C. A. Niemuller, H. J. Shaw and J. K. Hodges

Non-invasive monitoring of ovarian function in Asian elephants (Elephas maximus) by measurement of urinary 5\g=b\-pregnanetriol C. A. Niemuller, H. J. Shaw and J. K. Hodges ^Institute of Zoology, Zoological Society of London, Regent's Park, London, NWl 4RY, UK, and 2Division of Reproductive Biology, German Primate Centre, Kelinerweg 4, D-3400, Göttingen, Germany The development of an enzymeimmunoassay for 5\g=b\-pregnanetrioland its use for non\x=req-\ invasive monitoring of reproductive cycles in Asian elephants is described. Gas chromatography\p=n-\massspectrometry (GCMS) and high performance liquid chromatography (HPLC) confirmed the presence of 5\g=b\-pregnane-3\g=a\,17\g=a\,20\g=a\/\g=b\-triolsas the two most abundant urinary progesterone metabolites. The assay developed used the antiserum anti\x=req-\ 5\g=b\-pregnane-17\g=a\,20\g=a\-diol-3\g=a\-\g=g\lglucuronide but was designed to measure the free steroid in urine samples after hydrolysis and extraction. HPLC confirmed the presence of immunoreactive pregnanetriol in urine, but indicated that the measurement was nonspecific. Immunoreactive pregnanetriol concentrations were significantly correlated with the concentrations of both progesterone (r = 0.98, n = 269, P < 0.01) and 17\g=a\-hydroxyprogesterone (r = 0.95, n = 205, P < 0.01), the metabolic precursor of pregnanetriol. The mean \m=+-\SEM deviation of cycles as determined by measurements of plasma progesterone, 17\g=a\\x=req-\ hydroxyprogesterone and urinary pregnanetriol, respectively, were 15.54 \m=+-\1.5 (n = 23, where n = number of cycles), 15.21 \m=+-\1.7 (n = 15) and 15.45 \m=+-\0.94 weeks (n = 20). These results demonstrate that it is possible to monitor ovarian function in Asian elephants by the measurement of urinary immunoreactive pregnanetriol concentrations. Introduction Lothrop, 1990; Brown et al, 1991; Taya et al, 1991; Gross et al, 1991). Measurement of concentrations of The two of existing today (Elephas maximus circulating progesterone species elephant therefore the most reliable monitor of ovulatory cycles and Loxodonta africana) are classified as endangered (CITES, provides in and as such are widely used in many 1992), with current estimated wild populations of 50 000 and elephants zoological collections. Unfortunately, the on ovarian 500 000, For both species, habitat loss and dependence assessing respectively. function hormone has been a hindrance for have been major factors to the decline by plasma analysis poaching contributing those collections in which collection of blood and of the wild At the same time, zoological fragmentation populations. is not and cannot be used in studies monitor¬ zoos and reserves have become active in samples possible private increasingly the dynamics of wild animals. There is there¬ the development of management and breeding programmes ing reproductive fore a need to a non-invasive for endocrine for animals in the establishment of develop approach captivity, although assessment in On the basis of our conditions conducive to successful is often reproductive elephants. reproduction limited of ovarian this should difficult. knowledge elephant physiology, be most feasible the measurement of the last 10 hormonal methods for by urinary progesterone During years, monitoring metabolites. ovarian cycles in both Asian and African elephants have been There are no reports of the identity of urinary metabolites of described. Typically, the duration of an ovarian cycle is 14—16 in A recent attempt to monitor weeks and is characterized by an 8—10 week luteal phase and a progesterone elephants. ovarian function by measuring unmetabolized progesterone in 4-6 week interluteal or follicular phase, according to patterns of urine from Asiatic was unsuccessful and concentrations of (Hess et al, 1983; elephants (Mainka circulating progesterone 1990). measurement of Brannian et al, 1988; Plotka et al, 1988; Brown et al, 1991; Taya Lothrop, Although urinary pregnanediol or immunoreactivity has et al, 1991). Concentrations of oestradiol and of LH have also glucuronide 20ct-dihydroprogesterone valuable information on ovarian function in a variety been measured in but concentrations are generally very provided plasma, of mammalian (see, for Loskutoff et al, 1986; low or results inconsistent between studies, and their secretion species example, et al, 1990; Hindle et al, 1992; 1992 has not proved useful for indicating ovarian status (Hess et al, Kirkpatrick Hodges, for review), our own observations (J. Hindle and 1983; Brannian et al, 1988; Plotka et al, 1988; Mainka and unpublished C. Niemuller) indicated that this did not hold true for Asian the were therefore to 'Present address: RR 1 Cambridge, Ontario, N1R 5S2, Canada. elephants. The aims of present study Received II February 1993. (i) identify the major urinary progesterone metabolite(s) in Downloaded from Bioscientifica.com at 09/27/2021 11:34:10AM via free access Asian elephants (ii) develop a microtitre plate enzymeimmuno- separated into three distinct fractions with Sephadex LH-20 assay for its determination and (iii) evaluate its use as a non- (Pharmacia, St Albans, Herts.). The fractions were dried down, invasive method for monitoring reproduction in this species. reconstituted in ethanol and derivatized with methoxyamine hydrochloride and trimethylsilylimadazole. The final sample was reconstituted in 500 pi cyclohexane of which 1 pi was Materials and Methods injected into the gas chromatography mass spectrometer. Mass spectrometer (MS) profiles thus obtained were scanned Animals and sample collection and identification of unknown peaks attempted initially by retention times and by computer MS library search (Shackleton et Where possible, matched blood and urine samples were al, 1980). Peaks from gas chromatography (GC) were compared collected once a week for 1—3 years from 11 mature female with reference templates and re-run through the GC with straight elephants aged 12-25 years from four zoological collections. A chain alkanes to determine méthylène units for further identifi¬ 5-10 ml blood sample was collected from either the saphenous cation. This was followed wherever possible by coinjection of vein or an ear vein into heparinized tubes, centrifuged at 2000 g the unknown peaks with steroid reference standards to deter¬ for 10 min immediately after collection, and the plasma stored mine change in peak height and provide further confirmation of at 20°C. Mid-stream urine samples were collected during the the identity of the steroid metabolite. morning,— divided into 5 ml aliquots and stored frozen at -20°C. Oestrous behaviour in the eight females that had access to Preparation of [3H]pregnanetriol males was recorded by the elephant keepers on the basis of As were not commercially available, a increased interest by the bull and eventual with the [3H]pregnanetriols copulation [ H]-labelled form of one of the isomers was in our females. Three of the females monitored had no contact with prepared the reduction of males the duration of the study. Of the laboratory by enzymatic [3H]17a-hydroxy-5ß- throughout eight (Amersham, with 3a- females that had access to males, three were with the pregnane-3a,20a-diol Buckinghamshire) kept bull(s) and NADH (method but at The five cows had hydroxysteroid dehydrogenase provided daily separated night. remaining by A. P. Scott; Ministry of Agriculture, Fisheries and Food continuous access to a bull except during the winter months. (MAFF), Fisheries Laboratory, Lowestoft). The reagents were During this time, access to the male was restricted to daylight incubated for 2 h at room temperature, and the products ether hours and only for a cow in the latter half of the follicu¬ working extracted before thin layer Preliminary identi¬ lar as by analysis of As chromatography. phase predicted plasma progesterone. fication of as the soon as concentrations of to rise, 5ß-pregnane-3a,17a,20a-triol predominant plasma progesterone began labelled was confirmed by recrystallization of the major the cow had no further contact with the bull. product radioactive peak and pure standard to constant specific activity (SA). The tracer thus prepared had an activity of 8.8 pCi ml-1 Creatinine determination and was subsequently used to indicate steroid retention time on HPLC. All urine samples were analysed for creatinine concentration after the initial thawing by the method of Hodges and Green (1989) and as previously validated for elephants by Ramsey et HPLC al (1981) and Poole et al (1984), to correct for variations in Unconjugated neutral steroids were separated using HPLC. glomerular filtration rate of urine. The assay sensitivity was Sample preparation was done to the method of approximately 0.1 mg ml-1 and the intra- and interassay coef¬ according Hindle et al (1992). Urine (1 ml) were adjusted to 5 ficients of variation were 9.3 and 8.8%, (« = 30). samples pH respectively with buffer and hydrolysed with 1000 FU Urinary creatinine concentration ranged from below detection hydrolysis overnight (50 pi)-1 of hydrolysis enzyme (Sigma, sulfatase activity: (samples excluded) to 2 mg ml~\ with a mean of approximately 4500 U ml-1, 100 000 U ml-1). Since 0.6 mg ml^1. ß-glucuronidase activity: conjugated pregnanetriol was not available, efficiency of hydrolysis was monitored by the addition of [3H]pregnanediol ([3H]PdG) to urine monitored Gas chromatography mass spectrometry glucuronide pooled samples sep¬ arately. Samples were adjusted to pH = 7 with 3 mol NaOH 1~J Ten urine samples (each 20 ml) from three pregnant

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