for the

luteal phase reference sample were 8.9% and 8.34%, respectively.

Resumption of Ovarian Cyclicity

Cows with evidence of normal ovarian cyclicity during the first 60 days post

partum were those with: i) weekly plasma P4 COncentrations > 1 ng/mL for 2 or 3

consecutive samples followed by a decline in P4; Or ii) if P4 COncentration > 1 ng/mL was

followed by a marked decrease after PGF2a inj section and this followed by an increase in

P4 COncentration. Cows with a delayed resumption of ovarian cyclicity were those with

consistently low P4 COncentrations < 1 ng/mL during the first 60 days post partum

(Staples et al., 1990). For the purpose of this study, cows with P4 values above 1 ng/mL

for 4 or more consecutive samples were classified as cows with extended luteal phases

(Opsomer et al., 1999). First luteal phase was defined as the first rise in P4 above 1

ng/mL .

Reproductive and Health Management

All cows were subj ected to a pre-synchronization program. After days 30 to 3 5

postpartum cows received an inj section of prostaglandin F2a and then again at 44 to 49

days. Cows observed in heat after the second inj section of prostaglandin F2a were

inseminated, and those that did not demonstrate behavioral signs of estrus were enrolled

in a timed insemination program. This program involved the use of GnRH on Day 0, 7

days later prostaglandin F2a, a second GnRH on Day 9, and timed inseminated 16 to 18

hours later. All cows were examined for pregnancy at 42 to 49 days post insemination by

palpation of the uterus and its contents by the attending veterinarian.









Farm personnel examined cows for the detection of health problems several times

post partum following a pre-established protocol (Table 3-1). Cows were grouped by

days in milk and a group of fresh cows (less than 30 DIM) were kept together. Cows

were examined after each milking. On Day one cows were checked for retained fetal

membranes (fetal membranes visible at the vulva for more than 24hs after calving), udder

edema (by observation and palpation of the udder), and mastitis (by stripping all four

quarters). On Day 4, cows were checked for production (using Afimilk@ computerized

system), udder edema, metritis (Table 3-2) and rectal temperatures were taken. On day 7

cows were checked for Ketonuria (using Ketostixs@), metritis, body temperature, and

using a stethoscope, rumen movements, and displaced abomasum (DA) tympanicc sound

on the left side at simultaneous auscultation-percussion of the left paralumbar fossa). This

7-day check was repeated at day 10 and 15. In all checks, daily milk yield was monitored

for every cow for deviations in production.

Table 3-1. Protocol for examination of cows postpartum
Check day Health checks
1 Retained fetal membranes (RFM), udder edema, mastitis
4 Temp, udder edema, metritis, production, mastitis, manure consistency
7 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency
10 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency
15 Temp, ketosis, DA, rumen, metritis, mastitis, manure consistency


Table 3-2. Definitions of metritis done by farm personnel based on discharge and
palpation findings
Metritis code Definition
Ul Normal size and abnormal discharge.
U2 Abnormal size and abnormal discharge (pus discharge)
U3 Abnormal size and abnormal discharge (watery and foul smelling)
U4 Abnormal size, abnormal discharge and the cow looks sick









Cows with Ul and some with U2 were treated with an inj section of prostaglandin

F2a. Some cows with U2 and all with U3 were treated with an intrauterine infusion of

tetracycline (100 mg/mL) solution. Cows with U4 were treated with systemic antibiotics.

Other health codes recorded were calving outcomes (See table 3-3)

Table 3-3. Definition of calving outcomes
Calving code Definition
Pull 1 Easy pull, 1 person.
Pull 2 Difficult pull, 1 person
Pull 3 Easy for 2 persons
Pull 4 Difficult pull for 2 persons
Pull 5 Extreme pull.


Cows with pulls 4 and 5 were started with systemic antibiotics after calving.

All cows in the herd were monitored daily for deviations in milk (Afimilk@) production,

and milk conductivity for detection of mastitis following a pre-established criteria. (Table

3.4). All health events and treatments were recorded on a cow-side computer program

(Visi-Cow@) used by farm personnel.

Table 3-4. Criteria for monitoring production health and mastitis using Afimilk@ system
Population % Decrease in daily milk Increase in daily Increase daily milk
production (%) conductivity (%) production (%)
1 to 15 DIM ---- ---- < 10
1 to 40 DIM 15 12--
41 to 100 DIM 20 15--
> 100 DIM 25 18--


Statistical Analyses

The null hypothesis that risk of delayed ovarian cyclicity is the same in cows

classified as non-lame, moderately lame, or lame was tested using logistic regression. In

the analysis, non-lame cows were those with a score of 3 for one week only, or scores of

< 2. Cows classified as moderately lame were those with a score of 3 on two consecutive









weeks. Lame cows were those classified at least once with a locomotion score of 4 or 5.

Additional independent variables (lactation number, calving season, milk yield, dystocia,

retained placenta, metritis, ketosis, body condition score, use of PGF2a) were included in

the analysis to address possible modifying or confounding effects of these factors on risk

of delayed ovarian cyclicity. Stepwise forward regression was used, and a variable had to

be significant at the 0.20 level before it could enter the model. A variable remained in

the model when its significance level was < 0.10. Variables for lactation number and

calving season were forced into the model.

In the Einal model, adjusted odds ratios (OR) and 95% confidence intervals (CI)

were reported. The OR was used as an epidemiologic measure of association between a

variable (i.e., lameness) and the outcome of interest (i.e., delayed ovarian cyclicity). In

each variable, the reference category had an OR = 1. An assessed OR > 1.0 indicates that

the probability of delayed ovarian cyclicity increased, compared with cows in the

reference category. The attributable proportion was estimated as (OR 1)/OR, and

interpreted to represent the proportion of lame cows that experienced delayed ovarian

cyclicity because of lameness (Martin et al., 1987).

The null hypothesis that number of days post partum to first luteal phase did not

differ among groups of cows classified as non-lame, moderately lame, or lame was tested

by use of the Kruskal-Wallis nonparametric test (because the dependent variable failed to

meet assumptions of parametric testing), and multiple ANOVA for the dependent

variable of days to first luteal phase (ranked data) while simultaneously adjusting for

variables related to ovarian cyclicity (i.e., lactation number, calving season, ketosis, milk

yield). Significance was set at P < 0.05.












8








7 14 21 28 35 42 49 56 63 70 77
Days postpartum

Figure 1. Normal ovarian cyclicity













7 14 21 28 35 42 49 56 63 70 77 84
Days postpartum

Figure 2. Normal ovarian cyclicity for cows treated with PGF2


1

0.8

S0.6

S0.4

0.2

0


7 14 21 28 35 42 49 56 63 70 77 84
Days postpartum


Figure 3. Delayed resumption of ovarian cyclicity







68





6


PI2



7 14 21 28 35 42 49 56 63 70 77 84 91 98 105
Days postpartum

Figure 4. Extended luteal phase















CHAPTER 4
RESULTS

All 253 cows enrolled in the study were followed-up successfully during the

60-day study period. Two hundred and thirty-eight (94%) cows met the criteria for

ovarian delayed cyclicity used in this study. A visual examination of plasma P4

concentration patterns revealed that 15 cows (6%) experienced an extended luteal phase.

A total of 101 of 23 8 (42%) cows were classified as moderately lame (locomotion score

= 3) and 41 (17%) as lame (score = 4) (Table 4-1). The mean number of days post partum

when cows were classified as lame was 15 days (1 34 days). The most common lesions

observed were subacute laminitis (26/41 = 63%), and claw lesions such as sole ulcers and

white line disease (9/41 = 22%).

The overall incidence of delayed ovarian cyclicity was 11%. The incidence of

delayed ovarian cyclicity was higher in cows classified as moderately lame (14/101;

14%) or lame (7/41; 17%), compared to non-lame cows (6/96; 6%). In the univariable

analysis, cows classified as moderately lame were 2.4 times at higher risk of delayed

ovarian cyclicity compared to non-lame cows (OR = 2.4; 95% CI = 0.9 6.7; P = 0.07)

(Table 4-2). Cows classified as lame were 3.1 times at higher risk of delayed ovarian

cyclicity compared to non-lame cows (OR = 3.1; 95% CI = 0.9 9.9; P = 0.05).

In the multivariable analysis, lameness, lactation number, season, ketosis and milk

yield were retained in the final modeling process (Table 4-3). Addition oftwo-way

interaction terms did not contribute to the final model for risk of delayed ovarian

cyclicity, and these terms were removed from the model. Cows classified as moderately









lame were 2. 1 times at higher risk of delayed ovarian cyclicity, compared to non-lame

cows (OR = 2.1; 95% CI = 0.7 6.1; P = 0.15). Cows classified as lame were 3.5 times at

higher risk of delayed ovarian cyclicity compared to non-lame cows (OR = 3.5; 95% CI =

1.0 12.2; P = 0.04). The attributable proportions of cows that experienced delayed

ovarian cyclicity associated with moderate lameness and lameness were 0.52 and 0.71,

respectively (Table 4-4).

Overall, the time interval (median) from calving to first luteal activity in the study

population was 31 days. This time period was more prolonged in cows classified as lame

(median = 36; range = 17 to 97) or moderately lame (median = 32; range = 4 to 146),

compared with non-lame cows (median = 29; range = 2-172) (P < 0.05).





Table 4-1. Frequency distribution of cows classified as lame or non-lame using a
modification of the locomotion scoring system developed by Sprecher, 1997
Locomotion Clinical Assessment criteria Cows
score description n = 238
No. of cows (%)
0 Normal The cow stands and walks with a 3 (1)
level-back posture. Gait is normal.


The cow stands with a level-back
posture but develops an arched
back posture while walking. Gait
remains normal.

An arched-back posture is evident
both while standing and walking.
Normal gait.

An arched-back posture is evident
both while standing and walking.
Gait is affected and best described
as short strides with one or more
limbs.

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
limb s/feet.

In addition to criteria in LS4, the
cow demonstrates an inability or
extreme reluctance to bear weight
on one or more of her limbs/feet.


Barely lame


17 (7)




76 (32)



101 (42)






41 (17)






0 (0)


Mildly lame


Moderately lame


Lame


Severely lame










Table 4-2. Descriptive statistics and unadjusted odds ratios for risk of delayed ovarian
cyclicity in post-partum Holstein cows
Variable Delayed cyclicity Delayed cyclicity OR 95% CI P
Yes No
n = 27 n= 211
No. of cows (%) No. of cows (%)


Lameness group
Locomotion score <: 2


Lactation number

2
Season
Winter
Summer
Milk yield
Low
Medium
High
Dystocia
No
Yes
Retained placenta
No
Yes
Metritis
No
Yes
Mastitis
No
Yes
Ketosis
No
Yes
BCS at calving
< 2.75
2.75 3.5
> 3.5
BCS change (0.75)
No
Yes
Use of PGF2a,
No
Yes


6 (22)
14 (52)
7 (26)

10 (37)
17 (63)

18 (67)
9 (33)

9 (33)
16 (59)
2 (8)

20 (74)
3 (11)

23 (85)
4 (15)

16 (59)
11 (41)

25 (93)
2 (7)

18 (67)
9 (33)

3 (11)
20 (74)
4 (15)

20 (74)
7 (26)

17 (63)
10 (37)


90 (43)
87 (41)
34 (16)

77 (36)
134 (64)

123 (58)
88 (42)

50 (24)
102 (48)
56 (27)

163 (77)
17 (8)

181 (86)
30 (14)

132 (63)
79 (37)

174 (82)
37 (18)

177 (84)
34 (16)

34 (16)
151 (71)
26 (12)

173 (82)
38 (18)

125 (59)
86 (41)


1.0 Reference
2.4 0.9 6.7
3.1 0.9 9.9


NA
0.07
0.05


1.0 Reference NA
0.9 0.4 2.2 0.95

1.0 Reference NA
0.7 0.3 1.6 0.40


1.0 0.4 2.5
1.0 Reference
0.2 0.05 0.9


0.91
NA
0.04


Reference NA
0.3 5.3 0.58

Reference NA
0.3 3.2 0.93

Reference NA
0.5 2.5 0.73


1.0 Reference NA
0.3 0.09 1.6 0.19

1.0 Reference NA
2.6 1.0 6.2 0.03


0.6 0. 1 2.3
1.0 Reference
1.1 0.3 3.6


0.52
NA
0.79


Reference NA
0.6 4.0 0.32


1.0 Reference NA
0.8 0.3 1.9 0.70










Table 4-3. Final logistic regression model for risk of delayed ovarian cyclicity in
post- partum Holstein cows
Variable Adjusted odds 95% confidence interval P value
ratio

Lameness group
Locomotion score <;2 1.0 Reference NA
3 2.1 0.7 6. 1 0.15
4 3.5 1.0 -12.2 0.04

Lactation number
1 1.0 Reference NA

> 2 1.2 0.5 2.3 0.65

Season
Winter 1.0 Reference NA

Summer 0.9 0.3 2.3 0.90

Ketosis
No 1.0 Reference NA
Yes 2.7 1.0 7.0 0.03

Milk yield
Low 0.9 0.3 2.5 0.98
Medium 1.0 Reference NA

High 0.2 0.05 0.9 0.04
NA = Not applicable


Table 4-4. Attributable proportion of cows that experienced delayed resumption of
ovarian cyclicity
Locomotion score N OR Attributable proportion
< 2 6/96 1 NA

3 14/101 2.1 0.52
4 7/41 3.5 0.71















CHAPTER 5
DISCUSSION

The results of the study reported here support the hypothesis that lameness has a

detrimental effect on ovarian activity in Holstein cows during the early post partum

period. Cows classified as lame were 3.5 times at higher risk of delayed cyclicity,

compared to non-lame cows. Attributable proportion analysis indicated that delayed

ovarian cyclicity in lame cows would be reduced by 71% if lameness had been prevented.

In addition, cows classified as moderately lame were 2. 1 times at higher risk of delayed

ovarian cyclicity compared to non-lame cows (OR = 2. 1; 95% CI = 0.7 6. 1; P = 0. 15).

Even though this association was not statistically significant, the OR and the position of

the confidence interval (Szklo and Nieto, 2000) suggest that cows classified as

moderately lame were at high risk of delayed ovarian cyclicity. This observation is

further supported by the fact that the interval from calving to first luteal phase was more

prolonged in both lame cows (median = 36 days) or moderately lame cows (32 days)

compared with non-lame cows (29 days) (P < 0.05). Thus preventive measures (such as

examination of cows feet and, if necessary, use of corrective foot trimming techniques)

should be targeted at the group of moderately lame cows since as they represented 42%

of the study population. We examined a second logistic regression model which included

the 15 cows that experienced an extended luteal phase (in addition to the 238 cows that

met the criteria for ovarian cyclicity used in this study), and the effect of lameness on

delayed cyclicity did not disappear. Cows classified as moderately lame and lame were 2

(OR = 2.0; 95% CI = 0.7, 5.9; P = 0.17) and 3 times (OR = 3.0; 95% CI = 0.9, 10.3; P =










0.07) at higher risk of delayed cyclicity, respectively, compared to non-lame cows. To

our knowledge, only one previous study has examined the relationship between lameness

and ovarian activity. In a study conducted in 335 dairy cows on six high producing dairy

herds in Belgium, cows diagnosed with clinical mastitis, severe lameness, or pneumonia

by farmers were at higher risk of delayed ovarian cyclicity compared to cows classified

as clinically healthy (Opsomer et al., 2000). However, the actual number of cows

affected with clinical mastitis, severe lameness, or pneumonia was not reported.

The incidence of cows classified as moderately lame and lame during the first 35

days post partum was 42% and 17%, respectively. In a previous study involving 66 dairy

cows on a farm in Michigan (Sprecher et al., 1997), a locomotion scoring system similar

to that in our study was used for diagnosis of lameness. In the Michigan study, 27 (49%)

cows and 14 (24%) cows were classified as moderately lame and lame, respectively.

Results from that study are difficult to compare with results of the present study because

of differences in the scoring system. After testing the locomotion scoring system

(Sprecher et al., 1997) weekly for two months in the study herd, a new category was

added to include cows that were observed with an arched-back posture that was evident

both while standing and walking, but their gait seemed normal (score = 2, mildly lame);

76 (32%) cows were included in this category. In our analysis, this group of cows was

classified as non-lame. Assuming that this group of cows was misclassified as non-lame,

the incidence of cows classified as moderately lame (score = 3) would have been higher

(76 + 101 = 177 cows or 74%).

However, study results support our clinical observations and locomotion scoring

system for diagnosis oflameness. The possibility that cows classified as mildly lame









were misclassified is unlikely since the incidence of delayed ovarian cyclicity was lower

in mildly lame cows (3/76 or 4%) compared to moderately lame cows (14/101 or 14%). If

mildly lame cows were misclassified, there would have been an expected an incidence of

delayed ovarian cyclicity similar to that observed in cows classified as moderately lame.

Although we established an association between lameness and delayed ovarian

cyclicity, we failed to identify loss of body condition (or a modifying effect of lameness

and loss of body condition) as a significant risk factor associated with delayed ovarian

cyclicity. The risk of delayed ovarian cyclicity was 1.5 times higher in cows that had a

change in BCS > 0.75 in the first 50 days post partum compared to cows with a change in

BCS < 0.75. Therefore, this association was not significant

(OR = 1.5; 95% CI = 0.6 4.0; P = 0.32). The observed incidence of delayed ovarian

cyclicity in the study population was low (11%) compared to other studies (23 to 29%),

(Humboldt and Thibier, 1980; Bartlett et al., 1987; Staples et al., 1990), creating a sample

size limitation. In the previous study conducted in 335 dairy cows in Belgium, cows

losing more body condition during the first and second month after calving were at higher

risk of delayed ovarian cyclicity (Opsomer et al., 2000).

In our study, ketosis was, by itself, a risk factor for delayed resumption of ovarian

cyclicity. This result is in agreement with a study conducted in 84 dairy cows on 8 farms

in Switzerland (Reist et al., 2000) where blood serum and milk ketone body

concentrations during the first 6 weeks post partum were higher in cows classified as late

responders (i.e., cows started post partum ovarian cyclicity after 30 days) than in early

responders, with no significant differences in body condition scores between groups. It is

possible that lameness and ketosis may additionally interact with each other to affect the









risk of delayed ovarian cyclicity, but the small sample size was too small in the present

study did not allow detection of such an interaction.

Lameness can depress dry matter intake (Hassall et al., 1993; Galindo and Broom,

2002) and result in negative energy balance. It has been reported that negative energy

balance contributes to increased ketone body formation and delays the onset of ovarian

activity (Reist et al., 2000). A negative energy balance post partum not only contributes

to increased ketogenesis, but also delays the onset of ovarian cyclicity, especially if

energy deficiency is prolonged (Butler and Smith, 1989; Staples et al., 1990; Lucy et al.,

1992). Furthermore, results of the study reported here revealed that the risk of delayed

ovarian cyclicity was lower in high milk producing cows, compared to medium or low

producing cows. The results of previous studies suggested that low producing cows have

reduced inferior dry matter intake, a more negative energy balance, and are less likely to

restore ovarian activity during the first 63 days post partum compared to high producing

cows (Staples et al., 1990; Lucy et al., 1992).

Although is clear that lameness has an effect on resumption of ovarian cyclicity in

postpartum cows, we could not establish the cause of this effect. We hypothesized that

energy balance would be responsible for the delayed in resumption of ovarian cyclicity,

but our study could not support such pathway. Even though there are no studies reporting

a pathway to explain the effect of lameness on ovarian activity, we cannot ignore other

proposed mechanisms by which lameness may affect the hypothalamus-pituitary-ovarian

axis.

Dobson (2000) proposed that activation of the hypothalamus-pituitary-adrenal axis

by stressors reduces the pulasatility of GnRH/LH by actions at both the hypothalamus









and pituitary gland, depriving the ovarian follicle of adequate LH support. This will lead

to reduced estradiol production by slower growing follicles. A combination of a reduced

GnRH/LH pulsatility with a reduced production of estradiol, contributes to the delay and

reduced magnitude of the LH surge and a delayed or absence of ovulation (Dobson et al.,

1999; Dobson et al., 2000). Phogat (1997; 1999) provided evidence that the effect of

increased concentrations of ACTH either exogenously, or after transport, reduced the

amounts of LH released after challenges with small doses of GnRH, providing support

for additional effects at the pituitary level. Dobson (2000) proposed that in situations such

as during chronic stress of severe lameness or fever, the pulse GnRH/LH frequency will

be so slow that initial follicular growth will occur but will be unable to continue into the

later stages that depend on faster pulse frequencies. Thus the animal fails to maintain an

estrus cycle developing anestrous.

Another hypothesis is that the effect of lameness on cyclicity could be driven by

endotoxins released after an event of ruminal acidosis (Nocek, 1997). As shown by Peter

(1990) increases in cortisol concentrations after the infusion of endotoxin might block the

synthesis of estradiol at ovarian level resulting in failure of a preovulatory LH surge. This

may lead to anovulation or delayed ovarian cyclicity. Supporting the effects of

endotoxins, Bataglia (1997) found that the suppressive effects of endotoxins on the

reproductive axis can be mediated centrally through an inhibition of GnRH and thus LH

pulsatile secretion. Ruminal acidosis has been identified as risk factor for lameness

(Nocek, 1997; Hoblet et al., 2001). So if lame cows are suffering from acidosis with

endotoxin released by gram-negative bacteria, endotoxins can mediate the effect of






79


lameness in delayed resumption of ovarian cyclicity. At this point these hypotheses are

only speculative and need further research to be proved.















CHAPTER 6
CONCLUSION

Analysis of results of the study reported here support the hypothesis that lameness

has a detrimental effect on ovarian activity in Holstein cows during the early post partum

period. The locomotion scoring system used in this study is a useful management tool

that veterinarians and dairy farmers can adopt for early detection of lameness in dairy

cows. The use of corrective foot trimming techniques in moderately lame cows may help

reduce the risk of delayed ovarian cyclicity associated with the more severe forms of

lameness (i.e., score = 4 and 5) in dairy cows.
















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