Refinement of a Commercial Bench-Top Relaxin Assay for Pregnancy Diagnosis Using Urine from Domestic and Non-Domestic Felids
Relaxin, a 6 kDa polypeptide hormone, is excreted in the urine during pregnancy in several mammalian species.1,4 A recent study showed that detection of urinary relaxin using a bench-top serum assay (Witness® Relaxin kit, Synbiotics Corporation, San Diego, CA 92127 USA) can be diagnostic for pregnancy in domestic cats (Felis silvestris catus).2 In non-domestic felids, current methods used to diagnose pregnancy are invasive and/or nonspecific; however, with the exception of the Arabian leopard, (Panthera pardus), it is unknown if relaxin is present in measurable amounts in urine of most cat species during pregnancy or if the bench-top relaxin kit is applicable with urine across species.3 The objectives of this study were to 1) examine modifications in urine sample processing to improve the reliability of the relaxin kit in pregnant cats, 2) evaluate the impact of concentrating urine via filtration on relaxin detection, 3) assess the effect of sample freezing on relaxin concentrations, and 4) begin quantifying relaxin levels in urine of pregnant non-domestic felids. Urine and/or serum samples were collected from pregnant and nonpregnant domestic cats and non-domestic cat species (Pallas’ cat [Otocolobus manul], sand cat [Felis margarita], cheetah [Acinonyx jubatus], and lion [Panthera leo]) at several time points after breeding. Urine samples, subjected to various processing methods, were tested using the bench-top kit, and relaxin levels were later quantified via radioimmunoassay (RIA).3 For domestic cat urine samples, filtration and/or the addition of protein and phosphate buffer (or nonpregnant cat serum) improved the consistency of the relaxin kit for early pregnancy diagnosis. Urine freezing caused a slight (~13%) but significant (p<0.05) decrease in relaxin concentrations; however, all frozen/thawed urine samples from pregnant females still tested positive with the bench-top kit. In non-domestic felids, urinary relaxin concentrations in most pregnant females were variable but peak values were comparable to that of pregnant domestic cats, suggesting that relaxin is a reliable cross-species marker of pregnancy. Urinary relaxin was detectable using the bench-top kit in one non-domestic species, the Pallas’ cat, but multiple urine samples from other felid species tested negative on the kit assay, regardless of sample processing methods. These findings suggest that measurement of urinary relaxin is a promising approach as a non-invasive, non-stressful method of pregnancy diagnosis in exotic felids. Further assessment of urinary relaxin profiles among cat species and refinement of the bench-top relaxin kit are warranted to improve cross-species utility and provide zoos with an invaluable in-house tool for diagnosing pregnancy in their non-domestic felids.
The authors thank Helen Bateman and Dr. Genevieve Magarey for support with laboratory procedures and the animal keeper staff at the Cincinnati Zoo & Botanical Garden and the Denver Zoological Gardens for providing urine samples from non-domestic cats. We also would like to thank Dr. Florine de Haas van Dorsser for sharing information and advice on using the bench-top relaxin kit.
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2. de Haas van Dorsser, F.J., S. Lasano, and B.G. Steinetz. 2007. Pregnancy diagnosis in cats using a rapid, bench-top kit to detect relaxin in urine. Reprod. Domest. Anim. 42:111–112.
3. de Haas van Dorsser, F.J., W.F. Swanson, S. Lasano, and B.G. Steinetz. 2006. Development, validation, and application of a urinary relaxin radioimmunoassay for the diagnosis and monitoring of pregnancy in felids. Biol. Reprod. 74:1090–1095.
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