FECAL METABOLITES OF INFUSED C-14 PROGESTERONE IN DOMESTIC LIVESTOCK

Non-invasive monitoring of the reproductive function in animals is pos sible by measuring steroid metabolites in the faeces. After infusion o f C-14-steroids, differences between species concerning both the perce ntage and the delay of faecal excretion can be observed. In domestic l ivestock almost all faecal C-14-metabolites are found in an unconjugat ed form. The aim of this study was to characterize the main metabolite s present in faecal samples of sheep, ponies and pigs after infusion o f C-14-progesterone and to evaluate different extraction procedures. T his should help to standardize and improve faecal steroid analysis. Fa eces containing peak radioactivity were suspended in methanol/water an d Sep-Pak(R) C-18 cartridges combined with Sephadex(R) LH-20 columns w ere applied to clean up the samples. Fractions were subjected to strai ght phase high pressure liquid chromatography (HPLC) and thin layer ch romatography (TLC). Metabolites were classified by comparison with the elution patterns of steroid reference standards and by testing their immunoreactivity in different enzyme-immunoassays (EIAs). The results revealed considerable differences in the pattern of faecal progesteron e metabolites between species. A great number of metabolites (mainly m ono- and di-hydroxylated pregnanes as well as pregnanediones) was foun d and progesterone barely, if at all. In sheep, pregnanes containing a 20 alpha-hydroxy (OH) group dominated (about 45%), whereas in ponies metabolites with a 20 beta-OH group (about 44%; mainly one 5 alpha-pre gnanediol), and in pigs progestagens with a 20-oxo group (46-58%) pred ominated. Suspending the faeces with a high percentage of methanol, et hanol or iso-propanol yielded the best recoveries and enabled a practi cable extraction, without an evaporation step. As a wide range of meta bolites was formed, group-specific immunoassays using antibodies with high cross-reactions with pregnanes sharing a similar C-20 group shoul d be used for measuring faecal metabolites. To verify the data of the C-14-experiments, various progesterone metabolites were measured in fa ecal samples of superovulated ewes. In accordance with the results of the characterized progesterone metabolites, an EIA measuring 20 alpha- OH-pregnanes proved most suitable for non-invasive monitoring of the c orpus luteum function in sheep.