Changes in the milk metabolome of the Giant Panda (Ailuropoda melanoleuca) with time after birth: three phases in early lactation and progressive individual differences

Ursids (bears) in general, and giant pandas in particular, are highly altricial at birth. The components of bear milks and their changes with time may be uniquely adapted to nourish relatively immature neonates, protect them from pathogens, and support the maturation of neonatal digestive physiology. Serial milk samples collected from three giant pandas in early lactation were subjected to untargeted metabolite profiling and multivariate analysis. Changes in milk metabolites with time after birth were analysed by Principal Component Analysis, Hierarchical Cluster Analysis and further supported by Orthogonal Partial Least Square-Discriminant Analysis, revealing three phases of milk maturation: days 1–6 (Phase 1), days 7–20 (Phase 2), and beyond day 20 (Phase 3). While the compositions of Phase 1 milks were essentially indistinguishable among individuals, divergences emerged during the second week of lactation. OPLS regression analysis positioned against the growth rate of one cub tentatively inferred a correlation with changes in the abundance of a trisaccharide, isoglobotriose, previously observed to be a major oligosaccharide in ursid milks. Three artificial milk formulae used to feed giant panda cubs were also analysed, and were found to differ markedly in component content from natural panda milk. These findings have implications for the dependence of the ontogeny of all species of bears, and potentially other members of the Carnivora and beyond, on the complexity and sequential changes in maternal provision of micrometabolites in the immediate period after birth

Changeover from signalling to energy-provisioning lipids during transition from colostrum to mature milk in the giant panda (Ailuropoda melanoleuca)

Among the large placental mammals, ursids give birth to the most altricial neonates with the lowest neonatal:maternal body mass ratios. This is particularly exemplified by giant pandas. To examine whether there is compensation for the provision of developmentally important nutrients that other species groups may provide in utero, we examined changes in the lipids of colostrum and milk with time after birth in giant pandas. Lipids that are developmental signals or signal precursors, and those that are fundamental to nervous system construction, such as docosahexaenoic acid (DHA) and phosphatidylserines, appear early and then fall dramatically in concentration to a baseline at 20-30 days. The dynamics of lysophosphatidic acid and eicosanoids display similar patterns, but with progressive differences between mothers. Triglycerides occur at relatively low levels initially and increase in concentration until a plateau is reached at about 30 days. These patterns indicate an early provision of signalling lipids and their precursors, particularly lipids crucial to brain, retinal and central nervous system development, followed by a changeover to lipids for energy metabolism. Thus, in giant pandas, and possibly in all bears, lactation is adapted to provisioning a highly altricial neonate to a degree that suggests equivalence to an extension of gestation

Prolonged transition time between colostrum and mature milk in a bear, the giant panda, Ailuropoda melanoleuca

Bears produce the most altricial neonates of any placental mammal. We hypothesized that the transition from colostrum to mature milk in bears reflects a temporal and biochemical adaptation for altricial development and immune protection. Comparison of bear milks with milks of other eutherians yielded distinctive protein profiles. Proteomic and metabolomic analysis of serial milk samples collected from six giant pandas showed a prolonged transition from colostrum to main-phase lactation over approximately 30 days. Particularly striking are the persistence or sequential appearance of adaptive and innate immune factors. The endurance of immunoglobulin G suggests an unusual duration of trans-intestinal absorption of maternal antibodies, and is potentially relevant to the underdeveloped lymphoid system of giant panda neonates. Levels of certain milk oligosaccharides known to exert anti-microbial activities and/or that are conducive to the development of neonatal gut microbiomes underwent an almost complete changeover around days 20–30 postpartum, coincident with the maturation of the protein profile. A potential metabolic marker of starvation was detected, the prominence of which may reflect the natural postpartum period of anorexia in giant panda mothers. Early lactation in giant pandas, and possibly in other ursids, appears to be adapted for the unique requirements of unusually altricial eutherian neonates

The dominant compounds in giant panda milk in Phase 1 of early lactation.

<p>The 50 most abundant liquid chromatography-high resolution mass spectrometry (LC-HRMS) signals in Phase 1 (the first week of lactation). Relative abundances were estimated from areas under LC peaks. The compounds are listed in the order of their LC retention times (Rt). Italicised names indicate non-proton adducts and complex ions identified by MZMine 2.10, and confirmed by manually checking the raw LC-HRMS data. The metabolite annotations are based on the Metabolomics Standards Initiative (MSI) identification levels; level 1, retention times matched with authentic standards (labelled as ST); level 2: identified by MS/MS (labelled as MS); level 3: accurate mass; and level 4 unknown. The metabolites identified at levels 1 and 2 are also labelled with the compound identifiers (CID) codes from the PubChem database.</p><p>The dominant compounds in giant panda milk in Phase 1 of early lactation.</p

Progressive changes in milk composition and increasing individuality with time.

<p>Hierarchical cluster analysis (HCA) dendrogram (A) and principal components analysis (PCA) score plots (B-D) of milk samples collected serially from three giant pandas; Li Li (LL), circles; Yuan Yuan (YY), stars; Xiao Ya Tou (XYT), diamonds. Multivariate analysis using SIMCA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143417#sec002" target="_blank">Materials and Methods</a>) allows horizontal sectioning of the dendrogram in (A) at different levels of clustering as indicated by the horizontal dotted lines. This yields progressively more detailed PCA score plots that first reveal similarities in metabolomes with time in all three individual giant pandas (as in (B), only two main coloured classifications), and then progressive disparities between the individuals when analysed at deeper resolutions (as in (C) and (D), increasing number of coloured classes). The data representing quality control repetitions (YY-19d-1, 2 and 3) are circled in red and indicate good repeatability. Score plots of PCA (B, C and D): x-axis PC1 = 19.4% and y-axis PC2 = 9.95%.</p

Compounds in giant panda milk tentatively correlated with cub growth rate.

<p>(A) OPLS loading plot for 21 serially collected samples from giant panda Yuan Yuan (YY). Components selected for testing for correlation with rate of cub body weight change are highlighted in red; see main text for selection criteria. X-variables, Pareto scaled LC-HRMS data; Y-variable, rate of body weight change over time of YY’s cub. (B) Body mass changes for YY’s cub over the milk sampling period (see also Fig A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143417#pone.0143417.s001" target="_blank">S1 File</a>). (C, D, E, F) Changes in abundance with time of the compounds indicated with diamonds in (A) selected to illustrate diverse correlations with weight gain by the cub. Compound N258 showed positive correlation, whereas compound N254 showed negative correlation, and compound P318 exhibited a more complex pattern. Y-axes represent relative abundances of each compound as estimated from areas under peaks calculated from HILIC-HRMS data, x-axes represent time after birth. Putative initial identifications were N258, isoglobotriose; P318, methyl-imidazole acetate; N254, citrate; N338, 3-methyl-2-oxopentanate.</p

Changes in milk metabolome delineating discrete phases in early lactation.

<p>S-plot of orthogonal partial least squares discriminant analysis (OPLS-DA) of 55 giant panda milk samples collected serially after birth from three giant pandas. In an S-plot, the x variable is the relative magnitude of a variable, and the y variable is the variable confidence/reliability. So, data points falling in the upper right or lower left corners of the plot represent those features that are least likely to be the result of spurious correlations. Peaks with low magnitude/intensity falling in the centre of the plot near 0 are close to the noise level and exhibit high risks for spurious correlations. The 50 most abundant compounds in the early phase (before day 7; Phase 1) and the 20 most abundant in later milk (after day 7; Phases 2 and 3, cumulative) are highlighted in red and blue, respectively. The inset graphs describe the time-dependent changes in abundance of two oligosaccharide isomers N262 and N351 (identified as diamonds in the S-plot), subsequently identified as 3’-sialyllactose and 6’-sialyllactose, respectively.</p