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Giant pandas are mono-estrus seasonal breeders, with the breeding season typically occurring in the spring. Successful fertilization is followed by an embryonic diapause, of variable length, with birth in the late summer/autumn. There is a need for additional understanding of giant panda reproductive physiology, and the development of enhanced biomarkers for impending proestrus and peak fertility. We aimed to determine the utility of non-invasive androgen measurements in the detection of both proestrus and estrus. Urine from 20 cycles (−40 days to +10 days from peak estrus) from 5 female giant pandas was analyzed for estrogen, progestogens and androgens (via testosterone and DHEA assays), and hormone concentrations were corrected against urinary specific gravity. Across proestrus, estrogens increased while progestogens and androgens decreased – at the point of entry into proestrus, androgens (as detected by the testosterone assay) decreased prior to progestogens and gave 4 days advanced warning of proestrus. At the time of peak estrus, androgens (as detected by the DHEA assay) were significantly increased at the time of the decrease in estrogen metabolites from the peak, acting as an alternative confirmatory indicator of the fertile window. This novel finding allows for enlargement of the preparative window for captive breeding and facilitates panda management within breeding programmes. Androgens allow an enhanced monitoring of giant panda estrus, not only advancing the warning of impending proestrus, but also prospectively identifying peak fertility.

Mature female giant pandas usually ovulate once a year. This is followed by an obligatory luteal phase, consisting of a long-lasting corpus luteum dormancy phase (CLD; primary increase in progestogens) and a much shorter active luteal phase (AL; secondary increase in progestogens). Varying duration of both the dormant (embryonic diapause) and AL (post-embryo reactivation) phases has hampered unambiguous pregnancy length determination in giant pandas until today. Additionally, progestogen profiles have been considered not to differ between pregnant and pseudopregnant cycles. Only ceruloplasmin, 13,14-dihydro-15-keto-PGF2α (PGFM) and – more recently – estrogens have been assigned diagnostic power so far. Our study investigated the competence of metabolic (fecal output) and Urinary Specific Gravity (USpG)-normalized urinary endocrine (progestogens, PGFM, glucocorticoids (GCM) and ceruloplasmin) markers for pregnancy monitoring including defining the duration of the AL phase length. Research on 24 (6 pregnant, 8 pseudopregnant and 10 non-birth) cycles of 6 giant pandas revealed a fixed AL phase length of 42 days in giant pandas, e.g. representing 6 weeks of post- diapause development in case of pregnancy. Progestogen concentrations were significantly higher in pregnant cycles throughout the majority of the AL phase, with significant higher values during the AL phase in healthy twin compared to singleton pregnancies. GCM concentrations were also markedly higher in giant pandas expecting offspring, with a clear increase towards birth in the final 2 weeks of pregnancy. This increase in GCM was running in parallel with elevating estrogen and PGFM concentrations, and decreasing progestogens. In addition, during the AL phase, a more pronounced decrease in fecal output was obvious for pregnant females. The combined profiles of non-invasive metabolic and endocrine markers, the latter normalized based on USpG, showed a true pregnancy signature during the AL phase. The findings of this study are applicable to retrospective evaluations of non-birth cycles facilitating categorizing those into pseudopregnant or lost pregnancies, with USpG-normalization of the urinary endocrine markers as a prerequisite.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.