Pregnancy length and health in giant pandas: what can metabolic and urinary endocrine markers unveil?

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

Untargeted metabolomics reveals discriminative metabolic fingerprints in pregnant versus pseudo-pregnant giant pandas during the luteal phase

The complex reproductive biology of giant pandas imposes a challenge to their in situ and ex situ conservation programs. Conception is only possible during an annual window of 24-72 hours and the length of gestation is highly unpredictable because of embryonic diapause preceding implantation. The latter is supported by a low but modestly increased luteal progesterone production. Due to the relative inactivity of the corpora lutea (CLs), this is also referred to as the corpus luteum dormancy phase (CLD). The moment of implantation is preceded by a secondary rise in progesterone, e.g. the active luteal phase (AL). Furthermore, if no conception occurs after ovulation, giant pandas undergo obligatory pseudopregnancy with almost identical hormone profiles and behavioral changes compared to pregnant individuals, making pregnancy diagnosis only possible in the final 2-3 weeks of gestation via ultrasound or urinary hormone monitoring of estrogens and prostaglandins by enzyme immunoassays (EIAs). Because EIAs usually target only one family of hormone metabolites, little is yet known about changes in other metabolites that occur during (pseudo)pregnancy in this species. Therefore, this study aimed to obtain a complete view of metabolism of giant pandas during the different phases of their reproductive cycle. To this extent, non-invasively obtained urine samples were comprehensively analyzed using ultra high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HRMS). In total, 17 cycles from 7 giant pandas were included and divided into 3 groups: pregnant, pseudopregnant, and non-birth, with the latter including potentially lost pregnancies. Per cycle, 18 urine samples were selected, equally divided over 6 reproductive phases: anestrus, early CLD (CLD1), middle CLD (CLD2), late CLD (CLD3), early AL (EAL), and late AL (LAL). Urine (50 µL) was diluted 4 times with ultrapure water, separated on a Dionex UltiMate 3000 XRS UHPLC system (Acquity HSS T3 C18; 1.8 mm, 150 x 2.1 mm), and subsequently analyzed with a Q-Exactive Orbitrap mass spectrometer according to the polar metabolomics protocol from De Paepe et al. (2018)1. A total of 48,500 different metabolic features were obtained in the positive and negative ionization mode following pre-processing with IPO, CAMERA and xcms packages in R software. Feature abundances were corrected for urinary specific gravity (USpG) according to the formula of Wauters et al. (2018)2 to eliminate the effect of differences in hydration status of giant pandas throughout their cycle. Then, multivariate statistical analysis was performed showing good repeatability based on QC clustering (PCA-X). Pairwise comparisons between pregnant and pseudopregnant cycles were made per reproductive phase using orthogonal partial least squares discriminant analysis (OPLS-DA). A significant predictive model (7-fold validation) could be built for pregnant vs. pseudopregnant during LAL (Q2(Y) = 0.804, R2(Y) = 0.992, p = 0.01). The OPLS-DA models of the other reproductive phases could not be validated, however, several significantly discriminating metabolites could be detected per reproductive phase based on their variable importance of projection (VIP) score (VIP > 2), S-plot correlation (> ç0.5 ç) and Jack-knifed confidential interval (not across 0). The distribution of these metabolites was then checked across all reproductive phases in both groups using Welch two-sample t tests or Mann Whitney U tests. A total of 446 metabolites showed a significantly different distribution in at least 1 of the reproductive phases and were thus further retained. Metabolites that showed significantly different results in the anestrus phase were provisionally excluded to avoid potential group specific effects unrelated to reproductive status, leading to a final number of 293 significantly discriminating metabolites during LAL. Even in the CLD1, CLD2, CLD3, and EAL, 47, 45, 16 and 45 metabolites, respectively, could be retained, indicating that metabolic alterations in pregnant and pseudopregnant females start well before the LAL. These results offer the foundation to further unravel the metabolic pathways behind the corpus luteum dormancy and active luteal phase in pregnant versus pseudopregnant giant pandas. In addition, biomarkers for early pregnancy diagnosis may be identified. In the next phase, annotation of the retained metabolites, e.g. potential biomarkers, will be pursued via fragmentation analysis and molecular networking. Subsequently, pathway analysis will be performed to understand the biochemical origin of the metabolic profiles. This information will eventually assist in improving our knowledge about the reproductive biology of the giant panda and will as such improve their conservation management

Function, size and form of the gastrointestinal tract of the collared Pecari tajacu (Linnaeus 1758) and white-lipped peccary Tayassu pecari (Link 1795)

The peccary digestive tract is characterised by an elaborate forestomach. In order to further characterise the digestive function of peccaries, we report body mass, digestive organ mass, content mass of the gastrointestinal tract compartments and their length and width, as well as liver, parotis and mandibular gland mass. Our data on eleven collared and four white-lipped peccaries suggest that peccaries have a small relative stomach volume compared to other foregut fermenters, which implies a comparatively lower fermentative capacity and thus forage digestibility. The forestomach could enable peccaries to deal, in conjunction with their large parotis glands, with certain plant toxins (e.g. oxalic acid). The finding of sand being trapped in the forestomach blindsacs could indicate a disadvantage of the peccary forestomach design. The relevance of the forestomach to peccaries remains enigmatic