Reproduction in Domestic Animals

80 M Dehnhard, S Naidenko, A Frank, B Braun, F Go¨ ritz and K JewgenowBiological validationBiological validations are aimed to demonstrate thathormone metabolite measurements reflect the physiologicalevent in question. One possibility to validate anon-invasive hormone assay is to compare and correlatefaecal metabolite levels with hormone levels in blood(Heistermann et al. 1993; Barrett et al. 2002; Capezzutoet al. 2008). Even if blood sampling contradicts the noninvasiveapproach, veterinary check-ups and treatmentsshould be used to collect blood and fresh faecal samplessimultaneously.Appropriate techniques to physiologically validatenon-invasive methods are pharmacological stimulationsor inhibitions of steroid hormone release. These methodstypically involve the administration of high doses ofreleasing hormones such as GnRH to stimulate theproduction of gonadal sex steroids (Kretzschmar et al.2004) and adrenocorticotrophic hormone to stimulatethe adrenal gland to produce corticosteroids (Wasseret al. 2000).A biological validation can also be based on dataanalysis. The measured hormone pattern in the lynx(Fig. 1) should mirror the reproductive events of afemale. The predicted hormone pattern (derived fromother related felid species) should include an oestradiolpeak around mating, elevated progesterone levels duringpregnancy, followed by a decrease towards basal after4 weeks in pseudopregnant females and a more or lessimmediate decrease to baseline prior to parturition inpregnant females.Our results, however, revealed differences from thesepredicted patterns: oestrogens did not reflect follicularactivity peaking around ovulation (mating), but werestrongly correlated to the excretion of gestagens.Therefore, we assume that faecal oestradiol immunoreactivityreflect the activity of corpora lutea. Theprogesterone profiles of Eurasian lynxes were also notin accordance to typical felid hormone patterns. Wefound elevated progesterone (and oestradiol) metabolitelevels throughout pregnancy and thereafter. However,the composition of hormone metabolites after parturitionwas different from those during pregnancy. Particularlythe relation between the progesterone metaboliteseluting between fractions 7–9 and 13–14 differed markedlyduring pregnancy and the postpartum (p.p.) period(Fig. 2b–d). Comparing the oestrogen profiles from thedifferent reproductive phases (Fig. 3a–c) the relation ofconjugates vs the fractions including the unpolarmetabolites remained relatively constant whereas therelation between 17b-oestradiol and oestrone changeddramatically in the p.p. period in favour of oestrone(Fig. 3c).This might be contributed by different hormonesources during and after pregnancy (CL, placenta, oradrenals). To confirm this, an additional validation forCL function had to be performed. In case of theEurasian lynx, we performed a transrectal ultrasoundinvestigation by which we found corpora lutea. This is inagreement with the above mentioned high p.p. progesteronevalues, altogether supporting the hypothesis of ap.p. luteal activity. At present it is uncertain whether theCL are the result from retained CL of pregnancy orfrom a p.p. ovulation. In conclusion there is still noanalytical parameter available in faeces which can beused to monitor luteal activity in the lynx. Onealternative might be an extensive metabolite screeningusing LC-MS to detect suitable pregnancy markers inthe lynx. Then, these specific markers can be measuredcombining the selectivity of high pressure liquid chromatographywith mass spectrometric detection, in orderto be able to distinguish between pregnancy andpseudopregnancy.Currently the only pregnancy-specific method is thetransabdominal ultrasonographic or radiographic imagingof the uterus (Davidson et al. 1986), but thistechnique usually requires handling and anesthesia ofthe female, potentially stressing both the queen anddeveloping offspring.A second option are proteohormones produced by theplacenta, like relaxin. The cat placenta produces largequantities of relaxin, beginning approximately 20 daysof gestation (Addiego et al. 1987). As with dogs, relaxinhas not been detected in the serum of cycling orpseudopregnant cats (Stewart and Stabenfeldt 1985).However, the development of a urine-based relaxinpregnancy test would prove extremely useful for breedingmanagement of wildlife species. A radioimmuneassay (RIA) for relaxin was recently validated for domescat urine. Urinary relaxin was first detected betweendays 14 and 21 of gestation, whereas the levels peaked at42–49 days, followed by a decline during the last2 weeks prior to parturition (de Haas van Dorsser et al.2006). A similar urinary relaxin profile was demonstratedin the leopard (de Haas van Dorsser et al. 2006).These results indicate that measurement of urinaryrelaxin might turn out to be a reliable method forpregnancy determination in felids from as early as 3–4 weeks of gestation. First results analysing urinaryrelaxin in the Iberian lynx showed that relaxin is anappropriate analyte to differentiate between pregnantand pseudopregnant females particularly during thesecond half of pregnancy (BC Braun et al., unpublisheddata). Urinary relaxin-based pregnancy diagnosis mayprove useful in the breeding management of other felidspecies, and provides a foundation for future studies onpregnancy in captive exotic felids.Perhaps one of the greatest uses of steroid metabolitemonitoring will be in assisting reproductive managementby identification of ovarian cycle, oestrous, andpregnancy of females. Early pregnancy diagnosis, however,is complicated by the phenomenon of pseudopregnacnyin carnivoes. Particularly in felids, steroidanalyses did not provide an early pregnancy-specificdiagnosis. In the lynx, it is even impossible to use faecalP4 and estrogen metabolite analyses as an index ofpregnancy. However, there are evidences that theplacenta might contribute to steroid biosynthesis.Therefore, LC-MS based metabolite screenings comparingpregnant and pseudopregnant females offer anoption to identify pregnancy specific placental steroids.Their identification might allow the development of aspecific assay whose cross-species applicability had to beproved for practice in other felid species.Alternatively, sensitive urinary relaxin assays mightbe used to confirm pregnancy in the lynx. For theÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag