Elasmobranchs Flashcards

Question

What is the order most rays are in? What are the families of the following commonly kept rays: eagle rays, cownose rays, manta rays, southern rays, river stingrays, whiptail stingrays? What are the defining anatomic features of this group?

Answer 1

*Myliobatiformes (Cownose rays, Stingrays, Manta rays)* * Typical stingrays - dorsoventral compression w/ enlarged pectoral fins continuous w/ head * 5 gill openings ventral; eyes and spiracles on dorsal surface - water pulled in through spiracles for respiration - jaws protrusible and do not articular w/ neurocranium * No nictitating membrane, cornea attaches directly to skin; cranial vertebrae fused w/ pectoral girdle (synarcual), no anal fin, most have one or more venomous spines * *Aetobatidae, Myliobatidae, Mobulidae, Rhinopteridae* - eagle rays, mobulids, cownose rays - small dorsal fin at base of tail, head elevated, eyes and spiracles lateral on head; tooth-plates made up six-sided teeth in horizontal arrangement; capable of leaping into air. Mobulids have cephalic lobes - cranial extensions of pectoral fins to assist in feeding * Have gill rakers, mobulids lack venomous barbs * *Dasyatidae* - whiptail stingrays - inhabit FW and SW habitats; long, slender tail * *Potamotrygonidae* - river stingrays - all FW species, reduced rectal gland, venom more potent

Answer 2

*Torpeniniformes (Electric rays)* * Strong electric organs derived from branchial musculature; dorsoventrally flattened w/ rounded pectoral fins; slow-moving and do not use pectoral wings much for locomotion * Skin is soft, no denticles or thorns - eyes very small; well-developed caudal fin

Answer 3

*Pristiformes (Guitarfish, Sawfish)* * Guitarfish - stout tail, with two dorsal fins, caudal fin w/out a barb * Bowmouth guitarfish - similar but tail bilobed and rostrum unique * Sawfish - carpenter sharks - rostrum shaped like flat blade, non-replaceable “teeth” (modified dermal denticles) * Mouth and nares ventral, oral dentition varies but teeth mostly blunt-edged “cobblestones” in rows; prominent spiracles, no anal fins

Answer 4

**Fowler 9, Chap 48: Touch-Pools: The Other Side of the Hand** 1. Touch-pools provide unique immersive experience for guests to interact with collection 2. Should be designed to enhance welfare, minimize stressors, while balancing good guest experience **Setting Goals**- before design starts to guide habitat plans/species selection- animal welfare, education, conservation 1. Animal health/welfare standards compliant with AZA 2. Track morbidity/mortality and compare to display only exhibits 3. Long-term sustainable population plan 4. Meet guest needs and promote conservation discussion **Species Selection** - determine early to help optimize habitat, within framework of goals that were set 1. Meeting quarantine and environmental needs for all species, management if they outgrow habitat 2. Ease of monitoring individual health, mitigating morbidity and mortality, expected lifespan and breeding activity 3. Time on exhibit, desired behaviors for exhibit, rotating animals in and out 1. **Ideal species- stable populations with little turnover**- ELASMOBRANCHS (may outgrow), invertebrates \> teleosts 2. Determine training/enrichment plan early on (desensitize to touch, target feeding, husbandry behaviors **Planning** - all groups involved in building and managing should be involved in planning, allow additional time for training 1. Requires more space, should provide **refugia**, use suitable substrate/décor for target species 2. Allow interpreters to access animals and be seen by guests, use a cue if entering habitat itself 3. Life support system larger/more complex**- increased contaminants- high water turnover, biofilter accommodating peak attendance, good foam fractionation** (saltwater)**, UV light + Ozone** (monitor nitrate, iodate, iodide) 4. Regular period of full darkness, maintain appropriate temperature despite fluctuations in attendance/season 5. Off-exhibit holding area improves health/welfare- close to touch-tank to minimize transport time, same water system 6. **Hand-washing/drying stations**- low zoonotic risk, some guests may be immunocompromised, **30-60% compliance** 7. **Contingency planning: human fluid contamination, human injury, zoonotic concerns**, life support failure, morbidity/mortality, animal abnormality, welfare concerns **Monitoring**- more closely tracked than display-only habitats 1. Assess water quality parameters regularly- expect fluctuations in DO2, NH4, temp- include bacterial counts 2. Review feeding/behavior records regularly to address health concerns as needed 3. Routine exams, routine trimming of stingray barbs 4. Ensure collection in touch-pools is doing as well as display-only collection 5. **2-3 years to find optimal balance of animal health, life-support systems, guest dynamics** 6. Extensive training for staff- natural history, conservation/education goals, common questions, animal touch rules 1. Positive reinforcement for appropriate guest behavior 7. Evaluate guest metrics

Answer 5

* **Most common mineral deficiency in elasmobranchs = Iodine**. * Results in goiter. * Oral supplementation is recommended in systems where levels are below natural seawater and nitrate \> 10 mg/L. Commercially available supplementation tablets for elasmobranchs will meet these requirements. * **Ca and P major roles in growth and development of bone, teeth, cartilage, muscle contraction, and blood clotting in teleosts.** * Ca:P ratio in diet should be 1:1 or 2:1. * Inverted ratios lead to skeletal malformations, poor growth and muscle disorders. * Dietary Ca minimal contribution to daily requirements in teleosts, as fish have access to ionic Ca in sea water. * **Diets including whole bony fishes generally meet min Ca:P ratio.** * **Consider supplementing Ca if feeding chopped fish.** * Vitamins * **Nutritional deficiency in Vit C and Zn has been suggested as causative of spinal deformities in sand tiger sharks.** * **Health animals higher K, vit C, vit E, and Zn vs affected. Small sample size in that study.** * **Vit C and Zn are cofacts involved in collagen synthesis and cartilage development across taxa.** * While teleosts cannot synthesize vit C and require it in the diet, some research shows elasmobranchs may be able to synthesize it in the kidneys. Vit C can be supplemented. * Vit E declines over time through storage of fish. Elasmobranchs fed thawed fish are susceptible to deficiencies. * Thiamin (B1) – Integral for carbohydrate metabolism, digestion, growth, fertility, neuro function in fish. * Thiaminases found in certain fish and shellfish, render thiamin inactive when ingested. * Thiamin can also be lost by holding diet items too long in storage. * Wet or frozen diets risk thiamin deficiency. * Supplement thiamin when feeding frozen thawed fishes and shellfish. * CS of deficiency – Neuro symptoms, muscle spasms, spinning, jerky movements. * Supplementation is recommended. * Vit D and D essential for teleosts, probably also for elasmobranchs. * Potential accumulation in liver and adipose when over supplemented. * Excess vit A causes slow growth, anemia, abnormal vertebral growth and mortality. * Vit A and D likely met in aquarium elasmos that consume whole fatty fish. * Mollusks, crabs, crustaceans generally low in fat-soluble vitamins. * Nutrient values in elasmobranch blood: * Plasma Vit A largely influenced by diet or level of supplementation. * Plasma or serum vit A (retinol) good indicators of status. * Plasma vit E (a-tocopherol) may be directly related to diet or level fo supplementation. Variable based on spp. * Serum Ca generally only changes with prolonged deprivation or pathology. Plasma or serum P are a good determinant of phosphorus status. * Plasma/serum Mg reflective of dietary status, K may not be. * **At minimum – Supplementation with I, thiamin (B1) is recommended for elasmobranchs. Ca, Zn, Mn, vit C, vit A, and other nutrients can be considered.**

Answer 6

Quarantine Planning: * **Elasmobranch systems need appropriate space for normal swimming, gliding, and turning.** * **Large oval tanks are preferred.** * Jump barriers should be regularly inspected. * **Ozone and/or UV disinfection can reduce pathogen burdens.** * **Goitrogenic effects of ozone use and high nitrates are unlikely to be an issue in short term quarantine periods, supplement diet with iodide anyway.** * **Techniques for encouraging food intake:** * **Offer variety of foods – fish, crustaceans, mollusks, cephalopods.** * **Offer fresh rather than frozen.** * **Add fish blood to water prior to feeding.** * **Crushing or cutting stripes into food prior to feeding.** * **Using rod, grabber, or line to move food in water.** * **Offer live foods that clear quarantine.** * **Feed at different times to match feeding times in wild or previous institution.** * **Avoid aversive stimuli i.e. loud noises, people leaning over aquarium.** * **Isolate animals in suitable systems or parts of the system to reduce competition for food.** Physical Exams – Quarantine: * Endoscopic or laryngoscopic examination of buccal and branchial cavities can help ID parasites. * Determine sexual maturity and BCS. * Morphometrics – Body mass BM, total length TL, precaudal length, snout-to-vent length and/or disc width (DW). * Remove visible parasites, save images for future. * Gill biopsies – Potential for hemorrhage, lower diagnostic value vs teleosts, not often performed. * Fecal samples for helminths and coccidia from water or cloacal wash. * Coelomic aspirates, flushes recommended in cownose rays for coccidia. * Blood – Hematology, bioichemistry, blood gases, lactate and ancillary testing i.e. macronutrients, vitamins, hormone assessment. * Venipuncture – Ventral tail vein, posterior cardinal sinuses, wing or radial veins. * **Venipuncture sites affect PCV – Lower PCV from dorsal sinus (~8%). Sharks more tolerant of anaerobic conditions have lower HCT values, similar differences between the two sites. Ventral tail vein less affected vs the sinus. Total WBC count also affected. Heparin preferred.** * Elasmobranchs – Total dissolved solids = ~2x total protein. * Sodium, chloride, urea, osmolality high in marine adapted elasmos. * Serum or plasma may require dilution. * US – Free fluid may indicate inappropriate salinity, inanition, coelomitis, bacterial or parasitic infection. Female repro ultrasound is essential to check gestational status, ID pathology, and obtain baseline images of ovaries and oviducts. * Full necropsy for animals that die.

Answer 7

* Variation in Drug Effects: * **Body temperature, hepatic biotransformation, renal function, drug receptor type and distribution, injection site. Also gill surface area to BW ratio, lipid content, stress, health status, body condition changes.** * Most elasmobranchs – poikilothermic. * **Endothermic abilities – porbeagle, mako and white shark.** * **Regional heterothermy associated with variable vascular densities may result in unpredictable drug uptake.** * Liver size and composition is species dependent. * **Kidneys have higher filtration rate and different selectivity that influence drug elimination rates.** * **Renal-portal system may enhance nephrotoxicity or renal excretion of drugs.**

Answer 8

* Physical Restraint: * **Tonic immobility – Level of restraint highly variable between species, often starts with period of marked excitement, especially in batoids. Not considered to have any analgesic effects, has been linked to hyperesthesia. Additional analgesia required for painful or invasive procedures.** * **Hazards while handling and diving – Teeth, spines, tail barbs, venomous spines at leading edge of first dorsal fin (piked dogfish), abrasive skin, electric shock (Torpediniforme rays), saw blades (Pristiformes, sawfishes).** * Accident prevention – Procedures should be well planned, two operantions commanders (one for human safety, the other for the overall procedure and animal wellbeing). Staff briefing before the procedure. * Develop SOPs, PPE, emergency protocols, safety equipment (Kevlar, chain mail), safety glasses around rays, practice safety drills. * All staff should be trained in first aid. Blood loss from shark bites can be significant and must be stopped rapidly. * **Stingray envenomation can be painful, fragments of spine can remain in wounds. Heat labile proteins can be denatured through application of heat (hot water immersion as hot as the patient can tolerate without scalding).** * Diving – Occupational diving regulations (OSHA). DSO (diving safety officer) at each aquarium should understand and enforce relevant regulations. Additional safety divers are recommended for operations that require full attention i.e. repairs. Safety diver encourages elasmos to remain at a safe distance from working divers and maintains non-visual (sound tapping, etc) communication with working divers. * PVC with electrical tape around it – “shark wand” deterrent. * Can also isolate the diver from the elasmobranchs with cages, netting, etc. * **Grasp smaller sharks with one hand over top of the body and gripped in the region of the pectoral girdle, posterior to gills and anterior to pectoral fins. Can also hold around or behind the pelvic girdle for additional support.** * Do not hold around the abdomen or coelom. * **Handling electric rays – Keep all parts of body away from water in which rays are maintained, handle with fabricated tools made from electrically insulated materials i.e. wood, plastic. Wear thick rubber gloves with long cuffs.**

Answer 9

* Chemical Restraint: * Immersion – Disadvantage is drug volume needed. Not usually practical. * Benzocaine is similar to MS-222 with high potency, quick onset, high margin of safety, low cost, may cause bradycardia and increased resistance to circulatory flow. * Etomidate and metomidate – Rapid induction and recovery. * Halothane/O2/NO – Bubbled into water, human exposure hazard. * **O2 narcosis – Short procedures, 100% O2. Sedation likely due to CO2 elevations due to respiratory depression. Prolonged exposure results in hypercapnia and potentially life-threatening acidemia. Gas bubble disease does not occur since not administered under pressure.** * **Divers can put MS222 in a spray bottle/syringes and apply directly to gills, use food coloring.** * **MS222 75-90 mg/L most spp. Buffer in FW systems. And small SW systems.** * Injectable – **IV most reliable, rapid induction and short duration. IM dorsal saddle.** * **Ventral coccygeal vein. Spinal needle in large animals to avoid cartilage plug.** * IM injections – Differences between red and white muscle. Erratic/delayed. * Alfaxalone – Highly variable. * Azaperone – Reduces response to environment without sedation. * Carfentanil – No effect even with massive doses. * Detomidine – Little effect. Reversal split IV and IM smooths recovery. * Ketamine – Seizure-like muscle spasms occasionally seen. Only used in combos. * Metomidine – Combo with ketamine. * **Propofol – Works well, IV.** * Sodium pentobarbital – Has been used, may result in death. * Tiletamine/Zolazepam – Irritability, rapid swimming, unrestrained biting. * Combos: Xylazine/ketamine, medetomidine/ketamine. Erratic. * Doxapram causes dramatic arousal in anesthetized elasmobranchs. * **Concentrated midazolam solutions have been used in large elasmos i.e. mantas and whale sharks.**

Answer 10

* Diagnostics * **Venipuncture sites – Dorsal sinus, ventral coccygeal vein most common.** * Ventral coccygeal vein preferred because blood in the dorsal sinus pools rather than actively circulates. * Ventral coccygeal vein – Insert needle at 90 deg angle directly on midline, may have to advance through cartilage before reaching the vessel; spinal needles may be used. * Dorsal sinus – Insert needle on midline at 45 degree angle. * Sampling – **For sharks, heparin or EDTA can be used. ONLY heparin for stingray spp (EDTA causes rapid hemolysis in stingray spp). EDTA may also lower the concentration of calcium and other divalent ions.** * ***Hemocytometer count should be performed immediately after collection to prevent thrombocyte and WBC aggregation. Otherwise, preserve in 10% formalin for later evaluation.*** * ***Formalin can maintain cell morphology and prevent thrombocyte aggregation.***

Answer 11

* Important clin path info: * Marine elasmobranchs retain and reabsorb urea and other solutes so that plasma remains hyperosmotic to sea water. * **Normal BUN should range from 1000-1300 mg/dL.** * **Low values may indicate loss form renal dz or decreased production due to hepatic disease.** * Salt excretion occurs in kidney and rectal gland, compensates for influx of Na and Cl from the environment. * Na and Cl tend to be higher than in mammals. * **Differential counts generally should be 50-75% lymphocytes, 10-30% heterophils, 0-10% eos, 0-1% basos, 0-3% monos.** * Whale sharks – In captivity have higher cholesterol and triglycerides vs wild conspecifics. EHM2.

Answer 12

* Body condition assessment – * Epaxial muscles and muscles over pelvic girdle should be rounded and firm * Coelomic contours should be flat to slightly convex. * US can be used to evaluate liver size and echogenicity. Should be large, lipid-laden. Can calculate a liver-to-coelom ratio. * Urea and total proteins decrease with inappetance in some studies. * Glucose and electrolytes are generally well regulated during prolonged fasting.

Answer 13

* I**odine deficiency** captive and free ranging animals * Causes: ozone (converts iodide to iodate), high nitrates can inhibit uptake by thyroid, spitting out oral supplements * Ozone reduces the amount of environmental iodine available, which is critical for thyroid hormone synthesis. * **Thyroid enlargement - may appear as mass ventral midline caudal to mouth, grossly thickened or rounded, pale orange** * **Histo: diffuse hyperplastic goiter, diffuse colloid goiter, multinodular colloid goiter** * Note not all deficient animals will show gross or histologic evidence deficiency * Thyroid iodine content is reliable indicator of thyroid status and can be used to assess efficacy of iodine supplementation * Oversupplementation can cause iodine toxicity and gland enlargement; retinal necrosis

Answer 14

* Soft tissue mineralization - common in captive cownose rays * Progressive -\> weight loss, death * **May be related to inappropriate calcium supplementation in water or prey** * **Renal disease** * Lesions (domed or flat and ulcerated) - skin (distal pectoral fins, ventral torso), dorsal midline, tail; may have chalky white deposits on skin and internally in various organs * Histo: **basophilic mineral deposits in connective tissue**, smooth muscle, myocardium, basement membrane; **surrounded by granulomatous inflammation and fibroplasia (**von Kossa stain can confirm presence of mineral)

Answer 15

* Hypoxia * Causes: low DO secondary to equipment failure or high bioload or chronic gill damage * Species variation with susceptibility - epaulette sharks and sleeper sharks can survive in hypoxic environments * Buccal pumping: compensate with decreased activity and increased resp rate * Obligate ram ventilators (lamnid sharks): increase swim speed and mouth gape * **Acute hypoxia: good condition, no gross lesions or nonspecific congestion, histologic lesions may include acute necrosis in viscera (gill lamellae, renal tubules)**

Answer 16

* Stress (metabolic consequences) associated with capture and handling * Catecholamine release and inhibition of respiration * **Decreased pH and increased lactate of blood (metabolic acidosis); some sharks - concurrent respiratory acidosis, hyperglycemia** * No gross or histo lesions * May also develop **exertional rhabdomyolysis** in skeletal and cardiac muscle * Dogfish and tiger sharks less susceptible vs **Atlantic sharpnose and blacktip sharks more susceptible**

Answer 17

Toxic * Ozone toxicosis * Oxidizing agent - toxic to gill epithelium and erythrocytes -\> membrane peroxidation -\> formation of methemoglobin -\> death by hypoxia * No specific gross lesions, congestion * Histo: gill epithelium may show acute necrosis +/- gill congestion and lamellar atrophy * Gas bubble disease * Heavy metal toxicity (**copper, tin**) * Sublethal copper exposure - decreased erythrocytes and HCT, decreased plasma urea, increased lactate and acidosis in dogfish * Tin (tributyltin oxide in marine paint) - gill epithelial necrosis from peroxidation in yellow stingrays * **Fenbendazole toxicity** * Lethargy, inappetence, increased respiratory rate, leukopenia, anemia * Gross: ventral erythema and necrotic tips of tail or pectoral fins * Histo: necrosis of hematopoietic tissues - epigonal organ, gi mucosa basal cells * Die from sepsis from necrotic gi mucosa * Volatile organic chemicals - paint fumes * Acute mortality in sand tiger sharks (southern stingrays survived) * Gross: gill congestion * Histo: acute gill necrosis with thrombosis, laminar collapse and congestion * Red tide (*Karenia brevis*) * Mortality in blacktip sharks and Atlantic sharpnose sharks reported with bloom in FL * High brevetoxin concentration in liver, gill and skeletal muscle * Naturally bioaccumulate toxin, so diagnosis can be difficult * Sharks appear to be resistant to domoic acid toxicity

Answer 18

* Coronary arteriosclerosis (captive and free-ranging) * Correlates with larger size and may be age-related * Histo: loss of internal elastic lamina and proliferation of connective tissue and smooth muscle of the intima, reduction of vascular lumen; more common at branch points * Arteriosclerosis also seen in meningeal and ocular choroidal vessels * Histo: dense collagenous connective tissue within and around vessels * Cardiac valvular proliferative lesions and myocardial fibrosis * Geriatric animals may contribute to stress and mortality * Gross: white fronds around valve leaflets of conus arteriosus * Histo: papillary projections are mildly hyperplastic endothelium over collagenous or mucinous stroma. Fibrosis occurs specifically at the junction of the inner and outer myocardium of the ventricle

Answer 19

* **Trauma** * Male aggression, mating aggression - blunt force trauma during pursuit by male may cause hepatic rupture and hemocoelom in females with abundant lipid storage * Self trauma common - poor exhibit design * Ulceration of ventral pectoral girdle - excessive bottom resting that may indicate underlying disease - ischemic necrosis, ulcers, secondary infections * **Gastric eversion (mostly in sharks) is normal for voiding and cleaning, but can be fatal if stomach is not swallowed and replaced, prolapses through gill slits or entrapped in oropharyngeal cavity** * Spiral valve eversion - conspecific bite wounds may result in sepsis and death

Answer 20

* Curvature of pelvic fins in captive large *Carcharias* and *Carcharhinus* genus sharks * Unknown cause, but may be associated with redundant swimming pattern * The ram ventilators are not able to swim/ventilate as efficiently and may die from hypoxia * Fins curved ventrally and medially, thickened at curved portions, traumatic erosions/ulcers may be on dorsal aspects * Histo: increased mucinous and collagenous connective tissue between the dermis and curved ceratotrichia; fractured ceratotrichia in severe cases

Answer 21

* **Spinal deformities- frequent captive sand tigers** * Possible/suspected causes: spinal trauma, nutrient deficiency (zinc, potassium, vitamin E or C), short pool length, redundant swim behaviors * Could be capture related, especially if pound nets were used. * Scoliosis, kyphosis, subluxation, compressed and deformed vertebrae, spondylosis; between pectoral girdle and dorsal fin * Histo: poorly organized proliferation of hyaline cartilage from center of affected vertebrae, epaxial muscle: necrosis, hemorrhage, inflammation, atrophy, fibrosis, mineralization * Progressive swimming difficulty, euthanasia

Answer 22

* **Egg retention in sand tiger sharks (Carcharias Taurus).** * Wild C. Taurus females mature at 9-10 yrs age. * No pregnancies have been recorded for C. Taurus on display in the US excluding animals gravid prior to capture. * Continuously produce non-fertilized eggs, often released into tank. * Can be retained and lead to lethal consequences. * Eggs retained in one or both uterine horns eventually become necrotic, serving as a nidus for bacterial metritis and leading to septicemia and death. * CS – Depression, anorexia, diffuse dermal abscesses or draining fistulas. * Dx – Radiology, ultrasonography, endoscopy, nannulation and uterine lavage. * Abx along generally not effective. * Tx – Flushing out eggs with uterine lavage with antibiotic saline solution followed by parenteral antibiotics. * Common bacteria cultured from necrotic eggs – Pseudomonas, Enterococcus. * Two cervical openings which may be open or closed. Can manually dilate for egg removal. * Gastric air can be lost during this procedure, may need to manually reinflate

Answer 23

* **Cystic ovaries and mucometra in southern stingrays (Dasyatis Americana).** * Can produce 2-10 pups annually. * Impregnated within hours to days of parturition, consider any female to be pregnant. * Uterus never involutes and villus trophonemata do not atrophy, continue to produce thick mucoid histotroph and uterus remains dilated. * Induced ovulators – Copulation must occur to stimulate hormonal signals for ovulation. * If mature follicles are not ovulated they will remain on the ovary. * Many aquaria resort to single-sex exhibits; females will still produce large amounts of histotroph. Leads to marked mucometra. * CS – Distention with no pups on US. May develop pressure sores from resting on the bottom. Coelomic wall can rupture leading to salt water intrusion and death. * Tx – Temporary relief via cannulation of cervix and evacuation of uterus, will recur within months. * Left ovary continues to produce follicles despite absence of pregnancy. * Ovary will become cystic and get very large, can tear and result in hemorrhage and death. * Surgical ovariectomy in juvenile D. Americana can be employed to avoid this. * Size is critical to success of procedure. * Ideal size candidate is ~60 cm disc width. * Ovarian size should be evaluated pre-operatively with US. * Left paralumbar surgical approach –EHM2 has full procedure.

Answer 24

* **Dystocia in cownose rays (Rhinoptera bonasus).** * Dystocia can occur in any species, but cownose rays are the most common! * CS – Large girth without pupping. May be able to palpate in the urogenital sinus or by pushing at the dilated cervix. * Cloacal tissues may be bruised and edematous. * Episiotomy is often necessary for extraction through the cloaca. * It is recommended that pregnant R bonasus receive monthly physical and US examinations. Allows for monitoring of pregnant animals, management of the collection by segregating animals and intervention in event of pupping complications.

Answer 25

* **Southern stingrays (and other female elasmos) housed without males** * Coelomic distention, large left ovary (numerous mature follicles +/- cystic and inflamed follicles) and uterus with prominent trophonemata and histotroph accumulation, oviducts may contain mucosal cysts and fluid accumulation * Chronically affected - poor nutrition condition * Histo: ovarian tissue unremarkable, and stromal hyperplasia may occur, granulocytic inflammation and mural necrosis within cystic follicles, trophonemata elongated; affected rays have coelomic mesothelial hyperplasia

Answer 26

* Uterine impactions - multiple egg cases * May cause coelomic distention and rupture of dorsal hepatic lobe * Infection may be present as well (caseous exudate) - do not survive long * Not impaction: captive female sand tigers that do not breed - numerous follicles in uteri with marked distension (no abnormalities of uteri, unknown impact)

Answer 27

Neoplastic * Immunohistochemistry is often of little value - limited reagent cross reactivity * **Cutaneous papillomas** * Torso - bamboo, sand tiger, leopard, and white-tipped reef sharks * Gill slits and conjunctiva - nurse sharks * Multifocal lesions and multiple animals affected - suspected viral cause, but no viral particles identified in lesions * Lesions raised, polypoid or placque like with smooth surface * Histo: well differentiated hyperplastic epidermis, orderly maturation, overlying fibrovascular stroma * Malignant transformation not reported * **Lymphosarcoma** * May present as disseminated disease with visceral involvement and in some cases leukemia * Thymic lymphosarcoma * IHC using antibodies against mammalian CD20 may assist in B cell lymphoma diagnosis * **Cutaneous melanomas** * Multiple species, but most common in Rajiformes * Conclusive link between melanomas and UV light exposure has not been made * Plaque-like or raised, sessile and multilobular lesions with variable pigmentation * Histo: spindle cells arranged in streams, cells may contain cytoplasmic granular brown pigment * IHC MART1 was positive in cutaneous melanoma from nurse shark

Answer 28

* Herpesvirus - dermatitis in dusky smoothhounds * Lesions: raised round/oval, **white to gray, pigmented skin lesions** from 1 mm to \>1cm diameter with slight central depression and red margin, especially numerous on fins * Histo: intracellular edema, ballooning degeneration, necrosis, eosinophilic intranuclear and cytoplasmic inclusions * Spontaneous regression to foci of hyperpigmentation; Often resolves without intervention.

Answer 29

* **Iridovirus** – AKA Viral erythrocytic necrosis. young dusky smoothhounds, leopard sharks, possibly others * Erythrocytic necrosis -\> anemia * Young animals with no immunity to the virus most often affected. * Cytoplasmic inclusions in erythrocytes seen on blood smears

Answer 30

* Adenovirus-like * Young caught dusky smoothhounds * Fatal ulcerative dermatitis and branchitis * Large intranuclear inclusions with virions * Dermal ulcers coinfected with parasites; gill atrophy of secondary lamellae with intranuclear inclusions

Answer 31

* Sepsis * Skin and gill lesions commonly associated; but may have no gross lesions, only skin lesions, or foci of necrosis in organs * Meninges red and thick * Histo: bacteria in blood vessels or foci of necrosis with inflammatory cess, fibrin thrombi or aneurysms in capillary beds of corpus cavernosum and secondary lamellae, minimal autolysis * Tenacibaculum maritimum – Necrotic skin lesions in sand tiger sharks. * Common bacterial agents – Vibrio spp, Photobacterium damselae, Aeromonas spp, Pseudomonas spp, Citrobacter spp, Flavobacterium spp, Streptococcus agalactiae, Serratia marcescens. * *Vibrio* * ***V. harveyi*** **- urease producing sp. From 62% of elasmobranch sepsis cases (Stedman, unpublished) and most commonly recovered from blood of healthy free-ranging electric rays (Tao et al., 2014)** * Vibrio carchariae and Vibrio damsela – Disease and mortality in multiple shark spp; lemon sharks appear resistant.

Answer 32

* Aeromonas salmonicida – Hemorrhagic septicemia in blacktip reef sharks. * Flavobacterium spp – Neurologic dz in bonnethead sharks. * *Carnobacterium maltaromaticum*-like bacteria (gram positive bacilli) * Meningoencephalitis in stranded juvenile salmon sharks (homeotherms!) in California * Thick red meninges, liver - pinpoint white/yellow foci, abscess formation in ventricles * Histo: meninges - infiltrates of mononuclear cells and proteinaceous edema, congestion, hemorrhage and necrotic debris, bacteria, inflammation; hepatic - mononuclear cell infiltrates * **Branchial epitheliocystis (order Chlamydiales); Leopard shark.** * Gram negative, infect gills * Gills - pale, mottled * Secondary lamellae epithelial cells enlarged by intracellular cysts (10-200 um) with granular light basophilic material surrounded by eosinophilic membrane +/- mixed inflammatory infiltrates, fusion of lamellae * Can be incidental finding * Mycobacteriosis * Infection rarely reported, but most common infection is *M. chelonae* (single report of *M. avium* in epaulette shark) * C. Taurus drop jaw syndrome – Shark loses ability to close mouth and upper dental arcade is dropped into pre-bite posture. Localized infections of the base of the ligamentum levator palatoquarati. Unknown cause but has happened in many aquaria – EHM2.

Answer 33

* **Fusarium (*F. solani* species complex - FSSC) - most common fungal disease** * Mycotic dermatitis, affects the head and lateral line system, has caused fatal disease in juvenile bonnethead sharks. * Trauma predisposing factor - cephalofoil of captive bonnethead and scalloped hammerhead sharks * Lateral line involvement - demarcated ulcerative and/or hemorrhagic lesions, sometimes with white exudate * Lesions may extend into skeletal muscle and cartilage * Histo: necrosis, ulceration, infiltration of granulocytes and macrophages; angioinvasion with thrombosis and infarction possible; lateral line - vaculation, necrosis, epithelial hyperplasia, intraepithelial and luminal exudate * Nonpigmented branching septate fungal hyphae (difficult with HE, easy with fungal stains), culture, molecular diagnosis * Tx – Early and prolonged tx with voriconazole is recommended but often unrewarding.

Answer 34

* Other fungi in captive sp - *Exophiala, Paecilomyces* * Paecilomyces lilacinus – fungal infection in a hammerhead shark with systemic and terminal disease. * Skin and/or gill necrosis, erosion, ulceration, or systemic infection * Systemic infections may be present without apparent skin lesions * Coinfection with Exophiala pisciphila and Mucor circinelloides in a zebra shark also fatal. * Exophilia detected in a swell shark that displayed abnormal circular swimming patterns and mineralization of the cartilage of the skull and cervical vertebrae.

Answer 35

* Microsporidiosis * rare, but reported in captive leopard sharks * Histiocytic inflammation associated with cytoplasmic microsporidians in gill, spleen, pancreas, brain, and blood vessels * *Dasyatispora levantinae* - free ranging common stingrays * Spindle shaped raised white lesions filled with thick caseous material parallel to muscle fibers around disc margin * Severe infection may cause mortality

Answer 36

* **Monogeneans** - many species specific, may have wider host range in aquarium settings. * **Common monogeneans** * Monocotylidae i.e. Dendromonocotyle spp * Capsalidae i.e. **Benedeniella spp**, Entobdella spp, Neoentobdella spp. * Microbothriidae i.e. Dermophthirius spp and Neodermophthirius spp. * Hexabothriidae i.e. Erpocotyle spp and Heterocotyle spp. * Tx usually praziquantel immersion, organophosphate immersion, or temp or salinity changes. Take into account eggs, host range, and host sensitivity to tx. * **Dermophthirius nigrelli** – Wild lemon sharks. * **Dionchus penneri** – Chronic skin lesions in blacktip reef sharks. * Skin, gills, nasal, urogenital, gallbladder, coelom * Usually incidental in free-ranging * **Direct life cycle can cause problems in human care- warmer water** * **Lay eggs that are negatively buoyant and sink to form egg bank in substrate.** * **Ciliated larva (onchomiracidium) actively seeks a host. LC est 3-5 wks at 24C.** * **Spotted eagle rays, bonnethead sharks predisposed to fatalities** * Skin: multifocal thick gray to white lesions, erosion and ulceration may develop (rubbing, flashing) * **Thickened gill filaments with chronic gill parasitism and occasional yellow exudate, fusion of secondary lamellae, gill hyperplasia, mucoid and alarm cell metaplasia, partial collapse of gill tips, nodular metaplasia in primary filaments** * Adult monogeneans - large haptor organ, thick eosinophilic cuticle * **Capsalidae:** * Characterized by a posterior holdfast organ for attachment (haptor) and hooks. * Eat mucus and skin. * Clear to milky white in color. * **R. bonasus – Benedeniella posterocolpa.** * May be detectable on the cornea with a light source. * Can be seen falling off during FW or praziquantel tx. * Benedeniella distinguished from other capsalids by presence of a long vagina that opens on the left body.

Answer 37

* Digenean trematodes uncommon, but severe infections of rectal gland may disrupt osmoregulation and have been associated with vascular necrosis and hemorrhage in the brain * Cestodes common in free-ranging, species specific, most in spiral valve; can be encysted in mesentery or viscera as well. Typically considered incidental. * Paraorygmatobothrium spp in lemon sharks, Pedibothrium spp in nurse sharks, Crossobothrium spp in wild seven gill sharks, Tetraphyllidean spp in spadenose shark, Calliobothrium schneiderae in smooth-hound sharks. * Nematodes * Common in spiral valve * ***Huffmanela*** **ova (larvated, bipolar plugs) in skin and oral cavity of sharks - affected animals have black serpiginous tracks or discrete black foci** * **Adult migrates through the dermis and leaves behind a serial chain of heavily-tanned eggs visible through the epidermis.** * **Dx with skin scrape. Tx – Levamisole, may be self limiting.** * Spirurid larvae - meningoencephalitis in free ranging nurse sharks, also incidental * Spirurida and Dracunculoidea have been linked to vasculitis, bronchitis, pancreatitis, meningoencephalitis, oophoritis, metritis. Decisions to treat not always straight forward. * Myxozoans (generally incidental, biliary tract) and acanthocephalans reported. * Cestodes (GI, spiral valve), myxozoa (biliary), microsporidia (anywhere) in quarantine generally considered incidental.

Answer 38

* **Coccidia - Eimeria (*E. southwelli*)** * Most are small intestine and spiral valve parasites, only disease causing with heavy infections - spiral valve epithelial necrosis, sloughing and hemorrhage * ***Eimeria southwelli*** - coelomic epithelium of cownose rays and associated with high mortality in captive animals (unknown affect in free-ranging population). All myliobatid rays should be considered susceptible. * Coelomic distention (turbid effusion), weight loss, prominent cutaneous blood vessels, mesothelial hypertrophy and hyperplasia * Mesothelial cells - sporulated or unsporulated oocysts and meronts * Also reported in testes and within tubules of the kidney. * ID in coelomic fluid from coelomic pore flushes * Serosa of organs may appear roughened on necropsy. * Tx – Sulfadimethoxine, clindamycin, toltrazuril or ponazuril. Elimination of parasite is difficult. Manage rather than elimate.

Answer 39

* Fatal systemic scuticociliate infection reported * One case - ID was *Philasterides dicentrarchi* * Multifocal necrohemorrhagic hepatitis, cutaneous ecchymoses, turbid intracranial and coelomic fluid, and/or necrotizing pancreatitis * Histo: necrotizing vasculitis and intravascular fibrin thrombi associated with necrosis * Organisms: 20x50 um diameter, single hyperchromatic round macronucleus and cytoplasmic eosinophilic globules * Invasive Scuticociliatida-like (Uronema-like) ciliates have been associated with morbidity and mortality – Skin, gills, brain, liver of skates and demersal sharks.

Answer 40

* Ectoparasites: barnacles, amphipods, brachyurans, isopods, copepods, leeches; most not pathogenic * Larvae of *Gnathia* sp isopods feed on blood and tissue fluids - heavy infestations -\> anemia * ***Cirolana borealis*** **isopod** can penetrate body wall and skeletal muscle in sharks -\> feeds on cardiac and other tissues (myocardial necrosis and mild pericardial lymphocytic inflammation)-\> can cause death and potentially declines in free ranging populations * *Ommatokoita elongata* copepods can attach to cornea and can cause keratoconjunctivitis (common finding in Greenland sharks and Pacific sleeper sharks) * Copepods from orders Spiphonostomatoida and Poecilostomatoida are common around the head, buccal, branchial cavities, occasionally on fins, cloaca, and claspers. * Principally aesthetic issues. * LC can be broken in some spp during quarantine. * Branchiura i.e. Argulus spp are reported in dasyatid and potamotrygonid rays. * Tx – Physical removal, organophosphates, chitin inhibitors. * Leeches (family Piscicolidae) may transmit hemoparasites but generally nonpathogenic; they may enter the uterus and attach to the skin of embryos. May cause severe morbidity and mortality. * Tx – Physical removal, organophosphates, chitin inhibitors.

Answer 41

* Clipping stingray spines: * Restrain ray and tail during clipping. Do not let the spine get tangled in a net and tear out. Neoprene and leather gloves are NOT sufficient protection. * Cut spine close to the body with stout shears or wire cutters, avoid accidentally cutting the tail. Distal spine can be ejected when cut, so all staff must wear safety glasses. * After clipping place the spine in hot water to denature the toxin. * Assisted swimming/’walking’: * Elasmobranchs emerging from an anesthetic can be ‘walked’ to encourage circulation from the musculature. Can result in sequesteres anesthetic moving towards the CNS and re-anesthetize or disorient the animal. Recovery from anesthesia in general is unpredictable. * Inflating sand tiger tharks: Air can be introduced into the stomach via semi-rigid tubing connected to a compressed air supply. C. Taurus will periodically swallow air to regulate buoyancy normally, can lose air and become negatively buoyant, may need assistance with inflation.

Answer 42

* Reproductive technologies: Hormone analysis, hormone treatments to alter reproduction, semen collection, sperm cryopreservation, AI, artificial rearing of viviparous embryos. * Monitoring reproduction: * Breeding season for elasmos typically spring/summer. * Vitellogenesis and follicular growth generally over a period of months leading up to breeding season. * Some oviparous spp can deposit eggs year round. * Spermatogenesis also occurs seasonally leading up to breeding season. * Repro assessments should be performed regularly as part of routine health examinations. * Ultrasound: * Used routinely for evaluation of repro tract in elasmobranchs. * Hormone analysis: * Main hormones associated with repro cycles in elasmobranchs: * Females – Estradiol, progesterone, and testosterone. * Males – Testosterone. Especially useful since US is less useful in monitoring male cycles.

Answer 43

* Semen collection and sperm quality assessment: * Testes undergo annual seasonal changes to spermatogenesis, often accompanied by changes in gonadosomatic index GSI. * Seasonal changes in GSI correspond with annual breeding season in some spp (not all). * US is of limited use for monitoring repro cycles in males because relatively minor changes in size of the testes over the cycle. * Semen can be collected during the breeding season by exerting gentle pressure on the distal repro tract proximal to the cloaca or by passing a catheter into the ampulla of the ductus deferens via the urogenital papilla. * Light microscopy – Evaluation of sperm motility. * High osmotic pressure of elasmobranch body fluids -\> sperm should be assessed in solutions with an osmolality of approximately 900-1100 mOsm/kg for maximum activity. * Sperm viability staining: * Using dye that is membrane impermeant – Excluded by viable cells but able to stain nuclei of non-viable cells with compromised membranes. * Requires specialized equipment. * Motility assessment and viability are two different aspects of sperm function that should be used in combo to maximize the accuracy of sperm quality assessments.

Answer 44

* Methods for controlling reproduction: * Artificial insemination: * Mixed results depending on species, has been tried with fresh and stored semen, not a lot of published information. * Artificial rearing of embryos from viviparous spp: * Difficulties accurately replicating the elasmobranch uterine environment and embryo nutrition in species with matrotrophy. * Sperm cryopreservation: * Extender solutions. More studies needed. * Decreasing reproduction: * Maintaining constant water temps for some spp (C. Taurus). * House single sex populations. * Other issues i.e. repro disease development. * Depo-Provera (medroxyprogesterone acetate) has been used in D. Americana (not published). * Ovariectomy. * Deslorelin and other implants – Variable success.

Answer 45

* Treatment and Therapies: * Bamboo shark PK studies: * Cefovecin maintains serum concentrations for 4 days at 8 mg/kg SQ. * Florfenicol 40 mg/kg IM lasts 120 hours in serum and 72hr in CSF. * Blood transfusions – Cross-matching recommended prior to transfusion. * Nutritional support – Assist feeding or feeding gruel via gavage 2-5% body weight.

Answer 46

Treatment and Therapies: * Blood transfusions – Cross-matching recommended prior to transfusion. * Nutritional support – Assist feeding or feeding gruel via gavage 2-5% body weight.

Answer 47

* Common Antiparasitic Treatments – Quarantine (EHM2). * Praziquantel baths for monogeneans. * Manual dissolution of prazi is more common than chemical dissolution – potential effects of ethyl alcohol on biofilter, bacterial loads and DO. * DO should be monitored before and after immersion. * Prazi levels should be monitored, can rapidly degrade in some systems. * Georgia Aquarium Veterinary Services Water Quality Lab. * Some species can tolerate increased or decreased salinity. Adverse effects reported. * Organophosphates (trichlorfon) – Monogeneans, copepods, leeches. * Skin changes, inappetance, mortalities reported. * Species particularly sensitive to OP – eagle rays (Aetobatus spp), guitarfish (Rhinobatos spp and Rhina spp). * Chitin inhibitors may also be used (lufenuron or diflubenzuron) – copepods, leeches. * Copper is avoided in elasmos due to concerns over toxicity. * Long-term immersion has been successful in lemon sharks for monogeneans and cownose rays with monogenean infections. * Febnebdazole = mortalities in many spp. * Side effects may be more common when gavage fed (meds not lost during dosing). * Pyrantel and febantel associated with deaths in whitetip reef sharks. * Inflammatory response associated with death of histozoic nematodes. * Nematode tx should be carried out with caution!

Answer 48

* Odontaspididae – Carcharias taurus (sand tiger shark). * Generally hardy. * Atlantic – Copepods i.e. Anthosoma crissum which can be associated with deep gingival erosions, tooth loss. * 14 wks quarantine can break the lifecycle. * Juvenile porkfish can be used as cleaner fish. * Amyloodinium-like dinoflagellates can cause acute mortalities. * Chloroquine immersion resolved the infection. * Possible vital diseases – Diffuse crusting lesions on skin that resolved over 1 month and black, raised polypoid lesions that expanded and coalesced into gray lesions. * Inflammation of the Ampullae of Lorenzini has been observed, unknown etiology. * Scoliosis. * Predisposing factors – pound net capture, size and shape of tank prevents normal swimming. * Consider baseline rads. * May swallow air and interfere with US evaluations.

Answer 49

* Carcharhinidae * Carcharhinus plumbeus (Sandbar aka brown sharks). * Copepods Albion spp and Pandarus spp. * Eggs of nematode Huffmanela carcharhini create characteristic black tracks in the skin. * Lesions have been cleared with levamisole. * Prone to rostral and pectoral fin trauma. * Self-limiting prolapses of the cloaca and valvular intestine have been reported. * Carcharhinus melanopterus – Blacktip reef sharks * Sensitive to environmental stressors. * Focal dermatitis assoc with monogeneans – Dermophthirius melanopteri or D. penneri. * Cloacal and valvular intestine prolapse has been seen. * Prone to skin erosions. * Carcharhinus acronotus – Blacknose sharks. * High prevalence of IV nepatodiasis assoc with vascular occlusion, necrosis, inflammation in gills and brain. * Negaprion brevirostris – Lemon sharks. * Monogeneans – Dermophthirius nigrellii and Neodermophthirius harkemai on gills and skin. * CS – Rubbing, erratic swimming, gray plaques, ulcers. * Tx with trichlorfon immersion. * N. harkemai - May also have dark bands of hemorrhage around mouth and increased mucus around the head. Tx with copper sulfate and transfer to another system. * Tends to be more aggressive than other commonly displayed sharks. * Triaenodon obesus – Whitetip reef sharks. * Copepod – Paralebion elongates. * Morbidity from gill nematodes. Mortality following treatment due to inflammatory response. * Pack hunters, conspecific aggression reported.

Answer 50

* Triakidae * Triakis semifasciata – Leopard sharks. * Monogeneans – Erpocotyle spp on gills. * Copepods on skin. * Microsporidial parasites have caused morbidity and mortality. * Chamydiales associated with branchial hyperplasia and death. * Papillomaviruses have been identified, focal areas of depigmentation. * Conspecific aggression during breeding. * Spy-hopping in small systems. * Mustelus canis – Dusky smooth-hound * Iridovirus – Intravascular hemolysis (viral erythrocytic necrosis). * Herpes virus – Ulcerative dermatitis. * Adenovirus – Ulcerative dermatitis, epithelial and gill hyperplasia and mortality. * Intralesional ciliates and septicemia common. * High prevalence of biliary myxosporeosis and pancreatic nematodes. * Prone to trauma.

Answer 51

* Scyliorhinidae – Dogfish spp. * Cephaloscyllium ventriosum aka swell shark. * May take several hours to deflate their stomachs after handling, keep submerged whenever possible during PE. * Stegostomatidae * Stegostoma tigrinum - Zebra shark * Copepods Lepeophtheirus acutus on or around eyes and mouth, severe ulcerative keratitis. Tx with trichlorfon and dilflubenzuron immersion. * Scuticociliatida-like ciliates, necrotizing vasculitis and meningoencephalitis. * Orectolobidae * Orectolobus maculatus – Spotted wobbegong. * High prevalence of septicemia. * Require refugia and complex substrate. * Can be difficult to transfer to frozen diets, tend to prey on exhibit fishes. * Wild-caught females are often gravid, US should be part of the quarantine exam. * Strong rolling response, hard to manually restrain.

Answer 52

* Sphyrnidae * Sphyrna tiburo – Bonnethead sharks. * Prone to Fusarium solani dermatopathy. * Suboptimal temp and trauma increase risk. * CS – White pustules or ulcers along lateral line system and cephalofoil. * Progressive, sometimes systemic invasion. * Typically fatal, has been treated with voriconazole and temp modification. * Monogeneans – Erpocotyle tiburonis. * Huffmanella spp nematodes. Black tracks in skin. * Post-transport emaciation is common, inappetance. * Aggressive nutritional support is recommended until they are eating reliably. * Sensitive to small environmental changes. * Prone to cephalofoil and ocular trauma during shipping or quarantine.

Answer 53

* Heterodontidae * H. francisci – Horn sharks. * Invasive scuticociliatida-like ciliates associated with severe inflammation in skin, gills, brain, liver in Japanese bullhead sharks and port Jackson sharks. * CS – Lethargy, inappetance, mortality. * Breeding aggression between males if in groups during quarantine. * Refugia or hides are necessary. Caution with tight spaces that may cause entraptment and abrasions. * H. francisci have prominent spines, hazard during handling. * Ginglymostomatidae * Ginglymostoma cirratum – Nurse sharks. * Amyloodinium-like dinoflagellates on gills associated with acute mortality in quarantine. Chloroquine immersion resolved the signs. * Hemiscyllidae * Hemiscyllium ocellatum – Epaulette sharks. * Chiloscylium plagiosum – Whitespotted bamboo sharks. * Wild H. ocellatum have shown 100% prevalence of gnathiid isopod larvae on the skin around cloaca and gills, no morbidity observed. * Can tolerate low DO concentrations, relatively tolerant of FW dips. * Squalidae * Squalus acanthias – Spiny or piked dogfish. * High prevalence of biliary myxosporeosis and pancreatic nematodes. * High prevalence of mononuclear inflammation of the ampullae of Lorenzini.

Answer 54

* Rhinidae and Rhinobatidae * Rhina ancylostoma – Bowmouth guitarfish – Hardier than others. * Leeches i.e. Pontobdella spp common in wild-caught individuals. * Mechanical removal usually sufficient. * Inappetance common in quarantine, can offer uncommon food items i.e. lobster or crab. * Ulcerative lesions from shipping common. * Rajidae * Raja binoculata – Big scates. * Rajids prone to skin lesions, especially on tail, claspers, rostrum. * May become infected with furasium spp, scutilociliatida-like parasites, or tenacibaculum-like filamentous bacteria. * Filamentous bacteria has been treated with TMS immersion.

Answer 55

* Dasyatidae * Hypanus americanus – Southern stingrays. * Dasyatis sabina – Atl stingrays. * Neotrygon kuhlii - Bluespotted stingray * Taeniura lymma - Bluespotte ribbontail stingray * Commonly carry Monocotyle, Dendromonocotyle, Entobdella spp, and Neoentobdella spp monogeneans. * FW dip may remove monogeneans, relatively tolerant. * D. Americana and N. kuhlii high prevalence for septicemia. * Ultrasound of females is indicated, often gravid. * N. kuhlii, T. lymma may be inappetant, emaciated. * Tail trauma common. * Dasyatid rays have one or more venomous spines on the dorsal tail, typically trimmer or covered before restraint. * Many secrete mucus and frequent water changes may be required during PE. * Especially thick in leopard whiprays.

Answer 56

* Myliobatidae * Aetobatus narinari – Spotted eagle rays. * Commonly have heavy loads of gill monogeneans i.e. Dendromonocotyle spp. * Tx period is often life-long, regular prazi or FW dips. * Chlamydiales associated with branchial epitheliocystis. * Eimeria southwelli has been ID in asymptomatic and thin animals. * Hard to convert to frozen food. * Often need aggressive nutritional support in quarantine. * Dental plate overgrowth if whole clams and other bivalves not provided. * One or more venomous spines on the tail.

Answer 57

* R. bonasus – Cownose rays. * Often carry significant parasites. * Benedeniella posterocolpa monogeneans on ventral skin. * Tx repeated prazi or OP immersion, or long-term copper therapy. * Leech Branchellion torpedinis within buccal cavity. * Very large, infects wide range of demersal elasmos in aquaria. * Identified by presence of 33 pairs of frilly gill-like appendages laterally. * May cause profound hemorrhagic anemia. Even with a low burden. * Muscular, glandular proboscises feed on hosts that lack a keratinized epidermis. * Unlike most leeches that deposit cocoons of embryos onto hard surfaces, this leech releases small cocoons freely into the water that contains a single embryo. * Management implications. * Cocoons are discus-shaped, golden brown and sticky, become encased in sand grains. Can be viable months. * Monogenean-like reproductive mode confers extremely high intrinsic population growth. Cocoons also extremely resistant. * Treatment occurs primarily through aggressive exhibit maintenance and physical removal of leeches. * Severe cases – Remove manually and follow with bath of trichlorfon (Dylox). Pretreat with injectable atropine prior to bath with organophosphate. Supportive care for severe anemia. * Leeches can be problematic when placed in multispecies exhibits. * CS – Anemia, hypoproteinemia, inappetance, lethargy, secondary infection, mortalities. * Endoscopy of the buccal cavity is recommended with this species or prophy tx with OP. * Carry Eimeria southwelli coccidia in the coelom and GIT, can cause morbidity and mortality when stressed. * Coelomic aspirates or flushes show highest sensitivity for dx. Screening incoming animals warranted. * Tx – Sulfonamides, clincamycin, toltrazuril, ponazuril can reduce symptoms but does not often clean infection. * Side effects of tx may include cloacal prolapse and mortalities. * Thin tail limits venipuncture at that site, wing or radial veins useful.

Answer 58

* Potamotrygonidae * South American FW stingray – Potamotrygon motoro. * Prone to water mold infections on skin and around dental plates. * Tx – Prolonged low-dose salt immersion. * Argulus spp branchiurans on wild-caught individuals. * Physical removal sufficient. * Coccidia and nematodes common but not associated with morbidity or mortality. * Emaciation common. * Potent venom, one or more spines on tail.

Answer 59

Westmoreland, L. S., Archibald, K. E., Christiansen, E. F., Broadhurst, H. J., & Stoskopf, M. K. (2019). The mesopterygial vein: a reliable venipuncture site for intravascular access in batoids. *Journal of Zoo and Wildlife Medicine*, *50*(2), 369-374. Abstract: Intravascular access in batoid species is commonly achieved using the ventral coccygeal or radial wing vessels. However, these approaches can be difficult because of the presence of cartilage, lack of specific landmarks, species variation, and small vessel size in many species. This study used postmortem contrast radiography and gross dissection to develop landmarks for a new, dependable vascular access in three Myliobatiform species commonly maintained in captivity: Atlantic stingray (Hypanus sabinus), cownose ray (Rhinoptera bonasus), and smooth butterfly ray (Gymnura micrura). **The mesopterygial vein provides quick vascular access and is suitable for administration of large fluid volumes and intravascular drugs.** It is located immediately **ventrolateral to the metapterygium** cartilage, which sits adjacent to the coelomic cavity and supports the caudal half of the pectoral fin. Using **the pectoral girdle and cranial third of the metapterygium cartilage as landmarks**, vascular access can be achieved by directing a needle medially at approximately a 30 (adult cownose rays) or 45 angle (Atlantic stingrays, juvenile cownose rays, smooth butterfly rays) toward the metapterygium cartilage. Differences in the degree of needle direction are due to species and age-specific shapes of the metapterygium cartilage. **The mesopterygial vein is an alternate site of quick and reliable venous access in batoid species.** BACKGROUND INFO: * Cartilaginous skeleton supports pectoral fins, propterygium cranially, mesopterygium and then caudal metapterygium which are anchored via pectoral girdle * Radial wing veins drain blood medial to large propterygial vein cranially and mesopterygial vein caudally. * Metapterygial vein drains between the pectoral fin and body wall * All 3 of these go to brachial and subclavian veins to heart KEY POINTS: Mesopterygial vein located just ventrolateral to metapterygium cartilage in all 3 species * Ventral approach favorable with less tissue damage * Largest cranial 1/3, then decreased moving caudally * Technique: dorsal recumbency, palpate pectoral girdle centrally then palpate lateral margin of metapterygial cartilage in proximal pectoral fin (cranial 1/3). 45 degree angle for some species (30 for cownose). 22G 1inch needle/20g 2inch for adult cownose. * Clinically applicable, may induce tonic immobility in some species * Less muscle in ventral approach than dorsal approach, less chance for tissue damage * Very large vessel, away from barb * Separation of artery and vein in this site, less mixing of A-V blood * Helpful to use a spinal needle with stylet in larger species with thick connective sheath * Cranial 1/3 avoids more prominent lip found caudally TAKE HOME: Mesopterygial vein reliable for IV access in multiple species of batoidsC

Answer 60

Johnson III, J. G., Naples, L. M., Van Bonn, W. G., Kent, A. D., Mitchell, M. A., & Allender, M. C. (2017). Evaluation of health parameters in cownose rays (Rhinoptera bonasus) housed in a seasonal touch pool habitat compared with an off-exhibit habitat. *Journal of Zoo and Wildlife Medicine*, *48*(4), 954-960. Abstract: Cownose rays (*Rhinoptera bonasus*) are commonly displayed in zoo and aquarium touch pool exhibits; however, there is a gap in our understanding of how these practices might impact the health of these animals. **The aim of this study was to evaluate and compare selected health parameters in cownose rays housed in a seasonal outdoor exhibit touch pool system with abundant public contact and an indoor off-exhibit holding system with minimal human contact.** All animals underwent **physical examination, ultrasound, cloacal wash and cytology, and blood collection** for complete blood counts, point-of-care blood analysis, plasma protein electrophoresis, and plasma cholesterol electrophoresis in May and October 2014. **Physical examination, ultrasound, and cloacal wash cytology findings were all unremarkable for both** groups of animals. **Significant differences in health parameters among animals by location and time point were few and included decreased heart rate (*F* = 12.158, *P* = 0.001), increased lactate (*F* = 6.838, *P* = 0.012), and increased low-density lipoproteins (*F* = 19.961, *P* = 0.000) in touch pool animals over time.** Based on these results, cownose rays inhabiting a touch pool exhibit and an off-exhibit system remained in **comparable planes of health** based on routine diagnostic modalities with few differences in measured health parameters. * Key Points: * Touch pool vs not touch pool cownose rays – Only differences were decreased HR, increased lactate, increased LDL in touch pool animals. * Individuals with increased lactate did not have changes in other blood gas values, comparable to in-house ref ranges for others. * Clinical utility of LDL in elasmobranchs not defined. * Takeaway: Touch pool animals – lower HR, higher lactate, higher LDL.

Answer 61

Parkinson, L., Noblitt, S. D., Campbell, T., & Sladky, K. (2018). Comparison of two iodine quantification methods in an artificial seawater system housing white-spotted bamboo sharks (chiloscyllium plagiosum). *Journal of Zoo and Wildlife Medicine*, *49*(4), 952-958. **Abstract**: Iodine is an essential micronutrient for elasmobranchs in order to prevent goiter. **Preventing goiter requires bioavailable iodide: either oral iodide or maintaining adequate aquarium water iodide concentrations.** The objective of this study was to determine how oral and water supplementation affected iodine (I₂) and iodide (I-) concentrations in artificial seawater aquaria housing captive white-spotted bamboo sharks (*Chiloscyllium plagiosum*). Daily water samples were collected and free iodine (I2) was determined using ultraviolet-absorbance spectrophotometry (a relatively simple in-house assay) and total iodide (I-) via liquid chromatography (a more time- and expertise-intense quantification method) to learn the effects of supplementation. One water system received iodine and iodide supplementation in the form of 5% Lugol’s iodine solution added directly to the water, while a second water system received no supplementation. In addition, one tank of sharks in each water system received oral iodide supplementation. **Results indicated that oral supplementation provides greater increases in water concentrations of bioavailable iodide (I-) than direct water supplementation. In addition, the chromatographic results suggested that iodide is present in higher concentrations in the systems not receiving water supplementation. Increased iodide concentrations were detected in water samples after water changes and after oral iodide supplementation was administered, but total iodine (I₂) concentration changes were not detectable within the same time frame.** Key Points: * Seawater contains two species of dissolved inorganic iodine (iodide and iodate). * Iodide is the most biologically active form. Diffusion across gills and stomach, excretion kidney and rectal gland. * Iodide ranges from 0.001 – 0.03 ppm of a total iodine concentration of 0.06 ppm (goal in aquaria) * Goiter happens with low dietary or water iodine or goitrogenic compounds (perchlorate) in elasmobranchs * Ozone alters the speciation of iodine by reducing iodide to iodate. * In teleosts majority of iodine intake is from the water, not the food. * Results * Supplemented tanks had high iodine (I2, spectrophotometry) but lower I- (iodide, HPLC) * Chromatography was less reliable than spectrophotometry * Values higher on days after receiving VitaZu tablets Take home: Much higher concentrations of iodine species can be obtained with supplementation orally and in water. Adding Lugols iodine leads to less iodide (I-) being present in the water system as measured by chromatography, but when the spectrophotometric method was used to measure iodine (I2) it resulted in higher concentrations. **Commonly used spectrophotometric methods may significantly underrepresent the biologically available forms of iodine available in the water system.**

Answer 62

Kearns, P. J., Bowen, J. L., & Tlusty, M. F. (2017). The skin microbiome of cow‐nose rays (Rhinoptera bonasus) in an aquarium touch‐tank exhibit. *Zoo biology*, *36*(3), 226-230. **Abstract:** Public aquaria offer numerous educational opportunities for visitors while touch-tank exhibits offer guests the ability to directly interact with marine life via physical contact. Despite the popularity of touch-tanks, there is a paucity of research about animal health in these exhibits and, in particular, there is little research on the microbial communities in these highly interactive exhibits. Microbial community structure can have implications for both host health and habitat function. To better understand the microbiome of a touch-tank we used _high-throughput sequencing of the 16S rRNA gene to analyze the microbial community on the dorsal and ventral surfaces of cow-nose rays (Rhinoptera bonasus) as well as their environment in a frequently visited touch-tank exhibit at the New England Aquarium_. Our analyses revealed a distinct microbial community associated with the skin of the ray that had lower diversity than the surrounding habitat. The ray skin was dominated by three orders: Burkholderiales (∼55%), Flavobacteriales (∼19%), and Pseudomonadales (∼12%), taxonomic groups commonly associated with other fish species. Our results provide a survey of ray-associated bacterial communities in a touch-tank environment, thereby laying the foundation for future studies examining the role of potential challenges to ray microbiota and their associated health. * Ray skin microbiome = Burkholderiales; Flavobacteriales; Pseudomonadales * These orders of bacteria are known to produce antimicrobial compounds * Dorsal and ventral surfaces of ray were not different * Very small percentage of bacteria shared with human skin (\<1.5%) * No human pathogens detected * Environment was different from ray skin microbiome * Inlet water = Vibrio * Outlet water = Rhodobacterales and Thiotrichales * Biofilm = Rhodobacterales, Thiotrichales, Oceanospirillales * Sediment = Rhodobacterales, Alteromonadales **Conclusions:** Human interaction in touch pools does not introduce new bacteria to cownose ray skin, and rays are not a likely source of human pathogens

Answer 63

**TEMPORAL STABILITY OF IN VITRO VENOUS BLOOD GAS, pH, AND LACTATE VALUES OF COWNOSE RAYS (RHINOPTERA BONASUS) AND RED-EARED SLIDER TURTLES (PSEUDEMYS SCRIPTA ELEGANS)** Charles Innis, VMD, Dipl ABVP (RA), Emily Jones, MS, Julie Cavin, DVM, Ryan Knotek, BS, and John Mandelman, PhD _Abstract:_ This study assessed the in vitro temporal changes that occur in blood pH and lactate concentrations for an elasmobranch species and a chelonian species, as well as blood gases (partial pressures of carbon dioxide [pCO2] and oxygen [pO2]) for a chelonian species, with a portable clinical point-of-care analyzer. Blood samples were collected from 10 cownose rays (Rhinoptera bonasus) and 10 red-eared sliders (Pseudemys scripta elegans), stored on ice, and serially analyzed at six time points up to 90 min postcollection. **Results indicate that analysis should be conducted as soon as possible after blood collection for these species, with immediate analysis being preferred.** However, if analysis must be delayed, syringes may be capped, placed on ice, and analyzed at a later time. **Analysis within 90 min provided clinically acceptable results for pH and lactate in both species and for pCO2 in red-eared sliders, whereas substantial artifactual increases of pO2 were seen in red-eared sliders.** Journal of Zoo and Wildlife Medicine 51(1): 110–115, 2020 _Importance:_ low to medium- no huge takeaways but some of the findings could make good distractors _Introduction:_ * Important to understand factors that may affect results of samples taken in the field, especially when not able to be run immediately * Blood and pH parameters can change over time _Methods/results:_ * Assess the in vitro temporal changes that occur with blood pH, partial pressure of carbon dioxide (pCO2) and partial pressure of oxygen (pO2) and lactate measured by a point-of-care analyzer (iSTAT) for cownose rays and red eared sliders * Syringes coated with heparin before blood draw * Blood gases previously found to be inaccurate for cownose rays with the iSTAT, so not measured in this study * Compared values at time 5 min, 10, 15, 45, 90 with the values at time 0 _Discussion:_ * Most values changed over time but not in a significant manner that would change the clinician’s assessment of the results * Analysis within 90 minutes provides acceptable results for pH and lactate in rays * Analysis within 90 minutes provides acceptable results for pH, lactate and pCO2 but NOT pO2 (artifactual increases) in red eared sliders * Recommend running samples immediately when possible * If they have to be delayed, cap needle on syringe and place on ice

Answer 64

Delaune, A. J. M., Field, C. L., & Clauss, T. M. (2017). Whole blood transfusions to treat severe anemia in seven cownose rays (rhinoptera bonasus) and one short-tail stingray (dasyatis brevicaudata). *Journal of Zoo and Wildlife Medicine*, *48*(4), 1172-1180. **Abstract:** Blood transfusions can provide life-saving treatment to severely anemic animals. Due to limited availability and the difficulty of storing whole blood and blood products, such as fresh frozen plasma and packed red blood cells, exotic animals often receive fresh whole blood transfusions. Little is known about elasmobranch blood types and transfusions. Conspecific cross-matches within several different elasmobranch species were negative, indicating that in an emergency situation a single whole blood transfusion may be possible without causing a transfusion reaction. Experimental transfusions between healthy conspecific Atlantic rays (Dasyatis sabina) showed no adverse reactions and autotransfusions in marbled electric rays (Torpedo marmorata) were successful. There are no published reports of blood transfusions performed on clinically abnormal elasmobranchs. The following case series documents blood transfusions performed on seven cownose rays (Rhinoptera bonasus) and one short-tail stingray (Dasyatis brevicaudata**). All rays were treated with the same protocol, which included pretreatment with steroids and antibiotics followed by an intravenous transfusion of freshly collected, heparinized, whole blood**. **Three animals survived and currently exhibit no abnormal clinical signs. Two animals died 55 days and 100 days post transfusion. Three animals died 2–22 days post transfusion.** Although complications from blood transfusions could not be ruled out, all five animals that died had other health problems that likely contributed to their demise. All eight animals would almost certainly have died without a blood transfusion as they were severely anemic and moribund at the time of presentation. The methods described in this paper may be useful in the treatment of severely anemic elasmobranchs and this is the first report of blood transfusions in clinically abnormal elasmobranchs. * Elasmobranch blood types * Little is known * Crossmatches performed within species (sandtiger, sandbar, nurse, and white-spotted bamboo sharks) negative for agglutination and hemolysis à single whole blood transfusion from a healthy conspecific likely ok * Cross-matches between species * Sandbar and sandtiger sharks - negative * Sandtiger and zebra shark – positive * Spiny dogfish found to have different blood groups * Whole blood transfusion trial in Atlantic stingrays had no adverse reactions * Anemia induced in marbled electric rays subsequently transfused * Normal elasmobranch blood volume - 46-80 ml/kg * Case series * Whole blood transfusion in 7 severely anemic cownose rays and 1 short-tail stingray * Blood loss anemia from parasites * All PCV \<8% and clinically affected (in house normal PCV ~25%) * Transfusion protocol * Pretreat with prednisolone sodium succinate 3–4 mg/kg IM 30 min before * Admin vitamin B and iron * Ceftiofur also admin * 9-10ml/kg whole blood transfused IV via pectoral fin vein * 1 ml heparin sodium to 10ml whole blood from donors * Simplified major cross-match performed by mixing 2 drops of recipient serum with 1 drop of donor whole blood to screen for agglutination (macro and micro) * Performed after transfusion in all cases * No filter used * Delivered off the needle, no IVC * Admin over 20-40 min * Rays from cases 3–7 died post transfusion but unknown if secondary to tx or dz * Several died long after transfusion so less likely transfusion complication * Case 6 had cross-match agglutination but also septic – unknown if clinically significant * 3 cases survived * Takeaway: One-time intraspecific whole blood transfusion is likely safe; however, a pretransfusion cross-match recommended and must be performed if cross-species transfusion required

Answer 65

Retallack, H., Okihiro, M. S., Britton, E., Sommeran, S. V., & DeRisi, J. L. (2019). Metagenomic next-generation sequencing reveals Miamiensis avidus (Ciliophora: Scuticociliatida) in the 2017 epizootic of leopard sharks (Triakis semifasciata) in San Francisco Bay, California, USA. *Journal of wildlife diseases*, *55*(2), 375-386. **Abstract:** During March to August of 2017, hundreds of leopard sharks (Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, US. Similar mass stranding events occurred in 1967 and 2011, but analysis of those epizootics was incomplete, and no etiology was confirmed. Our investigation of the 2017 epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. ***We pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening.*** **We showed that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard (n¼12) and other shark species (n¼2) that stranded in San Francisco Bay but was absent in leopard sharks caught elsewhere.** This ciliated protozoan has been implicated in devastating outbreaks in teleost marine fish but not in wild elasmobranchs. Our results highlight the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease. * Leopard sharks in San Francisco Bay, CA * Die-offs over past 50 years: * 1967 -1,000 dead sharks, mainly leopard sharks, w/in 1 mo. * 2006 * 2011 (hundreds of leopard sharks) * Moribund sharks -described as confused and disoriented, with erratic behaviors and swimming patterns * 2017 1,000 estimated L sharks dead (sometimes as many as 30 daily) * Close to shore, disoriented and neuro behavior * Conventional approaches to epizootic investigation: environmental assessment, observation of animal behavior, and necropsy with cytology, histology, microbiological and chemical analyses * Molecular techniques like PCR have enabled rapid and specific pathogen identification * Limited to genetic information available * Metagenomic next-generation sequencing (mNGS): Analysis of all nucleotides in a sample * Scuticociliates are free-living opportunistic marine protozoa that belong to the subclass Scuticociliatida of the phylum Ciliophora * I**nflammatory meningitis** * **Meningoencephalitis: characterized by hemorrhage, cloudy CSF, and thickened meninges lesions** * **Prominent lesions at olfactory bulbs and lobes** * **Olfactory lamellae: often markedly hemorrhagic and inflamed** * Ciliated protozoan parasites (consistent with M. avidus): observed in the brains of 5 affected sharks, and in olfactory lamellae of 1 shark * Data does not exclude the possibility that M. avidus was not the primary or sole driver of disease and mortality. * Water temperature, salinity, toxins, or other pathogens could increase susceptibility to an opportunistic infection by M. avidus * In SF Bay, leopard sharks aggregate in large numbers in the warm shallow waters of bays and estuaries, with greater exposure to runoff that may contain toxins or decreased salinity. Takeaway: Miamiensis avidus = Inflammatory meiningoencephalitis, leopard sharks, SF Bay CA.

Answer 66

Sheldon, J. D., Allender, M. C., George, R. H., Bulman, F., & Abney, K. (2018). Reproductive hormone patterns in male and female cownose rays (Rhinoptera bonasus) in an aquarium setting and correlation to ultrasonographic staging. *Journal of Zoo and Wildlife Medicine*, *49*(3), 638-647. **Abstract**: Reproductive management of cownose rays (Rhinoptera bonasus) under professional care plays an important role in conservation of the species, but hormone and ultrasonographic analyses of their 12-mo reproductive cycle have not been documented previously. **Plasma reproductive hormone concentrations (17Bestradiol, progesterone, testosterone, and androstenedione) were measured monthly via radioimmunoassay for 1 yr in an aquarium-managed population of adult females (n ¼ 15) and males (n ¼ 5). Ultrasounds of the uterus were performed each month at the time of sample collection to identify gestation stage (0–5) based on a previously developed in-house staging system. Stages were correlated to hormone concentrations to track progression through pregnancy.** Thirteen females were reproductively active, and each produced one pup in March–June, similar to timing for free-ranging populations. **Female estradiol increased steadily throughout gestation from stages 0 to 5, while progesterone, testosterone, and androstenedione were increased only in early gestation (stages 1 and 2). Unlike month of year, gestation stage strongly predicted hormone concentration, but specific values to predict parturition date were not identified. Male testosterone and progesterone were higher in March–June (mating season) than July–January, while estradiol and androstenedione did not exhibit a seasonal pattern.** Aquarium-managed cownose rays have similar reproductive patterns to what is reported in wild populations. Ultrasonographic monitoring with serial hormone analysis and accurate mating records will provide the most useful information for managing a reproductive population of cownose rays in an aquarium setting. * Cownose ray reproduction * 11-12 month gestation, 1 pup * Aplacental viviparity – single embryo, left uterus - histotrophy * Annual synchronous reproductive cycles (ovulation & parturition mostly around April-May) * Males * Higher progesterone, testosterone, androstendione than females * Lower testosterone in winter months than in spring/summer months * Higher testosterone in those months correlates to increased spermatogenesis in wild males * Females * Estradiol rose through each stage of pregnancy * Progesterone, testosterone, and androstendione peaked in stages 1-2 * Higher testosterone in may through august reflects peak fertile periods, similar to Atlantic stingrays * Increasing estradiol throughout pregnancy seen in bonnetheads, Atlantic stingrays, spiny dogfish – may play a role in pregnancy maintenance & lecithotrophy, yolk sac, matrotrophy, and histotroph production * Progesterone rises postovulation in bonnetheads, early-midgestation in dogfish, and prior to ovulation & parturition in Atlantic stingrays Take home: **Estradiol increases throughout pregnancy. Males have higher testosterone and progesterone during mating season, and females have peaks in progesterone, testosterone, and androstendione during stages 1-2 (early preg).**

Answer 67

Mylniczenko, N. D., Sumigama, S., Wyffels, J. T., Wheaton, C. J., Guttridge, T. L., DiRocco, S., & Penfold, L. M. (2019). **Ultrasonographic and hormonal characterization of reproductive health and disease in wild, semiwild, and aquarium-housed southern stingrays (Hypanus americanus).** *American journal of veterinary research*, *80*(10), 931-942. Objective: To characterize physical examination, plasma biochemical, and ultrasonographic findings in aquarium-housed, managed semiwild, and wild southern stingrays (*Hypanus americanus*) with and without reproductive disease. Animals: Southern stingrays from aquarium (n = 48), lagoon (managed semiwild; 34), and wild (12) habitats. Procedures: Limited, opportunistic prosections were performed of presumed anatomically normal wild southern stingrays and compared with findings for aquarium-housed stingrays with reproductive disease. Ultrasonographic video data from both groups were used to assign a score (1 to 5) indicating increasing severity of ovarian and uterine reproductive disease. Plasma total 17β-estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T5) concentrations were measured with enzyme immunoassays validated for use in southern stingrays. Results: Ultrasonographic ovarian scores were significantly correlated with uterine scores. **No reproductive disease was detected in semiwild or wild stingrays, but 65% (31/48) of aquarium-housed stingrays had developing or advanced reproductive disease** (ie, ultrasonographic ovarian or uterine score of 4 or 5). **Significant correlations were identified between ovarian and uterine disease status and plasma concentrations of all steroid hormones except testosterone.** Conclusions and Clinical Relevance: **Findings suggested that ultrasonography and plasma hormone concentrations may be useful in the identification of reproductive disease and determination of disease severity in southern stingrays.** Background: * Southern stingrays – Slow to mature, produce low numbers of offspring. Aquariums developing sustainable breeding populations important for education, etc. * Mating can occur immediately after parturition. Females likely gravid for most of life span. * In aquaria, can reproduce biannually and are often housed in single-sex groups to prevent this. * Nonreproductive states for extended periods. * **Elasmobranchs produce P2, P4, T5, do not always regulate reproduction as expected.** * **P does not increase during early or midgestation.** * **Free P and T concentration peaks prior to parturition in Atl stingrays.** * **No P detectable in granulosa tissue throughout gestation in dogfish.** * **Other P4 metabolites and E metabolites may be important.** * **E2 prime regulatory steroid, and E1 also strong role in repro**. * Collected blood samples for plasma hormones, US data over 1 year. * Ultrasound scoring system on a 5 pt scale on basis of width of ovary, number of follicular layers, presence of cysts, overlap with uterus. * General opportunistic postmortem exams on 10 wild stingray cadavers. All gravid. Ovary against left lateral surface of epigonal organ. EO always overlapped uterus on the left side. Width of ovary did not exceed 4 cm. Individual ova rarely exceeded 2 cm. No cysts. Ovary did not overlap with uterus. Right oviduct visibly attenuated with no ova present. Oviduct assoc with epigonal tissue more caudal and smaller than contralateral epigonal tissue. * Right and left ostia originated ventral and lateral to esophagus, led dorsal then lateral into oviducts. Supported by a ligament connected to each oviduct. Right oviduct widened into vestigial right uterus. * Left oviduct expanded into nidamental gland aka oviducal or shell gland at cranial uterus. No obvious isthmus. Uterus on left attached to kidney, terminated into urogenital sinus analogous to mammalian cervix, lateral to single urogenital pore and into cloaca. US Scores: * 2 – Single layer of uniform follicles and occasional small follicles in periphery of ovary. * 3 – ovary appeared wider, multiple layers, various sizes of follicles and cysts. * 4, 5, - greater numbers of different-sized follicles with heterogeneous echogenicity (varying stages of vitellogenesis) and degeneration, cystic structures. * Semiwild or wild stingrays, only one in category 3, none in 4, 5. Hormones: * Plasma E1, E2, P4 increased with increasing ovarian scores. * E1 also increased with ovarian disease. Some exceptions. * No differences in T among ovarian scores. **Severity of repro dz positively correlated with plasma total E2, E1, P4.** 17β-estradiol (E2), estrone (E1), progesterone (P4) * Plasma total T5 high in stingrays assigned highest uterine score. * Regular pregnancy cycles with normal hormone fluctuations presumed to prevent accumulation of ova in southern stingrays, unbred females higher risk of disease. * Ova produce estrogens in elasmobranchs, increase in number of ova may create estrogen rich environment. * **E2 secretion increases dramatically in Atl stingrays during period when histotroph is produced. E2 in this study only high for highest uterine score, E1 increased with increasing uterine score.** * **Plasma conc of total E1 was higher than total E2, may be a more consistent steroid signal to monitor for indication of ovarian and uterine disease. E2 low regardless of disease in some individuals. Examining both probably best.** * Repro disease correlated with disk width, may be due to age and reproductive activity being related.

Answer 68

Donnelly, K. A., Stacy, N. I., Guttridge, T. L., Burns, C., & Mylniczenko, N. (2019). Evaluation of Comprehensive Coelomic Fluid Analysis through Coelomic Pore Sampling as a Novel Diagnostic Tool in Elasmobranchs. *Journal of aquatic animal health*, *31*(2), 173-185. Abstract: The objectives of this study were to describe a minimally invasive coelomic fluid sampling technique in elasmobranchs, to characterize the coelomic fluid composition in clinically normal and abnormal animals, and to compare findings from wild and managed populations. Fluid was collected via the coelomic pore in 89 individuals from 16 species spanning clinically normal and abnormal patients within a managed population (*n* = 54), a semi‐managed open‐lagoon population (*n* = 18), and a wild population (*n* = 17). Biochemical and cytological fluid analyses were performed on all samples, and bacterial and fungal culture, protein electrophoresis, and cholesterol electrophoresis were performed on a subset of samples. The presence of a variable volume of colorless to white and clear to slightly turbid coelomic fluid was consistent with a normal finding; however, the cytological and chemical makeup of coelomic fluid was found to provide additional clinically relevant information. The coelomic fluid from some of the abnormal samples (*n* = 37) contained white blood cells (*n* = 15) and concurrent bacteria (*n* = 7), the latter suggestive of bacterial coelomitis. Yolk was identified in both clinically normal and abnormal females. Of the biochemical parameters tested, calcium, chloride, cholesterol, osmolality, phosphorus, salinity, sodium, specific gravity, total protein, and urea nitrogen have clinical utility. **Abnormal samples were mostly associated with reproductive disease, but to a lesser extent with coelomitis and hemocoelom. The wild and semi‐managed groups had biochemical differences presumably reflective of the higher salinity of ocean water compared with that in the managed habitat. Aerobic bacteria were identified in normal (*n* = 7) and abnormal (*n* = 11) animals. Positive bacterial culture without inflammation may be normal.** This study contributes to a further understanding of elasmobranch coelomic fluid analysis and its use as a diagnostic modality for the evaluation of elasmobranch health. * Gross description variable – clear to white to slightly cloudy. Some green, blue, red. * Presence of yolk identified in all sample colors. * No association between sample color and fluid calcium. * Cytology – Salt crystals, mesothelial cells, RBC may be normal. Also yolk. * Abnormal population – RBC, WBC, mesothelial cells, yolk material, bacteria, salt crystals. * WBC should be considered abnormal. * Clinical chemistry – Significant differences between normal and abnormal animals. * Osmolality and UN significantly higher in abnormal animals. * Significantly higher TS in abnormal animals. * **Abnormal samples had higher Ca, chol, P, SG, and TS** when comparing normal managed and abnormal managed animals. * Across the four populations of southern stingrays (normal managed, abnormal managed, semi managed, and wild), significant differences in Ca, Cl, Na, osmolality, P, salinity, and UN. * Abnormal managed had significantly higher P than all other groups. * Salinity in managed normal group significantly lower vs semi-managed group. * Both managed populations significantly lower osmolality vs semi managed and wild. * Normal managed and semi managed groups had higher Ca. * Protein and CHOL electrophoresis. * NSF between normal and abnormal samples. Neither protein nor CHOL electrophoresis are recommended for evaluation of elasmobranch coelomic fluid. * Microbiology – Aerobic bacterial and fungal organisms isolated from normal and abnormal managed animals. * Urine dipstick – Findings generally supportive of cytology (WBCs, RBCs, etc). No nitrites, ketones, or glucose in any sample. Dipsticks not recommended for acquiring additional information. * Changes in color when used in conjunction in cytology, can be helpful in monitoring individual patients. * Coelomic pores are considered excretory, and coelomic walls an active part of the excretory system of these animals. * Sampling coelomic fluid via coelomic pore catheterization is a safe and effective technique. * Variable amounts of fluid is normal. * Microfauna without inflammatory cells may be considered normal vs WBC with bacteria supportive of coelomitis.

Answer 69

Tang, K. N., O'Connor, M. R., Landolfi, J., & Van Bonn, W. (2019). Safety and efficacy of milbemycin oxime and lufenuron to treat argulus spp. infestation in smooth back river stingrays (potamotrygon orbignyi) and magdalena river stingrays (potamotrygon magdalenae). *Journal of Zoo and Wildlife Medicine*, *50*(2), 383-388. Abstract**: Fish lice, ectoparasites of the genus Argulus**, are branchurian crustaceans that can significantly impact fish health by causing mechanical damage to cutaneous barriers and increasing susceptibility to other infections. While many treatments have been reported in teleosts and invertebrates, there are **no published treatments for elasmobranchs**. This study evaluated the safety and efficacy of a commercial formulation of **milbemycin oxime and lufenuron** in freshwater stingrays for treatment of Argulus spp. Seven juvenile Magdalena river stingrays (Potamotrygon magdalenae) and 10 juvenile smooth back river stingrays (Potamotrygon orbignyi) had severe infestations of Argulus spp. that were identified visually and microscopically. Animals were treated with milbemycin oxime and lufenuron (at 0.015 mg/L and 0.30 mg/L, respectively) in **a 6-hr immersion once weekly for two treatments**. They were visually examined for skin lesions as well as behavior and appetite daily by animal care staff. A subset of animals was euthanized and necropsied on days 8, 9, 43, and 78 after treatment initiation. There were **no Argulus spp. detected at the time of the second treatment**. Complete gross and histologic evaluations were completed for all animals. At all time points, **no gross abnormalities** were detected with the exception of thin body condition in some animals; **no Argulus spp. were noted.** Histologic lesions were all attributed to poor nutritional state at the time of acquisition. **No histologic evidence of acute or chronic toxicosis** was detected. The commercial formulation of milbemycin oxime and lufenuron, applied at the dose and for the exposure time used in this study, **effectively eradicated Argulus spp**. in a population of juvenile P. magdalenae and P. orbignyi, and did not cause mortality or clinical gross or histologic evidence of acute or chronic toxicity. Fish lice (*Argulus* sp.): branchurian crustaceans - Esp affect trout and carp species, but wide host range - Mechanical vectors (virus & bacteria = spring viremia of carp, *Flavobacterium columnare*) - Life cycle takes approx. 2 months. Peak abundance in summer and fall - Attach to back using suckers and hooks, compress epithelial, in a ring shape, vacuum effect, loss of cohesion, irritation, discomfort, hemorrhage. Can cause edema and anemia in severe infections - Dx by morphological ID: compound eyes, 2 large suckers that resemble eyes, shell-like carapace, 4 thoracic legs, jointed appendages, flattened saucer shape, 5-20 mm in size, visible to the naked eye Milbemycin: - Macrocyclic lactone effective on arthropods and nematodes (ineffective w/ cestodes and Platyhelminthes) - Binds to glutamate-gate Cl channels in neurons and myocytes in invertebrates -\> paralysis and death - Lethal doses have been described in fish + limited efficacy when given PO against nematodes Lufenuron: - Benzoyl urea pesticide - Inhibits chitin synthesis -\> prevents exoskeleton formation - Lethal doses have been described in fish Results/Discussion: Was effective after the first tx (day 8). No adverse effects **Take-home:** - **Immersion bath w/ lufenuron and milbemycin oxide** in stingrays **effective and safe against *Argulus*** - Lufenuron inhibits chitin synthesis, milbemycine oxide binds to Cl channels in neurons

Answer 70

Fayette, M. A., Rose, J. B., Hunter, R. P., Bowman, M. R., & Proudfoot, J. S. (2019). Naïve-pooled pharmacokinetics of ceftiofur crystalline free acid after single intramuscular administration in smooth dogfish (mustelus canis). *Journal of Zoo and Wildlife Medicine*, *50*(2), 466-469. Abstract: Pharmacokinetics study of ceftiofur crystalline free acid (CCFA) was conducted in 14 adult captive smooth dogfish (Mustelus canis). A **single dose of CCFA at 6.6 mg/kg** was administered intramuscularly. Blood samples were collected prior to treatment and at 1, 2, 6, 12, 24, 32, 48, 72, 96, 120, 144, and 168 hr posttreatment. **Naive pooling of data** from four sharks was used to generate the average plasma drug concentration at each time point. After concluding the study, additional blood samples were opportunistically collected from five randomly selected sharks at 1,920 hr. Plasma ceftiofur and desfuroylceftiofur metabolite concentrations were determined using reversed-phase high performance liquid chromatography (HPLC). Pharmacokinetic analysis was performed using a noncompartmental technique. Peak plasma concentration (Cmax) was 3.75 lg/ml with a time **to Cmax (Tmax) of 96** hr. Ceftiofur plasma concentrations were **maintained above 2 lg/ml for at least 168 hr** and were still quantifiable at 1,920 hr. **Results/Discussion:** 6.6 mg/kg IM, No adverse effects**.** Cmax peaked at 96h. Exceeded target MIC for at least 170h * Dosing interval recommendations not made bc could not determine terminal half-life **Take-home:** No adverse effects, reached target MIC

Answer 71

PHARMACOKINETICS OF A SINGLE DOSE OF INTRAMUSCULAR AND ORAL MELOXICAM IN YELLOW STINGRAYS (UROBATIS JAMAICENSIS) Journal of Zoo and Wildlife Medicine 53(1): 153–158, 2022 Abstract: Elasmobranchs are popular display animals in public aquaria and zoos, but medical management gaps remain in the understanding of the pharmacokinetics of analgesics and pain management in these species. Meloxicam is a nonsteroidal anti-inflammatory drug that has been evaluated intravenously and intramuscularly in teleosts, but has yet to be studied in any elasmobranch species. **The pharmacokinetics of meloxicam were determined in 17 yellow stingrays (Urobatis jamaicensis).** All stingrays were determined to be healthy from complete physical examinations and baseline bloodwork performed prior to study inclusion. **A single dose of 1 mg/kg meloxicam intramuscularly was administered to all rays, followed by a 2 mg/kg oral dose after an 8 wk washout period**. Blood samples were collected from the mesopterygial vein at baseline and nine time points up to 96 h after administration of meloxicam. Plasma concentrations were determined using reversed-phase highperformance liquid chromatography. Pharmacokinetic analysis was performed using a noncompartmental technique. The mean peak plasma concentrations for intramuscular and oral meloxicam were 1.29 and 0.42 lg/ ml, respectively. The mean terminal half-lives of meloxicam after intramuscular and oral administration were 5.75 and 15.46 h, respectively**. Based on these findings, the recommended meloxicam dosage and frequency for yellow stingrays is 2 mg/kg orally once daily. Due to rapid elimination with the intramuscular administration, maintaining clinically relevant plasma concentrations may be difficult using this route**. Further studies are needed to determine multidose pharmacokinetics of meloxicam in yellow stingrays, as well as single-dose and multidose pharmacokinetics in other elasmobranch species. Intro · The objective of this study was to determine the pharmacokinetic data of a single dose of intramuscular and oral meloxicam in yellow stingrays M&M · 17 healthy yellow stingrays (Urobatis jamaicensis) · Single dose of meloxicam 1 mg/kg IM followed by 2 mg/kg PO after 8-week washout Results · Easy to aadminister, no adverse effects noted 4 months post study · The mean peak plasma concentrations for intramuscular and oral meloxicam were 1.29 and 0.42 mcg/ ml, respectively. The mean terminal half-lives of meloxicam after intramuscular and oral administration were 5.75 and 15.46 h, respectively Discussion · Oral meloxicam administration in yellow stingrays produced variation in the pharmacokinetic analysis. The reason for this variation is undetermined without further study, but may be due to differences in plasma protein binding and biotransformation pathways · Both administration routes produced plasma concentrations · however, significant variability was appreciated with oral administration. Oral meloxicam maintained above therapeutic plasma concentrations for 16 h and intramuscular for only 6 h. · On the basis of these results, it is recommended that meloxicam be administered to yellow stingrays at 2 mg/kg orally once daily. · Intramuscular administration is not recommended at this time due to rapid elimination and unsustainable clinically relevant plasma concentrations.

Answer 72

Parkinson, L., Gaines, B., & Nollens, H. (2019). Effect of a nutrient enema on serum nutrient concentrations in white-spotted bamboo sharks (chiloscyllium plagiosum). *Journal of Zoo and Wildlife Medicine*, *50*(1), 55-61. Abstract: Ill and anorectic captive sharks present a unique challenge for husbandry and veterinary staff. Providing adequate fluid and nutritional support to sharks while minimizing handling remains difficult. **This study aimed to evaluate the ability of a nutrient enema to alter blood analyte concentrations.** Thirty-six healthy, fasted white-spotted bamboo sharks (Chiloscyllium plagiosum) were enrolled in the study with 18 sharks receiving a nutrient enema and 18 sharks receiving a non-nutrient saline enema. The metabolic state of sharks was evaluated via measurement of blood glucose, blood urea nitrogen, and b-hydroxybutyrate as well as other serum biochemistry parameters. Changes in sodium, chloride, calcium, b-hydroxybutyrate, glucose, total protein, and triglyceride concentrations were seen across time in both groups. Blood glucose absolute concentrations and changes over time differed between the nutrient and nonnutrient groups. **This pilot study indicates that it is possible to influence the glucose metabolism of healthy sharks via nutrient enema.** Further study is needed to better understand potential therapeutics for ill and anorectic sharks. **Study design:** 36 white-spotted bamboo sharks divided into two groups: non-nutrient (0.9% Sodium chloride) and nutrient enema (1:1 50% dextrose and AminoPlex solution [amino acids, electrolytes, B-complex vitamins, dextrose]) - Fasted 48-73h prior to enema - Enema with tube inserted 6 cm into cloaca and 6 ml of enema solution - Blood samples for glucose, BUN, BHB, TP, Na, K, Cl, Ca and triglycerides at timepoints Glucose concentrations were the only blood analyte that differed between saline and nutrient enema group, and only at hour 120 (significant decrease). All other analytes in the nutrient group mirrored changes observed in the saline group: * Ca increased at hours 3, 6, 9, and 20 then sharply decreased. * Cl significantly lower at hours 20, 72, 120. * Total protein significantly higher at hours 3, 6, 9, 20. * TG significantly higher than baseline at 72h and 120hr in both groups. * BHB not significantly different between groups throughout duration of study. * BUN, K no significant changes over time. **Results/Discussion:** - Blood parameters assumed to most likely reflect shark metabolism: glucose, BUN, BHB o BHB: ketone body produced in liver and released into circulation to provide energy; Play role of primary metabolic energy in shark and amplified role in starving o BUN : indicator of protein metabolism and osmoregulatory status. Urea normally maintained at high levels even during fasting to help maintain slightly hyperosmotic state o Blood glucose – enema did not provide sufficient nutrition to mimic a meal **Take-away:** Glucose only analyte that differed with nutrient enema vs saline and was lower at hour 120.

Answer 73

**DIAGNOSIS AND TREATMENT OF AN ENTEROCOCCUS FECALIS ABSCESS IN THE CRANIAL VAULT OF A SPOTTED EAGLE RAY (AETOBATUS NARINARI )\_Delaune et al\_JZWM 51(1): 2020** Abstract: An adult female spotted eagle ray (Aetobatus narinari) presented for medical evaluation due to a swelling located on the dorsal head. Ultrasound revealed that the swelling originated from a large pocket of fluid in the cranial vault. The swelling was aspirated, and purulent discharge was obtained; Enterococcus faecalis was cultured. An incision was made over the swelling in an attempt to drain fluid but was unsuccessful. Multiple aspirates were performed to drain the abscess, and the animal was treated with oxytetracycline injections. The initial incision sloughed and resulted in a large defect in the cranium that allowed exhibit water to come into the cranial vault and come in contact with the protective membrane of the brain. Forty-two days after initial presentation, the defect in the cranium was healed; fluid from the cranial vault was sampled and appeared normal. During and after treatment, the ray exhibited no abnormal neurologic signs. Key words: Abscess, brain, elasmobranch, medicine, spotted eagle ray (Aetobatus narinari), ultrasound **Importance:** Low to moderate; single case report with unknown etiology, but super interesting! Abstract sufficient **Case report:** Presentation: Adult female spotted eagle ray presented w/ hyporexia and swelling on dorsal head with no associated visible puncture wounds or abrasion Diagnostics: - Fine needle aspirate: purulent fluid - Ultrasound: large fluid pocket w/ abnormal flocculent material immediately cranial to the brain in the cranial vault + cystic lesion cranial to the cerebrum and caudal to the swelling - Culture of fluid: Enterococcus faecalis Treatment: - Initial: Vitamin C and ceftiofur - Oxytetracycline based on culture and sensitivity - Bioadhesive gel to devitalized tissue Case evolution: - 8 days after presentation, devitalized tissue sloughed off resulting in a full thickness defect and persistent opening into the cranium. US revealed exhibit water swirling around in the cranial vault - NEVER demonstrated neurologic signs, continued to eat and behave normally - Wound healed by secondary intention, almost completely closed by 40 days. Cystic structure rostral to the cerebrum remained unchanged - Died due to sig trauma from tank mate 14 mo later - Histopath: chronic granulomatous perivascular meningoencephalitis **Discussion:** - Inciting cause unknown. Possibly nonvisible penetrating wound or systemic infection that may have manifested as abscess in cranial vault as extradural fluid and peripheral bloodstream in some elasmobranch species appear to have no barriers - Unknown if cystic lesion contributed or was present before abscess - No neurologic signs: possibly bc tight membrane covering the brain inhibits communication btw extradural fluid and the brain **Take home:** Abscess in cranial vault of adult spotted eagle ray that resulted in severe cranial defect with exhibit water access to the cranial vault. Appeared to have grave prognosis but recovered with tx and never exhibited neurological signs

Answer 74

JZWM 2017 48(3) 902-905 **SQUAMOUS CELL CARCINOMA OF THE ROSTRAL MAXILLA IN AN ADULT CAPTIVE WHITESPOTTED BAMBOO SHARK (CHILOSCYLLIUM PLAGIOSUM)** Betsy E. Culp, D.V.M., Martin Haulena, D.V.M., Dipl. A.C.Z.M., M.Sc., Kelly Britt, D.V.M., Hannah Evans, B.A., and Stephen Raverty, D.V.M., Dipl. A.C.V.P., Ph.D., M.Sc. **Abstract**: An approximately 10-yr-old adult female whitespotted bamboo shark (Chiloscyllium plagiosum) presented with a smooth, **white, irregular, ulcerated, and expansile lesion** on the left lateral aspect of the **maxillary rostrum**. The lesion had short periods of abrupt and rapid proliferation and then remained static for several months. Cytology and culture were nonspecific and did not reveal any discernible etiologic agents or cellular atypia. The lesion was nonresponsive to parenteral antibiotics. **One year after the initial onset** of the lesion, the ulcer was 10 cm in diameter, a percentage **increase in size of 455%**. Due to a protracted clinical course and lack of response to medication and supportive care, coupled with an acute onset of neurologic signs and self-inflicted trauma, the shark was euthanized. Histopathology of the mass disclosed a **locally invasive squamous cell carcinoma with no evidence of metastasis**. **Key** **points**: * Mesenchymal tumors more commonly reported * Polygonal cells arranged as cords, broad fronds, trabeculae, and tightly packed pearls with moderate amounts of intervening stroma and mitotic figures **Take** **home**: First SCC in Orectolobiformes ‘‘carpet shark’’ order

Answer 75

Molter, Christine M., and Laura J. Baseler. "Clinical challenge: Diagnosis of Loimopapillosum sp. Branchitis in a southern stingray (Dasyatis Americana)." *Journal of Zoo and Wildlife Medicine* 48.4 (2017): 1267-1269. * Adult F southern stingray found dead * Necropsy findings * skin around gill slits erythematous, petechiae on cutaneous ventrum, ectoparasites attached to irregularly thickened gills, blood in oral cavity * chronic, severe, proliferative branchitis with lamellar fusion caused by Loimopapillosum sp. and a secondary mixed bacterial infection * Monogeneans * common parasites of skin and gills of freshwater and marine bony and cartilaginous fish * direct life cycle with unknown duration of each lifecycle stage * rapid reproduction rate * some species have eggs resistant to available treatments * poor water quality and overcrowding exacerbate infection * signs - dyspnea, hyperpnea, pruritus, lethargy, anorexia, behavioral changes, flashing, death * two subclasses à Monopisthocotylea and Polyopisthocotylea * Monopisthocotylea * simple haptor * feed on skin and gill epithelium and mucus * lesions - increased mucus on gills and skin, cutaneous hemorrhages, and hyperpigmentation and fraying of fins * gill histo - epithelial hyperplasia and necrosis, lamellar fusion, hemorrhage, increased mucus production, inflammatory cell infiltration * skin lesions - increased mucus production, dermatitis, epidermal hyperplasia * diagnosis - rigid endoscopy and gill biopsy * tx - freshwater and praziquantel baths * Polyopisthocotylea * complex haptor * feed on blood * can cause anemia

Answer 76

Greene, W., Brookshire, G., & Delaune, A. J. (2018). Hematologic and biochemical summary statistics in aquarium-housed spotted eagle rays (aetobatus narinari). *Journal of Zoo and Wildlife Medicine*, *49*(4), 912-924. Abstract: During this retrospective study, 18 plasma blood chemistry and 17 complete blood count (CBC) samples were analyzed from clinically healthy spotted eagle rays (Aetobatus narinari) at Georgia Aquarium in order to generate hematological ranges for complete blood count (CBC) and biochemical profiles. Summary statistics were generated according to the American Society for Veterinary Clinical Pathology guidelines for the determination of reference intervals in veterinary species.⁴ The mean packed cell volume (PCV) was 28.09% with a range of 23–35%. Mean total solids were 5.72 g/dl with a range of 5–7.0 g/dl. Lymphocytes were the dominant leukocyte observed on differential (67.35%), followed by fine eosinophilic granulocytes (FEGs) (15.41%), coarse eosinophilic granulocytes (CEGs) (10.24%), monocytes (1.88%), and basophils (1.24%). Chemistry samples were analyzed at two diagnostic laboratories, Michigan State University (MSU) and University of Miami (UMiami), and the results were compared. **Both labs have the capacity to run blood chemistries on zoo and aquatic species, but utilize different methods to obtain chemistry analyte values. UMiami uses a thin-film dry-slide technology, whereas MSU uses an ion-selective electrode (ISE) and Beckman Coulter AU 640 analyzer. There is poor agreement between the analyzers used by the two laboratories for both alkaline phosphatase and BUN**, because of proportional error. Establishing hematological ranges in spotted eagle rays and in elasmobranchs in general may enhance the understanding of the species and their health. This information may aid clinicians in deciding when and how to treat elasmobranchs. * Key Points: * Elasmobranch WBCs are unique in that they have both fine eosinophilic granulocytes (FEGs) and coarse eosinophilic granulocytes (CEGs) vs heterophils. * Lymphocytes dominant leukocyte on differential (~67%), similar to other elasmobranchs. * Takeaway: Spotted eagle rays similar to other elasmos in regards to CBC/chem values. Monitoring trends within individual recommended; laboratories can vary in results for chem analysis.

Answer 77

Natural environmental conditions and collaborative efforts provide the secret to success for sand tiger shark Carcharias taurus reproduction in aquaria. Wyffels, J., Coco, C., Schreiber, C., Palmer, D., Clauss, T., Bulman, F., George, R., Pelton, C., Feldheim, K. and Handsel, T. *Zoo Biology*, 2020;39(5), pp.355-363. Sand tiger sharks are an iconic large shark species held in aquaria worldwide. They rarely reproduce under managed care, with only seven aquaria reporting limited and sporadic success. For the first time in the Americas, a full‐term young was born in an aquarium. The young was the result of breeding among a group of sharks purposefully brought together in 2016 for reproduction. Sharks were maintained in natural seawater and exposed to natural light and seasonal temperature fluctuations similar to their in situ counterparts. Decreased food consumption associated with breeding season and gestation was observed. **Gestation time estimated from breeding observations and parturition was 321 days.** Although the neonate was stillborn, this was a significant achievement. The husbandry details described within will be useful for other aquaria striving to support the reproduction of sand tiger sharks. **Background** * Sand tiger shark reproduction: low fecundity, long reproductive cycle * Product 2 young: 1 per uterus, every 2-3 years * Adelphophagy (intrauterine cannibalism): embryos develop precocious dentition used to consume uterine siblings and egg cases with infertile ova * Egg cases not fertilized are shed 12-18 mo later * Gestation 280-290d **Key Points** * First sand tiger shark birth from natural breeding in NA * Natural seawater, UV sterilizer and filtration * Fed 3x/wk primarily fatty herring, Vitamin C tabs, Mazuri Vitazu shark/ray tabs * Water 21-22C rising to 24-25C, day length increasing * Food consumption decreased during mating, low during pregnancy, increased post-parturition * Male consumption highest in fall and water, reduced during mating season in spring * Repro behaviors occurred April/May to early June, may see semen in habitat, bite marks * Male reproductive behavior: nosing, tailing, snapping, clasper flexion, splaying, crossing * Female repro behavior: stalling, shielding, flaring, cupping, submission * Shared: biting, copulation * Ultrasound: vitellogenic follicles in ovary, ovulated as egg cases in uterus * Increased girth around 6 mo, fetus visible externally moving in uterus around 8-9 mo * Fetus was viable at mo 11 but mo 12 (321d) a full-term female was found dead at the bottom of the habitat * No yolk in digestive system and timeline suggests delayed parturition (~1 mo) **Conclusions** * With optimized husbandry, healthy sand tiger sharks can reproduce under managed care in a relatively short time-frame * Recommend developing a birth plan that includes intervention to assist deliver if gestation exceeds 300d.

Answer 78

RADIOGRAPHIC DETERMINATION OF GASTRIC EMPTYING AND GASTROINTESTINAL TRANSIT TIME IN COWNOSE RAYS (RHINOPTERA BONASUS) AND WHITESPOTTED BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM) AND THE EFFECT OF METOCLOPRAMIDE ON GASTROINTESTINAL MOTILITY Journal of Zoo and Wildlife Medicine 51(2): 326–333, 2020 Abstract: Gastrointestinal (GI) pathology is common in elasmobranchs; however, information regarding normal GI transit time and the effect of therapeutics on GI motility is lacking**. The objective of this study was to determine baseline gastric emptying and GI transit times in cownose rays (Rhinoptera bonasus) and whitespotted bamboo sharks (Chiloscyllium plagiosum) via radiographic barium sulfate contrast studies**. **Additionally, a pilot study was undertaken to determine the effect of metoclopramide on GI transit time in whitespotted bamboo sharks**. Eight cownose rays and eight whitespotted bamboo sharks were administered a 98% w/w barium sulfate suspension at 8 ml/kg via orogastric tube. Post-contrast radiographs were obtained at 2 min, 3, 6, 12, and 23 hr for rays; and 2 min, 3, 6, 9, 12, 16, 25, 30, 36, and every 12 hr until complete gastric emptying occurred for sharks. In cownose rays, the mean and standard error were established for time of initial spiral colon filling (3.4 6 0.4 hr), complete spiral colon opacification (12 6 0 hr), initial spiral colon emptying (21.6 6 1.4 hr), and complete gastric emptying (23 6 0 hr). In bamboo sharks, the mean and standard error were established for time of initial spiral colon filling (5.3 6 0.5 hr), complete spiral colon opacification (12.4 6 1.3 hr), initial spiral colon emptying (22.5 6 2.7 hr), and complete gastric emptying (39.9 6 3.3 hr). Cownose rays had a significantly shorter time to spiral colon filling and complete gastric emptying compared with bamboo sharks (P , 0.05). Whitespotted bamboo sharks (n ¼ 8) were administered metoclopramide (0.4 mg/kg orally once daily for 10 days) and the barium series was repeated. **Complete gastric emptying time was significantly shorter in treated sharks compared with control (P , 0.05), suggesting that metoclopramide may be a useful therapeutic for GI motility disorders in elasmobranchs**. Intro * GI pathology is common in elasmobranchs * Little info on normal GI transit times * The objective of this study was to establish baseline gastric emptying time and GI transit time of barium sulfate contrast in cownose rays and whitespotted bamboo sharks * A pilot study was also undertaken to determine the effect of metoclopramide on gastric emptying and GI transit time in the whitespotted bamboo shark M&M * 8 cownose rays and 8 whitespotted bamboo sharks were given 8 ml/kg barium PO and then radiographed in series to measure gastric emptying * 3 weeks after each bamboo shark was given metoclopramide 0.4 mg/kg PO SID x 10 days. On day 7 the barium series was repeated in the same manner Results and discussion * Results of this study reveal that transit time of barium through the elasmobranch GIT can be broken up into four distinct, easily identifiable time points including initial spiral colon filling, complete spiral colon opacification, initial spiral colon emptying, and complete gastric emptying. * Cownose rays displayed a significantly shorter mean initial spiral colon filling time (3.4 hr) and mean gastric emptying time (23 hr) as compared to whitespotted bamboo sharks (5.3 hr and 39.9 hr, respectively) * No differences when evaluated for sex or weight * With metoclopramide, treated sharks displayed a statistically shorter time to complete gastric emptying (27.6 hr) compared with non-treated sharks (39.9 hr) * Takeaway: GI transit time in cownose rays and whitespotted bamboo sharks consists of 4 distinct time points, GI transit time is shorter in cownose rays than bamboo sharks, and reglan orally may be useful in treating motility disorders in sharks

Answer 79

**DIAGNOSIS AND MANAGEMENT OF SUSPECTED CONGESTIVE HEART FAILURE SECONDARY TO DILATED CARDIOMYOPATHY IN A SAND TIGER SHARK (CARCHARIAS TAURUS) WITH ESTABLISHMENT OF PRELIMINARY NORMAL ECHOCARDIOGRAPHIC INDICES** Michael W. Hyatt, DVM, DACZM, and Trevor J. Gerlach, DVM, DACVIM Journal of Zoo and Wildlife Medicine 53(2): 363–372, 2022 Abstract: Elasmobranch cardiac anatomy and physiology has been well described; however, there is a dearth of information regarding cardiac disease. In support of a clinical case of suspected congestive heart failure in a 22-yrold male sand tiger shark (Carcharias taurus), a study was undertaken to identify feasible echocardiographic imaging planes and preliminary indices for this species. Eleven echocardiograms were performed on six apparently healthy sand tiger sharks. Echocardiographic parameters are presented using descriptive statistics, including mean, median, standard deviation (SD), minimum and maximum values. These data were utilized for the diagnosis and clinical management of the affected shark. The shark initially presented with increased respiratory effort, dependent, peripheral edema, and anemia. Echocardiography revealed atrial, ventricular, and sinus venosus dilation. As congestive heart failure secondary to dilated cardiomyopathy was strongly suspected, therapy was initiated with oral benazepril and torsemide, and later pimobendan. After a year of therapy, clinical signs resolved. Cardiac size and function improved on echocardiography with a reduction in sinus venosus dilation, maximum and minimum atrial and ventricular inner diameters, and an increase in atrial and ventricular fractional shortening. Cardiac disease in elasmobranchs may be underdiagnosed, so it may be necessary to develop standardized ultrasound techniques and cardiac measurements for each species of elasmobranch managed within zoos and aquaria. Key Points: - Elasmobranch heart – 2 chambers (atrium and ventricle) with accessory structures of sinus venosus located caudally and conus arteriosus cranially. -- Atrium and ventricle are cardiac muscle while sinus venosus and conus arteriosus are smooth muscle with cardiomyocytes. -- Contained within pericardium with variable amounts of pericardial fluid - Blood returns from systemic circulation to the heart via hepatic and cardinal veins emptying into sinus venosus 🡪 sinoatrial valve into the atrium 🡪 AV valve into the ventricle 🡪 out conal calve and conus arteriosus 🡪 ventral aorta to gills 🡪 dorsal aorta to systemic circulation - Heart rate stable during exercise – CO more heavily influenced by stroke volume in elasmobranchs -- Lack sympathetic innervation to heart 🡪 rely on parasympathetic innervation and circulating catecholamines - Aim of study was to describe clinical case and the results of normal sand tiger shark echos - Case report 🡪 sand tiger shark presented with increased respiratory effort (mouth gape and buccal pumping), dependent peripheral edema of claspers and base of pelvic fins -- Atrial, ventricular, and sinus venosus dilation with reduced atrial and ventricular contractility -- Hepatic veins emptying into sinus venosus dilated -- DCM with secondary congestive heart failure suspected -- No evidence of coelomic effusion, moderate anemia (11%) - Treatment – benazepril (ACE inhibitor), torsemide (pyridine-sulfonylurea loop diuretic), iron. -- Pimobendan (inodilator) added after no echo changes and progressive edema were seen -- Progressive clinical and echo improvements seen – peripheral edema and dyspnea resolved by 9 months, and echo parameters normalized within 1 year -- Compared to healthy group, prior to treatment, shark had increased atrial and ventricular maximum and minimum inner diameters, an increased atrium:conus arteriosus (A:CA) ratio, reduced atrial and ventricular shortening fractions, and increased sinus venosus measurements prior to initiation of therapy. -- Another case of suspected DCM in leopard shark 🡪 anemia, increased ventricular maximum inner diameter, and reduced shortening fraction though also had coelomic effusion, wt loss, and poor appetite -- Ddx for DCM 🡪 toxic insult, nutritional deficiency, metabolic derangements, genetic disease, and myocarditis, or the cause may have been idiopathic toxic insult, nutritional deficiency, metabolic derangements, genetic disease, and myocarditis, or the cause may have been idiopathic - In elasmobranch, cardiac output increases occur by increased stroke volume but indirectly regulate this through transfer of pericardial fluid into coelomic cavity Take-Home Message: - First case of clinical DCM with secondary CHF in a sand tiger shark. - - Clinical signs of dependent peripheral edema were consistent with CHF. - Study provides reference values for normal sand tiger sharks.

Answer 80

**Pharmacokinetics of meloxicam after a single 1.5 mg/kg intramuscular administration to nursehound sharks (scyliorhinus stellaris) and its effects on hematology and plasma biochemistry**. Morón-Elorza P, Rojo-Solís C, Álvaro-Álvarez T, Valls-Torres M, García-Párraga D, Encinas T. Journal of Zoo and Wildlife Medicine. 2022;53(2):393-401. A single-dose meloxicam pharmacokinetic (PK) study was performed with eight clinically healthy nursehound sharks (Scyliorhinus stellaris) maintained under human care. Meloxicam was administered IM at a dosage of 1.5 mg/kg to six animals; two animals were administered elasmobranch physiological saline solution (EPSS) IM as a negative control group. Blood samples were obtained prior to and at 12 predetermined times during the first 36 h after administration. Effects on hematology and plasma biochemistry were compared prior to and 24 h after administration. No animal died or showed clinical signs during the study. A significant increase in creatinine kinase and aspartate aminotransferase was found in both EPSS and meloxicam groups and could be considered a direct consequence of sampling and handling required for the PK study. **Observed mean time to maximum plasma concentration ± SEM was 2.58 ± 0.47 h **and observed mean maximum plasma concentration ± SEM was 806 ± 66 ng/ml; mean terminal half-life ± SEM was 15.97 ± 1.20 h; mean residency time ± SEM was 23.40 ± 2.25 h. Area under the plasma concentration–versus-time curve extrapolated to infinity ± SEM was 15.52 ± 1.70 h·µg/ml. **This study suggests that meloxicam 1.5 mg/kg IM in nursehound sharks is likely to result in clinically relevant plasma levels for periods of 24 h without producing significant alterations in blood analytics, although further PK studies with meloxicam IV in sharks are needed**. Future PK and pharmacodynamic studies with different drugs and doses are needed in elasmobranchs to establish safe and effective treatment protocols. Background - Shark WBC -- Fine eosinophilic granulocytes - granulocyte type I; heterophil-like cells -- Neutrophil - granulocyte type II; neutrophil-like cells -- Coarse eosinophilic granulocyte - granulocyte type III; eosinophil-like cells -- Granulated thrombocytes -- Lymphocytes -- Monocytes - In vitro equine studies - plasma meloxicam 150-250 ng/ml can effectively inhibit COX-2.3 Key Points - 1.5 mg/kg meloxicam (5 mg/ml) IM in epaxial mm. and blood drawn while manually restrained -- Preliminary study with 0.2 and 0.5 mg/kg IM and IV had low plasma concentrations - Rapid absorption, prolonged elimination, potentially therapeutic levels for at least 24hr -- Tmax and T1/2 were longer in sharks than previous study in fish (tilapia 1 mg/kg) - No change in CBC results 24hr after meloxicam - Elevated CK and AST, lower elevation in LDH after PK study in meloxicam and control/saline sharks - presumed from repeated handling +/- IM injection - No evidence of adverse effects or toxicity Conclusions - Single dose meloxicam 1.5 mg/kg in nursehound sharks had theoretically therapeutic plasma concentrations with no adverse effects or changes in BW, possible interdose period of 16-24hr -- Repeated handling caused elevations of muscle damage enzymes (CK, AST, LDH)

Answer 81

JZWM 2022 53(3):504-514 **Hematology And Plasma Biochemistry Reference Values Of Juvenile Undulate Rays (Raja undulata) Under Human Care** Morón-Elorza P, Steyrer C, Rojo-Solís C, Álvaro-Álvarez T, Valls-Torres M, Encinas T, García-Párraga D ABSTRACT: Despite the paucity of published literature on elasmobranch hematology and biochemistry, great interspecific diversity has been observed. Blood samples from 43 undulate rays (Raja undulata) (23 males, 20 females) hatched and raised at Oceanogràfic Aquarium, were analyzed for hematology and plasma biochemistry. Animals were divided into two age groups: 1 yr old (28 skates) and 2 yr old (15 skates). All individuals were clinically healthy on physical examination. Weight, total length, standard length, and disc width were recorded. No statistically significant differences were observed between male and female juvenile skates for the evaluated morphometric, hematologic, and plasma biochemical values. Once reference intervals (RI) were determined, blood samples from seven healthy adult skates housed at the same aquarium were collected for comparison. Statistically significant differences were observed in cholesterol, triglycerides, alkaline phosphatase, blood urea nitrogen, chloride, and sodium between juvenile and adult skates. This is the first article describing hematological and plasma biochemical RI for this species, increasing the clinical knowledge on elasmobranch blood analytics. These data will serve as a valuable diagnostic and research tool for professionals working with undulate rays and closer relatives in aquariums and in the field. Further studies using larger elasmobranch sample sizes are needed to determine reliable species-specific baseline health values and to evaluate the effect of intrinsic and extrinsic parameters on blood analytics more accurately. Background: - Undulate ray = endangered skate with oviparous reproduction, unlike viviparous rays -- Distribution: northeastern Atlantic Ocean, Mediterranean Sea Key Points: - RIs generated from reference population of 43 healthy juvenile undulate rays -- 23 males & 20 females -- Three age subgroups: 7-14mo (n = 28), 20-26mo (n = 15), adult (n = 7) - Hematology similar to previous studies in different elasmobranch -- Lymphocyte predominant WBC differential - Plasma biochemistry similar to previous studies in different elasmobranch -- Low blood glucose is a normal finding in healthy elasmobranchs -- ALP & cholesterol higher in rays < 26mo vs. adults -- Could be age-related variation (ALP) or differences in food availability, season, and/or repro status (cholesterol) TLDR: RIs for undulate rays; hematology & plasma biochemistry similar to other elasmobranchs

Answer 82

JZWM 2023 54(1):40-48 **Effects Of A Gnrh Vaccine And Deslorelin Acetate Implants In Male Freshwater Stingrays (Potamotrygon Sp.)** Sailler A, Laidebeure S, Lécu A ABSTRACT: Very little information is available in veterinary literature concerning chemical contraception in elasmobranchs. To decrease breeding and adverse reproductive behaviors, male Potamotrygon sp., housed in two zoologic institutions, were treated using methods used in other elasmobranchs. **Four animals received deslorelin acetate implants (Suprelorin 4.7 mg and 9.4 mg), four animals received a gonadotropin-releasing hormone vaccine (Improvac 50-100 µg) twice separated by 1 mon, and two animals were not treated to serve as controls**. Health checks, including blood sampling, coelomic ultrasound, and sperm analysis, were performed bimonthly and then monthly over almost 2 yr. **Microscopic examination of sperm never revealed any significant change in concentration or motility. Size of testes and seminal vesicles glands did not change significantly after treatment. Plasma testosterone concentrations were stable (∼1 ng/ml) in intact and vaccinated animals throughout the study period. Plasma testosterone level increased significantly after deslorelin implantation and remained very high for at least 13 mon, never returning to initial values. Peak concentration varied according to the deslorelin acetate concentration used. Aggression toward females continued despite the use of contraception. Histopathologic examination on dead stingrays revealed active testicular tissue**. These results suggest that deslorelin acetate implants and GnRH vaccine are ineffective at dosages used in our cases. **Implants caused a continuous stimulation of the hypothalamic-pituitary-gonadal axis that could be harmful for the animals**. Background: - Potamotrygonidae family = only Batoidea family in which all species require freshwater -- Native to South American rivers -- Seasonal breeders in the wild; repro cycle linked to variations of water levels & flooding -- Rising water level -> plasma testosterone increase -> spermatogenesis -- Estradiol doesn’t vary and progesterone remain high across different repro stages -- However, tend to breed all year round under managed care resulting in surplus -- Males can be aggressive and inflict severe and chronic wounds to females or other males - Castration risky because gonad is embedded in the epigonal organ and cannot be removed - Chemical contraception efficiency does not seem to be reliable in females - Maturation of spermatozoa in epididymis -> stored in a motile state in the seminal vesicles -- Alkaline glands & seminal vesicles have openings into cloaca via the urogenital papilla -- During ejaculation, spermatozoa in seminal vesicles and alkaline gland content gush from the cloaca and mix with clasper glands content - Many roles have been hypothesized for clasper glands secretions: -- Facilitating semen coagulation -- Enhancing storage viable sperm in the female oviducal gland after mating -- Completing the capacitation phase - Elasmobranchs lack a direct vascular connection from the hypothalamus to the pituitary gland -- Thus, GnRH is likely released into the general circulation -- Two gonadotropins, structurally similar to LH & FSH, are synthetized in the pituitary gland and are assumed to play similar regulatory role in elasmobranchs - Vertebrates possess up to 15 GnRH isoforms classified into three distinct paralogous lineages: -- GnRH1 gene = most jawed vertebrates, neurohormone stimulating gonadotropin release. -- GnRH2 gene = almost all vertebrates, structure conserved between species -- GnRH3 gene = reported in fish, including several elasmobranchs species -- GnRH2, GnRH3 are thought to be neuromodulatory factors, function not fully understood - GnRH vaccine -> stimulates anti-GnRH antibody production in mammals -> neutralizing endogenous GnRH -> FSH & LH inhibited -> suppressing testosterone and spermatogenesis -- Previously never been used in fish - Deslorelin = GnRH agonist -- Stimulates repro system before downregulation of HPG axis -- Trialed unsuccessfully in a male smooth stingray and male zebra shark -- Shown to modify plasma testosterone and aggressiveness in sand tiger sharks Key Points: - Male P. motoro reach sexual maturity at a smaller disc width than females - Plasma testosterone concentrations in two intact animals were stable over 1 yr -- Compatible with nonseasonal breeding suspected in freshwater stingrays managed under human care, as water levels and temperature remained identical whatever the season - Deslorelin clearly stimulated HPG axis, but a negative feedback did not occur -- A testosterone peak occurred between 9-16 d after implantation in three animals -- The flare-up effect was delayed compared w/mammals, but carried on for > 1 yr -- A deleterious effect of sustained elevated testosterone should be considered - No difference in testosterone levels between intact animals and those given GnRH vaccine -- Possible dose was not high enough to induce immunity against GnRH -- Possible vaccine is not immunogenic in potamotrygonids, regardless of the dosage -- Alternativley, an appropriate vaccinal reaction could happen, neutralizing GnRH1, whereas GnRH2 or GnRH3 could be neurohormones that take over from GnRH1 - Despite a Deslorelin-induced increase in plasma testosterone, there was no significant change in testis height and seminal vesicles diameter after treatment -- Seasonal sperm production is associated with drastic morphologic changes in the reproductive tract of Potamotrygonidae -- An increase in gonadosomatic indices and a depletion of hepatosomatic values are observed in active males TLDR: - GnRH vaccine and Deslorelin were ineffective for chemical castration of male freshwater stingrays. - Their efficacy on decreasing aggression could not be demonstrated. - Deslorelin stimulated the hypothalamic-pituitary-gonadal axis in all implanted animals, without any negative feedback