NON-INVASIVE MONITORING OF REPRODUCTION IN ASIAN ELEPHANTS (Eleohas maximus) BY URINARY ENDOCRINE ANALYSIS A Thesis Presented to The Faculty of Graduate Studies of University College London by CHERYL NffiMULLER In partial fulfillment of requirements for the degree of Doctor of Philosphy January, 1994 ® Cheryl Niemuller, 1994 ProQuest Number: 10016751 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10016751 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT NON-INVASIVE MONITORING OF REPRODUCTION IN ASIAN ELEPHANTS (Eleohas maximiis) BY URINARY ENDOCRINE ANALYSIS Cheryl Niemuller Supervisors: University College London Dr. H.J. Shaw 1994 Prof. J.K. Hodges The development of an enzymeimmunoassay for 5j8 pregnanetriol and its use for non-invasive monitoring of reproductive cycles and pregnancy in Asian elephants is described. Gas chromatography mass spectrometry (GCMS) and high performance liquid chromatography (HPLC) confirmed the presence of 5/3- pregnane-3a,17a,20a/i3 triols as the two most abundant urinary progesterone metabolites during pregnancy and the oestrous cycle. The assay developed utilized the antiserum anti-5)3-pregnane-17a,20a-diol-3a-yl glucuronide-carboxy- methyloxime-BSA and 4-pregnene-17a,20a-diol-3-one-HRP as the enzyme label. HPLC confirmed the presence of immunoreactive pregnanetriol in the urine but showed the measurement to be non-specific. Immunoreactive pregnanetriol concentrations were significantly correlated with the levels of both progesterone (r=0.98, n=269, p<0.01) and 17a hydroxy progesterone (r=0.95, n=205, p<0.01) the metabolic precursor of pregnanetriol throughout the ovarian cycle. The mean ± sem of cycle lengths as determined by measurements of plasma progesterone (P 4 ), 17a hydroxyprogesterone (17a OHP 4 ) and urinary pregnanetriol, respectively were 15.54 ±1.5 (n=23, where n=number of cycles), 15.21 ± 1.7 (n=15) and 15.45 ± 0.94 weeks (n=20). Mean concentrations of urinary pregnanetriol throughout pregnancy were not significantly greater than luteal phase values (457 ± 56.7 vs 357.9 ± 17.8). There was a cessation in ovarian cyclicity with levels remaining consistently elevated until 1 - 6 weeks prior to parturition, with the exception of a short decline occurring on average during weeks 6-9 of early pregnancy. Concentrations of both plasma progesterone and 17a hydroxyprogesterone were significantly elevated from early and mid pregnancy respectively, until parturition (p<0.001, n=496, p<0.(X)l, n=221). There was a significant change in the plasma 17a OHP4:P4 ratio between weeks 2-7 of gestation from greater than 0.7 to less than 0.7 as compared with non-conceptive cycles (p<0.05, N=5). This change in the ratio represents the earliest means to-date of determining pregnancy in this species. HPLC also confirmed the presence of oestrone as the major urinary oestrogen metabolite throughout pregnancy and the ovarian cycle. Comparision between hydrolysed and extracted urine samples prior to analysis in an oestrone RIA with samples assayed directly in an oestrone conjugate El A provided comparable results, thereby simplifying analyses. However, despite simplification of the oestrone assay, the data generated from both weekly and daily periovulatory samples were not consistent with the notable exception of the conceptive cycles. In this instance, 4/5 elephants demonstrated a rise in oestrone conjugate excretion the week prior to the rise in progesterone and presumed ovulation. In contrast, urinary concentrations of oestrone conjugate were significantly elevated over luteal phase levels from midpregnancy (week 30) onwards (p< =0.001, n=243), only declining to baseline concentrations after parturition. Thus, measurement of urinary oestrone conjugate may provide a useful, non- invasive method for determining pregnancy in elephants that are not monitored routinely. The overall length of gestation in the females monitored throughout their entire pregnancy was 95.2 ± 2.9 weeks (n=4). A 43% incidence of breech births was observed in this study, as was the rare phenomenon of twins and two spontaneous abortions. The offspring survivability was extremely poor at 47%. Finally, a conception rate of 17.6% was documented. These results demonstrate that it is possible to monitor reproduction in Asian elephants non-invasively by the measurement of urinary immunoreactive pregnanetriol and oestrone conjugate concentrations. These techniques will aid in the establishment and management of effective captive breeding programmes and have potential application to studying and monitoring the reproductive physiology of free-ranging elephants. ACKNOWLEDGEMENTS My supervisors Dr. H. J. Shaw and Prof. J.K. Hodges provided guidance and encouragement throughout the course of this thesis. I am grateful to Martin Smith, Bill Bliss and the keepers of Port Lympne Zoo, UK for their dedication and perseverance in collecting samples. Their willingness to share with me their love for their "charges” made the research much more personal. I am indebted to Brian Karmen and his staff of elephant keepers from the London Zoo, UK who renewed their faith in science thereby allowing me access to their elephants in order to study them. I am also thankful to Christine Dean, Jude Hewlett and Suzanne Jackson from the Veterinary department, who kindly collected blood samples for me. Simen van Dijk and his team of elephant keepers from the Noorder Dierenpark Zoo, Holland provided a wonderful European collaboration. His zoo carries the proud distinction of being the first place where an impending elephant parturition (with unknown conception date) was determined by urinary pregnanetriol analysis. I was pleased to be able to include former collaborators Don Moore and Chuck Doyle of Burnet Park Zoo, USA in this study. Their patience and understanding of the slow wheels of research did not impede their generosity in providing samples, for which I am truly grateful. My long standing partnership in elephant research with Charlie Gray and his dedicated crew from the African Lion Safari, Canada was able to continue, despite the formidable presence of the Atlantic. His skill and handling of the elephants as well as his knowledge regarding all things "elephant" are of a rare and wonderful ability. Drs. John Honour and Monica Schneider generously provided me with detailed instruction on the use of the GCMS as well as precious lab space to carry out my analysis. Drs. Jo Kindle and Robert Abayasekara provided invaluable advice on the intricacies of setting up and running HPLC. Glynne Williams kindly assisted me with the TLC and radiochromatogram scanning as well as ably sorting out various disasters on the HPLC. To my British compatriots Tessa Smith, Alison Paterson, Chris Faulkes and Helen Stanley: thanks for the wine and laughter. You all made life easier for this lonely foreigner. To my loyal supporter Daphne Green: you were a continuing source of companionship and encouragement in the lab which I will never forget. To my two special mentors. Prof. R.V. Short and Prof. R.M. Liptrap: I have thrived off our discussions, the more eclectic the better. Thank you for opening up the doors of science and teaching me to think past boundaries, see the imagined and truly believe. I cannot begin to thank my parents for their unanimous support, encouragement and love which has been constant throughout my University life. Without you, this work would not have been completed. Thank you for helping me to realise a dream. Finally, I owe a great debt of gratitude to Jonathan and our beautiful baby girl Kate, who both put it all into perspective and made the sacrifices all worthwhile. I shall never be able to express adequately in words my thanks to you. Ten years of elephant reproduction is more than anyone should have to listen to, especially when you prefer cows of a bovine nature. This study would not have been possible without the kind and generous support of Mr. John Aspinall of Howlett’s and Port Lympne Zoo’s Ltd., UK and Mr. Trevor Poole from the University Federation of Animal Welfare, UK. DECLARATION OF WORK PERFORMED I declare that with the exception of the items listed below, all work reported in this thesis was carried out by myself. Martin Smith and Bill Bliss from Port Lympne Zoo faithfully collected weekly urine, faecal and plasma samples from six elephants for five years. Charlie Gray, Jody Watkins and Mark Matassa from the African Lion Safari collected weekly plasma samples from four elephants for three years as well as daily urine and plasma samples during three periovulatory periods in two elephants. Chuck Doyle of Burnet Park Zoo, collected weekly plasma samples from two elephants for three years with matching urine samples for the last year and a half. Brian Harmen and either Christine Dean, Jude Howlett or Suzanne Jackson from London Zoo, carried out routine weekly collections of urine and plasma from four elephants for one year as well as daily sample collection during the perivovulatory period for one elephant. Simen van Dijk from the Noorderdierenpark Zoo, meticulously carried out weekly urine sample collections for eight months on three elephants. Daphne Green performed the final oestrone conjugate and pregnanetriol enzymeimmunoassays for two pregnant elephants, as well as analyzing the plasma periovulatory samples for oestrone. Sheila Boddy performed the oestradiol radioimmunoassay for analysis of the plasma and urine periovulatory samples.