Hindle, Joanne Elizabeth; (1991) Reproductive assessment in female African rhinoceroses by urinary steroid analysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Measurement of urinary pregnanediol glucuronide (PdG) and oestrone conjugates provides a reliable method of monitoring ovarian function and pregnancy in the Indian rhinoceros, but application is limited in the African species. The possibility of species differences in the metabolism and excretion of ovarian steroids was investigated by a metabolism study involving the i.v. injection of 100ccl each of 14C-labelled progesterone and oestradiol-17beta into an adult female white rhinoceros. Of the radiolabel injected, significant amounts were recovered in both urine (25%) and faeces (36%) collected over the following 4 days. Almost all (92%) of the label recovered in urine was accounted for in the Day 2 sample, of which 41% steroids were present in the conjugated form. 20a-Dihydroprogesterone (20a-DHP) was the only conjugated progesterone metabolite in urine with which radioactivity was associated and no radioactivity could be found to co-elute with pregnanediol after high performance liquid chromatography (HPLC) separation of conjugated neutral steroids. Whereas progesterone metabolites predominated in urine, isomers of oestradiol were the most abundant steroids in faecal extracts. The metabolism and excretion of endogenous ovarian steroids in African rhinoceroses was then examined by gas chromatography/mass spectrometry (GC/MS) and HPLC in conjunction with differential hydrolysis techniques. GC/MS identified pregnanediol as the major progesterone metabolite in the Indian rhinoceros, but neither PdG nor 20a-DHP was detected in urine from African rhinoceroses during any stage of the reproductive cycle. However, HPLC confirmed the presence of conjugated 20a-DHP and the absence of PdG in postoestrus urine. Oestrone-gIucuronide was the major urinary oestrogen in the black, whilst oestradiol-17-glucuronide predominated in the white rhinoceros. A sensitive enzyme immunoassay was developed for the measurement of 20a-DHP. This was initially validated by measurement of 20a-DHP in hydrolysed rhinoceros urine, which showed parallel diIution-response curves with the standards, and subsequently by cochromatography on HPLC. In hydrolysed urine from the black rhinoceros (9 cycles, 4 animals) levels of 20a-DHP and oestrone, indexed to creatinine (Cr), followed a cyclic pattern which correlated well with behavioural events. Levels of 20a-DHP were low for 3 days prior to mating during which oestrone levels rose to reach a peak. This was followed by a rapid rise in 20a-DHP to reach maximum levels (7*64+3*82 ng/mgCr) within 2 days. The pattern of 20a-DHP excretion in the white rhinoceros (6 cycles, 3 animals) differed in that the presumed follicular phase was longer than in the black species (12-17 d), and maximum levels of 20a-DHP (80-90 ng/mgCr) were not reached until approximately 7 days after peak oestradiol-17beta excretion at the time of oestrus. A mean cycle length of 22 days was recorded for the black rhinoceros whilst cycle lengths of 26 and 32 days were obtained for the northern and southern white respectively. In contrast to the ovarian cycle, PdG was present in late pregnancy urine from African rhinoceroses. Elevated levels were recorded during early pregnancy in the black rhinoceros, whilst concentrations did not increase for 5-6 months in the white species. Urinary oestrogen levels also rose throughout gestation. A gestation period of approximately 16 months was recorded for both species.

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