PRESENTATIONS

SESSION X

REPRODUCTION II

Oral imipramine and intravenous xylazine for pharmacologically-induced

ex copula ejaculation in an African elephant (Loxodonta africana)

Ray L Ball, DVM and Chris Massaro, Tampa’s Lowry Park Zoo

Semen collection-EEJ

Semen collection

• passive semen collection technique yields only stored sperm in a very low volume of seminal plasma

Improvement on manual collection?

• Repeatable

• Without anesthesia

• Potentially dangerous animal

• Labor/training effort

• Fresh ejaculate

• Pharmacologically-induced ex copula ejaculation in equids

Pharmacologically-induced ex copula ejaculation in equids

• stallions with limb weakness or pain that precludes safe mounting or ground collection

• injury of the penis

• inadequate libido

• failure of unknown etiology

Pharmacology of ejaculation

• Erection, emission, and ejaculation

– sympathetic nervous system

– alpha-adrenergic stimulation and smooth muscle contraction.

• Emission is thought to be primarily an a1 event

• Ejaculation being primarily mediated through a2 stimulation

Pharmacologically-induced ex copula ejaculation in equids

• Oral or intravenous imipramine

• Alpha-2 agonist

– Detomidine or xylazine

• prostaglandin F2a (PGF2a)

– 0.01–0.15 mg/kg

– smooth muscle stimulation

• Combinations of oral or intravenous imipramine with intravenous alpha-2 agonist.

Pharmacologically-induced ex copula ejaculation in equids

Imipramine

• tricyclic antidepressant – inhibits re up-take of several neurotransmitters,

including dopamine, norepinephrine, and serotonin

– alpha-adrenergic stimulation

• used in human for treatment of – aspermia

– premature ejaculation

– retro grade ejaculation

Xylazine

• Alpha 2 agonist sedative and muscle relaxant

• reversible

• affect contractions of genital smooth muscle in the horse

– and had been associated with the occasional side effect of induced-ejaculation

– Xylazine alone will induce ejaculation ~27% of treated horses

Imipramine and Xylazine

• xylazine alone typically has volume, sperm concentration, pH and total sperm number similar to those of ejaculates obtained in copula

• Treatment with imipramine appears to enhance contraction of the ampulla, and inhibit contraction of the accessory sex glands

– with the resulting ejaculates of lower volume, higher sperm concentration, higher total number of sperm, and lower pH than for in copula ejaculates

Imipramine and Xylazine

• imipramine at 3mg/kg orally

• two hours later xylazine 0.66mg/kg intravenously

• Ejaculate produced: – 1–3 min after xylazine

– 15 and 25 min after arousing from sedation

• titration of dose to individual stallions significantly improves the rate of ejaculation for any given treatment

Imipramine and Xylazine

• rate of successful ejaculation is affected by the level of arousal (calm or excited) of the stallion at the time of treatment

– with higher success in quiet and undisturbed stallions

• Teasing stallions before imipramine also reported useful

Imipramine/Xylazine in African elephant

• 23 year old

• 4223kg

Trial dosing

• 12500mg imipramine (2.95mg/kg) po

• followed by 100mg xylazine (0.024mg/kg) IV two hours later.

• very light tranquilization with his penis protruded

– 20 minutes the elephant moved out of the chute and recovered uneventfully.

– Later a few drops of fluid were reported to be seen dripping from his penis but these were not recovered.

Additional Trials

• 4 trials over 6 days

• imipramine ranging from 12500 to 20500 (2.95 – 4.85mg/kg)

• 170mg (0.04mg/kg) xylazine two hours later IV

• Held in ERD 20-30 minutes

• Moved out and monitored

• Each trial he head pressed and bellowed

– Ejaculation??

Results

• Varied

– Few drops with sperm to small AI dose

• Best results were ~30 minutes after recovery

• Light rectal massage

• 4ml of thick sperm rich coagulum

– Diluted with Hepes for evaluation

– Diluted to 12ml for AI attempt

TLPZ Experience

• Exhausted bull

– Horse attempts every 3 days

– TLPZ: 4 attempts in 6 days

• Teasing

– Best sample was when female had just ovulated

• Timing

– 30 minutes after recovery from sedation

• Titrating dose

Ejaculation

• Head pressing and bellowing – Behaviorally

suggestive of ejaculation

– Sample lost in long repro tract?

– US exam of ampulla subjectively shows size change

Next steps

• Prep bull

• Teasing

• Rectal massage before

– Timing of collection post-xylazine

• Move out

• Second rectal massage

– Catheterize while sedated after ejaculation?

– More complete US exams

Acknowledgements

TLPZ Elephant Team TLPZ Veterinary Staff

Dr. Trevor Gerlach Dr. Nico Maldonado

Sperm Cryopreservation and Seminal Plasma Analyses in Asian Elephants (Elephas maximus) Authors Wendy K. KisoA,B, Atsushi AsanoC, Alexander J. TravisC, Dennis L. SchmittA,D, Janine L. BrownE and Budhan S. PukazhenthiE

Author Affiliations A The Ringling Bros. Center for Elephant Conservation, Polk City, FL 33868, USA. B Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC 20008, USA. C Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. D The William H. Darr School of Agriculture, Missouri State University, Springfield, MO 65897, USA. E Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA. This presentation summarized key note findings from two recent manuscripts: Kiso WK, Asano A, Travis AJ, Schmitt DL, Brown JL, Pukazhenthi BS. Pretreatment of Asian

elephant (Elephas maximus) spermatozoa with cholesterol-loaded cyclodextrins and glycerol addition at 4°C improves cryosurvival. Reprod Fertil Dev 2012; 24:1134-1142.

Asian elephant spermatozoa are sensitive to chilling and do not respond well to cryopreservation. The objectives of the present study were to: (1) determine whether cholesterol content can be modified by preincubation of Asian elephant spermatozoa with cholesterol-loaded cyclodextrin (CLC); and (2) assess the effects of CLC concentration(s), temperature at time of glycerol addition (22C vs 4C) and dilution medium on post-thaw sperm survival. Spermatozoa incubated with 1.5 mg CLC exhibited increased (P < 0.05) cholesterol concentrations. Pretreatment of spermatozoa with 1.5 mg CLC resulted in improvements (P < 0.05) in all post-thaw parameters. Glycerol addition at 4C also improved all post-thaw parameters compared with 22C. Dilution of thawed spermatozoa in an egg yolk-based medium improved (P < 0.05) motility compared with Ham’s F-10 culture medium. In summary, our findings indicate that modifying cholesterol content within the plasma membrane improves the cryosurvival of Asian elephant spermatozoa. The development of an improved cryopreservation method that includes modification of membrane cholesterol and the addition of glycerol at 4C, as reported in the present study, is an important step towards utilization of cryopreserved spermatozoa in captive management of this species. As a result of this study, a Genome Resource Bank has been established for Asian elephants at the Ringling Bros. Center for Elephant Conservation. Kiso WK, Selvaraj V, Nagashima J, Asano A, Brown JL, Schmitt DL, Lesyzk J, Travis AJ,

Pukazhenthi BS. Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality. 2013; 8:e71033.

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein

profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility ( 65%) expressed higher percentages of spermatozoa with normal morphology (80.3 13.0 vs. 44.9 30.8%) and positive Spermac staining (51.9 14.5 vs. 7.5 14.4%), in addition to higher total volume (135.1 89.6 vs. 88.8 73.1 ml) and lower sperm concentration (473.0 511.2 vs. 1313.8 764.7 x 106 cells ml-1) compared to ejaculates exhibiting poor sperm motility ( 10%; P < 0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ~80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P < 0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.