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Hodges, J.K.; Hindle, J.E., 1988. Comparative aspects of steroid metabolism in rhinoceroses: implications for reproductive assessment: pp. 83-91, figs. 1-5

In: Dresser, B. et al. Proceedings of the 5th Internatioinal Conference on breeding endangered species in captivity. Cincinnati, Zoo and Botanical Garden: pp. i-xii, 1-722

Location: World
Subject: Reproduction - Management methods
Species: All Rhino Species

Original text on this topic:
Steroid metabolism in different species. The reasons for the poor breeding record in captivity are unclear, but undoubtedly one of the main contributing factors is the lack of understanding of the reproductive biology of rhinoceros and of the factors required for successful reproduction. Virtually nothing is known about the reproductive physiology of these species and yet the application of artificial breeding techniques, such as artificial insemination and embryo transfer which may well become a necessary part of captive management of rhinoceros in the near future, is critically dependent on such knowledge and on accurate and reliable methods for assessing reproductive status. In recent years, the development of improved methods for urinary hormone analysis has been extremely valuable in providing a practical and non-invasive approach to monitoring reproductive function in exotic mammals. Assays for urinary oestrone conjugates and pregnanediol glucuronide (PdG) are now well established and have been used successfully in the detection of ovulation and pregnancy in a wide range of species (see Lasley, 1985; Hodges, 1985; Hodges and Green, 1989 for references). Application of these techniques to reproductive assessment in rhinoceroses, however, has so far met with limited success (Kasman et al, 1986; Ramsay et al 1987).
The profile of conjugated oestrone and PdG excretion during the oestrous cycle in the Indian rhinoceros is shown in Figure 1. Here, the data derived from a study by Kasman et al (1986), clearly show a marked increase in conjugated oestrone prior to ovulation, with elevated levels of immunoreactive PdG characterising the luteal or post-ovulatory period. Unfortunately, a similar pattern of steroid excretion cannot be demonstrated in the African rhinoceroses and methods currently available for the measurement of conjugated oestrone and PdG have failed to provide useful information on ovarian function in both the Black and the White rhinoceros. Typical data for these species (Figures 2 and 3) show that levels of both urinary metabolises are much lower than those found in the Indian (and in many cases undetectable) with little evidence of a cyclical pattern of excretion or correlation with reproductive events. The existence of species differences in steroid hormone metabolism and route of excretion and the possibility that they may account for these observations has therefore been investigated.
Radiolabel Injection Study
A metabolism study was carried out in an adult female Southern White rhinoceros by administering 14C-labelled oestradiol and progesterone into the peripheral circulation. All faeces and urine excreted were collected separately over a four day period and the distribution of radioactivity determined. Bot h urinary (25%) and faecal (36%) routes of excretion contributed to the 61% of the administered label recovered. Of the label recovered in the urine, 92% was accounted for in the Day 2 sample, roughly half (43%) associated with metabol ites in the conjugated form and hal (49%) with those present as free steroids. Progestagenic (neutral) and oestrogenic (phenolic) steroids in each fraction were separated (Brown, 1955) and subjected to thin layer and high performance liquid chromatography (HPLC) in order to identify the inetabolites present.
Chromatographic analysis of the neutral fraction revealed a single peak of radioactivity suggesting 20*-hydroxyprogesterone to be the only conjugated progesterone metabolise present (Figure 4). The absence of radioactivity associated with the pregnanediol standard was notable. Two peaks of radioactivity were seen in the phenolic phase and although oestrone appeared to be an abundant metabolise, the form of the original conjugate is not known. The presence of oestradiol-17 is of interest since it has not previously been demonstrated in this species and although, in this sample, appears less abundant than oestrone, its measurement may provide a better indication of ovarian function. Together these results confim the existence of species differences in steroid metabolism and in particular provide an explanation for the failure to monitor ovarian status in African rhinoceroses by measurement of urinary PdG. It should however be remembered that our findings relate specifically to the White rhinoceros and it is not yet known whether they are applicable to the Black. The results also need to be confirmed by quantitative measurement of these metabolises during the ovarian cycle although, until the required assay validation is achieved, the uncertainty of whether such cycles are ovulatory remains a problem.
Progesterone Metabolites During Pregnancy
In contrast to the lack of success in monitoring ovarian function, measurement of PdG immunoreactivity appears to be informative in indicating pregnancy in African as well as Indian rhinoceroses. Levels of immunoreactive PdG during mid to late pregnancy in the Indian and both African species of rhinoceros are shown in Figure 5. All three profiles describe increased PdG immunoreactivity associated with pregnancy and a rapid fall in levels at the end of the gestation period. Absolute values for PdG immunoreactivity however vary considerably between species with levels in the two African species being 20- 100 fold lower than in the Indian. Values for the Indian rhinoceros are similar to those reported in a previous study by Kasman et al (1986). Both these and our own data (Hodges and Green, 1989) indicate a slow increase in PdG during early pregnancy with values becoming consistently higher than those in the luteal phase by approximately four months post-breeding. The limited data shown here for the African species do not indicate the onset of increased PdG excretion but Ramsay et al (1987), who reported elevated but highly variable levels of PdG imunoreactivity during late pregnancy in six Black rhinoceroses, have suggested that it occurs as late as 6-8 months of gestation.
Although the presence of PdG has been confirmed in late pregnancy, urine of both the Indian (Hindle et al, 1988) and Black (Ramsay et al, 1987) rhinoceroses using gas chromatography/ mass spectrometry (GC/MS) and HPLC analysis respectively, species differences in qualitative aspects of progesterone metabolism during pregnancy may exist. Thus, in contrast to the Indian rhinoceros in which PdG is the major progesterone metabolise, preliminary GC/MS analysis of urine from Black and White rhinoceroses suggests that metabolises other than PdG may be quantitatively more important and therefore more informative as a method of pregnancy detection. Nevertheless, data shown in Figure 5 clearly indicate that elevated levels of PdG immunoreactivity reflect the presence of a conceptus in all three species of rhinoceros and as such, its measurement provides the basis for a simple, non-invasive test for the mid-late stages of pregnancy.
Summary and Conclusions
This paper examines comparative aspects of steroid metabolism in rhinoceroses in relation to the application of urinary hormone analysis as a non-invasive method of reproductive assessment. Assays currently available for the measurement of conjugated oestrone and PdG have been successfully applied to the detection of ovulation and pregnancy in the Indian rhinoceros but, so far, have met with limited success in the African species. Results presented here provide evidence for species differences in the metabolism of oestrogen and progesterone which may account for these findings. 20a-hydroxyprogesterone and not PdG has been identified as the major metabolise of progesterone during the ovarian cycle in the White rhinoceros and possibly (by inference) also in the Black. Oestradiol-17a was detected as an abundant metabolise of oestrogen and may provide a more informative assessment of ovarian function than previously possible by the measurement of conjugated oestrone. PdG was detectable during late pregnancy in both African species of rhinoceros although other metabolises may be more abundant. Subject to the confirmation of these findings, the development of assays for the measurement of metabolises other than conjugated oestrone and PdG may provide new opportunities for monitoring reproductive function in African species of rhinoceros.

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