Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (<i>Acinonyx jubatus</i>)

Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify ‘biomarkers’ and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.在典型的动物园圈养种群中，约80%的猎豹（cheetah）始终无法成功繁育后代。在超过60%的繁育案例中，雌性猎豹虽被证实已排卵，但最终未能成功分娩。目前尚不清楚这些孕酮（progesterone）水平升高的非妊娠间期（即黄体期，luteal phase）究竟是受孕失败，还是妊娠过程中出现了胚胎或胎儿丢失。近年来，借助质谱法（mass spectrometry）识别特定生理状态（包括妊娠）相关生物标志物（biomarker）与调控机制的代谢组学分析（metabolic profiling）与蛋白质组学分析（proteome analysis）技术，在众多物种中已取得进展。本研究假设，猎豹排泄粪便中的蛋白质表达水平会随妊娠状态发生变化。为此我们开展了两项研究：1）明确雌性猎豹粪便中的全面蛋白质组图谱；2）筛选出可能参与早期妊娠建立与诊断的候选蛋白。本研究从妊娠组（n=8）、非妊娠黄体期组（n=5）以及非排卵对照组（n=5）的猎豹粪便样本中，共鉴定出570种独特蛋白质。与非妊娠组或对照组相比，妊娠组粪便样本中有4种候选蛋白显著上调（up-regulated），2种候选蛋白显著下调（down-regulated）。其中一种上调候选蛋白——免疫球蛋白J链（immunoglobulin J chain, IGJ，分泌型免疫系统的重要组成成分）——通过商用抗体借助免疫印迹法（immunoblotting）得以验证。研究结果显示，在交配后4周内，对23只受试雌性猎豹进行检测时，IGJ蛋白丰度升高可成功识别其中80%以上的妊娠个体。本研究发现的新型粪便妊娠标志物，有助于提升迁地保护与繁育源种群中猎豹生殖状态（尤其是妊娠状态）的判定能力。