Categories
Veterinary Medicine

Patent ductus arteriosus

In the fetus, the ductus arteriosus functions to bypass the pulmonary circulation. It is a short arterial connection arising from the sixth aortic arch which carries blood from the pulmonary artery to the aorta and systemic circulation. The ductus normally closes in the first few weeks after birth to form the ligamentum arteriosum. Closure is regulated by prostaglandin synthetase inhibition and also by the changes which occur in blood oxygen saturation.

Patent ducius arteriosus (PDA) occurs as a graded defect varying in severity according to the diameter of the dueial lumen. In some cases a ductus diverticulum forms when the ductal lumen closes at its pulmonary artery end but remains open over the rest of its length.

Pathophysioiogy

A ductus which remains patent after birth allows the shunting of blood during systole and diastole from the high pressure in the aorta to the pulmonary artery. patent ducius arteriosus therefore represents an artcriovenous fistula since oxygenated blood from the aorta mixes with venous blood of the pulmonary artery. Shunting of blood through the right side of the heart leads to pulmonary overcirculation, left ventricular volume overload, pulmonary venous and arterial hypertension and ultimately signs of left-sided congestive heart failure. Left ventricular and left atrial enlargement leads to mitral regurgitation which contributes to the volume overload and pulmonary venous engorgement. Pulmonary hypertension leads to pressure overload in the right ventricle and the direction of the shunt can reverse. The incidence of left heart failure and pulmonarv hypertension, and the rapidity of onset of clinical signs, is associated with the size of the ductal lumen. Occasionally patent ducius arteriosus occurs in association with other congenital cardiac defects, for example ventricular septal defect and pulmonic stenosis.

The incidence is highest in miniature poodles. German shepherds. Border collies. Shetland sheepdogs, pomeranians and Irish setters and a predisposition for females has been reported. A polygenic mode of inheritance is suspected in most of these breeds.

Clinical signs

Most dogs with patent ducius arteriosus show clinical signs before one year of age and only a few cases reach adulthood undiagnosed. The clinical signs are those of left-sided heart failure. A few animals may experience syncopal episodes. Advanced cases may show signs of biventricular failure. The characteristic feature of patent ducius arteriosus is the presence of a continuous ‘machinery-type’ murmur over the aortic / pulmonic valve region which may radiate to the thoracic inlet. Most * machinery-type* murmurs are confined to a very narrow region and are associated with a palpable precordial thrill over the cranial thorax; in many cases an additional systolic murmur associated with mitral regurgitation can be located over the mitral valve region. The femoral pulse becomes jerky (‘water hammer’ pulse) because of the sharp fall off in arterial pulse pressure.

Once pulmonary hypertension develops the direction of the shunt reverses. Initially the diastolic component of the murmur disappears but as the shunt reverses the murmur may disappear completely. Shunt reversal is associated with differential cyanosis. Since the patent ductus joins the aorta distal to the aortic arch the blood supply to the head and neck is preserved and only the caudal extremities become cyanotic giving rise to hindlimb weakness.

Electrocardiograpity

Wide P waves, tall R waves and prolonged QRS complexes reflect left atrial and left ventricular enlargement. The presence of deep Q waves and S waves in leads I, II, III and aVF is indicative of right ventricular enlargement. T waves are often deep and negative in leads II, III and aVF. Arrhythmias (atrial fibrillation and ventricular premature complexes) may be noted especially in older dogs showing severe signs of decompensation. The mean electrical axis, in most cases, is within normal limits.

Radiographic findings

Left atrial and left ventricular enlargement is usually associated with signs of pulmonary over-circulation (enlargement of both the pulmonary arteries and veins) and pulmonary oedema. Classically, three knuckles or bulges may be present on the dorsoventral projection ; these represent (I) the dilated aortic arch at the one o’clock position, (2) the enlarged pulmonary artery segment at the two o’clock position and (3) the enlarged left auricular appendage at the three o’clock position. Pulmonary hypertension may result in right ventricular enlargement.

Echocardiography

Echocardiography will confirm left atrial and left ventricular enlargement. In advanced cases there may be evidence of right ventricular enlargement and chronic volume over load may result in decreased left ventricular contractility. Sepcal motion may be exaggerated. The patent ductus is often difficult to image. Continuous flow disturbance or turbulence and high velocity retrograde flow toward the pulmonic valve can be detected if a pulsed Doppler sample gate is placed in the main pulmonary artery.

Angiocardiography and intracardiac pressure studies

A selective injection of contrast material into the ascending aorta or aortic root should result in simultaneous opacification of the pulmonary artery and aorta. A non-selective study, using a large diameter intravenous catheter placed into the jugular vein will demonstrate a left to right shunting patent ducius arteriosus only after contrast has reached the aorta; for this reason serial radiographs should be taken 1,5 and 10 s after the injection of the contrast agent. Pressure studies can be performed beforehand. Pressures in the right ventricle and pulmonary artery are increased with the pulmonary artery pressure being greater than that in the right ventricle. The pressures in the left heart may be normal. Pulmonary artery PO2, is usually increased and is greater than the PO2 in the right ventricle.

Prognosis

The prognosis for a young animal showing no clinical or radiographic signs of heart failure is good and surgical correction of the patent ducius arteriosus may result In near normal life expectancy. The prognosis becomes less favourable for dogs showing signs of cardiac decompensation, especially where echocardiography demonstrates decreased myocardial contractility.

Patent ductus arteriosus: Treatment

Treatment of patent ducius arteriosus usually involves double libation of the patent vessel. A large diameter patent ducius arteriosus may occasionally recanalize after 11 gat ion. Surgery is generally contraindicated if there is evidence of a right to left shunt. Congestive heart failure should be managed appropriately and the animal stabilized before surgery.

Categories
Animal Physiology

Endocrine function

Invertebrate endocrine systems

The endocrine or hormonal system (i.e. the use of body fluid-borne chemical messengers) together with the nervous system make up the control and coordinating systems of animals. However, there are major differences in the way in which control is achieved within the two systems. Firstly, the endocrine system works by transmitting chemical rather than electrical signals, although the nervous system utilizes chemical messengers at synapses. Secondly, the endocrine system has a much slower response time than the nervous system. An action potential is completed in 2-3 ms, but the action of hormones may take minutes or hours to be completed. Finally, endocrine action has a much longer duration of response. For example, reflexes in animals ― fast pre-programmed responses of the nervous system ― take a few milliseconds to be performed. Compare that with growth processes that are achieved utilizing the hormonal system that may take years to be completed. However, having stated that there are major differences between the two systems, it is becoming increasingly recognized that rather than working as two ‘independent’ systems, the nervous and endocrine systems work cooperatively to achieve a common goal. Indeed, some neurons will release neurotransmitters at their synapses that are then used to serve an endocrine function. Most animals have an endocrine system, and it controls many diverse physiological functions, e.g. metabolism, growth, reproduction, osmotic and ionic regulation, and so on.

Definition of endocrine systems

The classical idea of the endocrine system is that of cells, usually of a nonneural origin (although some neural tissue is considered to have an endocrine function), which secrete specific chemical messengers called hormones. The hormones are carried to their target organs (i.e. the organs where they exert their biological effect), usually some distance from their site of release, in the body fluids of the animal concerned. However, this classical view of endocrine organs and function has recently changed. For example, it is now known that some hormones do not need to enter the general circulatory system of animals in order for them to exert an effect, A good example of this is the role of histamine in controlling acid secretion in the vertebrate stomach, whereby various stimulatory factors converge on mast cells in the stomach (as well as parietal cells) leading to the release of histamine which, in turn, stimulates acid production. This type of ‘local’ hormone action is called paracrine control. In general terms, though, endocrine systems may be classified as one of two types. The first is the neuroendocrine system, also called the neurosecretory system or neurosecretory cells. In this case, neurons are specialized for the synthesis, storage and release of neurohonnones ― in reality, this is the neurotransmitter of the neuron concerned. The neurohormone, instead of being released into a synapse, is released into the general circulation from where it travels to its target organ. The neuroendocrine system is found in all invertebrates and vertebrates. In mammals, for example, renal excretion of water is controlled by the secretion of antidiuretic hormone (ADH) released from neurons whose cell bodies lie in the hypothalamic region of the brain and whose axons extend down to the posterior pituitary gland. In some cases, the release of neurohormones into the general circulation may influence other endocrine organs which then exert some biological effect. For example, in crabs, moulting is controlled by the neurohormone moult inhibiting hormone (MIH), which in turn inhibits the activity of a second endocrine gland which produces a hormone that promotes moulting. The widespread presence of neuroendocrine control systems in both invertebrates and vertebrates suggests that they evolved earlier in evolution than the second type of endocrine system, the classical endocrine system. In this case, hormones are released from specialized, nonneural tissue directly into the body fluids. The absence of ducts to transport the hormone from the gland to the circulating body fluids (e.g. plasma, haemolymph) gives rise to the term ductless gland, an alternative term by which endocrine glands are sometimes described. This contrasts with ducted glands (e.g. salivary glands), where an anatomical duct leading from the gland to the body fluids is present. Classical endocrine glands are only found in the higher invertebrates (e.g. some molluscs) and the vertebrates. This suggests that the appearance of this system occurred after the development of the neuroendocrine system. The presence of neuroendocrine control systems and paracrine control blurs the typical definition and concept of endocrine control, and it may be more appropriate not to consider the two control systems of neural and endocrine control as being so clearly distinct from each other.

Identification of endocrine organs

It can be difficult to determine whether a particular structure in an animal serves an endocrine function. The fact that there are no unique anatomical markers that serve to identify endocrine from nonendocrine tissue is just one reason. In order to overcome this problem, criteria have been established by which candidate tissues and their secretions may be classified as true endocrine organs.

(i) Removal of the candidate tissue or organ should produce deficiency symptoms. For example, if a tissue was suspected of producing a substance that maintained Na+ levels in body fluids, then removal would result in disruption of Na+ levels.

(ii) Reimplantation of the candidate tissue or organ should result in the reversal or prevention of the associated deficiency symptoms. In the case described above, Na” levels would return to their correct levels once the candidate tissue had been reimplanted in the animal concerned.

(iii) Administration of an extract of the tissue or organ should also result in reversal or prevention of the associated deficiency symptoms.

(iv) Finally, the suspected hormone must be purified, its structure determined and tested for biological activity. It must exert the same biological effect as that seen previously with the intact organ or tissue.

The chemical nature of hormones

Virtually all hormones from both invertebrate and vertebrate animals, fall into one of three major classes ― peptides or proteins, amino acid derivatives and steroids. There are exceptions to this, such as the range of C20 compounds known as the prostaglandins. The compounds serve many functions in animals and are beyond the scope of the present text. The chemical nature of the hormone is important because ultimately it decides how the hormone exerts its biological effect.

The mechanism of hormone action

Invertebrate endocrine systems

Vertebrate endocrine systems

Categories
Diseases

Treatment of Systemic Arterial Thromboembolism

Euthanasia In cats that are in acute pain that have a poor prognosis due to severe cardiomyopathy, euthanasia is a humane means of dealing with the problem. Systemic throm-boembolism is often a horrible complication of cardiomyopathy, the treatment options are all relatively poor, and rethrombosis is common. Consequently, although one should not automatically give up on a patient, one should not give false hope either.

Pain Control Cats that present in pain must be treated appropriately for their pain. Appropriate drugs include a fentanyl patch, oxymorphone (intramuscularly, intravenously, or subcutaneously) and butorphanol tartrate (subcutaneously). Aspirin does not produce adequate pain control. Oxymorphone may produce excitement in some cats. The analgesic properties of butorphanol are five times that of morphine, and it remains a nonscheduled drug by the Food and Drug Administration. Its respiratory depressant effects equal that of morphine. Consequently, one must be careful when administering this drug to a patient with dyspnea. Acepromazine may be administered intravenously in addition as an anxiolytic agent to cats that still appear distressed after the administration of the analgesic or to cats that become agitated after oxymorphone administration. The pain abates with time (usually hours) as sensory nerves undergo necrosis.

Palliative Therapy Beyond pain control, many cats with systemic arterial thromboembolism receive only supportive care or the administration of drugs with no proven benefit. The primary outcome of arterial occlusion depends upon the extent of occlusion and the time to spontaneous reperfusion. Poor prognostic factors for short-term survival include worsening azotemia, moderate to severe pulmonary edema or pleural effusion, malignant arrhythmias, severe hypothermia, disseminated intravascular coagulation, and evidence of multiorgan embolization. Long-term, cats may lose an affected leg because of ischemic necrosis, die of toxemia, remain paralyzed from peripheral nerve damage, or regain full or partial function of their legs. About 50% of cats that are not treated definitively will regain all or most caudal limb motor function within 1 to 6 weeks. Return of function presumably is due to the cat’s own thrombolytic system (e.g., plasmin) disrupting the thromboembolus. The degree and rapidity of the dissolution depend on the activity of a particular cat’s thrombolytic system and the size and quality of the thromboembolus. Usually, cats that have some evidence of caudal limb flow recover more rapidly than do cats with no evidence of flow. Presumably this is because the size of the thromboembolus in cats with some flow is smaller. With total occlusion, some cats recanalize within days (others never recanalize).This extreme variability makes it very difficult to render a prognosis for a particular patient at presentation.

Palliative therapy may be only cage rest and pain control or can include administering drugs such as heparin, aspirin, or arteriolar dilators along with the cage rest. No drugs administered for palliation have any proven benefit over cage rest alone.

Heparin is commonly administered in the hopes of preventing new thrombus formation on top of the existing thromboembolus or in the hope of preventing a new thrombus from forming in the left atrium. However, no evidence indicates that heparin is of benefit in cats with STE. Heparin does not aid in thrombolysis. Heparin can be administered at an initial dose of 100 U/lb intravenously followed by a maintenance dose of 30 to 100 U/lb subcutaneously every 6 hours. The dose should be tailored to each individual cat to increase the activated partial thromboplastin time (aPTT) to at least 1.5 times baseline.

Indomethacin is effective at preventing vasoconstriction distal to the thromboembolus when administered to experimental cats prior to creating an aortic thrombus. Theoretically aspirin could do the same thing. However, no evidence indicates that it improves collateral blood flow.

The administration of drugs that dilate systemic arterioles (e.g., hydralazine, acepromazine) has been advocated. These drugs act by relaxing the smooth muscle in systemic arterioles. The exact anatomy of collateral vessels is not well described, but they are probably larger vessels than arterioles. They contain smooth muscle, because they can open and close. The ability of arteriolar dilators to counteract serotonin and thromboxane A2-induced vasoconstriction is unknown.

Definitive Therapy Definitive treatments for systemic arterial thromboembolism in cats include administration of exogenous fibrinolytic agents, balloon embolectomy, rheolytic thrombectomy, and surgery. All definitive procedures are associated with high mortality and common recurrence of the thromboembolus days to months after the initial removal.

Surgery Surgical removal of systemic thromboemboli is generally thought to be associated with high mortality and is not frequently performed. Cats with systemic arterial thromboembolism commonly have underlying cardiac disease, and many are in heart failure. They are poor anesthetic risks, and reperfusion syndrome may be produced. In reperfusion syndrome the muscles of the legs prior to removal of the systemic arterial thromboembolism undergo necrosis, cellular breakdown, and the release of potassium and hydrogen ions from the cells into the interstitial spaces. Sudden reperfusion carries these ions into the systemic circulation, causing acute and often severe hyperkalemia and metabolic acidosis. Surgical intervention may be a viable option if careful monitoring and treatment for reperfusion syndrome with insulin and glucose, sodium bicarbonate, or calcium (or a combination of these therapies) can be initiated immediately if it occurs.

Balloon embolectomy The procedure of choice in human medicine is balloon embolectomy. The author has had limited experience with balloon embolectomy. In this procedure the femoral arteries are isolated and a small balloon embolec-tomy catheter is passed from one femoral artery into the aorta. The femoral arteries are not extremely difficult to isolate. The catheter is pushed past the thromboembolus, and the balloon is then inflated and the catheter withdrawn, along with throm-boembolic material. The catheter is passed sequentially, first in one artery and then in the other. Usually this sequence must be repeated several times. Reperfusion syndrome may occur with balloon embolectomy.

Thrombolytic therapy Thrombolytic therapy is a possible means of dealing with cats with systemic arterial thromboembolism using fibrinolytic agents. Thromboemboli in cats are composed of red cells, strands of fibrin, and possibly platelets. Fibrinolytic agents cleave plas-minogen to plasmin. Plasmin hydrolyzes fibrin, resulting in thrombolysis. Different agents vary in their ability to bind specifically to fibrin-bound plasminogen and in their half-lives. Efficacy and complication rates appear to be very similar in humans. Complications consist primarily of hemorrhage due to fibrinolysis and rethrombosis. Fibrinolytic agents can be very effective at lysing systemic thromboemboli. However, the author and colleagues currently treat few cats with systemic arterial thromboembolism with these agents because reperfusion syndrome and rethrombosis are common.

Tissue plasminogen activator (t-PA) and streptokinase have been used in cats. Tissue plasminogen activator is an intrinsic protein present in all mammals. Numerous reports exist of the use of t-PA for the lysis of thrombi as therapy for acute myocardial infarction, pulmonary thromboembolism, and peripheral vascular obstruction in humans and experimental animals. The activity of genetically engineered t-PA in feline plasma is 90% to 100% of that seen in human plasma. The half-life of t-PA is quite short. Heparin must be administered concomitandy to prevent acute rethrombosis but does not need to be administered with streptokinase because it has a longer half-life. A clinical trial with t-PA in cats with aortic thromboemboli has shown acute thrombolytic efficacy (shortened time to reperfusion and ambulation) associated with the administration of t-PA at a rate of 0.1 mg to 0.4 mg/lb/hr for a total dose of 0.4 to 4 mg/lb intravenously. Forty three percent of cats treated survived therapy and were walking within 48 hours of presentation. Post-t-PA angiograms demonstrated resolution of the primary vascular occlusion. Thus acutely, t-PA effectively decreases the time to reperfusion and return to function in cats with aortic thromboemboli. However, 50% of the cats died during therapy in this clinical trial, which raises extreme concerns regarding acute thrombolysis. Fatalities resulted from reperfusion syndrome (70%), congestive heart failure (15%), and sudden arrhythmic death, presumably the result of embolization of a small thrombus to a coronary artery (15%). Severe hemorrhage into the region distal to the systemic arterial thromboembolism causing anemia was also a common complication. The cats that successfully completed t-PA therapy exhibited signs of increasing neuromuscular function and ambulatory ability within 2 days of presentation. This contrasts with 1 to 6 weeks before seeing similar signs of improvement in most cats exhibiting spontaneous resolution.

Mortality due to reperfusion syndrome can be reduced if the patient can be observed continuously by an individual trained to identify clinical and electrocardiographic evidence of hyperkalemia, if intensive monitoring of electrolytes and blood pH can be performed, and if aggressive medical therapy for hyperkalemia and metabolic acidosis can be initiated very quickly. This means dedicated care 24 hours a day until the thromboembolus is lysed. Thrombolysis may occur within 3 hours or take as long as 48 hours.

If reperfusion syndrome was the only major complicating factor in cats treated with thrombolytic agents, continued use in selected patients might be warranted. However, 90% of the cats that were successfully treated in the aforementioned clinical trial

had another systemic arterial thromboembolism within 1 to 3 months. Rethromboembolism occurred despite aspirin, warfarin, or heparin administration. In addition, t-PA is expensive. Consequently, the author does not currently use t-PA for systemic arterial thromboembolism in cats.

Streptokinase has clinical efficacy very similar to t-PA in human patients with coronary artery thrombosis. Streptokinase is less expensive. No controlled clinical trials of streptokinase use for systemic arterial thromboembolism in cats are available, and the author’s clinical experience with the drug has been generally negative. There has been one small experimental study in which throm-bin was injected between two ligatures placed at the terminal aorta to create a soft thrombus, followed by removal of the ligatures. Streptokinase was administered as a loading dose at 90,000 III followed by 45,000 IU/hr for 3 hours. This dose produced evidence of systemic fibrinolysis in a separate group of normal cats but without evidence of severe fibrinolysis or bleeding. In most cats there was no angiographic change and no improvement in limb temperature. There was a tendency for the thrombus weight to be lower in the treated cats when compared with control cats at postmortem examination. However, lysis of a fresh thrombus created with thrombin is probably much different from trying to lyse an established thromboembolus. Streptokinase is usually unsuccessful, may hasten the death of some cats through bleeding complications, and should not be routinely used.

Rheolytic thrombectomy Rheolytic thrombectomy is an experimental catheter-based system used for the dissolution of the thromboembolus using a high-velocity water jet at the end of the catheter that breaks up the thromboembolus and sucks it back into the system using the Venturi effect. Anesthesia is required and blood transfusion is almost always needed. The catheter is passed from the carotid artery to the region of the thromboembolus. The author and colleagues have used it in six cats. The procedure successfully removed the thromboembolus in five cats but only three left the hospital. Time from onset of clinical signs to thrombectomy was several hours to 8 days. The cat that had the procedure 8 days after the event had residual neurologic deficits but was the longest survivor. Interestingly, reperfusion syndrome has not been a common complication.

Adjunctive therapy Cats with systemic arterial thromboembolism are commonly in heart failure at the time of presentation. Medical therapy with furosemide and an angiotensin-converting enzyme inhibitor is often indicated. Cats that are in pain usually do not eat or drink. Fluid therapy is warranted but must not aggravate or produce heart failure

Cats that take a long time to recover caudal limb function or that only attain partial function may develop regions of skin and muscle necrosis, especially on the distal limbs. These regions may need to be debrided surgically. Cats that lose the function of only one leg or that do not regain function of one leg benefit from amputating that leg. Cats that have perma-nendy lost muscle function distal to the hock may benefit from arthrodesis.

Prognosis The short-term prognosis for life is guarded in cats without heart failure. Cats with a rectal temperature lower than 98.9° F had a worse prognosis in one study. Ifi The long-term prognosis is highly variable and depends on the ability to control the heart failure and the events surrounding the STE. One of the most common causes of death within the first 24 hours is euthanasia. In one study, cats lived between 3 and 30 months after the initial episode. The average survival time was about 10 to 12 months. In another study, MST for cats that recovered and were discharged from the hospital was approximately 4 months but was only about 2.5 months in cats that were also being treated for heart failure. The long-term prognosis for limb function depends on the ability of the cat to lyse its own clot or on the success of intervention. Many biologic variables determine whether or not reperfusion will spontaneously occur. A significant percentage of cats will develop a new thromboembolus, days to months after recovery.

Prophylaxis Feline patients with myocardial disease, especially those with an enlarged left atrium, should be considered at risk for developing intracardiac thrombi and signs of peripheral arterial thromboembolism, although the incidence appears to be low. Preventing peripheral thrombosis is, in theory, one of the most important therapeutic objectives for the veterinarian managing cats with severe myocardial disease. The ideal means of preventing thrombosis is resolution of the underlying myocardial disease. This is usually only possible in a cat with dilated cardiomyopathy secondary to taurine deficiency.

At-present, the only option available is to manipulate the patient’s coagulation system in an attempt to alter the delicate balance between the pathways that promote clotting and those that inhibit thrombus formation to reduce the patient’s thrombogenic potential. At this time, antiplatclet and anticoagulant therapies are the only means of preventing thrombus formation in cats with myocardial disease. Unfortunately, they are often ineffective and, in the case of warfarin, can produce serious side effects. Experience is similar in human medicine.

Antiplatelet therapy Prostaglandins enhance platelet aggregation via activation of cyclic adenosine monophosphate (cAMP). Aspirin (acetylsalicylic acid) acetylates platelet cyclooxygenase, preventing the formation of thromboxane A2, a potent prostaglandin-like platelet aggregating substance. The inhibition of platelet cyclooxygenase is irreversible, and bleeding time is restored to normal only after the production of new platelets. The inhibition of endothelial cyclooxygenase is reversible. The dose of aspirin recommended in cats is 10 mg/lb every third day. Whether or not this dose allows endothelial cyclooxygenase to recover or not in cats is undetermined. At this dose, aspirin has a half-life of 45 hours in the cat. In humans, doses as low as 20 to 100 mg/day inhibit platelet cyclooxygenase; however, no evidence suggests that this low dose has any more benefit than conventional daily doses of 625 to 1250 mg. In one study in cats, no difference was seen in thromboembolus recurrence between cats on low dose (5 mg/cat every 72 hours) and high dose (greater than or equal to 40 mg/cat every 72 hours). No evidence indicates that any dose of aspirin is effective at preventing the formation of an intracardiac thrombus in cats with myocardial disease. Clinical impression of aspirin’s efficacy varies between clinicians. Cats that have already experienced one systemic arterial thromboembolism are the only appropriate population in which to study the efficacy of an agent meant to prevent STE. Aspirin does not prevent recurrence of peripheral thromboembolism in this population.

Glycoprotein IIb and IIIa, an integrin present on platelet surfaces, is a receptor for fibrinogen, fibronectin, and von Willebrand factor. It mediates aggregation, adhesion, and spreading of platelets. The binding of prothrombin to glycoprotein IIb and IIIa also enhances the conversion of prothrombin to thrombin. Glycoprotein IIb and IIIa antagonists have been developed, and one (abciximab) increased mucosal bleeding time and reduced thrombus area when combined with aspirin (when compared with aspirin and placebo).

Anticoagulant therapy Available anticoagulants include heparin, the low molecular weight heparins, and warfarin. Heparin binds to a lysine site on AT, producing a conformational change at the arginine-reactive site that converts AT from a slow, progressive thrombin inhibitor to a very rapid inhibitor of thrombin and factor Xa. AT binds covalently to the active serine centers of coagulation enzymes. Factor Xa bound to platelets and thrombin bound to fibrin are protected from activation by the heparin-antithrombin III complex. Heparin may be administered intravenously or subcutaneously. Repeated intramuscular injection is discouraged because local hemorrhage may result. Some owners can administer heparin subcutaneously at home but the method is not ideal. The author and colleagues have noted rethrombosis with heparin therapy in some cats with cardiac disease. The dose of heparin for preventing thrombosis in cats is unknown.

Low molecular weight heparins include nadroparin calcium, enoxaparin sodium, dalteparin, ardeparin, tinzaparin, reviparin, and danaparoid sodium. The low molecular weight heparins have fewer bleeding complications in experimental animals, improved pharmacokinetics over heparin, are administered subcutaneously, and do not require monitoring in most situations. Although heparin does not reduce red cell aggregation in slow-moving blood, heparin and low molecular weight heparins are effective at preventing deep vein thrombosis in humans. Consequently, they may be beneficial in preventing intracardiac thrombus formation in cats with cardiomyopathy. No controlled studies are available. The author empirically uses enoxaparin sodium at a dose of 2.2 mg/lb every 12 hours subcutaneously in cats that have recovered from an systemic arterial thromboembolism or that have a severely enlarged left atrium and SEC.

Warfarin sodium is an oral anticoagulant (). Warfarin exerts no anticoagulant effect in vitro. In vivo, inhibitory effects on synthesis of clotting factors begin immediately. However, clotting is unaffected until already existing clotting factors decline. Therefore a delay occurs between initial administration and effect on the prothrombin time (FT). Historically, oral warfarin therapy has been monitored with the PT.This test measures the activity of factors II, VII, and X. The factor depressed most quickly and profoundly (usually factor VII) determines the FT during the initial days of therapy. The FT is performed by measuring the clotting time of platelet-poor plasma after the addition of thromboplastin and calcium, a combination of tissue factor and phospholipid. Intra- and interlaboratory variation in the FT was a significant problem for laboratories in the past, when crude extracts of human placenta or rabbit brain were the only source of thromboplastin. The international normalized ratio (INR), developed by the WHO in the early 1980s, is designed to eliminate problems in oral anticoagulant therapy caused by variability in the sensitivity of different commercial sources and different lots of thromboplastin. The INR is used worldwide by most laboratories performing oral anticoagulation monitoring and is routinely incorporated into dose planning for human patients receiving warfarin. When the anticoagulant effect is excessive, it can be counteracted by administering vitamin K,. However, once synthesis of factors 11, VII, IX, and X is reinstituted, time must elapse before factors achieve concentrations in the plasma that will adequately reverse the bleeding tendency. If serious bleeding occurs during therapy with warfarin, it may be stopped immediately by administering fresh blood or plasma that contains the missing clotting factors. Other drugs can modify the anticoagulant actions of warfarin by altering the bioavailability of vitamin K by altering the absorption, distribution, or elimination of the coumarins; by affecting synthesis or degradation of clotting factors; or by altering protein binding of the warfarin. The maintenance dose should be evaluated daily during the initial titration (3 days), then every other day (twice), and then weekly until a safe and stable dose regimen is determined. The therapeutic effect should be reevaluated periodically (at least once per month). The recommended initial dose is 0.1 to 0.2 mg per cat every 24 hours orally to a 6 to 10 lb/cat. The dose may then be increased to maintain an INR of 2.0 to 3.0. It can take up to 1 week for new steady state conditions to be achieved. The efficacy of warfarin at preventing recurrent thrombosis in cats with cardiac disease has been reported. M-s In one report, out of 23 cats examined retrospectively, 10 experienced a new thromboembolic episode while being administered warfarin. Two of these cats had at least two new episodes. In the other report, eight of 18 cats on warfarin experienced a new thromboembolic episode. This may be some improvement over the 75% recurrence rate reported for aspirin alone after t-PA therapy, but these results are still disappointing.i:,h In the first report, four cases also died suddenly (which could have been caused by thromboembolLsm). Three of these cats did not have postmortem examinations. The one cat that did have a postmortem examination had a thrombus present in its left atrium. One cat also died of a renal infarct that produced renal failure. Four cats appeared to have bleeding complications. In the second report, one cat died of a hemoabdomen and one was suspected to have an acute intracranial hemorrhage resulting in death. Consequendy, it appears that warfarin therapy can produce fatal complications. However, it should be noted that these studies were performed without using the INR for monitoring.

Categories
Drugs

Aglepristone (Alizin, Alizine)

Injectable Progesterone Blocker

Highlights Of Prescribing Information

Injectable progesterone blocker indicated for pregnancy termination in bitches; may also be of benefit in inducing parturition or in treating pyometra complex in dogs & progesterone-dependent mammary hyperplasia in cats

Not currently available in USA; marketed for use in dogs in Europe, South America, etc.

Localized injection site reactions are most commonly noted adverse effect; other adverse effects reported in >5% of patients include: anorexia (25%), excitation (23%), depression (21%), & diarrhea (13%)

What Is Aglepristone Used For?

Aglepristone is labeled (in the U.K. and elsewhere) for pregnancy termination in bitches up to 45 days after mating.

In dogs, aglepristone may prove useful in inducing parturition or treating pyometra complex (often in combination with a prostaglandin F analog such as cloprostenol).

In cats, it may be of benefit for pregnancy termination (one study documented 87% efficacy when administered at the recommended dog dose at day 25) or in treating mammary hyperplasias or pyometras.

Pharmacology/Actions

Aglepristone is a synthetic steroid that binds to the progesterone (P4) receptors thereby preventing biological effects from progesterone. It has an affinity for uterine progesterone receptors approximately three times that of progesterone. As progesterone is necessary for maintaining pregnancy, pregnancy can be terminated or parturition induced. Abortion occurs within 7 days of administration.

Benign feline mammary hyperplasias (fibroadenomatous hyperplasia; FAHs) are usually under the influence of progesterone and aglepristone can be used to medically treat this condition.

When used for treating pyometra in dogs, aglepristone can cause opening of the cervix and resumption of miometral contractility.

Within 24 hours of administration, aglepristone does not appreciably affect circulating plasma levels of progesterone, cortisol, prostaglandins or oxytocin. Plasma levels of prolactin are increased within 12 hours when used in dogs during mid-pregnancy which is probably the cause of mammary gland congestion often seen in these dogs.

Aglepristone also binds to glucocorticoid receptors but has no glucocorticoid activity; it can prevent endogenous or exogenously administered glucocorticoids from binding and acting at these sites.

Pharmacokinetics

In dogs, after injecting two doses of 10 mg/kg 24 hours apart, peak serum levels occur about 2.5 days later and mean residence time is about 6 days. The majority (90%) of the drug is excreted via the feces.

Before you take Aglepristone

Contraindications / Precautions / Warnings

Aglepristone is contraindicated in patients who have documented hypersensitivity to it and during pregnancy, unless used for pregnancy termination or inducing parturition.

Because of its antagonistic effects on glucocorticoid receptors, the drug should not be used in patients with hypoadrenocorticism or in dogs with a genetic predisposition to hypoadrenocorticism.

The manufacturer does not recommend using the product in patients in poor health, with diabetes, or with impaired hepatic or renal function as there is no data documenting its safety with these conditions.

Adverse Effects

As the product is in an oil-alcohol base, localized pain and inflammatory reactions (edema, skin thickening, ulceration, and localized lymph node enlargement) can be noted at the injection site. Resolution of pain generally occurs shortly after injection; other injection site reactions usually resolve within 2-4 weeks. The manufacturer recommends light massage of the injection site after administration. Larger dogs should not receive more than 5 mL at any one subcutaneous injection site. One source states that severe injection reactions can be avoided if the drug is administered into the scruff of the neck.

Systemic adverse effects reported from field trials include: anorexia (25%), excitation (23%), depression (21%), vomiting (2%), diarrhea (13%) and uterine infections (3.4%). Transient changes in hematologic (RBC, WBC indices) or biochemical (BUN, creatinine, chloride, potassium, sodium, liver enzymes) laboratory parameters were seen in <5% of dogs treated.

When used for pregnancy termination, a brown mucoid vaginal discharge can be seen approximately 24 hours before fetal expulsion. This discharge can persist for an additional 3-5 days. If used in bitches after the 20th day of gestation, abortion maybe accompanied with other signs associated with parturition (e.g., inappetance, restlessness, mammary congestion).

Bitches may return to estrus in as little as 45 days after pregnancy termination.

Overdosage / Acute Toxicity

When administered at 3X (30mg/kg) recommended doses, bitches demonstrated no untoward systemic effects. Localized reactions were noted at the injection site, presumably due to the larger volumes injected.

How to use Aglepristone

WARNING: As accidental injection of this product can induce abortion; it should not be administered or handled by pregnant women. Accidental injection can also cause severe pain, intense swelling and ischemic necrosis that can lead to serious sequelae, including loss of a digit. In cases of accidental injection, prompt medical attention must be sought.

Aglepristone dosage for dogs:

To terminate pregnancy (up to day 45):

a) 10 mg/kg (0.33 mL/kg) subcutaneous injection only. Repeat one time, 24 hours after the first injection. A maximum of 5 mL should be injected at any one site. Light massage of the injection site is recommended after administration. (Label information; Alizin — Virbac U.K.)

To induce parturition:

a) After day 58 of pregnancy: 15 mg/kg subcutaneously one time. 24 hours after aglepristone injection, give oxytocin 0.15 Units/kg every 2 hours until the end of parturition. ()

b) On or after day 58 of pregnancy: 15 mg/kg subcutaneously; repeat in 9 hours. In treated group, expulsion of first pup occurred between 32 and 56 hours after treatment. Use standard protocols to assist with birth (including oxytocin to assist in pup expulsion if necessary) or to intervene if parturition does not proceed. ()

As an adjunct to treating pyometra/metritis:

a) For closed cervix: 6 mg/kg twice daily on the first day followed by the same dose once daily on days 2, 3, and 4. Some prefer using larger doses (10 mg/kg) once daily on days 1, 3,and 8, then follow up also on days 15 and 28 depending on the bitch’s condition. ()

b) For metritis: 10 mg/kg subcutaneously once daily on days 1,2 and 8.

For open or closed pyometra: aglepristone 10 mg/kg subcutaneously once daily on days 1,2 and 8 and cloprostenol 1 meg/ kg subcutaneously on days 3 to 7. Bitches with closed pyometra or with elevated temperature or dehydration should also receive intravenous fluids and antibiotics (e.g., amoxicillin/clavulanate at 24 mg/kg/day on days 1 – 5). If pyometra has not resolved, additional aglepristone doses should be given on days 14 and 28. ()

Aglepristone dosage for cats:

For treating mammary fibroadenomatous hyperplasia: a) 20 mg/kg aglepristone subcutaneously once weekly until resolution of signs. Cats who present with heart rates greater than 200 BPM should receive atenolol at 6.25 mg (total dose) until heart rate is less than 200 BPM with regression in size of the mammary glands. ()

Monitoring

■ Clinical efficacy

■ For pregnancy termination: ultrasound 10 days after treatment and at least 30 days after mating

■ Adverse effects (see above)

Client Information

■ Only veterinary professionals should handle and administer this product

■ When used for pregnancy termination in the bitch, clients should understand that aglepristone might only be 95% effective in terminating pregnancy when used between days 26-45

■ A brown mucoid vaginal discharge can be seen approximately 24 hours before fetal expulsion

■ Bitch may exhibit the following after treatment: lack of appetite, excitement, restlessness or depression, vomiting, or diarrhea

■ Clients should be instructed to contact veterinarian if bitch exhibits a purulent or hemorrhagic discharge after treatment or if vaginal discharge persists 3 weeks after treatment

Chemistry / Synonyms

Aglepristone is a synthetic steroid. The manufactured injectable dosage form is in a clear, yellow, oily, non-aqueous vehicle that contains arachis oil and ethanol. No additional antimicrobial agent is added to the injection.

Aglepristone may also be known as RU-534, Alizine, or Alizin.

Storage / Stability/Compatibility

Aglepristone injection should be stored below 25°C and protected from light. The manufacturer recommends using the product within 28 days of withdrawing the first dose.

Although no incompatibilities have been reported, due to the product’s oil/alcohol vehicle formulation it should not be mixed with any other medication.

Dosage Forms / Regulatory Status

Veterinary-Labeled Products:

Note: Not presently available or approved for use in the USA. In several countries:

Aglepristone 30 mg/mL in 5 mL and 10 mL vials; Alizine or Alizin (Virbac); (Rx)

The FDA may allow legal importation of this medication for compassionate use in animals; for more information, see the Instructions for Legally Importing Drugs for Compassionate Use in the USA found in the appendix.

Human-Labeled Products: None

Categories
Horses

Criteria For Induction Of Parturition

Before labor is induced in a mare, the ability of the fetus to survive extrauterine life must be confirmed. Several physiologic processes occur within the fetus before delivery to ensure that the foal will be viable after birth. The equine fetus is unique in that final maturation occurs only 24 to 48 hours before delivery. Consequently, the equine fetus is at substantially greater risk of dysmaturity/prematurity if delivered at an inappropriate time.

Several indicators have been identified that suggest fetal and maternal “readiness for birth.” Gestational length (>330 days) is often erroneously used by those considering induction of parturition in the mare. The normal gestation period in the mare is highly variable between animals and ranges from 320 to 360 (-340) days. Most mares tend to have a similar gestational length from year to year; thus historic information can be very useful. However day length can affect gestational length so that mares foaling during short days typically have a longer gestation than mares foaling during long days. Therefore all fetuses are not necessarily mature at 330 days from the last breeding. The length of a mare’s gestation should only be used in conjunction with other signs when the decision is being made to induce parturition.

Mammary development and colostrum production in the mare are presently the most reliable indicators of fetal maturity and “readiness for birth.” Colostrum is paramount to the survival of the neonate both as a source of nourishment and immunoglobulins. Furthermore, concentration of mammary secretion electrolytes has been well correlated with fetal maturity in horses. Calcium concentration rises sharply in mammary secretions of most normal mares 24 to 40 hours before foaling. Additionally, sodium concentration typically is much higher than potassium until 3 to 5 days before birth, at which time the sodium to potassium ratio inverts. Changes in mammary secretion electrolytes (calcium, sodium, and potassium) have been compared with neonatal parameters that are indicative of adequate maturity at birth. A rise in mammary secretion calcium greater than 10 mmol/L and inversion of the sodium-potassium ratio are well correlated with fetal maturity in foals.

Precise measurement of mammary secretion electrolyte concentrations requires a flame spectrophotometer or a laboratory chemistry analyzer. With these systems, elevation of mammary secretion calcium to greater than 40 mg/dL and potassium concentration greater than sodium (i.e., potassium >30 mEq/ml and sodium <30 mEq/ml) usually indicates fetal maturity in the normal equine pregnancy. Stall-side tests are available to measure calcium (Ca++) or calcium carbonate (CaC03) concentration. Test kits that measure mammary secretion Ca++ typically use pads on a test strip that change from green to red (Predict-A-Foal, Animal Health Care Products, Vernon, Calif.) or titrate a diluted sample until an indicator dye changes from pink to blue (Titret, CHEMetrics, Calverton, Va.; Sofchek, Environmental Test Systems, Elkhart, Ind.). The dilution kits are somewhat more labor-intensive than the test strip kits. Of the commercially available mammary secretion test kits, the Titret test kit is the most reliable and repeatable test for predicting foaling within 24 hours. In one study, the mammary secretion CaC03 was between 300 and 500 ppm in most mares that foaled within 12 to 18 hours of testing. Mares with mammary secretion CaC03 less than 200 ppm had less than a 1% chance of foaling within 24 hours of testing.

As with other induction criteria, care must be taken when changes in mammary secretion electrolyte concentrations are interpreted. Changes occur most often at night, which is when the majority of mares foal. Therefore samples taken early in the day may not reflect electrolyte changes that occur in the evening or at night shortly before parturition. Mares foaling for the first time may show rapid or no change in electrolyte concentrations before foaling. “Maiden” mares often do not have significant mammary development and colostrum production until immediately before parturition. Conversely, mares with twins or placental pathology may precociously develop a mammary gland, and mammary secretion calcium levels may rise prematurely. Thus although highly reliable for predicting fetal maturity and impending parturition in the normal, multiparous mare, mammary secretion electrolytes may be less useful for maiden mares or mares with abnormal pregnancies.

The importance of cervical dilation before the induction of parturition in the mare has been a point of great controversy. Numerous studies cited in the human medical literature associate poor cervical relaxation with failed induction, prolonged labor, and a high rate of cesarean deliveries. Reports in the veterinary medical literature suggest that inductions may proceed successfully in a mare with a tightly closed, mucous-covered cervix as late as the end of first stage labor. In one study, mares with spontaneously dilated cervixes (determined by digital examination per vagina) before induction delivered their foals more quickly than those mares with a closed cervix. Foals delivered rapidly stood and nursed more quickly and had fewer signs of intrapartum asphyxia (e.g., hypercapnia, maladjustment) than foals that experienced prolonged delivery. Prostaglandin E2 (PGE2) has recently been used to promote cervical relaxation before induction of mares. PGE2 is routinely administered to women to dilate the cervix before induction of labor. PGE2 enhances cervical dilation and eases delivery in treated mares. Foals delivered from PGE2-treated mares suckle more quickly than foals from control mares. Results from these two studies indicate that cervical relaxation before parturition can positively affect neonatal health.

In summary, no one criterion effectively predicts the success of an induced parturition in the mare. Adequate udder development, changes in mammary secretion electrolytes, and cervical softening are all important considerations before induction.

Categories
Horses

Uterine Torsion

The causes of uterine torsion in the mare are not well-defined. The condition is much more common in cattle; in that species a large, term fetus has been implicated as a major risk factor. The majority of uterine torsions in cows occur at term and most are thought to be a direct result of fetal positional changes during late first-stage and early second-stage labor. A striking difference between the mare and the cow is that more than 50% of uterine torsions in mares occur before the end of gestation. In this author’s clinical experience the vast majority occur before term, and other authors have reported on cases from as early as 8 months of gestation. Owners who work closely with their mares may mention that they have observed excessive fetal movements in the flank area. In a recent equine obstetrical study, 80% of term fetuses were found to be in dorsosacral position when the uterine torsion was corrected. This finding suggests that fetal righting reflexes may have played a role in creating the torsion. This author believes that vigorous fetal movements during the latter stages of gestation are likely to be a significant factor in the etiology of this condition in the mare ().

Diagnosis of Uterine Torsion

Correction of Uterine Torsion

Prognosis For The Fetus

The prognosis for cases of equine uterine torsion depends on the degree of vascular compromise. Severity and duration of the condition will affect placental circulation and subsequent fetal viability. In this author’s experience, if the fetus is alive and the uterine wall is not severely congested and edematous, then the prognosis for both the mare’s survival and for the birth of a live foal at term is good. The concept of progestin supplementation after the first 100 days of gestation remains controversial. Luteolysis can occur during early pregnancy as a result of endotoxin-mediated prostaglandin F2a release, and progestin supplementation has been shown to be effective in maintaining pregnancies. In late gestation a viable placenta should produce adequate amounts of progestins. If the fetoplacental unit is compromised to the extent that it is incapable of producing sufficient progestins to maintain pregnancy then it is probable that insufficient oxygen and nutrients will be available to support the rapidly growing late gestation fetus anyway. However, progestin supplementation during late gestation may still be indicated to ensure myometrial quiescence, and thus maintenance of the placental attachment. Recent studies support the premise that progestins may suppress myometrial activity by inhibition of endogenous prostaglandin F2a production. Although supplementation after a uterine torsion would be in the last 2 to 3 months of gestation, reports exist of mares retaining a nonviable (died at 3 to 5 months gestation) fetus while being administered progestins. Thus if progestin supplementation is administered to a mare after correction of a uterine torsion, fetal viability should be monitored at regular intervals.

Categories
Horses

Oocyte Transfer

Oocyte transfer is the placement of a donor’s oocyte into the oviduct of a recipient. The recipient can be inseminated within the uterus or within the oviduct. Placement of the oocyte and sperm within the recipient’s oviduct is more accurately termed gamete intrafallopian transfer (GIFT).

The first successful oocyte transfer was done in 1989; however, the technique was not used for commercial transfers until the late 1990s. Oocyte transfer is currently used to produce offspring in subfertile mares in which embryo transfer is not successful because of various reproductive problems. These problems include ovulatory failure, oviductal blockage, recurrent or severe uterine infections, and cervical tears or scarring. In some cases, the cause of reproductive failure cannot be diagnosed; however, oocyte transfer can be successful.

Sychronization Of Donors And Recipients

Oocytes are collected from preovulatory follicles between 24 and 36 hours after the administration of human chorionic gonadotropic (hCG; 1500-2500 IU, IV) to a donor mare or between 0 and 14 hours before anticipated ovulation. Criteria for hCG administration are as follows:

• Follicles greater than 35 mm in diameter

• Relaxed cervical and uterine tone

• Uterine edema or estrous behavior present for 2 or more days

Some mares, especially older mares, do not consistently respond to hCG. In these cases, this author uses a combination of the gonadotropin-releasing hormone (GnRH) analog, deslorelin acetate (2.1 mg implant; Ovuplant), followed by an injection of hCG (2000 IU, IV) between 4 and 5 hours later.

Oocytes collected 36 hours after hCG administration to the donor are transferred immediately into a recipient’s oviduct. Oocytes collected 24 hours after drug administration to the donor are cultured in vitro for 12 to 16 hours before transfer. The advantage of collection of oocytes between 32 and 36 hours after hCG administration to the donor is that the oocytes do not require culture in vitro. However, donors could ovulate follicles before oocytes are collected. The collection and culture of oocytes at 24 hours after hCG administration to the donor are often easier to schedule; the oocyte can be collected in the afternoon and transferred the next morning. This method requires expensive equipment and training for tissue culture, however. In a modification of these procedures, oocytes are collected 24 hours after hCG and immediately transferred into the recipient’s oviduct to allow maturation to complete within the oviduct. With this latter method, recipients are inseminated 16 hours after transfer.

Oocyte Collection

Oocytes are usually collected by one of two methods. In one method, the ovary is held per rectum against the ipsilateral flank of the mare. A puncture is made through the skin and a trocar is advanced into the abdominal cavity. The ovary is held against the end of the trocar while a needle is advanced through the trocar and into the follicular antrum.

In this author’s laboratory oocytes are collected by using transvaginal, ultrasound-guided follicular aspirations. For this procedure, a linear or curvilinear ultrasound transducer is used with the transducer housed in a casing with a needle guide. Before the procedure, the rectum is evacuated and the vulvar area is cleaned. The mare is sedated (xylazine HC1, 0.4 mg/kg, and butorphanol tartrate, 0.01 mg/kg, IV) and a substance to relax the rectum (propantheline bromide, 0.04 mg/kg, IV) is administered. A twitch is applied. The probe is covered with a nontoxic lubricant and placed within the anterior vagina lateral to the posterior cervix and ipsilateral to the follicle to be aspirated. The follicle is positioned per rectum and stabilized with the apex of the follicle juxtaposed to the needle guide. A needle is advanced through the needle guide to puncture the vaginal and follicular walls. In this author’s laboratory, a 12-gauge, double-lumen needle is used (Cook Veterinary Products, Spencer, Ind.). The follicular fluid is aspirated from the follicle by using a pump set at a pressure of -150 mm Hg. After removal of follicular fluid, the lumen of the follicle is lavaged with 50 to 100 ml of flush (typically modified Dulbecco’s phosphate-buffered solution or Emcare [ICP, Auckland, New Zealand]) that contains fetal calf serum (1%) or bovine serum albumin (0.4%) and heparin (10 IU/ml).

Equipment used to handle the oocyte is warmed to 38.5° C before use because the oocyte is sensitive to temperature changes. On collection, the follicular aspirate and flush are poured into large search dishes and examined under a dissecting microscope to locate the oocyte. Aspirations of preovulatory follicles are often bloody because the follicle has increased vascularity as ovulation approaches. The oocyte is approximately 100 μm in diameter and is surrounded by a large mass of nurse ceils — the cumulus complex. Cumulus cells, or the corona radiata, appear as a ring surrounding the oocyte. When the oocyte matures, the cumulus complex becomes less distinct. The corona radiata appears clear in the bloody flush solution and can be observed by the naked eye.

Oocyte Evaluation And Culture

On collection, cumulus oocyte complexes (COC) are evaluated for cumulus expansion (graded from compact to fully expanded) and for signs of atresia. Oocytes are determined to be in a stage of atresia when the COC is clumped and/or sparse, the corona radiata is fully expanded, or when the ooplasm is shrunken and dark or severely mottled. Oocytes with a fully expanded cumulus (marked separation of cumulus cells with expansion of the corona radiata) are considered mature and are transferred as soon as possible into a recipient’s oviduct. Oocytes with a moderately expanded cumulus complex (translucent COC with moderate separation of cumulus cells and incomplete expansion of corona radiata) are cultured before transfer. On occasion, the donor does not respond to hCG and the follicle does not begin to mature. Consequently, the granulosa cells that line the follicle are collected in small, compact sheets, and the oocyte is frequently not retrieved. If an immature (compact COC with little or no separation of cumulus cells) oocyte is collected, special culture conditions are required, including a maturation medium with additions of hormones and growth factors.

On identification and evaluation, the oocyte is washed and placed in a transfer or collection medium. A commonly used medium for the culture of maturing oocytes is tissue culture medium (TCM) 199 with additions of 10% fetal calf serum, 0.2 mM pyruvate, and 25 mg/ml gentamicin sulfate. A carbon dioxide (C02) incubator must be used to establish the proper culture conditions of 38.5° C in an atmosphere of 5% or 6% C02 and air.

Oocyte Transfer

Mares that will receive oocytes should be young (preferably 4-10 years of age) with a normal reproductive tract. Oocytes are transferred surgically; therefore, adequate exposure of the ovary is essential to facilitate transfers. Mares with short, thick flanks and short broad ligaments are not good candidates for recipients. Both cycling and noncycling mares have been used as oocyte recipients. When cyclic mares are used, they must be synchronized with the donor; thus, hCG is administered to the estrous donor and recipient at the same time of day. Before the mare can be used as a suitable recipient, her own oocyte must be aspirated. Anestrus and early transitional mares are suitable noncyclic recipients. During the breeding season, a high dose of a GnRH analog or injections of progesterone and estrogen (150 mg progesterone and 10 mg estradiol) can be administered to reduce follicular development in potential recipients. Noncyclic recipients are given injections of estradiol (2-5 mg daily for 3-7 days) before transfer and progesterone (150-200 mg daily) after transfer. In mares that are not having estrus cycles, pregnancies must be maintained through the use of exogenous progesterone.

Oocytes are transferred through a flank laparotomy into standing sedated mares. Recipients are placed in a stock and a presurgical sedative (xylazine HC1, 0.3 mg/kg, and butorphanol tartrate, 0.01 mg/kg, IV) is administered. The surgical area is clipped, scrubbed, and blocked with a 2% lidocaine solution. Immediately before surgery, additional sedation is administered (detomidine HC1, 9 mg/kg, and butorphanol tartrate, 0.01 mg/kg, IV). An incision is made through the skin approximately midway between the last rib and tuber coxae, and the muscle layers are separated through a grid approach. The ovary and oviduct are exteriorized through the incision. The oocyte is pulled into a fire-polished, glass pipette, and the pipette is carefully threaded into the infundibular os of the oviduct and advanced approximately 3 cm. The oocyte is transferred with less than 0.05 ml of medium.

Insemination Of Recipients

In a commercial oocyte transfer program, use of stallions with good fertility is essential. Cooled and transported semen is often provided. When fresh semen from fertile stallions and oocytes from young mares was used in different experiments, insemination of the recipient only before (12 hours) or only after (2 hours) oocyte transfers resulted in embryo development rates of 82% (9/11) and 57% (8/14), respectively. In a commercial oocyte program, mares were older with histories of reproductive failure and cooled semen from numerous stallions of variable fertility was used. Pregnancy rates when recipients were inseminated before or before and after oocyte transfer were significantly higher than when recipients were only inseminated after transfer (18/45, 40%; 27/53, 51% and 0/10, respectively). These results suggest that the insemination of a recipient before transfer with 5 X 108 to 1 x 109 progressively motile sperm from a fertile stallion is sufficient. However, if fertility of the stallion is not optimal, insemination of the recipient before and after transfer may be beneficial.

After insemination and transfer, the recipient’s uterus is examined by ultrasonography to detect intrauterine fluid collections. The uterine response to insemination often appears to be more severe when recipients are inseminated after transfer than when they are inseminated only before transfer. The uterus is evaluated and treated once or twice daily until no fluid is imaged. Recipients with accumulations of intrauterine fluid are treated similar to ovulating mares, with administration of oxytocin or prostaglandins to stimulate uterine contractions or with uterine lavage and infusion.

Future Of Oocyte Transfer

Oocyte transfer has proved to be a valuable method of obtaining pregnancies from mares that cannot carry their own foal or produce embryos for transfer. Because the mare does not have to ovulate or provide a suitable environment for fertilization or embryo development, the oocyte donor is only required to develop a preovulatory follicle with a viable oocyte.

The transfer of oocytes and a low number of sperm (200,000 motile sperm) into the oviduct of recipients has been successful. Pregnancies could be produced with GIFT when sperm numbers are limited, such as from subfertile stallions and from sex-selected or frozen sperm.

Through the use of this technique at this author’s laboratory, pregnancies have been recently produced from oocytes that were frozen and thawed and from oocytes that were collected from the excised and shipped ovaries of dead mares. These advances provide excellent methods to preserve the genetics of valuable mares.

Categories
Horses

Selection And Management Of Recipients

Selection and management of recipient mares for an embryo transfer program is the most important factor affecting pregnancy rates. On farms handling only one or two donors, recipient mares may be purchased from local backyard horse owners who are familiar with the mare’s reproductive history. However, acquiring a large number of recipient mares requires that mares be purchased from local sale barns. Thus the reproductive history of these mares is unknown. In either case the recipient mare should meet the following criteria: 900 to 1200 pounds; 3 to 10 years of age; and broken to halter. The effect of size of recipient on the subsequent size of the foal has not truly been determined. However, the size of the donor mare should be matched with the recipient as nearly as possible. This may be difficult when obtaining embryos from large warmbloods or draft horses.

Typically nonlactating mares are easier to use in an embryo transfer program than a mare that is lactating. If a lactating mare is not being used, the animals should not be used as recipients until at least the second postpartum cycle. Numerous types of recipient mares can be used: ovarian-intact cycling mares; ovariectomized mares; mares in anestrus; and mares during the transitional period. This author prefers to use ovarian intact normal cycling mares. However, pregnancy rates using ovariectomized, progesterone-treated mares have been shown to be similar to ovarian-intact mares.

Occasionally, early in the year a scarcity of normal cycling mares occurs. The alternative at that time of the year is to use either an anestrous mare or a transitional mare. In this author’s experience transitional mares are more appropriate to use than truly anestrous mares. Mares in transition should be selected based on the presence of endometrial folds. This indicates that estrogen is being secreted. Transitional mares can then be placed on progesterone at the time of the donor mares ovulation. The suggested progestin treatment for either ovariectomized mares or transitional mares includes altrenogest (Regumate) daily or 150 mg of progesterone injected daily. With the use of ovariectomized mares, progesterone treatment must continue until the placenta begins to produce progesterone at approximately 100 to 120 days. With transitional mares, progesterone treatment may be terminated once the mare has ovulated and developed secondary corpora lutea during early gestation.

The recipient mare should be examined by rectal palpation and ultrasonography before purchase. The external genitalia are observed for normal conformation. Those mares with poor external conformation that may predispose them to wind sucking are generally rejected. Mares are then palpated per rectum and the size and tone of the uterus, cervix, and ovary are determined. The uterus and ovary are then examined with ultrasonography. Evidence of pathology such as uterine fluid, uterine cyst, ovarian abnormalities, or the presence of air or debris in the uterus would render the mare unsuitable for purchase as an embryo recipient. In addition, any mare found to be pregnant is not purchased unless the pregnancy is less than 30 days.

Approximately 15% to 20% of the mares initially presented are rejected. Mares that pass the initial examination are given a breeding soundness exam similar to the exam of the donor mare. Recipients are vaccinated for influenza, tetanus, and rhinopneumonitis and are quarantined from other mares for at least a period of 30 days. Those mares that are in thin condition are fed a concentrate ration and a free-choice alfalfa hay. The majority of recipients are purchased in late fall and placed on a 16-hour lighting regimen beginning December 1. Starting approximately February 1, mares are palpated and examined with ultrasonography twice per week until a follicle greater than 35 mm is obtained. Mares with follicles greater than 30 mm are examined daily with ultrasonography until ovulation. Ideally, recipient mares should have one or two normal estrous cycles prior to being used as a recipient. Mares are excluded as potential recipients if they consistently have erratic or abnormal estrous cycles.

Hormonal manipulation of the recipient mare’s estrous cycle is an important component of an embryo transfer program. The degree of hormonal manipulation is dependent upon the size of the embryo transfer operation. Smaller operations that deal with only one or two donors use more hormonal manipulation than larger operations that may have a large number of donors and recipients. Small operations should place the donor and one or two recipients on progesterone for 8 to 10 days and then administer prostaglandins on the last day of treatment. The progesterone can either be altrenogest used daily or injectable progesterone at a level of 150 mg daily for the same length of time. It is not uncommon to use a combination of progesterone and estrogen (150 mg progesterone, 10 mg estradiol-17β) daily for 8 to 10 days followed by prostaglandins.

The donors and recipients will ovulate 7 to 10 days after prostaglandin treatment. Generally, having the recipient ovulate either 1 day before or up to 3 days after the donor mare is desirable. This can be accomplished by using hCG (Chorulon) or GnRH (Ovuplant) to induce ovulation in either the recipient or donor mare to provide optimal synchrony of ovulation. In a larger embryo transfer station it is common to manipulate the cycle by using only prostaglandins, hCG, or GnRH. Typically the ovulation dates of the recipient are recorded and once a donor mare ovulates then a recipient is selected that has ovulated either 1 day before or up to 3 days after the donor. If a mare is not used as the recipient she is then given prostaglandins 9 or 10 days after her ovulation and induced to return to estrus.

Each recipient mare is given a final examination 5 days after ovulation before to being used as the recipient. Mares are classified as acceptable, marginal, or nonacceptable based on this 5-day exam. The 5-day exam includes palpation per rectum for uterine and cervical tone, and ultrasonography of the uterus and ovaries. An acceptable recipient should have a round, tubular, firm uterus and a closed cervix. She also would have the absence of endometrial folds, a normal sized uterus, and the presence of a visible corpus luteum. Mares generally are placed in the marginal category based on a decrease in uterine tone or cervical tone or perhaps the presence of grade 1 endometrial folds. Unacceptable recipients typically have poor uterine tone, a soft-open cervix, or presence of endometrial folds and/or fluid in the uterus. A retrospective examination of this author’s commercial embryo transfer program has revealed that the 5-day check is the best predictor of whether or not a recipient mare will become pregnant.

Embryos are transferred either surgically by flank incision or nonsurgically. Most of the embryo transfer stations are now using nonsurgical transfer methods. The details of the transfer methods are presented in the subsequent chapter. Mares are examined with ultrasonography for pregnancy detection 4 or 5 days after transfer. Mares that are diagnosed pregnant are reexamined on days 16, 25, 35, and 50. Mares not confirmed pregnant on the initial examination (day 12) are reexamined 2 days later. If the ultrasound scan continues to be negative the mare is considered not pregnant and given prostaglandin to induce estrus. Unless the embryo was extremely small (<150 microns) the majority of mares that are to be pregnant have a visible vesicle at 12 days of gestation. Those mares in which the vesicle does not appear until 14 or 16 days of gestation have delayed embryonic development and are more likely to suffer early embryonic loss. The initial ultrasound examination allows the breeder to decide whether to rebreed the donor and attempt a second embryo recovery. The ultrasound exam at 25 days determines whether a fetus is present with a viable heartbeat. The majority of losses that do appear in embryo transfer recipients occur between days 12 and 35. However, early embryonic loss before 50 days of gestation appears to be no greater in embryo transfer recipients than other pregnant mares that are inseminated with either fresh or cooled semen. Mares that fail to become pregnant after an embryo transfer are generally used a second time but not a third. The pregnancy rates on mares receiving an embryo on a second attempt are no different than those that receive an embryo only one time and become pregnant.

Pregnant recipients should be fed a maintenance ration similar to other broodmares during the first two thirds of gestation and then administered extra energy in the form of concentrate rations during the final one third of pregnancy. Recipients should be monitored closely around the time of impending parturition. Management procedures identical to those used for foaling broodmares should be used. No greater difficulty in foaling embryo transfer recipients than normal broodmares has been found. The influence of the size of the recipient versus size of donor on ease of foaling has not been adequately studied, although this does not appear nearly as critical in horses as it does in cattle.

In summary, a relatively high pregnancy rate can be anticipated in an embryo transfer program if management of the donor and recipient mares are maximized. Attention should be given to selection of both donor and recipient, nutrition, proper monitoring of the donor and recipient with palpation per rectum and ultrasonography, careful assessment of the recipient, and management of the recipient after embryo transfer. Day 12 pregnancy rates for either fresh or cooled semen should be 75% to 80% and those at 50 days of gestation should be 65% to 70%.

Categories
Horses

Preparation Of The Mare For Artificial Insemination

The ultimate goal of insemination is to provide semen in a time frame that coordinates the availability of capacitated spermatozoa with the arrival of the transported oocyte within the mare’s oviduct. It is also the responsibility of the veterinarian to ensure the optimum intrauterine environment that supports the developing embryo. With these objectives in mind, preparation of the mare begins well in advance of the actual anticipated breeding date.

Too often, the fertility of the mare is somewhat overlooked and she is selected as a broodmare candidate simply due to her availability. Truly, her potential fertility needs to be as carefully evaluated as the fertility of the stallion. A breeding soundness examination (BSE) should be performed at the beginning of the breeding season that is based on her age and parity. The BSE may range from a rectal and ultrasonographic examination of the reproductive tract to a cytology, culture and/or biopsy of the endometrium to a videoendoscopic examination of the endometrium. Inclusion of an ultrasound examination at every rectal exam enhances the continual education of the veterinarian and aids in detection of many changes that are not palpable, thereby increasing pregnancy rates.

In young maiden mares, especially those that have never raced, a rectal and ultrasonographic exam may constitutea a sufficiently adequate prebreeding examination. In this author’s opinion, in the foaling mare a cytologic evaluation of the endometrium should be included. This test is a simple stall-side procedure that provides immediate information on the status of the lining of the endometrium. If significant numbers of polymorphonuclear leukocytes (PMNs) are present on the smear, the mare has endometritis and its etiology needs to be investigated before proceeding with insemination. Bacteria and yeast forms also may be detected with a cytologic exam.

During the estrous phase of the heat cycle, an ultrasound examination of the reproductive tract usually supports the rectal palpation findings of a dominant, growing follicle, in addition to the classic “spokewheel” pattern of endometrial edema, which may be subjectively quantified. The ultrasound examination also may demonstrate the presence of echogenic particles within the follicle in addition to increasing echogenicity of its wall — both indicative parameters of ovulation within the next 24 hours. Although the duration of heat in the mare may be variable, most mares ovulate near the end of this estrous phase. Interestingly, the edema is less apparent on ultrasound just before ovulation. Because the timing of insemination with respect to ovulation is so critical, the presence or absence of this edema can be a powerful tool used to optimize pregnancy rates through the control of ovulation timing. A more detailed discussion is provided in site.

A number of agents that shorten the interval to ovulation in the mare have been investigated. The most effective agents possess luteinizing hormone (LH) activity with varying degrees of follicle stimulating hormone (FSH) activity. Human chorionic gonadotropin (hCG) with its potent LH-like activity, is, at the time, the least expensive and perhaps the most popular agent used for the induction of ovulation. It has been reported to be effective at doses ranging from 1000 to 5000 IU given intramuscularly, intravenously, or subcutaneously. The author tends to base the dosage on size of the mare; very large breeds receive larger doses and breeds such as the Miniature Horse or small ponies receive the minimum dose of hCG.

The use of hCG in the mare is somewhat controversial. First, with its repeated use, antibody development has been documented in several studies; however, the clinical impression of many practitioners is not in agreement with these studies. Secondly, mare owners often complain that their mares experience pain associated with administration of some hCG products. Finally, the reliability of hCG in its ability to hasten the interval to ovulation, especially in the older or compromised mare, has been questioned. Regardless of its potential disadvantages, hCG remains a popular, inexpensive and effective means of inducing ovulation in the majority of mares.

Synthetic gonadotropin-releasing hormone (GnRH) analogs (deslorelin acetate and buserelin) also have proved effective in inducing ovulation. Although the use of these latter agents may delay slightly the mare’s return to estrus if she fails to conceive, in this author’s experience these GnRH analogs are more reliable in inducing ovulation, especially in mares more prone to ovulation failure. These include older mares, mares in vernal transition, and mares concomitantly treated with prostaglandin inhibiting agents such as many of the antiinflammatory drugs.

Ovulation-inducing agents are far more reliable and effective if given at the appropriate time during estrus. If endometrial folds are apparent on the ultrasound examination, and a dominant softening follicle is present (usually >30 mm in diameter) hCG, deslorelin acetate, and buserelin are expected to hasten ovulation on average of 36, 41 to 48, and 24 to 48 hours, respectively, after administration of the induction agent. Samper (see readings list) reported that 98% of mares with maximal endometrial edema given hCG or deslorelin would consistently ovulate with 48 hours of administration.

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Horses

Mare Behavior Problems

This post briefly outlines the four most common complaints concerning reproductive behavior in mares: (1) failure to show estrus or to stand for breeding, (2) maternal behavior problems, (3) stallionlike behavior, and (4) estrus cycle-related performance problems in mares.

Failure To Show Estrus Or To Stand For Breeding

Research and clinical experience consistently indicate that most mares show estrus, or some detectable and reliable change in behavior consistent with estrus, in association with ovulation. A stallion given free access to the mare probably would have no difficulty detecting estrus and proceeding with normal breeding. A trained and careful observer would see changes in response to prolonged interaction with a male. Therefore “failure to show estrus” or “silent ovulation” in most cases represents management failure to adequately elicit and/or detect estrus under farm conditions. Difficulty detecting estrus also is complicated in certain individual mares that may naturally show good estrus for only a few hours.

The recommendation for detection of estrus in mares is teasing for at least 5 minutes, preferably with the mare at liberty to approach the stallion, along a fence line or with the stallion in a teasing pen. This enables a fuller range of mare estrus behavior and avoids submissive behavior evoked by forced encounter with the stallion. Sometimes it helps to tease with two or more stallions (sequentially for at least 5 min each).

Some mares show estrus during teasing and then fail to stand for mounting. Normal fertile mares pastured with stallions often are observed to go through periods of alternating solicitation and rejection of the stallion. This natural tendency for ambivalence may account for some of failure to stand for breeding in hand-breeding. Another factor in failure to stand for breeding appears related to severe restraint of the mare and limited precopulatory interaction with the stallion at the time of breeding.

Maternal And Foal Behavior And Problems

Stallionlike Behavior

Heterotypical behavior, that is, abnormal behavior typical of the opposite sex, in mares includes fighting with stallions; elimination-marking behavior (olfactory investigation, flehmen, and marking of excrement); herding teasing; and mounting mares. It is caused by exposure to androgens or high levels of estrogens that convert to androgen. The most common source of androgens in mares are granulosa cell tumors and administered steroids. Removal of the source of androgens generally leads to cessation of stallionlike behavior within weeks to months.

Stallionlike behavior occasionally is observed during mid pregnancy. At one time this was attributed to androgens in a male fetus, but it has been observed since in mares carrying females.

Estrus Cycle-Related Performance Problems

Temperament and performance of mares can vary with the ovarian cycle, with some mares showing more or less desirable behavior during diestrus, estrus, or anestrus. Complaints require careful, detailed analysis of the specific desirable and undesirable behavior in relation to ovarian activity. Careful evaluation of complaints may reveal a physical, handling, or training problem that may be either unrelated to the ovarian cycle or that may worsen with estrus as many physical problems do. In evaluating complaints, a common finding is that owners and trainers are unaware of the specific behavioral elements of estrus and diestrus, often confusing the two states, and sometimes assuming estrus equals bad behavior. When it is confirmed that problem behaviors are associated with the ovarian cycle, improvement can be achieved with suppression or manipulation of the cycle using progesterone, hCG, and prostaglandin as recommended in “Induction of Ovulation.” A large percentage of such complaints involve submissive cowering, leaning away, and urine squirting that are easily misinterpreted as estrus. This pattern of behavior often is called “starting gate estrus” because it is common in young anxious race fillies in the starting gate.