The Acute Phase Protein Ceruloplasmin as a Non-Invasive Marker of Pseudopregnancy, Pregnancy, and Pregnancy Loss in the Giant Panda

and Pregnancy Loss in the Giant Panda. PLoS ONE 6(7): e21159. doi:10.1371/journal.pone.0021159 The Acute Phase Protein Ceruloplasmin as a Non- Invasive Marker of Pseudopregnancy, Pregnancy, and Pregnancy Loss in the Giant Panda Erin L. Willis 0 David C. Kersey 0 Barbara S. Durrant 0 Andrew J. Kouba 0 John P. Lydon, Baylor College of Medicine, United States of America 0 1 Department of Conservation and Research, Memphis Zoological Society, Memphis, Tennessee, United States of America, 2 Department of Reproductive Sciences, Center for Species Survival, Smithsonian Conservation Biology Institute , National Zoological Park , Front Royal, Virginia, United States of America, 3 Reproductive Physiology Division, Institute for Conservation Research, San Diego Zoo Global , Escondido, California , United States of America After ovulation, non-pregnant female giant pandas experience pseudopregnancy. During pseudopregnancy, non-pregnant females exhibit physiological and behavioral changes similar to pregnancy. Monitoring hormonal patterns that are usually different in pregnant mammals are not effective at determining pregnancy status in many animals that undergo pseudopregnancy, including the giant panda. Therefore, a physiological test to distinguish between pregnancy and pseudopregnancy in pandas has eluded scientists for decades. We examined other potential markers of pregnancy and found that activity of the acute phase protein ceruloplasmin increases in urine of giant pandas in response to pregnancy. Results indicate that in term pregnancies, levels of active urinary ceruloplasmin were elevated the first week of pregnancy and remain elevated until 20-24 days prior to parturition, while no increase was observed during the luteal phase in known pseudopregnancies. Active ceruloplasmin also increased during ultrasound-confirmed lost pregnancies; however, the pattern was different compared to term pregnancies, particularly during the late luteal phase. In four out of the five additional reproductive cycles included in the current study where females were bred but no birth occurred, active ceruloplasmin in urine increased during the luteal phase. Similar to the known lost pregnancies, the temporal pattern of change in urinary ceruloplasmin during the luteal phase deviated from the term pregnancies suggesting that these cycles may have also been lost pregnancies. Among giant pandas in captivity, it has been presumed that there is a high rate of pregnancy loss and our results are the first to provide evidence supporting this notion. Funding: Support for this work was provided by the Memphis Zoo Department of Conservation and ResearchsTM Giant Panda Physiology and Nutrition Research Program. The director of the Memphis Zoos Department of Conservation and Research partipated in the decision to fund the project, and in the preparation of and decision to publish the manuscript. Competing Interests: The Memphis Zoo has filed a provisional patent application associated with the applications of urinary ceruloplasmin as a non-invasive marker of pregnancy and pregnancy loss. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. With over 300 pandas in captivity around the world, substantial progress has been made in the ex-situ (captive) conservation of this species in recent years, but as with any small population, careful management is required to maintain genetic diversity and to prevent inbreeding [1]. Although significant progress has been made in growing the captive population, giant pandas remain a challenge to breed in captivity due to the single estrous period per year with sexual receptivity lasting only one to three days, mate incompatibility, lack of sexual interest, and failure to give birth following optimal timing of insemination. Female giant pandas spontaneously ovulate [2] and undergo a phenomenon known as pseudopregnancy if not pregnant, wherein a females reproductive hormones are similar in concentration and length during the nonpregnant luteal phase as during pregnancy. This makes pregnancy determination impossible by diagnostic hormonal tests typically used in other mammals [2,3]. In many other species, pregnancy can be diagnosed from hormonal monitoring of the luteal steroid, progesterone, or its excreted metabolites in urine and feces. However, progestagen patterns between pregnant and pseudopregnant pandas are indistinguishable [2,3,4,5,6]. Furthermore, these luteal phases are unpredictable in length. They consist of a variable primary phase lasting anywhere from around 60 to 122 days, which is characterized by a slight increase in progestagens above baseline levels [4,5]. The primary rise of progestagens is then followed by a more consistent secondary phase which comprises a substantial increase in progestagens above baseline lasting 4050 days [4,5]. In a pregnant giant panda, the embryo remains quiescent in embryonic diapause until the secondary rise of progestagens when implantation is suspected to occur and the fetus begins to grow rapidly [2,3,7,8,9]. Pregnant females do not show marked changes in behaviors until the last two weeks of gestation, but these behaviors are often inconsistent among females and pseudopregnant females may also exhibit similar changes in behavior prior to the end of the luteal phase [4]. In a limited number of cases, ultrasonography has been helpful in the detection of a fetus or gestational sac but is only applicable when used late in pregnancy at about 2 to 3 weeks prior to parturition, following the delayed implantation [7,8]. Even then, fetal detection can be challenging and requires the skill of an expert ultrasonographer as well as the cooperation of the animal. While ultrasonography has provided some evidence of failed pregnancies due to embryonic loss in giant pandas [4,8,10], if no indications of pregnancy are observed by ultrasound and a cub is not born after progestagens return to baseline, it is usually assumed the female was not pregnant. However, because ultrasound can only detect a fetus in late gestation and females are often uncooperative at this time, many lost pregnancies could very easily go undetected. During one term pregnancy, thermal imaging also showed promise for detecting growing fetal tissue and determining litter size in giant pandas at an earlier stage than ultrasound [11]. However, this technique still does not differentiate pseudopregnancy from pregnancy until after the time of implantation. For decades, researchers all over the world have been searching for a magic bullet that would provide a pregnancy test for giant pandas and other exotic wildlife that undergo pseudopregnancy. To date, all tests utilizing steroid hormones and their metabolites, as well as other hormones such as relaxin, have proven ineffective for distinguishing between pregnancy and pseudopregnancy [2,3,5,12]. Thus, the lack of a reliable pregnancy test continues to limit our abil (...truncated)