Preservation, extraction and analytical methods for faecal progesterone metabolite in cows and immune response to pregnanolone hemisuccinate in rabbits
Monitoring reproductive function using faecal progesterone metabolite evaluations is a well-established technique in gazelles, horses, baboon, pigs, rhinoceros, elephants and should be extensively applied in domestic animals. Some factors however, prevent the application of this non-invasive...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/84076/1/FPV%202019%2022%20-%20ir.pdf |
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| Summary: | Monitoring reproductive function using faecal progesterone metabolite evaluations is
a well-established technique in gazelles, horses, baboon, pigs, rhinoceros, elephants and should be
extensively applied in domestic animals. Some factors however, prevent the application of this
non-invasive technique to monitor reproduction in animals. These factors include
paucity of practical information on stability of faecal progesterone metabolites
under different preservation and extraction methods as well as sensitivity and specificity of
analytical techniques. Therefore, general objective of this study was to evaluate
preservation, extraction and analytical techniques for progesterone metabolite in faeces
of cows; determine plasma progesterone and progesterone metabolite profiles in pregnant and
non-pregnant cows; modify existing high performance liquid chromatography (HPLC) method for
steroid hormones for the simultaneous detection of multiple progesterone metabolites in faeces of
cows and to determine the immune responses and cellular changes following administration of low
dose of pregnanolone hemisuccinate in rabbits. Progesterone metabolites were extracted from
faecal samples using already established procedures and their concentrations together
with plasma progesterone levels were determined using radioimmunoassay. A mobile phase
made of distilled water and acetonitrile (70:30 v/v) and a stationary phase made up of C18 column
coupled to an Agilent HPLC 1100 series module was modified from existing HPLC method of steroid
hormones to detect progesterone metabolites in faeces of cows. This modified HPLC method
was hypothesized to be able to serve as an alternative to use of immunoassay on the
analysis of progesterone metabolite in animals. Male rabbits were parenterally
administered with pregnanolone hemisuccinate on day 0 and were given a booster dose on day 14
so as to produce group specific antibody to progesterone metabolites. Antibody response was
determined using IgG and IgM evaluations using ELISA. The results show that there is no
statistically significant difference in progesterone metabolite concentration in faeces that
were dried at 70°C (9.33±5.05 ng/g), 50°C (5.66±4.19 ng /g) or at 90°C (5.29±2.51 ng/g). Similarly, radioimmunoassay
technique was used to determine the stability of progesterone metabolites in oven dried and fresh
faecal samples. Faecal progesterone metabolite concentrations were found to be higher when
extracted from oven-dried samples (2.06±1.58 ng /g) as compared to extraction from fresh faecal
samples (0.49±0.30 ng /g). This difference was found to be statistically significantly (P<0.05).
When un-preserved faeces were left exposed to environmental conditions for several days,
progesterone metabolite concentrations therein were observed to rise from 11.04±7.68 ng/g at 0
hrs to maximum value of 31.71±10.33 ng /g at 48 hrs and thereafter decline to 36.62±12.30 ng/g
after 216 hrs. There was no statistically significant difference (P>0.05) among these
values. The relationship between faecal progesterone metabolites concentration and plasma
progesterone concentration in cows were also investigated in this study. The results showed a
correlation between plasma progesterone and faecal progesterone metabolites in pregnant
(r=0.211, n=8, p=0.23) and in non-pregnant cows (r=0.209, n=8, p=0.35). A High-Performance
Liquid Chromatography (HPLC) method for steroid hormones commonly employed in other species of
animals was modified and used in this study for cows using 5β-pregnan-3α-ol-20-one and
3β-Hydroxy-5α- pregnan-20-one sulphate pyridine salt as internal standards. UV detection
was employed to monitor the eluent from a stationary phase at an excitation wavelength of 254 nm
and an emission wavelength of 360 nm. The linearity of the calibration curve was obtained in the
concentration range of 500-7500 µg/µL for 5β-pregnan-3α-ol-20- one and 3-15 µg/µL for
3β-Hydroxy-5α-pregnan-20-one sulphate pyridine salt respectively. The co-efficient of
determination was found to be 0.9977 for 5β-pregnan- 3α-ol-20-one and 0.9776 for
3β-Hydroxy-5α-pregnan-20-one sulphate pyridine salt. The production of group specific antibodies
for progesterone metabolites using the immunogen in rabbits was not achieved in this study. This
could have been due to minute amount of the immunogen used or a defective bioconjugation method or
due to inherent molecular properties of 5β-pregnan-3α-ol-20-one. Histopathological,
haematological and biochemical parameters did not significantly differ between control and
immunised rabbits in this study. This study provides practical and reliable information on faecal
progesterone metabolite levels exposed to different storage, preservation and extraction
parameters in cows. Best results for the analysis of faecal progesterone metabolites in cows are
obtained when faeces are dried at 70°C and are extracted soon after voiding. The correlation
observed between faecal progesterone metabolites and plasma progesterone concentrations shows that
faecal progesterone metabolites concentration can be used for determination of reproductive
status in cows. The HPLC method described in this study is simple, practical and rapid and can be
applied for determination of progesterone metabolite concentration. It is therefore recommended
that the information provided by this study be utilised in the non- invasive study of
reproductive function in cows. These techniques are simple, reliable and can be performed within a
short time. Group specific antibodies were not produced using
5β-pregnan-3α-ol-20-one-hemisuccinate, it is recommended that a modification in the
bioconjugation method used or immunisation procedure be performed. Furthermore, other
progesterone metabolites could be used to produce group specific antibodies and thereafter used
in immunoassays to produce a more specific and
sensitive analysis. |
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