Reproduction in Domestic Animals

76 M Dehnhard, S Naidenko, A Frank, B Braun, F Go¨ ritz and K JewgenowRadiometabolism studyTo identify relevant progesterone metabolites, whichreflect the corpus luteum (CL) activity, a radiometabolismstudy was performed: for that purpose a solution(0.25 ml) containing 250 lCi [ 3 H] progesterone(70–110 Ci ⁄ mmol, TRK413; Amersham Bioscience,Freiburg, Germany) in ethanol was used. Sterile 0.9%NaCl solution (2.25 ml) was added to the radiolabelledsolution and the total volume was injected into thecephalic vein of a 15-year-old female lynx. Prior toinjection, the animal was sedated by an i.m. injectionwith a mixture of 3 ml of Rometar (2% solution ofXylazine hydrochloride) and 1 ml of 10% Ketaminehydrochloride (both from Spofa, Prague, Czech Republic)corresponding to a dosage 2.4 and 4 mg ⁄ kg bodyweight for Xylazine and Ketamine, respectively.Following radiolabel injection, all voided faecal sampleswere collected separately for 4 days in plastic bagsfrom the cage and the floor of the enclosure immediatelyafter defecation and stored at )20°C. Aliquots of eachsample were extracted for progesterone and oestrogendetermination and for radioactivity counting. The secondsample, collected on day 2 after injection, containedthe highest amount of radioactivity (89%) and it wasused for HPLC analyses. All radioactive counting wasconducted in a Perkin Elmer MicroBeta Trilux counter(Perkin Elmer, Rodgau, Germany).Tritiated steroids are usually of very low radiotoxicitybecause of very low energy beta emission. The specificradio activity of injected 3 H-steroids, however, is quitehigh, so the additional mass of these radioactivehormones will not substantially increase the totalconcentration of hormone in the body. Furthermore,the amount of radiolabel used for this study is far belowmaximum permitted level. Nevertheless, in Germanyradiometabolism studies in animals require a priorpermission of the local Animal Experiment EthicalCommittee.HPLC analysis of metabolitesFor separation and characterization of faecal steroidmetabolites, 50 ll portions of faecal extracts were used.For gestagen metabolite analysis a reversed-phase UltrasepES100 ⁄ RP – 18 ⁄ 6 lm HPLC column (4 · 250 mm;Sepserv, Berlin, Germany) was used. Metabolites wereseparated with a methanol + water mixture (78 + 22)at a flow rate of 1 ml ⁄ min. Fractions of 0.33 ml werecollected at 20 s intervals and diluted with one volumeof water, before 20 ll of the fractions were added intothe assay systems. The elution positions of authenticprogesterone (4-pregnen-3,20-dione; P4), 5a-pregnan-3,20-dione (DHP), 5a-pregnan-3b-ol-20-one (5a-P), 5bpregnan-3a,20a-diol(pregnanediol, PD) on this columnhad been previously determined in separate HPLC runs.For faecal oestrogen metabolite separation an AllureBiphenyl 5 lm HPLC column (3.2 · 150 mm; Restek,Bad Homburg, Germany) was used. Metabolites wereseparated with a acetonitrile + water mixture (43 + 57)at a flow rate of 1 ml ⁄ min. Fractions were collected andadded to the assay as described above. The elutionpositions of authentic 1,3,5(10)-oestratrien-3,17-one(oestrone), 1,3,5(10)-oestratrien-3,17a-diol (17a-oestradiol),and 1,3,5(10)-oestratrien-3,17b-diol (17b-oestradiol)on this column had been determined in separateHPLC runs after their injection.ProgesteroneProgesterone (P4) analyses were carried out with anin-house microtitre plate enzyme immunoassay asdescribed earlier (Go¨ ritz et al. 2001) using a commercialP4 antibody (Sigma P1922, raised in rats to progesterone)and 4-pregnen-3,20-dione-3-CMO-peroxidaselabel. The cross-reactivities to progestins were asfollows: 4-pregnen-3,20-dione (progesterone), 100%;5a-pregnan-3,20-dione, 31%; 5a-pregnan-3b-ol-20-one,18%; 5-pregnen-3b-ol-20-one, 12%; 4-pregnen-3aol-20-one,4.2%; 0.05). Intra- and inter-assay coefficients of variationfor two biological samples with low and highconcentrations were 5.0 and 5.1% (n = 10) and 13.7 and22.8% (n = 10), respectively.OestrogensFaecal oestrogen analyses were carried out also with anin-house microtitre plate enzyme immunoassay using apolyclonal antibody raised in rabbits to 1,3,5(10)-oestratrien-3,17b-diol-17-HS-BSA and 1,3,5(10)-oestratrien-3,17b-diol-17-HS-peroxidaselabel (Meyer et al.1997). The cross-reactivities to oestrogens were asfollows: 1,3,5(10)-oestratrien-3,17b-diol (17b-oestradiol)100%, 1,3,5(10)-oestratrien-3,17-one (oestrone) 100%,1,3,5(10)-oestratrien-3,17a-diol (17a-oestradiol) 66%,1,3,5(10)-oestratrien-3,16a,17b-triol (oestriol) 1.5%, and 0.05).Intra- and inter-assay coefficients of variation for twobiological samples with low and high concentrationswere 5.8 and 12.3% (n = 10) and 17.0 and 9.7% (n =11), respectively.Results and DiscussionMonitoring of faecal steroid hormonesWe analysed faecal samples of pregnant (n = 15),pseudo-pregnant (n = 7) female Eurasian lynxes duringa 3-year study period. Figure 1a,b shows the faecalprogesterone and oestrogen metabolite profiles in apregnant and a pseudopregnant female. There is atendency towards higher gestagen and oestrogen metaboliteconcentration during pregnancy. However, nodistinct difference between profiles from the pregnant(a) and the pseudo-pregnant (b) female were obtained.In addition, both steroid metabolites showed a postpartumincrease with no difference between the pregnantand the pseudo-pregnant female. Surprisingly a highlyÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag