Pasteurellosis or hemorrhagic septicemia is an often fatal disease in Asian elephants, caused by Pasteurella multocida and Mannheimia hemolictica. If diagnosed in its early stage , antimicrobial and supportive treatment should be started immediately. Elephants in certain range countries are vaccinated twice a year with commercial vaccines used in cattle. In 2020 a mass die-off of free ranging African elephants occured in Zimbabwe and Botswana, caused by Pasteurella Bisgaard taxon 45 (multocida). To infectious diseases Pasteurellosis Pasteurellosis or hemorrhagic septicemia in Asian elephants has been report in several countries in Asia, where it is a common disease in water buffalo and other ruminants. In elephants it can cause a severe generalized disease, which is often fatal. The causative pathogens are Pasteurella multocida and Mannheimia (Pasteurella) haemolytica. They are nonmotile, facultative anaerobic and may exhibit bipolar staining with Giemsa or Wright’s stain. Transmission of the pathogen can be through direct or and indirect contact and possibly through biting insects and wound contamination. It is generally assumed that Pasteurella spp. are secondary pathogens that may strike when the immune system is challenged by a primary disease or stressful condition. Higher magnification of lung impression smear showed bipolar characteristic Pasteurella sp. ( Harish, 2009) Inapparent infections have not been reported, though vaccination-induced antibodies against P. multocida can be detected using an indirect ELISA (Tankaew, 2017). This indirect ELISA was more sensitive in elephants than the indirect hemagglutination assays (IHA), which is the WHO recommended diagnostic test in farm animals. The epidemiology of hemorrhagic septicemia is not fully known. However, multiple cases were reported following periods of consecutive droughts from 2010 to 2012 and sudden heavy monsoon rains in India in 2013 (Chandranaik, 2016). Clinical symptoms Clinical symptoms may vary, ranging from its presence in foot abscess to an acute fatal disease. The FAO manual for elephant managers describes the following signs, which might be associated with hemorrhagic septicemia: There is a high fever. Take the elephant's temperature. (See page 71.) If it is over 37.8° C or 100° F, that is a sign of danger. The breath exhaled from the mouth and trunk is very hot. There is a bright red at the eyes, mouth, the end of the trunk, and other soft tissue. Swelling (edema) is found in body parts such as the throat, the shoulders, the base of the tail, the anal flap, on the belly under the legs. The elephant is listless, the trunk rests on the ground, and the ears do not flap. The elephant does not eat. The elephant frequently opens its mouth to "yawn". The body trembles and has spasms because breathing is difficult. The urine is cloudy and richly coloured. In fatal cases pneumonia, hemorrhagic tracheitis, haemorrhages on the heart, and/or lesions of acute septicemia in all other vital organs have been described (Harish, 2009; Srivastav, 2017). Pasteurellosis presents in varies forms, so many other diseases must be considered in a differential diagnosis, including anthrax, trauma, foreign-body reactions, staphylococcosis, salmonellosis, and pneumonia caused by various agents. Diagnosis Isolation and identification of Pasteurella spp. or Mannheimia haemolyticum combined with a disease presence leads to the diagnosis of hemorrhagic septicemia. If diagnosed, one should always consider that there might be involvement of another primary disease process! Confirmation of the diagnosis by PCR may lead to the source of the infection if other elephants of other animal species are involved. Treatment Immediate treatment is required when hemorrhagic septicemia is suspected. Pasteurella sp. are usually susceptible to amoxycillin, trimethoprim+sulfa an fluoroquinolones (like enrofloxacin). Antimicrobial therapy can be initiated using one of the above mentioned drugs, however samples should be taken for culture and as soon as the sensitivity of the pathogens have been determined, the therapy should be adjusted according to the antibiogram results. Supportive therapy consists of administration of fluids (rectally and/or intravenously). NSAIDs should be given if the general condition or pain reactions indicate their use. For dosages go to: https://elephantcare.org/resources/formulary/drug-index/ . Make sure that no mahouts or other people who have been in contact with the ill elephant have any contact with the healthy ele phants. The healthy elephants should be taken to a place where they have no contact with dung, urine, or uneaten food of the infected elephant. Feed the elephant with items of high nutritional value, such as bananas, unhusked rice, sugarcane, and high quality browse . When an elephant dies of hemorrhagic septicemia, the carcass must be buried or burned. The carcass should not be butchered for meat or to remove the tusks to sell because this can spread the disease to other elephants and to other animals. The FAO has elaborated an action plan for additional measures in case of hemorrhagic septicemia in elephants ( see Elephant Care Manual for Mahouts and Camp Managers ): Immediately separate the infected elephant and keep it as far away as possible from other animals. Take the infected elephant to a clean, quiet and shady spot that is easily cleaned and where run-off water and waste, such as dung and uneaten food, do not contaminate other areas. Most importantly, ensure that the water source for sick animals and healthy animals is separate. If there is only one source of drinking water, it is likely contaminated. If so, try to get the healthy animals to a new source of water. You might even have to truck water in, but you must ensure your animals are drinking pure water free of infection. Prevention In several Asian range countries elephants are vaccinated with the vaccines available for cattle, like an inactivated aluminium-precipitated vaccine used in (non-pregnant) elephants over 6 months that used to work in the timber industry in Myanmar (5ml, subcutaneous, twice a year).(pers. Comm. Khyne U Mar, 2023). Contact with susceptible farm animals, especially water-buffaloes and cattle should be avoided. Outbreak of Pasteurellosis in free ranging African elephants During a period of 4 months in 2020 a total of 35 African elephants were found dead in north-western Zimbabwe. The estimated age of the dead elephants ranged from 18 months–30 years. Elephants of both sexes were found dead (16 males, 9 females). The carcasses were in average body condition with hepatomegaly and splenomegaly as the most prominent gross pathological findings, with variable hemorrhages across the epicardium, liver, lungs, intestinal serosae, hepatic and splenic lymph nodes, and in one case, the diaphragm. Histopathological lesions in elephants were similar and consisted of acute multifocal heterophilic and necrotizing inflammation in liver, spleen, and lymph node, with presence of intralesional Gram-negative bacterial colonies of coccobacillary morphology. Specifically, one elephant displayed necrotizing lesions in spleen and liver, with the additional presence of fibrinocellular and bacterial emboli in the pulmonary vasculature. Presence of Gram-negative bacterial colonies without associated morphological changes was observed in veins and capillaries, prominently in the encephalon. Acute multifocal heterophilic and necrotizing lymphadenitis, hepatitis and splenitis with intralesional Gram-negative coccobacilli was observed in one. Most blood smears (n = 13/15) stained with Giemsa contained small to moderate numbers of bacteria with a bipolar, short-rod, or coccobacilli morphology (0.5–2 µm), and intracellular bacteria were observed. Of 15 sampled elephants, six showed molecular evidence of septicemic infection by Bisgaard taxon 45. There was no evidence of toxins, including those from cyanobacteria, or for any viral infection. The failure to identify Bisgaard taxon 45 in samples from all 15 elephants is likely due sample quality and delays in testing. The authors propose that stress from a combination of heat, drought, and population density were likely contributing factors in this outbreak. Food and water resources normally wane as temperatures rise during the dry season, and elephants must travel increasing distances between water points and foraging areas. The source of infection and route of transmission remain unknown in this outbreak. For more details about this mass die-off, click here . References Chandranaik BM., Shivashankar BP., Giridhar P., and Nagaraju DN. 2016. Molecular characterisation and serotyping of Pasteurella multocida isolates from Asiatic elephants (Elephas maximus ). Eur J Wildl Res (2016) 62:681–685 FAO: Elephant Care Manual for Mahouts and Camp Managers. Foggin, C.M., Rosen, L.E., Henton, M.M. et al. Pasteurella sp. associated with fatal septicaemia in six African elephants. Nat Commun 14, 6398 (2023). https://doi.org/10.1038/s41467-023-41987-z Harish, B.R., B.M. Shivaraj, B.M. Chandranaik, M.D. Venkatesh & C. Renukaprasad. 2009. Hemorrhagic Septicemia in Asian Elephants (Elephas maximus ) in Karnataka state, India. Journal of Threatened Taxa 1(3): 194- 195 . Preecha Phuangkum P., Lair RC., and Angkawanith T. 2002. Elephant Care Manual for Mahouts and Camp Managers. FAO. ISBN: 974-7946-71-8. Shrivastav AB., Rokde A., Agarwal S., and Shrivastav G. 2017. Pasturollesis: Complication of Metastatic Supporative Pneumonia Severe Stress in Asian Elephant (Elephas maximus ). Indian Journal of Veterinary Sciences & Bio technology (2017) Volume 12, Issue 4, 93-94. Tankaew P., Singh-La T., Titaram C., Punyapornwittaya V., Vongchan P., Sawada T., Sthitmatee N. 2017. Evaluation of an In-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants Journal of Microbiological Methods. Vol.134, pp30-34. Weston P. 2023. It took years to solve the mystery elephant deaths. Now, the threat is spreading. The Guardian, 2023 10 23.

Monitoring fecal quality in elephants includes regular control on parasites, consistency and fiber length. Fecal quality reflexes molar condition, food passage speed and presence of parasites. Intestinal infections can cause abnormal feces. Increased fibre length can indicate poor mastigation due to abnormal molar wear. Fecal quality control Compiled by Christian & Linda Schiffmann The physiology of defecation in elephants Elephants are megaherbivores with a mean retention time of about 24 hours (Rees 1982; Hackenberger 1987). Elephants do defecate on average around 12-16 times per 24 hours with a mean amount of five fecal boluses per defecation (Coe 1972; Ratnasooriya et al. 1994). Being hind gut fermenters, the plant fibers of their diet are shed in the feces exactly the size as they have been swallowed after being chewed. Therefore, fecal particle size is correlated to chewing efficiency in elephants. With respect to this physiology, fecal quality offers a valuable opportunity to assess an elephant’s digestive health. Accordingly, regular fecal check is strongly recommended as an integral part of continuous health monitoring in elephants under human care. Aspects to be assessed through regular fecal checks We recommend checking the following criteria for elephant feces in the indicated intervals: fecal consistency, structure and bolus size – on a daily basis fecal particle size – monthly coproscopy in order to detect intestinal parasites – 1-4 times a year (depending on regional parasite prevalence and housing conditions). In addition, fecal samples can be used to determine glucocorticoid metabolites and reproductive hormones, if this information is relevant for management decisions or scientific research. Fecal consistency, structure and bolus size Although fecal consistency and structure may slightly vary depending on diet composition, it usually allows the building of well-shaped and evenly sized boluses (Fig. 1a). Ideally, the boluses are compact and can be taken from the ground without breaking into pieces. They have a brown color which can tend towards slight green or yellow depending on the roughage fed. Gastrointestinal disorders as well as short-term stress may lead to reduced fecal consistency resulting in varying degrees of diarrhea (Fig. 1b-e). Not well-masticated food stuff (e.g. seeds, fruits) can be observed in the fecal boluses (Fig. 1f), as well as foreign materials which have been swallowed intentionally or unintentionally. These indicators provide information on which material the elephant has access to and on his (potentially abnormal) feeding behavior. Fecal bolus size varies with age and therefore size of an elephant (Coe 1972; Morrison et al. 2005; Leopardi et al. 2013). Elephants with improper chewing efficiency (e.g. due to molar issues), may show heavily enlarged fecal boluses as a consequence of poorly masticated roughage (Fig. 2). Such mega boluses may cause abdominal pain or even lead to constipation. In addition to the bolus size, also the total amount of feces during a 24h cycle should be checked. Showing an increased or decreased amount of feces may hint to digestive disorders or inappropriate feed intake in an elephant. Even the distribution of fecal boluses in the habitat of an elephant may tell you a story. As an example, geriatric individuals suffering from severe degenerative joint disease may not interrupt their lying rest to defecate, which can be recognized by the pattern of their defecations (Fig. 3). Taking this together, macroscopic fecal check as a daily routine can provide experts in charge with a multitude of valuable information on elephants’ (digestive) health status. Figure 1. Fecal consistency in elephants may vary from well-shaped firm boluses in a healthy situation (a) to different degrees of diarrhea with reduced fecal consistency (b-e). Unchewed parts such as corn may offer additional information on an elephant’s food intake on the previous day (f). Picture a: Photo Courtesy of Martin Kristen. Pictures c,d and e Photo Courtesy Patrycja Kasprzak. Figure 2. Mega boluses of different sizes (cell phone for size comparison) and in relation to regularly formed fecal boluses (upper right). Note the large plant fibers contained in the mega boluses (left). Photo Courtesy of Martin Kristen. Figure 3. Defecation pattern of an elephant suffering from severe degenerative joint disease and therefore avoiding to interrupt its lying rest for defecation. Healthy elephants do get up from recumbency in order to defecate. Fecal particle size Chewing efficacy has been shown to vary during an elephant's lifetime, presumably due to continuous changes in the molar grinding surface caused by the physiological process of molar progression (Schiffmann et al. 2019). Fecal particle size as an indicator for chewing efficacy can be used to monitor these physiological changes over time and to detect pathological alterations (Fig. 4). A simple sieving method allows the determination of fecal particles by focusing on the largest fibers. This method is practical in the field and has been shown to correlate well with a sophisticated approach in the lab (Schiffmann et al. 2023). A concise description of the procedure is given in Figure 5. It is recommended to document the detected size of the ten largest fibers photographically (Fig. 6). This enables the monitoring of fiber length of an individual elephant over time. Of course, this information should be completed with parallel monitoring of the molar status, which can also be documented photographically (Fig. 7). Figure 4. Obvious difference in fecal particle size between two female Asian elephants on the same diet, but with varying chewing efficacy. While one female with a healthy molar status was able to chew roughage as needed (a), the other female suffered from molar issues and struggled to chew properly (b). Figure 5. Guidance for a simple sieving protocol to determine fecal particle size in elephants. Figure 6. The size of the ten largest fibers can easily be documented photographically. Figure 7. Ideally, molar status in the upper and lower jaw should be monitored and documented on a regular basis. We recommend a frequency of three months for photographic documentation. Coproscopy for the detection of intestinal parasites Basically, African as well as Asian elephants are susceptible for clinical gastrointestinal parasite infestation. Most relevant gastrointestinal parasites for elephants do belong to the trematoda, cestoda, nematoda and protozoa (Vimalraj and Jayathangaraj 2013; Abeysekara et al. 2018; Chel et al. 2020). Severe parasite loads are reported in free-ranging as well as captive elephants in the range countries with the potential for fatal outcomes (Kinsella et al. 2004; Obanda et al. 2011; Nishanth et al. 2012; Hing et al. 2013; Vimalraj and Jayathangaraj 2013; Lynsdale et al. 2017; Abeysekara et al. 2018; Kingori et al. 2020). They seem less relevant in modern zoos. Prevalence of gastrointestinal parasites seems significantly higher in free-ranging elephants compared to elephants living in human care (Abeysekara et al. 2018; Abhijith et al. 2018). The composition of parasites seems to shift from helminth-dominated in free-ranging elephants to mostly protozoa in captive elephants, presumably due to regular anthelminthic treatment in human care (Abeysekara et al. 2018). Based on these reports, the recommended monitoring interval heavily depends on the geographic region and the housing conditions in particular regarding feed hygiene (Fig. 8). In Western zoos annual to biannual coproscopy seems a reasonable protocol, if appropriate hygienic conditions are ensured. Figure 8. Providing roughage in top-feeders (e.g. hay nets) can significantly reduce contamination of food items and ensure hygienic conditions. Especially for Asian elephants, these feeders should not be too high (like on this photo) as this may place too much strain on the flexibility of the spine, which could result in damage to the spinal joints. In order to cover the intestinal parasite species relevant for elephants, the fecal sample should be examined both by sedimentation as well as flotation. Table 1 gives an overview on which detection method is appropriate for which parasite species. Various treatment options are available (Fowler and Mikota 2006). If anthelmintic treatments are administered a subsequent coproscopy should be conducted to confirm the effectiveness of the treatment (Lynsdale et al. 2015). Table 1. Appropriate detection methods for the parasite classes most relevant in elephants Summary Regular fecal checks provide a non-invasive, simple and cheap opportunity to monitor an elephant’s molar and digestive health. Fecal consistency, structure and fiber length provide valuable information and should be checked on a regular basis. In combination with parasitology in the lab on an interval appropriate for the parasite prevalence in the region, these fecal checks present an important part of health monitoring in elephants under human care. References Abeysekara N, Rajapakse RPVJ, Rajakaruna RS (2018) Comparative cross-sectional survey on gastrointestinal parasites of captive, semi-captive, and wild elephants of Sri Lanka. Journal of Threatened Taxa, 10, 11583-11594. http://threatenedtaxa.org/index.php/JoTT/article/view/3406 Abeysinghe KS, Perera ANF, Fernando P (2012) Developing a practical and reliable protocol to assess nematode infections in Asian elephants. Gajah, 37, 22-26. Abhijith TV, Ashokkumar M, Dencin RT, George C (2018) Gastrointestinal parasites of Asian elephants (Elephas maximus L. 1798) in south Wayanad forest division, Kerala, India. Journal of Parasitic Diseases, 42, 382-390. https://link.springer.com/article/10.1007/s12639-018-1012-0 Baines L, Morgan ER, Ofthile M, Evans K (2015) Occurrence and seasonality of internal parasite infection in elephants, Loxodonta africana, in the Okavango Delta, Botswana. International Journal for Parasitology: Parasites and Wildlife, 4, 43-48. Chel HM, Iwaki T, Hmoon MM, Thaw YN, Soe NC, Win SY, Bawm S, Htun LL, Win MM, Oo ZM, Masum MA, Ichii O, Nakao R, Nonaka N, Katakura K (2020) Morphological and molecular identification of cyathostomine gastrointestinal nematodes of Murshida and Quilonia species from Asian elephants in Myanmar. International Journal for Parasitology: Parasites and Wildlife, 11, 294-301. Coe M (1972) Defaecation by African elephants (Loxodonta africana africana (Blumenbach)). East African Wildlife Journal, 10, 165-174. Fowler ME, Mikota SK (2006) Biology, Medicine, and Surgery of Elephants. Iowa, USA, Blackwell Publishing. Hackenberger MK (1987). Diet digestibilities and ingesta transit times of captive Asian (Elephas maximus) and African elephants (Loxodonta africana). Guelph, University of Guelph. MSC Thesis. Hing S, Othman N, Nathan SKSS, Fox M, Fisher M, Goossens B (2013) First parasitological survey of endangered Bornean elephants Elephas maximus borneensis. Endangered Species Research, 21, 223-230. http://www.int-res.com/abstracts/esr/v21/n3/p223-230/ Kingori E, Obanda V, Chiyo PI, Soriguer RC, Morrondo P, Angelone S (2020) Patterns of helminth infection in Kenyan elephant populations. Parasites & Vectors, 13, 145. Kinsella JM, Deem SL, Blake S, Freeman A (2004) Endoparasites of African Forest Elephants (Loxodonta africana cyclotis) from the Republic of Congo and Central African Republic. Comparative Parasitology, 71, 104-110. Leopardi S, Keratimanochaya T, Solmi F, Roberts J (2013). Estimation of a linear model capable to predict age of Asian elephants (Elephas maximus indicus) using dung bolus circumference. Proceedings of the International Conference on Diseases of Zoo and Wild Animals, Vienna, Austria. Lynsdale CL, Franco dos Santos DJ, Hayward AD, Mar KU, Htut W, Aung HH, Soe AT, Lummaa V (2015) A standardised faecal collection protocol for intestinal helminth egg counts in Asian elephants, Elephas maximus. International Journal for Parasitology: Parasites and Wildlife, 4, 307-315. http://www.sciencedirect.com/science/article/pii/S2213224415300031 Lynsdale CL, Mumby HS, Hayward AD, Mar KU, Lummaa V (2017) Parasite-associated mortality in a long-lived mammal: Variation with host age, sex, and reproduction. Ecology and Evolution, 7, 10904-10915. Morrison TA, Chiyo PI, Moss CJ, Alberts SC (2005) Measures of dung bolus size for known-age African elephants (Loxodonta africana): implications for age estimation. Journal of Zoology, 266, 89-94. Nishanth B, Srinivasan SR, Jayathangaraj MG, Sridhar R (2012) Incidence of endoparasitism in free-ranging elephants of Tamil Nadu State. Tamilnadu Journal of Veterinary & Animal Sciences, 8, 171-173. Obanda V, Iwaki T, Mutinda NM, Gakuya F (2011) Gastrointestinal parasites and associated pathological lesions in starving free-ranging African elephants. South African Journal of Wildlife Research, 41, 167-172. Punya MS, Shyma VH, Reshnu VC, Vijayakumar K, Vinodkumar K, Ambily R, Zachariah A (2021) Gastrointestinal parasites of captive Asian elephants in Kerala. Journal of Veterinary and Animal Sciences, 52, 312-315. Ratnasooriya WD, Molligoda PS, Molligoda WHM, Fernando SBU, Premakumara GAS (1994) Absence of synchronization either in defaecation or urination of the Sri Lankan elephant (Elephas maximus maximus) in captivity. Ceylon Journal of Science, 23, 47-51. Rees PA (1982) Gross assimilation efficiency and food passage time in the African elephant. African Journal of Ecology, 20, 193-198. Schiffmann C, Hatt JM, Hoby S, Codron D, Clauss M (2019) Elephant body mass cyclicity suggests effect of molar progression on chewing efficiency. Mammalian Biology, 96, 81-86. Schiffmann C, Schiffmann L, Bonillo J, Blukeviciute I, Gozalbes Aparicio E, Paniagua J, Ribera G, Ruiz M, Torro M, Clauss M (2023) A simple approach to monitor faecal particle size in the Asian elephant - A proof of concept study. Gajah, 56, 30-35. Thewarage LD, Dissanayake DSB, Perera US, Bandara AT, Perera BVP, Wickramasinghe S, Rajapakse RPVJ (2020) Morphology and molecular characterization of Parabronema smithii (Cobbold, 1882) (Nematoda: Habronematidae) from wild Asian elephant (Elephas maximus maximus) of Sri Lanka. Acta Parasitologica, 65, 504-517. Vanitha V, Thiyagesan K, Baskaran N (2011) Prevalence of intestinal parasites among captive Asian Elephants Elephas maximus: effect of season, host demography, and management systems in Tamil Nadu, India. Journal of Threatened Taxa, 3, 1527-1534. http://threatenedtaxa.org/ZooPrintJournal/2011/February/vanitha.htm Vimalraj PG, Jayathangaraj MG (2013) Endoparasitic infections in free-ranging Asiatic elephants of Mudumalai and Anamalai Wildlife Sanctuary. Journal of Parasitic Diseases, 39, 474-476. To page top

Esophagus obstructions are not uncommon in elephants. Early intervention can prevent esophagitis and deterioration of the elephant's condition. Standing sedation (detomine + butorphanol or xylazine) and the use of a (home-made) mouth gag are needed to get access to the pharynx. A (home-made) endoscope is required to visualize the esophagus wall. To non-infectious diseases Treatment of esophageal obstruction in elephants The diagnosis 'esophageal obstruction' is based on the clinical signs (anorexia, regurgitation of water and/or food) and esophagoscopy performed under standing sedation . A firm, large bore-hole tube and endoscopic camera are advanced into the esophagus as described here . Sometimes it can be difficult to insert the tube into the esophagus due to the lack of space between the molars. Opening the mouth with a mouth gag may facilitate this manoever. Success of the treatment largely depends on the material that has blocked the esophagus passage. Packed, poorly chewed roughage particles can occupy the entire esophagus, even obstructing completely the insertion of the diagnositic tube. Although never reported in elephants, reduction of the spasm of the esophagus might be obtained by the administration of oxytocin (dosage in horses 0.1-0.2 IU/kg BW given in a slow intravenous bolus (Meyer 2000). The antispasmodic (spasmolytic) and anticholinergic drug Buscopan can be used at the dose of 0.3 mg/kg body weight (0.14 mg/lb), slowly IV. Maintaining hydration is important as the elephant may not have taken water for a while. Rectal administration of luke-warm fluids is the easiest and fastest way to restore the body fluids. After manually removal of the feces from the distal part of the rectum, a firm tube should be inserted deep into the rectum and luke-warm water should be administered at a dose of 10-20 ml/kg BW. After this procedure the tail must be pushed down for 1 minute to prevent water from being squeezed out by the animal. Once the tube and camera are advanced into the esophagus, the obstruction can be visualized. Luke-warm water can be flushed into the esophagus, preferably after inserting another small diameter tube. The original large tube can then be used for draining the excessive water. Flushing should be done with care, avoiding regurgitation of the fluid into the trachea. If water does not have the expected effect, liquid paraffin can be tried respecting the same precautions. Esophageal obstruction in elephants Case report 1 A fatal case occured in a 3-yrs-old female African elephant (Wood, 1992). The animal regurgitated water and was unable to swallow food. 30 seconds after attempts to drink water, the water was ejected from the mouth together with a quantity of thick, stringy white mucus. ( Click here to see a video of water regurgitation in an elephant with esophageal spasm). The animal was drenched with 1 liter of mineral oil, which did not resolve the problem. After 24 hours the elephant was anesthetized with xylazine and etorphin. It was rolled in right lateral recumbency with the trunk placed on sacking to prevent the aspiration of dirt and fluid. An attempt to manually remove the obstruction failed. Some undigested food could be removed from the pharynx. A large-bore equine stomach tube was passed and several liters of warm water were flushed in the esophagus, which drained out of the mouth again. Flushing with obstetric lubricants had the same result. The tube could not be advanced. The animal was given antibiotics and phenylbutazone (IM) and 25 liters of electrolytes (IV). During the next 24 hours the animal's condition deteriorated and another anesthesia was performed. The previous treatment was repeated and seemed to result in free passage of fluid into the stomach. An endoscope was advanced into the esophagus, but was too short to visualize the distal part of the esophagus. ( Click here for an example of an easily available endoscope, which serves well if advanced into the esophagus while protected by a (stomach) tube. During the following day water was given in 5 liter amounts without resulting in regurgitation. Unfortunately the animal died 48 hours after the second anesthesia. At necropsy an apple was found in the distal part of the esophagus. The esophagus tissue around the apple showed marked necrosis and 2 perforations. Case report 2 Another case describes the blockage of the esophagus in a 15-yrs-old male Asian elephant (Oo, 2018). This animal vomited 2-3 minutes after drinking some water. Body temperature was normal. Supportive treatment was given, consisting of dextrose saline (1000 ml) every 5 h to compensate for the loss of water and electrolyte and multivitamin (80 ml). On the next day a locally made mouth-gag instrument was placed into the elephant’s mouth and the upper alimentary tract was examined by palpation. A very hard mass was felt inside the throat. This mass could be removed and was found to be a bolus consisting of a mixture of sugar cane and rice, weighing 1.6 kg. Click here to read the complete manuscript. Case report 3 A 42-year-old female Indian elephant (Elephas maximus indicus) developed a sudden onset of excessive salivation and dysphagia (Phair 2014). Esophageal obstruction was suspected; possibly related to palm frond ingestion. Esophageal endoscopy revealed a mat of plant material in the distal esophagus. An initial attempt at relieving the obstruction was unsuccessful, but subsequent use of custom-made instruments along with insufflation and hydropulsion enabled partial removal of the material. Postimmobilization care included aggressive intravenous and rectal fluids, anti-inflammatory and antibiotic administration, and fasting. Despite treatment, the dysphagia persisted and the elephant was euthanized due to lack of improvement and grave prognosis. Postmortem examination revealed remaining plant material in the esophagus, complicated by an esophageal dissection, mural hematoma, and secondary bacterial infection. Iatrogenic trauma may have contributed to the extent of esophageal injury. Although treatment was ultimately unsuccessful, the supportive care employed could potentially aid recovery in cases of less severe esophageal trauma. Case report 4 In the Case report section you can find another report of an esophageal impaction in a 4.5-yrs-old African elephant. Click here to read the full report. References MEYER G. A., RASHMIR-RAVEN A.,HELMS R. J., and BRASHIER M. 2000. The effect of oxytocin on contractility of the equine oesophagus: a potential treatment for oesophageal obstruction. Equine vet. J. (2000) 32 (2) 151-155. Oo Z.M., Aung T.T., Aung M.M., Nada N and Than M. 2018. Esophageal Blockage in a Captive Asian Elephant . Gajah 48, 38-39. Phair, K. A., Sutherland-Smith, M., Pye, G. W., Pessier, A. P., & Clippinger, T. L. 2014. ESOPHAGEAL DISSECTION AND HEMATOMA ASSOCIATED WITH OBSTRUCTION IN AN INDIAN ELEPHANT (ELEPHAS MAXIMUS INDICUS). Journal of Zoo and Wildlife Medicine, 45(2), 423–427. doi:10.1638/2013-0177r.1 Wood, D.T. 1992 Oesophageal choke in an African elephant. Veterinary Record 131, 536-537. Esophageal obstruction Esophageals obstruction or “choke” symptoms can be associated with: Congenital abnormalities of the upper digestive system; symptoms usually show up when the animal is weaned and starts eating solid food. Mega-esophagus, which can be congenital or acquired; Foreign bodies that get stuck in the upper digestive system; Impaction or blockage of the stomach/duodenum; Abnormal function of the cardiac sphincter which is the valve allowing food to flow into the stomach. Blockage of the upper digestive system can cause damage to the esophagus resulting in strictures and narrowing, which makes the problem worse or even necrosis and rupture of the esophagus. Regurgitation can result in food and liquid entering the trachea and the lungs. This causes a foreign body pneumonia, which in chronic cases can result in the eventual death of the animal. Esophagus spasm should be distinguished from esophagus obstruction, though the passage of food and water can be obstructed as well. Warning: damaging and even perforation of the esophageal wall as a result of attempts to advance a large-bore tube into the esophangus have been reported!

This page describes the technique of blood collection and hematology with special attention to white blood cell counts in elephants (because of different monocytes, manual counting is required in elephants). Hematology Blood collection Hematology Blood chemistry Reference values blood Blood collection There are 3 anatomical sites on the elephants body where blood can be collected: One of the ear veins (or arteries for arterial blood sample) One of the branches of the saphenous veins (hindlegs) Cephalic vein (front legs) How to collect a blood sample: Most elephants can be trained for this procedure using positive reinforcement training. If not trained, standing sedation (or general anesthesia) in case of free ranging elephants) will be required. When alpha-2 agonists are used , vasoconstriction may hinder the access to the ear veins, especially in young calves. Combining alpha-2 agonists with butorphanol may help to increase the filling of these veins. When the environmental temperature is low, the ear veins may collapse. Flushing the inner side of the ear with large amounts of warm water (or packing the area with heated bean or rice bags) can increase the filling of these veins. The collection site should be clean and dry before blood is collected. Ear veins : press firmly on the site where you can see the shape of the vein. Press until you see the vein becoming larger in diameter. Use a small butterfly needle and collect the blood in a vacuum blood tube. If not available, you can use a small needle (23G) and a syringe and empty the syringe in the blood collection tube after removing the needle. To avoid damaging the blood cells (hemolysis), the tube should be filled slowly while flushing the blood carefully against the wall of the tube, that is slightly tilted. Saphenous and cephalic veins : these veins are larger in diameter than the ear veins, but covered by a thicker skin. A 19G or 21G needle (preferably connected to a vacuum tube) can be used and should be inserted perpendicular to the skin. Indication of the blood collection sites on the front leg (left photo, Vena cephalica) and the inner side of the hind leg (middle and right photo, Vena saphena). Blood collection from the inner side of the ear using a butterfly needle and vaccum tube. Blood cellection from the inner side of the hind leg of a well-trained adult Asian elephant bull using a vacuum bottle for collecting large amounts. Courtesy: Rotterdam Zoo Hematology This chapter includes a lab manual that was developed for a Healthcare and Welfare Workshop for elephant veterinarians given in Myanmar in 2018, organized by Elephant Care Asia (an initiative or elephant Care International - http://elephantcare.org/ . Get PDF of the manual PLEASE NOTE: white blood cell differentiation in elephants must always be done by MANUAL DIFFERENTIATION Erythrocytes: Erythrocytes in elephant whole blood EDTA samples can best be counted on an automated blood cell analyzer. Such an analyzer automatically measures the hematocrit . After centrifugation of the blood sample, a plasma evaluation should always be performed. The morphology of the erythocytes should be examined microscopically in a (preferably fresh) blood smear , stained with Wright-Giemsa. If no automated cell counter is available, manual erythrocyte count is a second option. Total white Blood count (WBC): Automated analyzers have limited value in elephant hematology. However, they can be used to measure the total amount of white blood cell. Automated cell counters come in several forms. Those used for other mammalians can only be used for total WBC in elephants. Manual WBC count can be done using a Hemocytometer Counting Chamber . Differential White Blood Cell (WBC) count: Automated analyzers cannot be used to differentiate the white blood cells of elephants. This should ALWAYS be done by manual counting the different cell types on blood smears , stained with Wright-Giemsa. See also the Manual Differential WBC Count . Platelets: Estimating the platelet count is best done by calculating the average of platelets counts in 10 fields x 15,000, which gives the Estimated platelet count/µL . See also: Manual platelet count (Cornell University) . Reference values: The table below shows the normal hematology values for Asian and African elephants (Wiedner, E. 2015). References Perryn K.L. et al. 2020. Biological variation of hematology and biochemistry parameters for Asian elephant (Elephas maximus), and applicability of population-derived reference intervals. Journal of Zoo and Wildlife Medicine 51(3): 643–651 Steyrer C, Miller M, Hewlett J, Buss P and Hooijberg EH (2021) Reference Intervals for Hematology and Clinical Chemistry for the African Elephant (Loxodonta africana). Front. Vet. Sci. 8:599387 Weisbrod T.C., Isaza R., Cray C., Adler L., and Stacy N.I. 2021. The importance of manual white blood cell differential counts and platelet estimates in elephant hematology: blood film review is essential. Veterinary Quarterly, 41:1, 30-35, DOI: 10.1080/01652176.2020.1867329. (Click here for the complete text). Wiedner E. 2015. Proboscidea. In: Fowler's Zoo and Wild animal Medicine 8. Species 360 - ZIMS 2023. Reference values To hematology gallery Back to Top To lab diagnosis Hematology Blood collection

This case report describes an open fracture of the mandible in a young Asian elephant, that resulted in a severe infection. The elephant was humanely euthanized. To bone fractures Case report Mandibular fracture Date: 2020 Place: 13th Asian Society of Conservation Medicine Virtual Conference History •Coconut-sized abscess on left mandible •Asymmetric buccal margins •Tongue ulceration •Periodontitis •Endodontitis •Foul-smelling caseous material •Malocclusion Diagnostic results •Radiography 70kVp, 10mAs •Lateral and intraoral bisecting angle •Soft tissue swelling •Callus formation Conclusion: Left sided comminuted fracture at the body of the mandible. Unilateral mandibular fracture on left side → Malocclusion → Failure of 𝑀3 extrusion on right side → Impeded eruption of caudal molar on right side → Abnormal eruption of 𝑀5 on right side → Excessive molar abrasion on the left side → Compromised mechanical digestion of food → Anorexia → Malnutrition Treatment The animal was not a release candidate, and keeping the animal in captivity for a prolonged period nor permanently was not an option. Therefore, the authorities opted for euthanasia. Read the poster To page top

The hormonal changes (progesterone, estrogen and luteinising hormone (LH) play a key role in the estrous cycle. Transrectal ultrasound examination can visualize the ovaries, uterus and (vestibular) vagina. Monitoring the estrous cycle is done with gestagen assays in blood (ADVIA Centaur XP or miniVidas), urine (beta-pregnanetriol) or feces (usually locally designed assays). To reproduction Estrous cycle Schematic overview of the endocrine and ovarian events during the estrous cycle (courtesy Imke Lüders). LUF= Luteinizing follicles LH = Luteinizing Hormone Pm = Progestagens CL = Corpus Luteum ovCL = CL from ovulation acCLs = accessory corpora lutea Progestagens in elephants include a large group of progesterone-like molecules. The major part of progestagens found in the blood of elephants does not react in the usual human assays. When starting monitoring progestagens, one should always validate first the assay by providing serum samples with known progestagen concentrations of different levels. Even the use of the same hardware, is not always a guarantee that the results are reliable. Companies permanently develop news progesterone assays for the same machine, which are more specific for human progestagens and less for elephant progestagens. The estrous cycle can be divided in 2 major phases, the luteal phase and the non-luteal or follicular phase . The luteal phase starts after ovulation and lasts on average 10 weeks. It is characterized by elevated progestagen blood levels. The follicular phase starts when the progestagen blood concentration has dropped to what we call in this document the “base line” value. Progesterone serum levels in mmol/l during the estrous cycle in an elephant. Note the 2 LH-peaks (red arrows). Progesterone analysis Many lab assays fail to accurately measure progestagens in the blood during the follicular phase in elephants. Some machines calculate these low concentrations rather then measuring them exactly. Moreover, the progestagen concentrations that are provided by the different laboratories, vary greatly depend on the machine and technique used. It is therefore very important to use the same technique and machine for monitoring the estrous cycle of an individual elephant. Note: progestagen concentrations measured by one assay may differ up to a factor 10 from the results obtained when another assay is used. Automated analysers that have proven to give reliable results for both species are: ADVIA Centaur XP (Siemens) with ranges between 0.21 mmol/l and 10 mmol/l. miniVidas (Biomérieux) with ranges between 0.79 mmol/l and 15 mmol/l. Laboratory System canine progesterone assay: point-of care (=on the spot) assay to measure 4-pregnen-3,20-dione drop as prediction for parturition (Molenaar et al. 2022). During the follicular phase, approximately 18-20 days prior to ovulation, a 1-day LH-peak concentration can be distinguished, which may result in a temporary rise of progesterone. This first LH-peak results in ovarian follicles that will not ovulate. These follicles produce progesterone untill they go into gregression in the luteal phase. A second LH-peak occurs just prior to ovulation. This LH-peak induced ovulation in one follicle. The remaining corpus luteum will also produce progesterone and maintain the luteal phase for about 10 weeks. To monitor the estrous cycle in elephants gestagens can be measured in blood (progesterone), urine (pregnanetriols, Primate Center Göttingen, Germany) or feces (progesterone, Chester Zoo, UK). Frequency of sampling should be once a week for blood and urine and 3 times per week for feces. Urine samples Urinary hormone analyses are performed at the Endocrinology Lab at the German Primate Centre in Goettingen, Germany. All facilities in Europe are welcome to send samples on a voluntary basis and assays are run every week. The service for the Asian elephants started in 1994 and that for African elephants in 1996, so comparative data are available for more than 20 years. It is important to note that in urine not progesterone itself but its metabolites are measured. These metabolites differ between the two elephant species. Whilst in Asian elephants pregnanetriol (P3) is the most abundant metabolite in urine, it is 5α-pregnane-3-ol-20–one (5α-P-3-OH) in African elephants. This means that different assays are needed for Asian and African elephants. The pregnanetriol concentration in the urine is always compared with urinary creatinine. If the creatinine level is too low, a new sample should be submitted. Urine can be collected in many different ways depending on keeping and housing system. Elephants can be trained to urinate on command. Only 2 ml urine are needed for the analysis. It is good to use plastic tubes that close well, best with screw lid. Labelling of the sample is essential! Labels must be waterproof and show the name of animal and the date of collection. If the samples are not sent within 2 days after collection, they need to be frozen soon after collection. See also the Practical guide for urine collection (Ann-Kathrin Oerke) . Fecal samples The estrous cycle can also be monitored by measuring gestagens in fecal samples (3 samples per week). Currently Chester zoo offers this service. If you are interested in this method of monitoring the estrous cycle, please contact Sue Walker (s.walker@chesterzoo.org) or Rebecca Mogey (r.mogey@chesterzoo.org). References Molenaar FM, Rowcliffe M, and Lakey A. 2022. Adaptation of a point-of-care canine progesterone test for use of parturition prediction in captive Asian elephants (Elephas maximus): proof of concept. J. of Zoo & Wildl. Med. 53(4): 791–796, 2022. Practical guide for urine collection (Ann-Kathrin Oerke) . Veterinary guidelines for reproduction-related management in captive female elephants . EAZA 2020. To page top

A longitudinal crack or fissure in an elephant tusk can be treated by the application of a carbon cast if the pulp tissue is not exposted. The procedure can be performed in a well-trained animal without sedation or using standing sedation. case report Tusk fissure treatment Place: Rotterdam Zoo Date: 2019 Data provided by: Willem Schaftenaar History A 22-yrs old bull elephant developed a longitudinal crack (fissure) in its left tusk, presumably due to excessivly hitting the tusk against the steel door of his enclosure during musth. The fissure had a length of 20 cm and was located at the dorsal side of the tusk, extended below the sulcus. The fissure was getting longer all the time. It was not known whether the pulp tissue was exposed. Treatment A standing sedation was performed using detomidine and butorphanol. The eyes were covered by cotton gauze patches. Radiographs were taken to determine whether the fissure was in contact with the pulp tissue. Unfortunately it was not possible to visualize the fissure in the proximal part of the tusk. Radiograph of the left tusk, showing the pulp cavity (white dashed line) and the fissure (yellow dashed line). The depth of the fissure was carefully explored by grinding the borders away using a hand grinder (Dremel). It became clear that the fissure had not reached the pulp tissue. Consequently the fissure was etched with a hypochloride solution for bonding and the proximal 8 cm was filled with glass-ionomer cement. Following the procedure described before ( https://www.youtube.com/watch?v=Do13hcb8wJI ), a 30 cm long fiberglass-reinforced carbon cast was made around the tusk in order to prevent the fissure to get larger. When after grinding it became evident that the pulp tissue was not exposed, the proximal 8 cm of the cleaned-out crack was filled with glass-ionomer cement The carbon cast is being positioned. Note that the plastic tube is placed to make the cast vacuum durig the drying process. Treatment results During the months following the treatment, the tusk continued to grow. The proximal end of the cast was partly damaged when he was rasping the tusk on hard objects. It was provisionally repaired with a 2-component resin. After 10 months the distance between sulcus and proximal end of the fissure was 5 cm. Assuming that a normal tusks grows approximately 1 cm per month, the fissure must have been at least 5 cm inside the sulcus at the time of treatment. The fissure did not increase in size during the following 20 months. 10 months after the treatment, the distance between the sulcus and the fissure is 5 cm. Three years after the treatment it was clear that the fissure had not become longer (Photo: courtesy Eindhoven Zoo) References: https://www.youtube.com/watch?v=Do13hcb8wJI Sim R.R. et al. 2017. Use of composite materials as a component of tusk fracture management en an Asian elephant (Elephas maximus) and an African elephant (Loxodonta africana). Journal of Zoo and Wildlife Medicine 48(3): 891–896. To page top

Serum chemistry data in elephants can provide valuable information about organ function (liver, kidney, muscles, intestines, pancreas) and disease conditions (inflammation, infection), hormone levels and toxins. Proteins, AST, ALT, GGT, creatinine, bilirubin, CK, LDH, Ca, P, glucose, Na, Cl, K are part of the comprehensive chemistry panel. To lab diagnosis Blood chemistry Reference values blood Serum/plasma chemistry Refractometry Serum/plasma Chemistry Serum quality Ser um quality Serum chemistry data may help determining the function of certain organ systems. The reliability of the results largely depend on the quality of the sample. Before running any biochemistry tests, the blood should be well clotted, preferably without hemolysis (shown as red colorat ion of the serum). Chemistry data are usually obtained from serum. Some tests can also be run usi ng plasma. Before using plasma, this option should be checked with the test instructions. Test tubes to be used: red-topped serum-tubes with or without a clotting activator. The color of the serum should be light yellow. The figure below shows different serum characteristic: Hemolysis: Red to brown color due to hemolysis (destruction of erythrocytes). This can have a pathological origin or can result from poor sampling/handling; the red-brown color can also be caused by myoglobin after massive muscle damage (rhabdomyolysis) Milky white color due to presence of fat particles in the serum (physiologic shortly after eating or pathological condition) Yellow color due to the presence of bilirubin (liver damage ->icterus). Blueish-red color due to methemoglobin (low venous oxygen saturation). Lipemia Lipemia, which can be a natural occurrence if the elephant has just eaten, can alter several test results. Calcium, phosphorus, total bilirubin or hemoglobin may be falsely elevated. When using a refractometer to measure the total protein remember that the serum must be clear. If not the value may be falsely elevated. Albumin, sodium and potassium may be falsely lower. Lipemia also enhances hemolysis which in turn can affect lab results. But there is a solution: If you refrigerate your sample the lipemic portion will separate and you can use the clear aliquot below the lipid layer. Normal and lipemic elephant serum Reference values Normal serum chemistry values are determined by species, age, gender and reproductive status of the elephant. It is important to have an understanding of the limitations of laboratory values. The term “reference value” is now considered a more appropriate term than “normal value.” Ideally reference values should be established from studies using a minimum of 30 healthy animals and stated selection criteria. Few elephant studies have been conducted to meet this standard. One report describes chemistry results for different genders of Asian elephants used for logging in Myanmar (Santo, 2020). Moreover, many reference ranges are laboratory specific. So it is best to use one laboratory that can help to develop reference ranges for your elephants. Another important point is that a test result that falls outside of the reference is not necessarily clinically significant. Lab values are information that must be used with all the other information that you have when you are faced with a sick elephant. Establishing a baseline during health and performing sequential tests during illness will give the most reliable information. You also want to use a lab that has good quality controls. If you change labs it is advisable to get new healthy baselines. V ery odd results should always be double checked at the same lab. To page top One study in 10 healthy Asian elephants showed that most Asian elephant hematology and biochemistry parameters are highly individual, requiring individual normal values for accurate interpretation (Perrin, 2020). Test result units Another complicating factor when looking at serum chemistry values is that there is a lack of uniformity regarding units and this can be confusing. Most U.S labs use conventional units whereas in Europe they use SI units. There are conversion factors to go from one system to the other but the conversion factor is test specific – so each test has a different conversion factor. You can find SI conversion calculators online, i.g. ht tps://www.amamanualofstyle.com/page/si-conversion-calculator. Liver The liver plays an important role in the following processes: Protein synthesis and degradation (albumin, clotting factors) Carbohydrate and lipid metabolism Breakdown of hemoglobin Storage (fat soluble vitamins) Detoxification Liver enzymes: Aspartate aminotransferase (AST; SGOT) Alkaline phosphatase (ALP) γ-glutamyl transferase (GGT) Bilirubin Bile acids (?) BSP excretion (bromsulphthalein) AST (SGOT) Aspartate aminotransferase, previously known as serum glutamic oxaloacetic transaminase (SGOT) occurs in all cells. Highest levels are in the liver, cardiac muscle, and skeletal muscle. If AST is elevated then you should also look at the creatine kinase (CK) value. If the CK is normal then AST is likely of liver origin. If the CK is elevated or there is obvious muscle trauma then AST may be of muscle origin. Also AST may falsely increase if the sample is hemolyzed. ALP Alkaline phosphatase is also found in all cells with the highest levels in liver, bone, kidney, intestine, and placenta. ALP is not a sensitive indicator of liver disease in the horse and this probably holds true for elephants although research would be needed to confirm this assumption. ALP may increase with disorders such as rickets. ALP levels are normally higher in young animals including elephants. ALP may indicate colostrum absorption. Non-steroidal anti-inflammatory drugs may cause ALP to elevate. GGT Gamma glutamyl transferase (GGT) is liver specific in horses and pigs. Whether it is liver specific in elephants is unknown. GGT is an indicator of cholestasis (the interruption of bile excretion). GGT has been shown to increase in musth bulls and it has been used together with ALP as an indicator to evaluate passive transfer of antibodies to neonates via colostrum. Bilirubin There is not much information about bilirubin in elephants. There are two forms: unconjugated and conjugated bilirubin. The unconjugated is the main form in horses so this may be true for elephants but we don’t know for sure. Unconjugated (indirect) bilirubin is mainly hemoglobin released from old erythrocytes. It is bound to albumin and transported to the liver where it is conjugated. Conjugated (direct) bilirubin is secreted into bile, transferred to the intestine, converted to urobilinogen by intestinal bacteria, and excreted. Elevated bilirubin may be caused by hemolysis; hepatocellular disease that results in reduced functional mass; and intra - or extrahepatic cholestasis or bile duct obstruction. Unconjugated bilirubin predominates in horses with hyperbilirubinemia regardless of etiology whereas in ruminants unconjugated bilirubin is typical. In one report, elevated total bilirubin (4.94 mg/dl) was observed in a female Asian elephant with colic caused by over-zealous feeding of produce. Values for indirect and direct bilirubin were 3.7 mg/dl and 1.2 mg/dl respectively. Tests of hepatic uptake, conjugation and excretion of bilirubin. Diagram from Lattimer, K.S., Mahaffey, E.A., and Prasse, K.W. 2003. Clinical Pathology 4th edition. Blackwell. P.199. Bile acids Bile acids assist with fat digestion. In most species, bile acids are stored in the gall bladder and released into the intestine. However, elephants do not have a gall bladder. There is controversy whether elephants have bile acids. In several cases bile acids were shown to increase in elephants with TB. Bromsulphthalein (BSP) excretion test In the BSP test a dye is injected IV and measured at several points in time post-injection. In the horse, the half-life is 3.5 minutes. Slow clearance time may indicate cholestasis. Although levels have been shown to increase with liver flukes in elephants, it is not a very practical test. Kidney Blo od Urea Nitrogen ( BUN) and creatinine are the main enzymes used to evaluate kidney function in mammals. BUN Elevations in blood urea nitrogen concentration may be due to prerenal causes like inadequate renal perfusion, shock, or diminished blood volume; renal causes like glomerular-nephritis; or postrenal causes like urinary tract obstruction. Blood Urea Nitrogen makes up approximately 75% of the total non-protein nitrogen (NPN) fraction of the blood. BUN is the major end product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver from ammonia which is produced by amino acid deamination. Urea is excreted mostly by the kidneys, but minimal amounts are also excreted in sweat and degraded in the intestines by bacterial action. Creatinine Creatinine only elevates when disease is severe and there is marked kidney damage. Unfortunately, in elephants these enzymes are not always useful to predict kidney disease. Creatinine may be lower in young elephants; higher in musth bulls. Serum osmolality/urine osmolality. The osmolality reflects the total number of electrolytes in a fluid. To keep the number constant, the kidney excretes the surplus that is present in the liquid fraction of the blood and the osmolality ratio between these two fluids should be <1. If the kidneys fail to maintain this equilibrium, the serum-urine osmolality ratio becomes > 1. Serum dimethyl arginine (sDMA). In domestic animals sDMA is a marker for endothelial dysfunction and early diagnose of renal disease (declining glomerular filtration rate). In one case report it was associated with kidney failure (polycystic kidney disease). To page top These values are from an Asian bull elephant with capture myopathy. The elevations in SGOT and CK are dramatic. ALT (SGPT) did not increase as much but is probably significant. The elevations in BUN and creatinine may have been related to capture myopathy however this bull was chained to a tree and not given access to food or water post-capture so these changes may reflect dehydration. Muscles Muscle enzymes are: Creatine Kinase (CK) Lactate Dehydrogenase (LDH) Aspartate Aminotransferase (AST; SGOT) Alanine Aminotransferase (ALT; SGPT) Evaluating muscle enzymes can help to diagnose muscle pathology. Conditions that may cause elevated muscle enzyme levels include prolonged recumbency, rhabdomyolysis (also called over-exertion, or tying up syndrome), and clostridial myositis. Bacterial endocarditis, and aortic thrombosis are other causes as well as Vit E/Se deficiency and capture myopathy. Working elephants may be at risk for muscle over-exertion disorders especially as the planet heats up. In all of these conditions, muscle cell membranes rupture and enzymes are released into the blood. Conditions that may lead to increased muscle enzymes in serum are: Prolonged recumbency Rhabdomyolysis Clostridial myositis Bacterial endocarditis Aortic thrombosis Vitamin E/ selenium deficiency Capture myopathy Creatine kinase (CK) CK is critical to muscle energy production. Highest levels are in skeletal muscle, cardiac muscle, and brain. Most CK in the serum is of muscle origin and it is the most sensitive indicator of muscle damage. CK rises quickly – within hours. It also returns to normal quickly as long as there is no on-going damage. Levels that remain high indicate an on-going disease process. Hemolysis interferes with the test and causes falsely elevated values. Lactate d ehydrogenase (LDH) LDH is present in all tissues. Muscle, liver, and red blood cells are the usual sources. LDH is not as useful for determining muscle damage because it is not muscle specific. If LDH is elevated and there is no muscle injury then liver problems should be considered. Like CK, LDH will be falsely elevated in the presence of hemolysis. Aspartate aminotransferase (AST) AST was previously known as serum glutamic oxaloacetic transaminase (SGOT). Muscle and liver are the major sources. It is another enzyme to check if muscle damage is suspected. Alanine aminotransferase (ALT) ALT, also known as serum glutamic pyruvic transaminase or SGPT, is considered muscle specific in large domestic animals. Increased levels have been associated with myopathies in a number of species. ALT will increase in recumbent elephants that are down for a long time. Severe muscle damage can occur in case of Capture Myopathy: To page top Calcium Ionized calcium This electrolyte that is involved in many chemical reactions in the body. The active form is ionized calcium (Ca2+) and this parameter gives the best impression of the available calcium. To measure ionized calcium, special heparinized tubes are needed . Ionized calcium should be > 1.5 mmol/L (>5 mg/dL). Low ionized calcium levels are associated with prolonged parturition and dystocia. Total calcium Non-ionized calcium is predominantly conjugated with albumins and expressed as total calcium. Higher levels of total calcium have been reported in very young elephants and in elephants with TB and severe kidney disease. Many pregnant captive elephants develop subclinical hypocalcemia if calcium is not supplemented during pregnancy. When parturition starts, the demand of calcium is high as it essential for uterine contractions that open the cervix and help expel the calf. Calcium is also high in milk. Calcium supplementation during pregnancy is recommended to form a stock supply. However, releasing stock-calcium ions from the bones is a very slow process, so the blood itself should contain enough calc ium conjugated to albumin to supply the uterus and body muscles for these hours of high demand. Total calcium levels lower than 1.5 mmol/l (6 mg/dl) result in recumbency and levels less than 1 mmol/l (4 mg/dl) will result in death. Low total calcium levels are associated with prolonged parturition and dystocia. Total calcium should be 2.8 +/- 0.2 mmol/L (11 +/- 0.8 mg/dL). Other minerals and electrolytes Sodium (Na) Potassium (K) Chloride (Cl) The reference ranges of these electrolyte in elephants are similar to other species and can best be compared with horse values. To page top Proteins Elephants have a higher to tal protein serum level than most mammals; albu min is lower, and globulins are higher. The ratio of albumin to globulin was shown to be lower in one study that compared elephants with and without TB however the number of TB positive elephants in the study was low. Protein electrophoresis separates the albumin and globulin fractions. There are not many reports in elephants. Protein electrophoresis may be useful to monitor inflammatory conditions or problems like TB, herpes, and others. Acute Phase Proteins Acute phase proteins (APPs) are components of the innate immune system that are markers for infection, inflammation, neoplasia, and tissue injury in humans and domestic animals. APPs are produced by the liver in response to cytokines released from leukocytes. They are initially released into serum 24–96 hr following an acute inflammatory stimulus, where they function to promote healing, reestablish homeostasis, and inhibit microbial growth. The main ones are C-reactive protein (CRP) , serum amyloid (SAA) and haptoglobin (HA) . In one study serum samples from 35 healthy Asian elephants were analyzed for these 3 APPS and levels between the values in healthy and unhealthy elephants were compared (Isaza et al., 2014). From this and other studies it seems that SAA may be the most responsive APP in elephants. The APP values in this study in Asian elephants are shown here: C-reactive protein 12.4-122 nmol/l (1.3-12.8 mg/l) Serum amyloid 0-47 mg/l Haptoglobin 0-1.1 mg/l Glucose Glycolysis of glucose in serum or plasma will decrease the glucose level starting shortly after blood collection. Serum or plasma should therefore b e separated from the red blood cells within 30 minutes after collection. Glycolysis can be prevented by using a Na-fluoride tube for blood collection. Significantly lower levels have been noted in one study comparing TB culture positive and negative elephants. In horses, hypoglycemia may be seen with hepatic failure or bacteremia. There is one report of diabetes mellitus in a 50-yr-old Asian elephant (van der Kolk 2011). Hyperadrenocorticism and hyperthyroidism have not been reported in elephants. Increased blood glucose in elephants is likely to be transient. Some of the causes of transient hyperglycemia could be a cute severe colic acute stress, p ost-postprandial, certain drugs (s teroids, x ylazine, p henothiazine) . Hypoglycemia can occur in n eonatal elephants and in cases associated with m alnutrition, m alabsorption, s epsis, e xtreme physical exertion, a dvanced liver disease or n eoplasia. Amylase and lipase Ranges for these enzymes vary tremendously depending on the methodology and the lab so at this point in time they are not very useful tests for elephants. Amylase may increase in case of pancreas, gastro-intestinal, liver and kidney disease Lipase may increase in case of pancreas or kidney disease. One case of pancreatitis in an elephant has been described so far (pers. comm. Susan Mikota, 2023). Lactate Lactate is an important serum parameter to monitor severe, life-threatening conditions in elephants, like septicemia, Disseminated Intravascular Coagulopathy in a EEHV-HD case. Normal values are between 0--0.11 mmol/L (0-1 mg/dL). Values >0.44 mmol/L (4 mg/dL) are indicative for perfusion problems due to DIC. EEHV-HD patients often have lactate value > 0.22 mmol/L (2 mg/dL) (Wiedner, pers. comm. 2022). A comprehensive elephant serum chemistry panel should include: Total protein Albumin BUN Creatinine AST (sGOT) ALT (sGPT) GGT Bilirubin Bile acids (?) CK LDH Na Cl K Ca P Lactate To page top References Steyrer C, Miller M, Hewlett J, Buss P and Hooijberg EH (2021) Reference Intervals for Hematology and Clinical Chemistry for the African Elephant (Loxodonta africana). Front. Vet. Sci. 8:599387 Santos DJ, Franco dos J, John, Nyein UK, and Lummaa VM. 2020. Sex differences in the reference intervals of health parameters in semi-capt ive Asian elephants ( Elephas maximus ) from Myanmar. J.Zoo&Wildl Med 51(1): 25–38 Debbie JG and Clausen B. 1975. Some hematological values of free-ran ging African elephants. Journal of Wildlife Diseases, 11(1):79-82. Isaza R, Wiedner E, Hiser S, Cray C. 2014. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus ). J. of Vet. Diagn. Invest. 1-6 Perrin, KL, Kristensen AT, Gray C, Nielsen SS, Bertelsen MF, Kjelgaard-Hansen M. 2020. Biological variation of hematology and biochemistry parameters for the Asian elephant (Elephas maximus ), and applicability of population derived reference intervals. Journal of Zoo and Wildlife Medicine, 51(3) : 643-651. Van der Kolk JH, Hoyer MJ, Verstappen FALM, Wolters SABI, Treskes M, Grinwis GCM and Kik MJL (2011). Diabetes mellitus in a 50-year-old captive Asian elephant (Elaphas maximus ) bull, Veterinary Quarterly, 31:2, 99-101. Https://doi.org/10.1080/01652176.2011.585793 To page top

Hiccup in elephants has been reported anecdotically several times in conjunction with renal disease (kidney failure), hypocalcemia and low environmental temperature. To non-infectious diseases Case report 'Hiccup' in an adult female Asian elephant Date: 2008 History Adult female Asian elephant showing hiccup-like periodic movements of the abdomen. This phenomenon occured only during the cold period of the year. Treatment The calcium concentration in the food was increased. Unfortunately no calcium blood levels are available. Treatment results The hiccup was not observed anymore during the cold seasons following the increase of the calcium intake. A similar result was obtained in a herd mate of the same age. To page top Hiccup hypocalcemia Hiccup in an adult female Asian elephant with polystic nephritis Case report Date: 2016 Place: Planckendael Zoo Data provided by: Francis Vercammen DVM History Adult female Asian elephant, mid term pregnancy of 6th calf showing hiccups since several months. Chronic weight loss (600 kg decrease), hypercalcemia (35% increase). At transrectal ultrasonographic examination, muticystic nephrophathy was diagnozed. Treatment The diet was adapted (low protein,high energy). A healthy calf was born a term. However, during lactation the condition of the dam deteriorated and the animal was humanely euthanized. Further reading: Vercammen F, Lueders I, Schaftenaar W. 2020. Multisystemic disease in a pluriparous Asian elephant (Elephas maximus). Proc Zoo Wildlife Health Conf 2020 Hiccup kidney disease To page top

Only a handful of fetotomies have been performed in elephants. The case described here is the only one in which the dam survived. Wound healing is similar to the vaginal vestibulotomy, however, wound infection may occur due to the heavy manipulations during the long procedure and the passage of sharp bony fragments of the fetus through the created opening. To Reproduction To dermatology Case report Fetotomy and wound treatment Date: 2008 Place: Rotterdam Zoo Data provided by: Willem Schaftenaar History A 37 years old matriarch Asian elephant failed to deliver her 5th calf. At 13 months from the failed parturition, a herd mate of this animal delivered a healthy full-term calf. The calf tried to nurse from the matriarch and this elephant actively encouraged the calf to do so. Within 6 hours of this behavior, the matriarch displayed intense labor. After 24 hr, the hind legs of the retained calf could be palpated transrectally in the vagina, and several sharp fetal bone fragments (dorsal spines, fractured ribs) were palpated as they almost penetrated the vaginal wall. At that time, the animal was completely exhausted and contractions had ceased. With a failed attempt to extract the fetal carcass through the intact birth canal using two Krey-Schöttler fetotomy hooks, a decision was made to perform a vagino-vestibulotomy. Onset of labor 6 hours after the birth of a calf in the herd Six hours after the birth of a calf in the herd, labour in the matriach started, 13 months after the first signs of labour had come to a stop. Treatment The animal was chained on one front leg and the opposite hind leg. 150 mg xylazine was slowly hand-injected intravenously. During the entitre procedure (11 hours) extra doses of 75 mg xylazine were given intravenously every 50-60 minutes. Four 4 local depositions of 20 ml Lidocain 2% (with adrenaline) were injected in the midline below the anus. Epidural anesthesia was not used, however new insides have proven that this is an important method to reduce movement of the tail and decreases pain perception in the perineal region. The animal remained standing throughout the entire procedure. A 15cm skin incision was made in the midline. A rubber (cattle) stomach tube with a 10x60mm window at the tip was inserted in the vestibulum vaginae and advanced till the tip reached the dorsal edge of the incision. The vestibulum vaginae was incised over the window of the tube. The legs of the calf could be visualized in the horizontal part of the vagina. At least 4 fractured ribs were found, probably fractured by the natural contractions of the uterus duting the past 24 hours. The fractured ends had penetrated the wall of the vestibulum on the left caudo-ventral side, but not completely perforated, resulting in several tears of the mucosa (20cm long). The roof of the vestibulum vaginae had some mucosa tears too, also not perforating the wall totally. The carcass was cut into more then 100 large pieces; many ribs were removed one by one to avoid damage to the wall of the vestibulum vaginae. A complete fetotomy was performed using a Thygesen fetotome (Utrecht model). Finger knifes, Krey Schüttler hooks, saw directors and calf delivery chaines were used to cut the carcass in smaller lieces and pull them out. Large pieces were pulled out with extra support by a calving-pully for cattle. A long blunt "eye-hook" (used to grab a dead cattle or horse fetus in the orbit) was modified at the spot making it a prolonged knife (like a "finger knife" for fetotomy). Muscles were split from their bone attachment using this knife and blunt fingers. Lumbs of the carcass that were too big to pass through the fetotomy opening, were removed via the normal birth canal. After 3 hours 40 ml Duphaspasmin (11.58 mg isoxsuprinelactate per ml, Fort Dodge NL8514) ws injected i.m. followed 30 minutes later by another 40 ml Duphaspasmin i.m. as well as 100 ml Amoxicilline 20% (200 mg amoxicilline trihydrate/ml, NL 2795) i.m. After 9 ¾ hours the entire carcass of the fetus was removed. 5 ml Oxytocin (oxytocine 10 IU/ml, NL3852) was given intravenously. The uterus was flushed with large amonts of luke-warm water using a plastic hoose pipe. The water was drained as much as possible. Ten liters of 0.09% NaCl was brought into the uterus using a cattle stomach tube and a funnel. Thirty minutes after the previous injection, another 5 ml Oxytocin (oxytocine 10 IU/ml) was given i.v. The uterus started to show moderate contraction on the ventral side. The rest of the uterus was still filled with air. A custom-made balloon (100 ml volume) catheter was advanced into the urinary bladder. The mucosa of the vestibulum vaginae was sutered with 2/0 PDS, continuous Cushing stitches. The overlying muscular tissue and connective tissue was sutured in 2 layers using) Vicryl, continuous stitches. Only the dorsal 5 cm of the skin was sutured intradermally using 1 Vicryl, single stitches. The mucosa of the vulva and the distal part of the vaginal vestibule had been severed by the passage of large lumps of the carcass with sharp bony edges. A 84 kg female calf carcass was removed in more than 100 pieces. The carpal joints were in flexed position and could not be bent unless the flexor tendons were cut thorught. This condition is known as arthrogryposis. Whether this had caused the initial dystocia remains inclear. Antibiotic treatment (150 ml amoxicilline 20%, i.m. SID) was given for 9 days. The balloon catheter came out one day after the fetotomy. During the following weeks the vulva and vaginal vestibule became heavily infected and large pieces of necrotic tissues were lost. After 5 days transrectal ultrasonographic examination demonstrated that there was still a lot of detritus in the uterus. Five ml. of oxytocine (10 IU/ml) was given i.m. Surprisingly the uterus was still responsive to oxytocine, as aftre a few hours a lot of fluid was discharged through the vulva. When this was repeated 2 days later, no reaction of the uterus was observed. Making an incision in the vaginal vestibule guided by a plastic tube (with a window cut out) advanced in the vaginal vestibule. The Thygesen fetotome is advanced into the horizontal birth canal. Most parts of the fetus were removed through the vulva, which allowed a relatively small incision of the vaginal vestibule Krey-Schüttler hook Thygesen Fetotome Giggli saw director Custom-made balloon catheter Finger knife Pieces of the carcass were put together to check whether bones were missing. One femoral condyl was missing. It was found the following day. Note the arthrogryposis in the right carpal joint. The left carpal joint is stretchted after cutting through the flexor tendons. Treatment results All sutures came off after 14 days, which facilitated the daily treatment of the distal part of the birth canal: flushing with 50-100 liters of 0.09% saline solution through the surgical wound. This treatment was continued for several weeks. Six weeks after the fetotomy, the elephant was sedated again with xylazine, and the necrotic area of the surgery wound was debrided. In the following weeks, the surgery wound and the wounds in the distal part of the birth canal started to heal nicely. Ten weeks after the fetotomy the surgery wound had reduced in size from 15cm to 10 cm. Flushing with saline water was discontinued. At 14 weeks the surgery wound was 5.7 cm long. At 5 months the skin and mucosa of the vaginal vestibule had fused completely, leaving a 4 cm opening. The elephant was sedated again with xylazine and the skin-mucosa fusion was surgically disconnected. Over a circular area of 4 cm around the opening, the skin was seprated from the vaginal vestibule. The vaginal vestibulum was closed in 2 layers using 2/0 Moncryl; first layer: Schmieden suture; second layer: Lambert suture. The skin was left open. All sutures came off after 5 days. It was decided to allow the wound to heal per secundam. The wound was flushed with saline solution on a daily base. Two months later, the wound diameter was 4 cm and the epithelization between skin and mucosa was complete again. One year after the fetotomy the opening was still 4 cm. Standing sedation was performed using xylazine. The tissues around the fistula were injected with a total amount of 17 ml Lidocain 2% (with adrenaline). A small strip of the epithelium layer that formed the edges of the fistula was cut off and the subcutis was incised, separating the vestibulo-vaginal wall from the skin over 2-3 cm. The vestibulo-vaginal wall was closed using Vicryl 1, continous Utrecht-uterus suture. Antiseptic, silver impregnated gauze was inserted in the subcutaneous space. The gauze was sutured to the ventral edge of the wound using Vicryl 1 to facilitate later removal. The skin was closed using a continuous intracutaneous suture (Vicryl 1). Three extra single matrass sutures (Vicryl 6) and 1 single suture were applied as an extra support for the intracutaneous sutures. The ventral part of the fistula was left open to allow later removal of the gauze. 9 days after wound dressing: all sutures came off. The vaginal wound is retracting in the subcutaneous space (thus enlarging the wound surface). Diameter of skin wound: 6 cm. 15 months after fetotomy:wound almost closed, leaving a permanent opening of 3 mm, Wound healing Severely swollen vulva 3 days after the fetotomy. Two months after the fetotomy, the wound starts healing nicely. Necrotic tissue protruding through the vulva, 12 days after fetotomy. Five months after the fetotomy, epithelization of the edges of the skin and mucosa has interrupted wound healing Five months after the fetotomy, wound dressing is performed. The vaginal vestibule is closed. Photo taken before closing the skin. Five days after the surgical wound correction, all sutures came off. One year after the fetotomy, a second wound dressing is performed. One week later, the wound opened and the subcutaneous drain came off. Eighteen months after the fetotomy, the wound was closed, leaving only a 3 mm opening (black structure above the pink connective tissue area. Conclusion of the author: the skin should not be sutured after a vestibulotomy or a fetotomy in elephants, as it delays the healing process. Closing the vaginal vestibule is also questionable, as the mucosa around the surgical wound is heavily contaminated by the strong manipulations needed for the procedure, resulting in wound infection. Healing per secundam of the entire wound is recommended, no matter how hard it is for a vet to leave such a big wound open! To page top

Reproductive tumors and cysts are frequently seen in aging nulliparous elephants. Diagnosis is based on ultrasound examination. The anomalies may finally result in infertility. If leiomyomas need treatment (because of blood loss), Gonadotrophin Releasing Hormone (GnRH) vaccins can be used to down-regulate the estrous cycle, which will result in reduction of the size of the leiomyomas. To reproduction Reproductive Tumors and cysts Prevalence of tumors and cysts Abegglen et al. (2022) published an overview of reproductive tumors in elephants: "Asian elephants in particular are often diagnosed with benign uterine tumors called leiomyomas or fibroids (the term typically used to describe these lesions in humans). While malignant tumors overall are rare in elephants, when they do occur, the reproductive tract is disproportionately affected. Benign and malignant reproductive tract tumors are known to affect reproduction and pregnancy in other animals, and even tumors outside of the reproductive tract can have significant negative impacts on reproductive success." In a literature study of the same authors the following reproductive anomalies were found in (aged) Asian elephants : Uterine leiomyoma Uterine adenocarcinoma Anaplastic carcinoma (uterus) Carcinoma in situ in endometrial polyp Peripheral neuroectodermal tumor (uterus) Angiosarcoma (uterus) Anaplastic sarcoma (pelvic mass of presumed uterine origin) Ovarian carcinoma Ovarian cysts Hyperplastic endometrial disease Vestibular cysts Vaginal leiomyoma Hyperplastic, polyploidy or papillomatous mucosal lesions of vagina/vulva Vagina polyps Vulvar polyps Uterine polyps Uterus undifferentiated malignant neoplasm The vast majority of reproductive pathologies in Asian elephants consists of uterine leiomyomas, followed by hyperplastic endometrial disease and cyst s. Reproductive tumors and cystic changed found in African elephants are: Endometrial cysts Ovarian carcinoma Bilateral multilocular serous ovarian cystadenoma Hyperplastic endometrial disease Vestibular cysts Vestibular polyps Vagina polyps Polyps and Hyperplastic endometrial disease are the most frequently encountered reproductive anomalies. Symptoms and diagnosis Most elephants suffering from the above mentioned disorders are aged and nulliparous . There are no hard data about the impact of these abnormalities on the reproduction. No doubt that large areas of affected uterine surface may reduce implantation options for the embryo. The same applies to hyperplastic endometrial disease. Large polyps in the distal reproductive tract can impede copulation by blocking the passage of the penis. Symptoms are rarely observed. Occasionally blood loss can be observed in severe cases of leiomyomas. Diagnosis The only way to diagnoses these reproductive disorders is by transrectal ultrasound examination. Location and severity of the abnormalities can be evaluated when they can be reached during this examination. Figure 1: Transrectal examination in a 12 yr-old Asian elephant showing the early stage of an embryo in the left uterine horn and a large leiomyoma in the right uterine horn, clearly compressing the endometrium. The embryo developed normally and the elephant produced several calves after this diagnose was made. Figure 2: Leiomyoma (55 cm diameter) in an Asian elephant that produced 6 calves. (Courtecy: Planckendael Zoo) Figure 3: Transrectal examination in an adult African elephant showing a cyst in the cervix uteri. Figure 4: Transrectal sonogram (4-2MHZ) of the uterus (UT) with a cystic (CY) degenerated endometrium. The rectal wall (RW) appears as a moderate echoic strip on the top of the sonogram (Fowler & Mikota 2006) Treatment of severe cases of leiomyomas Female elephants suffering of chronic pathological conditions in the reproductive tract (leiomyomas) may benefit from a permanent shut-down of the estrous cycle by the administration of gonadotrophin releasing hormones (GnRH) vaccins. The following schedule should be used: A minimum of 450 μg of a GnRH vaccine (i.e. 3ml of Improvac©, Zoetis Animal Health)* deep intramuscularly once per month for 3 months. Thereafter booster vaccinations are given every 6 months (please note that some individuals may not respond and thus, require higher doses of up to 1000 μg protein conjugate or more frequent injection, in this case, please contact the v et advisors). Other available commercial products/brand names are Improvest©, Equity© and Bopriva©, depending on country. References Abegglen, L.M.; Harrison, T.M.; Moresco, A.; Fowles, J.S.; Troan, B.V.; Kiso,W.K.; Schmitt,D.; Boddy, A.M.; Schiffman, J.D. 2022. Of Elephants and Other Mammals: A Comparative Review of Reproductive Tumors and Potential Impact on Conservation. Animals 2022, 12, 2005. https:// doi.org/10.3390/ani121520052022 Reproductive Tumors and Potential Impact on Conservation Boedeker et al. 2012. Effects of a gonadotropin-releasing hormone vaccine on ovarian cyclicity and uterine morphology of an Asian elephant (Elephas maximus). Journal of Zoo and Wildlife Medicine 43(3): 603–614, 2012. Landolfi JA, Gaffney PM, McManamon R, et al. Reproductive tract neoplasia in adult female Asian elephants (Elephas maximus). Veterinary Pathology. 2021;58(6):1131-1141. https://doi : 10.1177/03009858211031843 Lueders et al. 2019. Use of gonadotrophin releasing hormone (GnRH) vaccines for behavioural and reproductive control in managed Asian elephant Elephas maximus and African elephant Loxodonta africana populations. Int. Zoo Yb. (2019) 53: 138–150. Thitaram et al. 2018. Monitoring and controlling ovarian activity in elephants. Theriogenology 109, 42-47.

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