Non-invasive monitoring of reproduction in Asian elephants ('Elephas maximus') by urinary endocrine analysis

The development of an enzymeimmunoassay for 5β 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 5β- pregnane-3α, 17α, 20α/β triols as the two most abundant urinary progesterone metabolites during pregnancy and the oestrous cycle. The assay developed utilized the antiserum anti-β-pregnane-17α, 20α-diol-3α-yl glucuronide-carboxy- methyloxime-BSA and 4-pregnene-17α, 20α-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, <0.01) and 17α 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 (P₄), 17α hydroxyprogesterone (17α OHP₄) 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 17α hydroxyprogesterone were significantly elevated from early and mid pregnancy respectively, until parturition (p<0.001, n=496, p<0.001, n=221). There was a significant change in the plasma 17α 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 EIA 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

Prolonged luteal lifespan and pseudopregnancy in Asian elephants (Elephas maximus)

Pseudopregnancy is a physiological occurrence in mammals which have copulation induced ovulation, but is rarely described in spontaneous ovulating species. In this study, three cases of prolonged luteal lifespan are reported in non-pregnant Asian elephants (Elephas maximus). Case 1 was a 25-year-old female that had produced three calves previously; Case 2 was a nulliparous and 32-year-old at the start of the pseudopregnancy episode; and Case 3 occurred in a 49-year-old nulliparous elephant. Serum progesterone metabolite concentrations remained elevated for 10 months in Case 1. Urinary progestagens were high for >16 months in Case 2 and for five months in Case 3. In Case 1, multiple persistent corpora lutea were visualized monthly by ultrasonography. In all three cases, uterine leiomyoma were present and progestagen concentrations decreased spontaneously. In Case 1, the elephant became pregnant 3 years later, whilst with Case 2, the female resumed estrous cycling normally, and for the Case 3 female, there was continuation with another prolonged luteal phase before ovarian function was purposely suppressed. These examples indicate that persistently elevated progestagen concentrations may not always be indicative of pregnancy in elephants. The reasons for prolonged luteal lifespan are not understood, although serum prolactin concentrations quantified in the Case 1 female were elevated compared to values from previous reports and two other herd mates. Furthermore, all three elephants had varying degrees of uterine pathologies. It is believed that the resulting damage to the endometrium may have led to a reaction similar to implantation, which includes prolactin secretion. Prolactin may exert luteotropic properties and is thought to initiate luteal rescue during pregnancy in elephants.Japan Society for the Promotion of Science (JSPS) for their support of this research which allowed sample analysis in Tokyo, Japan for prolactin (grant # PE 11016).http://www.elsevier.com/locate/anireprosci2019-10-01hj2018Anatomy and Physiolog