Non‐invasive Monitoring of Hormones: A Tool to Improve Reproduction in Captive Breeding of the Eurasian Lynx

Abstract

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the world's most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Histological and endocrine characterisation of the annual luteal activity in Eurasian lynx (Lynx lynx)

“…Interestingly, the highest oestrogen concentrations were found in CLs of pregnant lynxes, signalling an involvement of this steroid in the maintenance of pregnancy. Non-invasive monitoring of steroids in urine and faecal samples of Eurasian and Iberian lynxes corroborates the special role of oestrogens during pregnancy in lynxes; showing elevated profiles of urinary and faecal oestrogen metabolites in pregnant animals, which were correlated to progestin metabolites (Dehnhard et al 2008). In contrast to P 4 , elevated oestrogens were not reflected in serum oestradiol (E 2 ) levels (Gö ritz et al 2009).…”

“…Duplicates of the extracts (20 and 10 ml respectively) were tested for both P 4 and oestrogens using in-house 96 well microtitre plate enzyme immunoassays previously validated for the Eurasian lynx (Dehnhard et al 2008). P 4 analyses were carried out as described earlier ) using a commercial P 4 antibody (Sigma P1922, raised in rats) and 4-pregnen-3,20-dione-3-CMO-peroxidase label.…”

Fecal Progestagens to Detect and Monitor Pregnancy in Captive Female Cheetahs (Acinonyx jubatus)

“…A previous study in domestic cats described that progesterone was excreted primarily as conjugated metabolites and unconjugated pregnanolone epimers (primarily 5β-pregnan-3α/β-ol-one) and that progesterone is not detected [15]. Fecal progesterone metabolites in the female Eurasian lynx also contained 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione [8]. Progesterone has not been usually detected in the feces of wild felid species, such as the tiger (Panthera tigris), lion (P. leo) [16], leopard cat (Felis bengalensis), clouded leopard (Neofelis nebulosa) and snow leopard (Uncia uncia) [15].…”

“…However, information about the types of fecal hormone metabolites among the reproductive stages is limited. It has been reported that in the Eurasian lynx (Lynx lynx), the excretion pattern of fecal progestagen metabolites is different among pregnancy stages [8]. Understanding the excretion pattern, such as the components of fecal hormone metabolites, among reproductive stages is important for validating the assay method used for monitoring the stages.…”

Using PGFM (13,14-dihydro-15-keto-prostaglandin F2α) as a non-invasive pregnancy marker for felids

“…In contrast to other felids, steroid-based monitoring of ovarian luteal function is impossible using fecal and urinary progestagen metabolites, therefore a reliable pregnancy diagnosis method has never before been developed [8,25]. The Witness relaxin pregnancy test [13] has been used for diagnostic purposes using urine samples collected between Days 26 and 46 from pregnant Iberian lynx [10].…”