 Reference Base  Faecal progesterone metabolite analysis for non-invasive ... |
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Schwarzenberger, F.; Walzer, C.; Tomasova, K.; Vahala, J.; Meister, J.; Goodrowe, K.L.; Zima, J.; Strauss, G.; Lynch, M., 1998. Faecal progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white rhinoceros (Ceratotherium simum). Animal Reproduction Science 53: 173-190
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| Location: |
Captive |
| Subject: |
Reproduction |
| Species: |
White Rhino |
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Behavioural observations of captive white rhino indicated oestrous cycle lengths ranging from 30-90 days, whereas Owen Smith reported an oestrous cycle length of about 30 days for animals in South Africa. Wagner (1986) said 42 days. Another study using urinary steroid analysis indicated the oestrous cycle was 25 and 32 in lengths for northern and southern subspecies respectively. Finally, Radcliffe et al. 1997 using combined ultrasonography and faecal progesterone metabolite monitoring in one white rhino showed an initial period of ca. 3 months in which there was some ovarian activity, but no ovulation, followed by 2 non-conceptive cycles (31-35 days in length) and 2 conceptive cycles which ended in early embryonic death after day 28. The conceptive periods were 73-78 days in length, and the luteal structures persisted 42-48 days.
This study hoped to validate group-specific enzyme-immuno-assays for measuring faecal immunoreactive pregnanes containing group 20-oxo-P. - IA = enzymeimmunoassay; HPLC = high performance liquid chromatography; FP = folicular phase; LP = luteal phase of reproductive cycle.
Results
HPLC separation of faecal 20-oxo-pregnanes. Elotion profiles between FP and LP samples were comparable, qualitativley, but not quantitatively. During the LP, four major immuno-reactive 20-oxo-P peaks were detected, one of which coeluted with [3H] progesterone. Comparison with corresponding reference steroids together with the different cross-reactivities in the two EIAs indicated that the faecal immunoreactive peaks not coeluting with [3H] progesterone are the following 5 -reduced pregnanes: 5 -pregnane-3,20-dione, 5 -pregnane-3 -ol-20-one and 5 -pregnane-3 -ol-20-one, which eluted in fractions 7-8, 21-23 and 25-27, respectively.
Faecal 20-oxo-P profiles. Concentrations of faecal 20-oxo-P were similar between the two antibodies tested; therefore, only results of the EIA using the antibody against 5 -pregnane-3 -ol-20-one 3HS:BSA are shown. The concentration of 20-oxo-P within the faeces did not differ significantly between the outer layer and the central portion of the faecal balls. Therefore, mean values for the respective days were calculated and used in Fig. 3.
Oestrous cycle characteristics and LP 20-oxo-P concentrations varied among and, to a certain extent, within animals. In general, white rhinoceroses could be classified into four categories on the basis of oestrous cycle length and LP 20-oxo-P concentrations:
(1) females with regular, ca. 10 weeks oestrous cycles and high luteal phase 20-oxo-P concentrations (> 800 ng/g of faeces);
(2) females with oestrous cycles ranging in length from 4-10 weeks with luteal phase 20-oxo-P concentrations of 250-750 ng/g of faeces;
(3) females with no apparent oestrous cycle regularity, but some luteal activity (20-oxo-P values 100-200 ng/g faeces); and
(4) females exhibiting no luteal activity (20-oxo-P values <100 ng/g faeces).
Only 2 of 21 animals exhibited category 1 ovarian activity, although one of those animals also exhibited category 2 activity during part of the study. That female had been moved from the Munich to the Salzburg Zoo in the summer of 1991. Samples collected in October 1991 indicated an initial short, category 2 cycle followed by 10-week category 1 cycles until May 1994 (except for one shorter cycle in September/October 1993). In June, July and August 1994, this female displayed three cycles of approximately 1 month duration (30, 26 and 31 days, respectively) before re-establishing 10-week cycles. After September 1994, LP 20-oxo-P values appear to decline, and, although concentrations were at the higher end of category 2, oestrous cycle activity in this female was categorised as 2 since June 1994. The duration of category 1 cycles (n = 10; December 1991-May 1994, except the period of February-June 1992, where no samples were available) was calculated to be 68.5 + 3.5 days., the length of the FP and LP were 12.4 + 0.9 and 55.9 ? 3.2 days, respectively. The category 2 cycles (September 1994-October 1996 except January, February 1995; n = 1 1) averaged 68.9 + 3.3. 14.6 + 2.0 and 53.5 + 2.3 days for the cycle length, the FP and the LP, respectively. Oestrus behaviour was observed primarily at the end of the LP.
The female in Fig. 4 also displayed category 1-type oestrous cycles. Because faecal samples from this animal were not collected as regularly as those from other females, an estimated cycle length of 70 days was determined by dividing the number of luteal phases (n = 5) into the total number of days monitored (beginning of October 1994 until mid-September 1995).
Examples of category 2 oestrous cycles are shown in Fig. 5. The oestrous cycle length varied within individual animals (4-> 10 weeks) and results often differed between consecutive years in the same animal. LP and FP lengths varied between 5- > 70 (n = 31) and 5- > 39 days (n = 30), respectively. When the LPs was categorised as being < 35 days (n = 21) or > 45 days (n = 6), the mean values were 16.8 ? 2.0 and 58.7 + 4.8 days, respectively. Mean FP length for category 2 females (n = 26) was 16.7 + 1.7 days.
Representative category 3 animals, are shown in Fig. 6, and category 4 females are depicted in Fig. 7. The animal Nabire (Fig. 7) exhibited two category 2 luteal phases in 1994 and was mated during this period, but did not conceive. Concentration of faecal 20-oxo-P in one pregnant animal during the 4th and 5th months of gestation were 5046 ? 844 (n = 7) and 6432 + 662 (n = 11) ng/g of faeces, respectively, and were considerably higher than those observed during the non-pregnant luteal phase.
Oestrous cycle induction. Faecal 20-oxo-P concentrations were low before and during each CMA treatment (Fig. 8); CMA metabolises did not affect steroid measurements. Faecal 20-oxo-P concentrations then increased approximately 10 days after hCG injection, resulting in a LP of 18 and 17 days, respectively. In response to the first treatment, 2 days of oestrous behaviour were observed 70 days after hCG injection.
Discussion
The objective of this study was to characterise long-term ovarian activity using faecal
steroid analysis to try and resolve the conflicts associated with determining the oestrous cycle length in the white rhinoceros. Still, despite the continuous monitoring of some females for over 4 years, results indicate that it may be difficult to make generalisations with respect to 'normal' cycle length. Although transition between categories was flowing and some females did at certain periods fall into different groups, animals could be classified in four major categories. On the basis of regularity of interovulatory intervals (category 1 vs. category 2 animals), the 'normal' oestrous cycle in the white rhinoceros appears to be 10 weeks in length, which is considerably longer than the 25 days observed in the black and Sumatran rhinoceros, and the 45 days reported for the Indian rhinoceros. Our finding of an oestrous cycle length of 70 days contrasts with recent studies in white rhinoceroses which suggested an oestrous cycle lengths of 1 months. However, these studies were based on only one or two cycles per female, and there were several examples in our study where similar results of an ca. 1 month cycle length would have been suggested, if we had only collected for 1-2 months.
Only two of the 21 white rhinoceroses investigated exhibited ovarian cycles characterised by high luteal phase 20-oxo-P values and regular 10-week interovulatory intervals over periods of more than 1 year. Ovarian activity in category 2 animals alternated between 4 and 10 week cycles, and LP 20-oxo-P concentrations were considerably (5-10 fold) lower than those of category 1 females. These LP concentration differences between categories are likely caused by differences in progesterone production. Sample collection site within the faecal ball did not significantly effect results and differences in faecal water content, as well as food fibre content within physiological ranges are too minimal to explain a 5-10 fold concentration difference between categories. There was also no age effect with respect to categorising of ovarian activity and season also was not a factor because at least two females cycled year round.
Defining the 70 day cycles in category 1 animals as resulting from persistent corpora lutea is not an adequate explanation because these interovulatory intervals were too regular. Persistent corpus luteum activity in mares without uterine anomalies or infections is characterised by highly variable LP lengths ranging between 35 and 95 days. Conversely, the presence of an inflammatory uterine infection generally reduces progesterone concentrations during dioestrous and causes early regression of the corpus luteum. Evidence of intrauterine fluid accumulation has been identified using ultrasound twice during early embryonic resorption in one 33-year old white rhinoceros. The two conceptions observed by these authors were characterised by interovulatory intervals of ca. 11-12 weeks. Early embryonic death in both pregnancies occurred 28 days post ovulation, but faecal progestagens were elevated for ca. 44 days. This study demonstrates that category 2 animals can conceive, whereas the fertility potential of category 1 females remains to be determined.
In the study of Radcliffe et al. (1997) the first 3 months of evaluation were characterised by short luteal phases with low faecal progestagen concentrations and no ovulations. Instead, the ovary contained a large follicle with luminal fibrous bands, which disappeared gradually analogous to an equine haemorrhagic follicle. In another study, a persistent follicular cyst-like structure was monitored by rectal ultrasonography over a 34 day observation period in one white rhinoceros (Adams et al., 1991). Again, no ovulation occurred and no corpus luteum was observed. These findings suggest that category 3 and 4 animals could have cystic structures that may or may not become luteinized. Results of these and the present studies also suggest that anovulation may be a major problem in the captive white rhinoceros population. Animals in categories 1 and 2 were apparently the only animals in which ovulation occurred regularly, whereas nearly two-thirds of the remaining animals (category 3 and 4 animals) did not ovulate.
Although our treatment regimen using chlormadinone acetate followed by an hCG injection failed to induce continuous cyclicity, results indicate that ovulation and subsequent corpus luteum activity can be induced in white rhinoceroses. The LP 20~oxo-P concentrations increased ca. 10 days after hCG injection. These findings corroborate the ultrasound findings during natural ovulation, which was detected 7-9 days before a substantial rise in faecal progestagens. Because ovulation of a preovulatory follicle usually occurs within 48 h after hCG injection, these two days plus the 7-9 days for the development of a steroidogenically functional corpus luteum add up to the 10 days observed in our study. Also comparable to Radcliffe et al. (1997) mounting by a bull or mating in several occasions in our study occurred ca. 10 days before the next LP.
Elution profiles of immunoreactive 20-oxo-P were comparable to those of pregnant black rhinoceroses, excluding a small peak coeluting with [3Hlprogesterone. The finding of different immunoreactive 5 .-pregnanes agrees with studies on faecal metabolises in several other mammalian species, but contrasts with a study by Hindle and Hodges (1990) who administered radiolabelled progesterone to a white rhinoceros and found only one radioactive peak of in the faeces which coeluted with progesterone after HPLC-separation. In contrast to faecal samples in black rhinoceroses, where pregnanes containing a 20-oxo-group or a 2Oct-OH-group seem to be equally important, faecal pregnanes containing a 20-oxo-group dominate over those containing a 200t-OH-group in the white rhinoceros (Schwarzenberger, unpublished observations). This finding is also supported by concentration differences in faecal samples of both species. Luteal phase 20-oxo-P concentrations in faeces of the white rhinoceros exceed 1000 ng/g, compared to ca. 500 ng/g in faeces of the black rhinoceros.
Our preliminary results of the faecal progestagen concentration in the one pregnant white rhinoceros suggested that foetoplacental progesterone metabolite production can he used for pregnancy diagnosis as in other rhinoceros species. Gestation length in the white rhinoceros is ca. 15 months and the faecal 20-oxo-P values during the 4th and 5th month of pregnancy were already considerably higher than luteal phase concentrations. Hodges and Green (1989) also reported high urinary pregnanediol concentrations during the second half of gestation in a white rhinoceros.
In conclusion, on the basis of differences in oestrous cycle length and LP 20-oxo-P concentrations among animals, white rhinoceroses could be classified into four categories. Animals in category 1 and 2 appeared to ovulate, resulting in either regular oestrous cycles of 10 weeks in duration (n = 2) or variable cycles of 4-10 weeks in length (n = 6). In contrast, faecal 20-oxo-P values in almost two thirds (n = 13) of the white rhinoceroses in this study indicated erratic or missing luteal activity, apparently attributable to anovulation.
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