2009: Ecbolic and tocolytic agents in bovine reproduction Ecbolic and tocolytic agents in bovine reproduction 1 M. A. Edmondson 2 Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, 3 Auburn, AL, USA 4 Abstract 5 The use and efficacy of ecbolic and tocolytic agents to treat and manage 6 reproductive conditions in the bovine have been controversial topics at times. This is in 7 part due to our limited understanding and lack of research with regard to the pregnant and 8 post partum uterus in the cattle. This article reviews the anatomy and physiology of the 9 bovine uterus and research evaluating the use and efficacy of various ecbolic and 10 tocolytic compounds. 11 Keywords: Bovine uterus, ecbolic, tocolytic 12 Introduction 13 Ecbolic agents have been administered in an attempt to treat and prevent many 14 reproductive conditions in the periparturient cow which most commonly include retained 15 fetal membranes, post-partum endometritis and metritis. Tocolytic agents have been used 16 to inhibit uterine contractility in a variety of clinical situations which include delaying 17 parturition and facilitating obstetrical manipulations. To further understand the uses and 18 possible roles of ecbolics and tocolytics in bovine practice, one must first understand the 19 anatomy and physiology of the uterus and the pharmacology of these agents. 20 Anatomy and physiology of the uterus 21 The uterine wall is composed of three layers. The innermost layer, the 22 endometrium, lines the lumen of the organ and consists of columnar epithelium and 23 underlying stromal tissues. The middle layer, the myometrium, which consists 24 predominately of smooth muscle cells, also contains blood and lymph vessels, nerves, 25 309 immune cells, and connective tissue. The outer layer, the serosa, is a thin layer which 26 covers most of the uterus and is composed of mesothelial cells. The individual smooth 27 muscle cells of the myometrium are the physiologic units of uterine contraction. The 28 muscle fibers of the outer longitudinal layer are arranged parallel and those of the inner 29 circular layer are arranged concentrically around the long axis of the uterus. Advancing 30 gestation is accompanied with hypertrophy and hyperplasia of the uterine smooth muscle. 31 The increase in size can be up to three- to fivefold by the end of gestation.1,2 32 Smooth muscles are innervated by the sympathetic nervous system and have α 33 and β receptors. The α receptors are responsible for muscular contractions while the β 34 receptors are responsible for relaxation. The β1 receptors are confined to the heart, 35 adipose tissue, and small intestine. Stimulation of β1 receptors leads to increased cardiac 36 automaticity, positive chronotropic and inotropic effects, and elevated free fatty acids. 37 The β2 receptors are found in the smooth muscle of the uterus (myometrium), vascular 38 smooth muscle, and bronchioles. Stimulation of β2 receptors causes relaxation of the 39 uterus, vasodilation, and bronchodilation. Activation of these β2 receptors leads to an 40 elevation of in cAMP, mediated through adenylate cyclase. These increased levels of 41 cAMP prevent myosin light-chain kinase (MLCK) activity through both decreased 42 phosphorylation and inhibition of release of stored intracellular calcium thus inhibiting 43 uterine contraction.1,2 44 Uterine contractility 45 The contractile activity of the uterus is directly related to the electrical activity in 46 the smooth muscle cells. This activity is characterized by cyclic depolarization and 47 repolarization of the plasma membrane and action potentials. Contraction of the smooth 48 310 muscle cells occurs by the interaction of the myofilaments which are composed of 49 myosin and actin. The sarcoplasmic reticulum also plays a key role as it is the site of 50 calcium storage. In the resting stage, the intracellular calcium in the smooth muscle is 51 low. Contraction of the smooth muscle is preceded by an increase in free intracellular 52 calcium levels. Calcium ions bind with calmodulin which then activates myosin kinase. 53 Myosin kinase in turns phosphorylates a myosin head. The phosphorylated myosin head 54 then binds with an actin filament, thus inducing smooth muscle contraction.2 55 During pregnancy, the uterine smooth muscle is relatively quiescent, displaying 56 weak, localized, and poorly coordinated contractions. In contrast, during parturition, the 57 contractions are forceful, sustained, regular, and well-synchronized. Factors responsible 58 for initiating the process of parturition include 1) an increase in the number of gap 59 junctions which allows cell coupling and interactions, 2) decreased production of nitric 60 oxide thereby inhibiting uterine relaxation, and 3) stretch of the myometrium which 61 enhances contractility.2 62 Ecbolic agents (oxytocics) 63 Ecbolic agents or oxytocics are compounds that hasten uterine evacuation by 64 stimulating uterine contractions of the myometrium. The best known and most widely 65 used ecbolics are oxytocin and prostaglandins (PG). Ecbolics have been used for the 66 treatment of retained placentas and for the treatment and prevention of metritis. 67 Oxytocin 68 In 1906, Sir Henry Dale reported that an extract of the neurohypophysis had 69 oxytocic effects. The use of this extract to induce labor was first reported as early as 70 1911. The name given to this compound was oxytocin, derived from the Greek word 71 311 meaning “swift birth.” Oxytocin is a neuropeptide hormone produced by magnocellular 72 cells in the hypothalamus, transported to the posterior pituitary via axons, and stored and 73 secreted by the posterior pituitary. Another major function of oxytocin in mammals is 74 stimulation of milk letdown from the mammary glands. In the 1950s Du Vigneaud and 75 others established the structure and synthesized oxytocin. Oxytocin was the first peptide 76 to be synthesized.3-5 77 Oxytocin is a potent stimulus for uterine contractions. The action of oxytocin is 78 mediated by binding to specific oxytocin receptors present in the uterus when the uterus 79 is under the influence of estrogen. Thus oxytocin is considered to be effective in causing 80 uterine contraction and uterine evacuation if administered within 48 to 72 hours 81 postpartum. The binding of oxytocin to its receptors causes an increase in intracellular 82 calcium which results in contraction of myometrial cells from the uterus. Oxytocin has 83 three specific actions on the uterus which include 1) induction of myometrial 84 contractions, 2) release of PGF2α from the endometrium, and 3) release of PGE2 from 85 cervical mucosa. Most formulations of oxytocin contain 20 United States Pharmacopeia 86 (USP) units/mL with package inserts recommending up to 100 USP units. This dosage 87 recommendation seems high considering that 1.0 IU of oxytocin achieves physiologic 88 levels comparable to those seen during milking. Thus, an oxytocin dosage of 10 IU is a 89 supra-physiologic dosage.6 90 Prostaglandins 91 Prostaglandins were discovered in the early 1930s as substances present in human 92 seminal plasma that induced, and sometimes relaxed, uterine muscle activity in vitro. 93 Von Euler named these substances prostaglandins believing that they came from the 94 312 prostate gland. Although, it was later discovered that the seminal vesicles were the major 95 source of prostaglandins; thus, prostaglandins is really a misnomer. In 1957, Bergstrom 96 and Sjovall reported the first isolation of prostaglandins and determined their structures. 97 By the 1960s, Sammuelsson and colleagues began describing the prostaglandin metabolic 98 pathways. In 1971, Vane reported that aspirin inhibited PG production and later showed 99 that non-steroidal anti-inflammatory drugs acted via PG inhibition. Bergstrom, 100 Sammuelsson, and Vane were awarded the Nobel Prize in 1982 for their work with PGs.5 101 When biologically active PGs are released into the blood they are metabolized by 102 enzymes in the liver, kidney, and particularly the lung. After one passage of PGF2α 103 through the lungs, over 90% of the PG is metabolized. This is one reason why most PGs 104 have very short half-lives in blood (<1 min). In addition to the lung, the placenta also has 105 very high concentrations of PG dehydrogenase. Thus, it is very unlikely the biologically 106 active PGs can cross the placenta and affect the fetus. Prostaglandins are capable of 107 causing muscle relaxation and muscle contraction depending upon the branch of the 108 receptors (relaxant or stimulatory receptors). The relaxant receptors act via adenylate 109 cyclase to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) and 110 phosphokinase A (PKA) activity. This in turns relaxes smooth muscle via elevated 111 cAMP which induces sequestration of intracellular calcium. The stimulant receptors act 112 via enhancing intracellular phosphokinase C (PKC) activity and intracellular calcium. 113 The activity through this receptor stimulates smooth muscle contraction by elevating 114 intracellular calcium levels. This discussion will focus on the stimulatory effects of PGs, 115 specifically PGF2α, on the uterus.5,6 116 313 During the immediate postpartum period, serum concentrations of PGF2α and its 117 metabolites are elevated. These elevations are thought to facilitate uterine involution. 118 Prostaglandin F2α has many actions, including luteolysis, stimulation of myometrium, and 119 constriction of blood vessels. In addition, PGs also have a relaxant effect on the cervix. 120 Thus, PGF2α is used to treat a variety of medical conditions. These conditions include 1) 121 induction of parturition, 2) lysis of corpus luteum (CL) for cases of pyometra, 3) lysis of 122 CL for management of the estrous cycle, 4) induction of abortion, and 5) evacuation of 123 the uterus in cases of metritis or endometritis. Prostaglandins can be used to induce 124 parturition and abortion by its uterotonic effect and by its luteolytic effect. 125 Prostaglandins used in veterinary medicine include cloprostenol sodium, dinoprost 126 tromethamine, fenprostalene, fluprostinol sodium, alfaprostol, and luprostiol.6 127 Cloprostenol sodium. Cloprostenol (Estrumate®, Intervet/Schering-Plough 128 Animal Health Corp., Summit, NJ, USA) is a powerful luteolytic agent and causes rapid 129 regression of the CL and arrests its secretory activity. Cloprostenol is available in a 130 concentration of 250 mcg/mL and is a PG analog that is administered by intramuscular 131 injection for all indications in both beef and dairy cattle. Cloprostenol is used in beef or 132 dairy cattle to induce luteolysis. It is recommended by the manufacturer for unobserved 133 or undetected estrus in cows cycling normally, pyometra or chronic endometritis, 134 expulsion of mummified fetus, luteal cysts, induced abortions after mismating and to 135 schedule estrus and ovulation for controlled breeding.7 136 Dinoprost tromethamine. Dinoprost tromethamine (Lutalyse®, Pfizer Animal 137 Health, New York, NY) is the naturally-occurring PGF2α as the tromethamine salt. Each 138 mL contains 5mg of dinoprost and is luteolytic in cattle at 25 mg (5 mL) administered 139 314 intramuscularly. Dinoprost tromethamine is labeled for estrus synchronization, treatment 140 of unobserved (silent) estrus and pyometra (chronic endometritis) in cattle; and for 141 abortion of feedlot and other non-lactating cattle.7 142 Other PGs. Alfaprostol and luprostiol are prostaglandin analogs that are used 143 mainly outside of the United States. These drugs have similar effects and uses as the 144 other synthetic prostaglandins. 145 Xylazine 146 Xylazine produces uterine contraction by stimulation of α2-adrenergic receptors in 147 the uterus. The pregnant bovine uterus appears to be more sensitive to xylazine-induced 148 contractility than the non-pregnant uterus, particularly after 270 days of gestation. This 149 enhanced susceptibility may involve hormone-related changes in α-adrenergic receptor 150 populations in myometrial tissue. In one study, administration of xylazine (10mg 151 intravenously) significantly increased uterine motility during late gestation. Thus, the use 152 of xylazine in the last month of gestation is contraindicated in cattle because of the 153 increased tendency for induction of premature parturition. Xylazine has no practical uses 154 as an ecbolic due to its sedative and muscle relaxant properties.6,7 155 Estrogen 156 Estrogen has been used in an attempt to initiate and/or strengthen myometrial 157 contractions. However, the use of estrogen is controversial. Because estrogen levels 158 normally decrease dramatically once the calf is expelled, it appears that normal uterine 159 involution can proceed without the influence of estrogen in the normal cow. Studies have 160 shown no beneficial effects on the prevention of metritis or reproductive performance, 161 and that the use of estrogen may actually have a negative effect on subsequent fertility.8 162 315 It is believed that contractions induced by estrogen may force septic uterine contents not 163 only through the cervix but also into the uterine tubes which results in severe bilateral 164 salpingitis.9 Research has shown that estrogen treatment postpartum has a negative 165 impact on uterine motility where the normal uterine contractions changed to a sustained 166 contraction or spasm.8 In addition, one study demonstrated that the use of oxytocin in an 167 estrogen-primed uterus did not increase the contraction frequency and thus did not 168 enhance the myometrial effect of oxytocin. Thus, scientific evidence does not support 169 the use of exogenous estrogens in the postpartum cow. 170 Use of ecbolics to treat retained fetal membranes and uterine infections 171 Two common problems that are encountered in periparturient dairy cattle and 172 occasionally in beef cattle are retention of fetal membranes and metritis. Retained fetal 173 membranes is one of the most important factors that predisposes cattle to uterine 174 infections. Cattle that have retained fetal membranes are six times more likely to develop 175 a uterine infection than are cows without retained fetal membranes. Primary retention of 176 the fetal membranes results from lack of detachment from the maternal caruncles, 177 whereas secondary retention is related to mechanical difficulty in expelling the already 178 detached fetal membranes. Greater than three-fourths of cows expel the placenta by 6 179 hours post-partum with the majority of the remaining cows expelling the placenta before 180 12 hours postpartum. Because the incidence of retained fetal membranes and postpartum 181 disease varies with parity, the definition of retained fetal membranes may also be age- or 182 parity-dependent. Suggestions have been made to define retained fetal membranes from 183 8 to 48 hours; however, 12 hours is widely used to define retained fetal membranes.9 184 316 Detachment of the placenta in the cow involves separation of the cotyledon villi 185 from the caruncular crypts without tearing of either fetal or maternal epithelia. For 186 appropriate separation of the cotyledon villi from the caruncular crypts, proteolytic 187 enzymes (collagenases) act to open the cotyledon thereby releasing the caruncle. 188 Collagenase activity of cotyledon villi during delivery is increased in healthy cows and 189 decreased in cows with retained fetal membranes. The cellular sources of collagenase 190 and proteolytic enzymes responsible for placental release in the cow are unknown. 191 However in laboratory animals and humans, myometrial cells, fibroblasts, and leukocytes 192 have been identified as sources of collagenase in the uterus. By day 6 postpartum, the 193 caruncle is disorganized; by day 15, caruncles are completely sloughed as a result of 194 necrosis. Retained fetal membranes are detached by caruncle necrosis within 6 to 10 195 days and not later than 17 days postpartum. The surface of the endometrium is covered 196 by new epithelium by day 26 to 30 postpartum. After placental detachment, uterine 197 involution is completed in about 39 days in normal cows and 50 days in cows with 198 retained fetal membranes. Lack of uterine motility is not considered a reason for primary 199 retention because uterine motility is normal or above normal in cows with retained fetal 200 membranes.9 201 Factors reportedly contributing to the development of retained fetal membranes 202 include periparturient hypocalcemia, dystocia, abortion, twinning, stillbirth, and 203 induction of parturition. There are several management approaches that have been used 204 for cows with retained fetal membranes of which many are controversial and lack 205 scientific evidence to support their use. These options include no intervention, manual 206 removal of the fetal membranes, antimicrobial therapy, and hormone therapy. No 207 317 intervention in an otherwise healthy cow is a common practice that allows the fetal 208 membranes to liquefy and necrose until they are passed. These cows should be 209 monitored closely for any signs of septicemia or toxemia in which case systemic therapy 210 is necessary. Manual removal of the fetal membranes was once practiced but has fallen 211 from favor due to the likelihood of causing trauma to the uterine wall, the high incidence 212 of leaving tags of the fetal membranes within the uterus, and iatrogenic contamination of 213 the uterus. Intrauterine antibiotic therapy is beyond the scope of this discussion but will 214 be discussed in a subsequent presentation. Hormone therapy is still one of the most 215 common methods for managing cows with retained fetal membranes.6,9,10 216 The majority of cattle experience bacterial contamination of the uterus at the time 217 of parturition. In the normal cow, the uterus is cleared of this bacterial contamination by 218 four weeks postpartum. When these bacteria are not cleared by the cow’s defense 219 mechanisms, a uterine infection ensues. Numerous bacteria have been isolated from the 220 cow’s postpartum uterus, some of which may be incidental and not cause problems. 221 Uterine infections are most commonly due to Arcanobacterium pyogenes. The gram 222 negative anaerobes Fusobacterium necrophorum and Bacteroides melaninogenicus are 223 frequently associated with A. pyogenes. Other organisms that may be associated with 224 uterine disease in the cow include Pseudomonas aeruginosa, staphylococci, hemolytic 225 streptococci, coliforms, etc. Clostridium sp. may occasionally infect the uterus and cause 226 a severe gangrenous metritis or tetanus. Uterine infections are associated with retained 227 fetal membranes, dystocia, and delivery of twins. Metritis is the result of severe 228 inflammation involving all layers of the uterus – endometrial mucosa and submucosa, 229 muscularis, and serosa. Metritis usually develops during the first week after calving and 230 318 is associated with dystocia, retained fetal membranes, and calving trauma. Affected 231 cattle may be septic and present with fever, depression, and anorexia and a copious fetid 232 vaginal discharge may also be present. Endometritis is characterized by inflammation of 233 the endometrium extending no deeper than the stratum spongiosum. Cows with 234 endometritis are usually not systemically ill, and bacteria are usually eliminated after a 235 few estrous cycles. Pyometra is a collection of purulent exudate within the uterus with 236 the persistence of the corpus luteum, and suspension of the estrous cycle. Pyometra 237 usually develops in cows that have their first postpartum ovulation before bacterial 238 contamination of the uterus has been eliminated. The corpus luteum that is associated 239 with the infection persists because intrauterine fluid prevents luteolysis. Thus 240 progesterone persists and suppressed uterine defense mechanisms.9,10 241 Several different hormones have been used in an attempt to manage retained fetal 242 membranes and uterine infections with the ecbolic agents oxytocin and PG being the 243 most common. Oxytocin appears to stimulate myometrial contraction by 1) direct 244 activation of receptors on myometrial cells and 2) indirect stimulation of contraction 245 through the release of stimulatory PGs from the endometrium. Circulating oxytocin 246 binds to myometrial receptors which leads to rapid uterine contraction and an increase in 247 PGF2α levels. It is believed that PGF2α stimulates the release of more oxytocin and also 248 enhances the sensitivity of the myometrium to oxytocin.3 As little as 2.5 IU of oxytocin 249 intravenously will cause the proximal ends of the uterine horns to respond within 30 to 50 250 seconds when progesterone levels are low in a cycling cow, and this increase in 251 myometrial activity persists for up to 80 minutes.11 Studies such as this in cycling cows 252 have supported the idea that the myometrium is only responsive to oxytocin when 253 319 estrogen is dominant; whether oxytocin is effective in cows with toxic metritis is 254 unclear.6 One study indicated that as little as 5 units of oxytocin intravenously can 255 initiate a more intense rhythm of contraction in cows with retained fetal membranes.12 256 Other studies refute this evidence and suggest that oxytocin was of no benefit to 257 postpartum cows with retained fetal membranes; however, in these two studies a dose of 258 60 to 100 units of oxytocin was administered which causes a spasm of the uterus versus a 259 progressive contraction.13-15 It also appears that the traditional dose of oxytocin (40 260 units) when administered intravenously causes an initial tetanic spasm of the uterus.16 261 Most of the studies demonstrating the positive effect of exogenous oxytocin have used 262 the intravenous route of administration instead of the more commonly used intramuscular 263 route of administration. However, one study did show that the myometrial response 264 following administration of 20 to 30 units of oxytocin was similar following 265 administration via intravenous, intramuscular, and subcutaneous routes of 266 administration.16 A day two to three protocol of repeated 20 unit (1.0 mL) oxytocin 267 injections administered at least three hours apart or three doses evenly spaced between 268 milkings, etc has been suggested.6 Although the frequent administration with low dose 269 oxytocin appears to be impractical in most situations, it would appear to induce a more 270 physiologic response than current therapeutic protocols which use infrequent 271 administration at supra-physiologic dosages which induce tetanic uterine spasms. The 272 most physiologic uterotonic dose of oxytocin has not been determined. 273 Despite much research, the ability of exogenous PGs to have a direct effect on 274 periparturient uterine activity in cattle has been a controversial issue among researchers 275 and clinicians. Although a few studies indicate that PGF2α may reduce the incidence of 276 320 retained fetal membranes, subsequent studies have failed to confirm these results and 277 many report that exogenous PG has no effect. Many of these studies lack sufficient 278 numbers of animals, lack control animals, and used concurrent medications which make 279 interpretation of the results difficult. It appears that suboptimal uterine contraction is 280 rarely the cause of retained fetal membranes in a nontoxic cow. Studies have shown that 281 the presence of retained fetal membranes alone doubles the rate and increases the 282 frequency of uterine contractions. In another study, cows that had evidence of uterine 283 infection (fetid, sanguine-purulent lochia) at up to day 15 post partum had significantly 284 higher concentrations of PGF metabolite (PGFM) than did cows that had a mucopurulent 285 to purulent lochia.17 In addition, studies indicate that a single intramuscular injection of 286 PG before the formation of a functional corpus luteum will have no beneficial effect in 287 the post-partum cow.12,16 Even when the PG dose was doubled, there was still no 288 increase in uterine tone. However, luteolytic doses of PGF2α (25 mg) administered by 289 rapid intravenous injection on day 2 postpartum did cause an increase in uterine 290 contractions.16 However, by day four postpartum the stimulatory effect was noticeably 291 decreased. Intravenous administration of PG also has significant side effects (dyspnea, 292 salivation, milk ejection, frequent urination) that make it impractical to use particularly in 293 a toxic cow. Only when luteal tissue is present on the ovary is it widely accepted that 294 exogenous administration of PG has a beneficial effect on the postpartum cow. 295 Intramuscular injection of PGF2α may not be uterotonic because the PGF2α is metabolized 296 almost entirely into PGFM upon a single passage through the lungs. Using PG to lyse the 297 CL allows for removal of the immunosuppressive effects of luteal progesterone which 298 may aid in the resolution of chronic postpartum endometritis.6 Currently, there is no 299 321 scientific evidence that intramuscular or subcutaneous injections of either natural or 300 synthetic PGF2α aids in the expulsion of retained fetal membranes. In addition, 301 administration of PG during the immediate postpartum period has not been shown to 302 have an effect on the rate of uterine involution.18 303 Some studies indicate that the use of PG may improve overall reproductive 304 performance in cows that are not affected by periparturient diseases. In addition, cows 305 affected with dystocia, retained fetal membranes, or both that were treated with PGF2α 306 early post partum followed by a second treatment 14 days later experienced a higher 307 conception rates to first service than non-treated cows.19 308 Conclusion 309 Although more research is needed on the postpartum cow uterus, there is no 310 proven scientific evidence that supports the routine use of ecbolic agents as a treatment 311 for the pathologic postpartum uterus. Based on scientific research, exogenous estrogen 312 and PG at published doses appear unable to stimulate the appropriate rhythmic 313 contractions necessary to empty the pathologic post partum uterus. There is some 314 evidence that supports the use of exogenous oxytocin to stimulate uterine contractions 315 that are similar to those contractions observed during stage II of labor. However, these 316 studies used intravenous oxytocin rather than the more common intramuscular route of 317 administration. 318 Tocolysis 319 Tocolysis is derived from Greek with “tokos” meaning childbirth and “lysis” 320 meaning capable of dissolving. A tocolytic agent is a compound that is capable of 321 inhibiting uterine contractions. Tocolytics were originally designed for use in human 322 322 medicine to interrupt premature labor and have, over time, been used more commonly in 323 veterinary medicine. The use of tocolytics to inhibit uterine contraction has a number of 324 potential clinical applications in cattle. Delaying parturition for controlled calving may 325 be useful if parturition were occurring at a time that decreased fetal survival (nocturnal 326 delivery). Obstetrical manipulations such as correction of malpresentation and 327 malposition, repulsion and rotation of the fetus, correction of uterine torsion, ease of 328 extraperitoneal lifting of uterus during cesarean section, and replacement of uterine 329 torsion may be aided by the use of tocolytic agents. Some believe that these drugs may 330 also be useful in the area of embryo transfer. 331 Tocolytic Agents 332 A variety of tocolytics have been used for the aforementioned applications to 333 cause uterine relaxation. Ethyl alcohol, magnesium sulfate, progesterone, prostaglandin 334 synthetase inhibitors, calcium channel blockers, epinephrine, and β sympathomimetics 335 have all been used to induce uterine quiescence. However, the unpredictable efficacy and 336 adverse side effects make some of these drugs less acceptable than others for the 337 induction of uterine relaxation.20 338 Ethanol 339 In the mid 20th century, ethanol was a commonly used tocolytic agent in human 340 patients to halt pre-term labor. It is believed to effectively inhibit the secretion of 341 oxytocin and interfere with prostaglandin synthesis. Ethanol was given intravenously in 342 humans at a rate to maintain a blood alcohol level of 0.9 to 1.6 mg/liter. Of course, side 343 effects were observed which included nausea, vomiting, depression, intoxication of 344 323 mother and fetus, and acidosis. Research has since demonstrated that ethanol is not 345 effective in delaying parturition.21 346 Magnesium sulfate 347 Magnesium sulfate has also been used as a tocolytic. Magnesium sulfate is a 348 central nervous depressant which blocks neuromuscular transmission and lowers 349 acetylcholine. In 1959, the tocolytic properties of magnesium sulfate were first 350 described. The exact mechanism by which magnesium sulfate exerts its tocolytic effects 351 is unknown. However, one possible mechanism may be its ability to block nerve 352 transmission and/or by its actions as a calcium antagonist. Magnesium sulfate has been 353 given intravenously as a 10% solution to delay parturition for 24 to 48 hours. Most 354 human studies do not indicate a significant ability for magnesium sulfate to prolong 355 pregnancy. The use of magnesium sulfate as a tocolytic is no longer recommended due 356 to its lack of effect at preventing preterm deliveries and because of its association with a 357 higher risk of perinatal death.22 358 Progesterone 359 The actions of progesterone on the pregnant myometrium include relaxation of 360 myometrial smooth muscle, blocking the action of oxytocin, and inhibition of gap 361 junctions. Progesterone decreases the concentration of myometrial oxytocin receptors 362 which counteracts the effect of estrogens. Progesterone also inhibits PG production by 363 the placenta. In human research, progesterone has been found useful for the maintenance 364 of tocolysis to increase gestational age at delivery or as a preventative agent in women 365 with high-risk pregnancies.23 Progesterone has been shown to enhance the tocolytic 366 effect of some of the beta sympathomimetics (ritodrine) when used in human patients.24 367 324 Progesterone prolongs gestation length when administered during advanced pregnancy 368 and, as a result, chances of dystocia increase due to additional weight gain of the fetus. 369 Prostaglandin synthetase inhibitors 370 Prostaglandins are known to be important mediators in uterine contractility. At 371 the time of parturition, there are increased concentrations of arachidonic acid, PG E2 and 372 PGF2α. Prostaglandins increase intracellular free calcium levels which may increase the 373 frequency of uterine contractions. Thus, nonsteroidal anti-inflammatory drugs that 374 inhibit PG synthesis in the uterus have been considered for use as tocolytics. 375 Indomethacin, a product used in human medicine, has been studied, although with a small 376 sample size, in human patients and was found to delay parturition for 48 hours. 377 Indomethacin is relatively safe as far as the maternal side effects, but crosses the placenta 378 and causes concerns regarding fetal pulmonary hypertension, gastrointestinal 379 inflammation, and hemorrhage.22 Flunixin meglumine (Banamine®, Intervet/Schering-380 Plough Animal Health), has been considered for use as a tocolytic agent in cattle based 381 on its ability to block PG synthesis. Although the effects of flunixin meglumine have not 382 been evaluated in a controlled study, the label actually warns against use of flunixin 383 meglumine in late gestation as it is “known to have the potential to delay parturition 384 through a tocolytic effect”.7 One study evaluated the effect of flunixin meglumine on 385 uterine contractility on a small group of postpartum cows and concluded that flunixin 386 meglumine inhibited PG production by more than 80% and decreased spontaneous 387 uterine motility.25 The effects of flunixin meglumine on uterine contractility have not 388 been evaluated in the pregnant cow. 389 Calcium channel blockers 390 325 Calcium channel blockers have been used for their tocolytic effects in human 391 medicine since the 1980’s. Calcium channel blockers work to inhibit uterine contraction 392 by blocking the influx of calcium into the cells of the myometrium through disruption of 393 the voltage-operated calcium channels.22 Some studies indicate that nifedipine is as 394 effective as magnesium sulfate and beta agonists with fewer side effects. Limited 395 research has shown that the calcium channel blocker, nifedipine, is capable of blocking 396 xylazine-induced uterine contractions in goats. One study found that nifedipine at 80 397 mcg/kg given intravenously was able to delay parturition in sheep for six to seven 398 hours.26 Side effects associated with the use of calcium channel blockers for tocolysis 399 include fluid retention and decreased cardiac output which can result in pulmonary 400 edema.22 401 Epinephrine 402 Epinephrine is an adrenergic that has both α and β activity; therefore, it is capable 403 of relaxing smooth muscle. The use of epinephrine in veterinary medicine has been 404 primarily limited to emergency situations to treat anaphylactic shock or cardiac 405 resuscitation and because of its vasoconstrictive properties as an additive to local 406 anesthetics to decrease absorption and prolong effect.7 However, epinephrine has also 407 been administered to cattle at 10 cc per cow of the 1:1000 solution as a slow intravenous 408 infusion to cause uterine relaxation and quiescence.6,20 This use of epinephrine in cattle 409 has been primarily to facilitate obstetrical procedures and conditions such as cesarean 410 section, fetotomy, uterine prolapse, and uterine torsion. Uterine relaxation is almost 411 immediate following intravenous administration. Side effects which may be seen with 412 326 rapid administration and overdose of epinephrine include severe increases in blood 413 pressure, cardiac arrhythmias, pulmonary edema, and dyspnea.20 414 β sympathomimetics 415 As previously mentioned stimulation of β1 receptors leads to increased cardiac 416 automaticity, positive chronotropic and inotropic effects, and elevated free fatty acids 417 while stimulation of β2 receptors causes relaxation of the uterus, vasodilation, and 418 bronchodilation. Although β sympathomimetic tocolytics may be β2 selective, they do 419 retain some β1 activity which accounts for the side effects that may be observed.22 An 420 ideal β tocolytic would be completely β2 selective. However, no such drug exists. 421 Therefore, the benefit of uterine relaxation is often accompanied by side effects 422 attributable to β1 activity. Maternal side effects with β sympathomimetics are associated 423 with cardiovascular complications which include tachycardia, arrhythmias, and ischemia. 424 In humans, the most common complication is pulmonary edema which occurs in 425 approximately 5% of patients.22 426 Clenbuterol and isoxsuprine have been widely used to induce uterine relaxation. 427 Isoxsuprine was the first beta sympathomimetic used for tocolysis. One study 428 demonstrated that isoxsuprine was able to induce tocolysis within 10 to 15 minutes of 429 administration with a duration of one to 1.5 hours.27 However, the lack of discrimination 430 between β1 and β2 receptors and the resulting tachycardia have limited its use. 431 Clenbuterol is a specific β2-adrenergic agonist and thus has fewer side effects on 432 extrauterine tissues than does isoxsuprine. Clenbuterol also has the longest duration of 433 action (eight to 10 hours) of any of the β sympathomimetics.28 Terbutaline is another 434 specific β2-adrenergic agonist that has been used as a tocolytic in humans. Limited 435 327 research has demonstrated the tocolytic effects of terbutaline in rats, sheep, and buffalo, 436 and preliminary pharmacokinetic data suggests that it may be a useful tocolytic in 437 cattle.29 Terbutaline shares many of the side effects associated with other beta 438 sympathomimetics which include tachycardia, cardiac arrhythmias, muscle 439 fasciculations, hypotension, and hyperglycemia of both dam and fetus.29 Although the 440 beta sympathomimetic tocolytics do have some side effects, these side effects are 441 considered to be a minor concern, particularly when using the more specific β2-442 adrenergic agonist. Due to the more predictable efficacy and minor side effects, the beta 443 sympathomimetics are generally considered to the best choice for tocolysis.20 444 However, it is important to remember that none of these drugs are approved for 445 use in food animals in the United States. High doses of clenbuterol have been used as 446 repartitioning agents to promote protein deposition while lowering fat deposition which 447 improved carcass composition. This use of clenbuterol has been associated with acute 448 poisonings in humans who consumed meat from clenbuterol-fed animals. In 1990 in 449 Spain, 135 people had to be hospitalized after consuming tainted veal and liver. In 1994, 450 another 140 people suffered from dizziness, heart palpitations, breathing difficulty, 451 tremors, and headaches. In addition, clenbuterol was banned in the United States, 452 Europe, and Canada in 1997 due to reports of aplastic anemia due to human intoxication 453 with clenbuterol subsequent to its use as a repartitioning agent in cattle.30 The use of 454 clenbuterol in food-producing animals remains illegal in the United States. However, 455 abuse of clenbuterol in show cattle in United States has been reported.30 456 Use of tocolytic agents 457 328 Tocolytic agents have been used historically to treat or to assist in the treatment of 458 numerous conditions in cattle. These uses include 1) threatened abortion/preterm labor, 459 2) controlled calving/nocturnal delivery, 3) reduction in neonatal morbidity and mortality 460 associated with dystocia, 4) aid in obstetrical operations such as cesarean section and 461 fetotomy, 5) treatment of uterine prolapse, 6) treatment of uterine torsion, and 7) embryo 462 technologies. Research on the use of tocolytics in ruminants has revealed several factors 463 that may affect tocolysis. The parity of animal may be important to consider as heifers 464 were found to respond faster to clenbuterol and have a longer duration of tocolysis when 465 compared to cows.31 The amount of cervical dilation and the position of the fetus may 466 also have an affect on tocolyis. In animals where the cervix was fully dilated or fetal feet 467 were found to be passing into cervical area, clenbuterol was only able to delay labor for a 468 maximum of a few hours.32 Another study evaluated the use of clenbuterol for 469 postponing parturition at various stages in cows. This study found that cows treated 470 during the second stage of labor postponed calving by two hours. In addition, cows 471 treated during the first stage of labor calved 5.2 to 9.7 hours later than control animals.33 472 Another study evaluated the use of clenbuterol in beef heifers. This study found that 473 administration of clenbuterol during the first stage of labor (cervical dilation of 5 cm) was 474 able to delay parturition by increasing the length of the first stage of labor with no 475 adverse effects on the fetus or the dam.28 One report suggests that the use of clenbuterol 476 will reduce neonatal morbidity and mortality in dystocia, will aid in obstetrical operations 477 such as cesarean section and fetotomy. The authors of this study also reported less 478 requirement of epidural anesthesia when clenbuterol was administered versus controls, 479 easier correction of malpresentation and malposition, correction of uterine torsion and 480 329 uterine prolapse, and no increase in the incidence of retained fetal membranes in bovine 481 dytocias.34 Another study evaluated the use of clenbuterol in seventeen cows undergoing 482 cesarean section and concluded that the use of clenbuterol in these animals resulted in 483 decreased uterine tone to the uterus which allowed for easier exteriorization and suturing 484 of the uterus.35,36 485 Conflicting reports exist as to the usefulness of tocolytics with regard to embryo 486 technologies. Some have speculated that relaxation of the uterus would improve embryo 487 recovery when used in donor animals and increase pregnancy rates in the recipient 488 animals. However, research and subjective evaluation have not shown any significant 489 improvement in recovery rates of embryos or pregnancy rates in recipients. 490 Conclusions 491 It is important to remember that the much of the research on the efficacy and side 492 effects of tocolytic drugs have been conducted in the field of human medicine. There are 493 limited studies in ruminants which are burdened by the lack of critical evaluation and 494 subjective interpretation of the studies’ results. Among the β-sympathomimetic agents 495 used in reproduction, only clenbuterol and isoxsuprine have been widely used in clinical 496 management of obstetrical disorders apart from embryo biotechnology with encouraging 497 therapeutic results. The efficacy of these drugs is mostly assessed clinically. However 498 successful the beta sympathomimetics may be as tocolytics, their use is still extra-label 499 and the use of clenbuterol is illegal. Thus more research regarding new tocolytic agents, 500 efficacy, and the pharmacokinetics of these drugs needs to be studied in detail to ensure 501 wide use with awareness of adverse effects of drug metabolites, if any. 502 503 330 References 504 505 1. Dailey RA: Female reproductive system, nonhuman mammal. In: Knobil E, Neill J, 506 editors. Encyclopedia of reproduction, vol. 2. St. Louis: Elsevier; 1998. p. 229-239. 507 2. Jain V, Saade GR, Gartfield RE: Uterine contraction. In: Knobil E, Neill J, editors. 508 Encyclopedia of reproduction, vol. 4. St. Louis: Elsevier; 1998. p. 932-942. 509 3. DeVries AC, Carter CS: Oxytocin. In: Knobil E, Neill J, editors. Encyclopedia of 510 reproduction, vol. 3. St. Louis: Elsevier; 1998. p. 630-634. 511 4. Irwin JC, Giudice LC: Decidua. In: Knobil E, Neill J, editors. Encyclopedia of 512 reproduction, vol. 1. St. Louis: Elsevier; 1998. p. 823-835. 513 5. Mehendale R, Wilson L: Oxytocics. In: Knobil E, Neill J, editors. Encyclopedia of 514 reproduction, vol. 3. St. Louis: Elsevier; 1998. p. 620-629. 515 6. Frazer GS: A rational basis for therapy in the sick postpartum cow. Vet Clin North 516 Am Food Anim Pract 2005; 21:523-568. 517 7. Plumb DC: Veterinary drug handbook. 6th ed. Hoboken: Wiley; 2008. 518 8. Burton MJ, Dzuik HE, Fahning ML, et al: Effects of oestradiol cypionate on 519 spontaneous and oxytocin-stimulated postpartum myometrial activity in the cow. Br 520 Vet J 1990;146:309-315. 521 331 9. Risco CA, Youngquist RS, Shore MD: Postpartum uterine infections. In: 522 Youngquist RS, Threflall WR, editors. Current therapy in large animal 523 theriogenology. 2nd ed. St. Louis: Saunders; 2007. p. 339-344. 524 10. Smith BI, Risco CA: Management of periparturient disprders in dairy cattle.Vet 525 Clin North Am Food Anim Pract 2005;21:503-521. 526 11. Ruckebusch Y, Bayard F: Motility of the oviduct and uterus of the cow during the 527 estrous cycle. J Reprod Fertil 1975;43:23-32. 528 12. Kundig H, Thun R, Zerobin K: Uterine motility in the cow during late pregnancy, 529 parturition and peurperium. II. Drug influence. Schweiz Arch Tierheilkd 530 1990;132:515-524. 531 13. Hickey GJ, White ME, Wickenden RP: Effects of oxytocin on placental retention 532 following dystocia. Vet Rec 1984;114:189-190. 533 14. Miller BJ, Lodge JR: Effect of oxytocin on retained placentas. J Anim Sci 534 1981;53:350. 535 15. Miller BJ, Lodge JR: Postpartum oxytocin treatment for prevention of retained 536 placentas. Theriogenology 1982;17:237-243. 537 16. Burton MJ: Uterine motility in periparturient dairy cattle [dissertation]. St. Paul: 538 University of Minnesota; 1986. 539 332 17. Mateus L, Lopes da Costa L, Diniz P, et al: Relationeship between endotoxin and 540 prostaglandin (PGE2 and PGFM) concentrations and ovarian function in 541 dairy cows with puerperal endometritis. Anim Reprod Sci 2003;76:143-154. 542 18. Thatcher WW: Role of prostaglandin during the periparturient period in the cow. 543 Proc Annu Meeting Soc Therio 1988; p. 55. 544 19. Risco CA, Hernandez J: Comparison pof ceftiofur hydrochloride and estradiol 545 cypionate for metritis prevention and reproductive performance in dairy 546 cows affected with retained fetal membranes. Theriogenology 2003;60:47. 547 20. Gilbert RO, Schwark WS: Pharmacologic considerations in the management of 548 peripartum conditions in the cow. Vet Clin North Am Food Anim Pract 549 1992;8:29-56. 550 21. Keirse MJ: The history of tocolysis. BJOG 2003;110:94-97. 551 22. Rode ME, Macones GA: Tocolytic agents. In: Knobil E, Neill J, editors. 552 Encyclopedia of reproduction, vol. 4. St. Louis: Elsevier; p. 813-819. 553 23. O'Brien JM, Lweis DF: Progestins for the prevention of spontaneous preterm birth: 554 review and implications of recent studies. J Reprod Med 2009;54:73-87. 555 24. Chanrachakul B, Pipkin FB, Warren AY, et al: Progesterone enhances the tocolytic 556 effect of ritodrine in isolated pregnant human myometrium. Am J Obstet 557 Gynecol 2005;192:458-463. 558 333 25. Thun R, Kundig H, Zerobin K, et al. Uterine motility of cattle during late 559 pregnancy, labor, and puerperium. III. Use of flunixin meglumine and 560 endocrine changes. Schweiz Arch Tierheilkd 1993;135:333-344. 561 26. Perez R, Garcia M, Arias P, et al: Inhibition of xylazine induced uterine 562 contractility by clenbuterol and nifedipine. Res Vet Sci 1997;63:73-76. 563 27. Horvath G, Bacsfay N. Experience with the use of a uterine muscle relaxant 564 preparation. Acta Vet Acad Sci Hung 2009;29:65-69. 565 28. Putnam MR, Rice LE, Wettemann RP, et al: Clenbuterol (planipart) for the 566 postponement of parturition in cattle. Theriogenology 1985;24:385-393. 567 29. Boileau MJ, Washburn KE, Clarke CR, et al: Terbutaline pharmacokinetics in 568 cows: preliminary data. Can Vet J 2007;71:70-73. 569 30. Mitchell GA, Dunnavan G: Illegal use of B-adrenergic agaonists in the United 570 States.J Anim Sci 1998;76:208-211. 571 31. Zerobin K, Kundig H: The control of myometrial functions during parturition with a 572 beta 2-mimetic compound (planipart).Theriogenology1980;14:21-35. 573 32. Arbeiter K, Holler W: Control of birth: about the influence on partus, puerpartum, 574 and rate of conception following the injection of 575 flumethasone/dexamethasone and planipart in cattle. Dtsch Tierarztl 576 Wochenschr 1980;87:249-251. 577 334 33. Greene HJ: Clinical study of the use of clenbuterol for postponing parturition in 578 cows. Vet Rec 1981;109:283-285. 579 34. Menard L: The use of clenbuterol in large animal obstetrics: manual correction of 580 bovine dystocias. Can Vet J 1994;35:289-292. 581 35. DeNooij PP: The use of clenbuterol for obstetrical procedures in forty cows and one 582 horse. Can Vet J 1984;25:357-359. 583 36. Hassett LJ, Sloss V: The use of clenbuterol to produce relaxation of the 584 myometrium during caesarean operation in cattle. 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