Human Milk from Tandem Feeding Dyads Does Not Differ in Metabolite and Metataxonomic Features When Compared to Single Nursling Dyads under Six Months of Age

Abstract

Given the long-term advantages of exclusive breastfeeding to infants and their mothers, there is both an individual and public health benefit to its promotion and support. Data on the composition of human milk over the course of a full period of lactation for a single nursling is sparse, but data on human milk composition during tandem feeding (feeding children of different ages from different pregnancies) is almost entirely absent. This leaves an important knowledge gap that potentially endangers the ability of parents to make a fully informed choice on infant feeding. We compared the metataxonomic and metabolite fingerprints of human milk samples from 15 tandem feeding dyads to that collected from ten exclusively breastfeeding single nursling dyads where the nursling is under six months of age. Uniquely, our cohort also included three tandem feeding nursling dyads where each child showed a preferential side for feeding-allowing a direct comparison between human milk compositions for different aged nurslings. Across our analysis of volume, total fat, estimation of total microbial load, metabolite fingerprinting, and metataxonomics, we showed no statistically significant differences between tandem feeding and single nursling dyads. This included comparisons of preferential side nurslings of different ages. Together, our findings support the practice of tandem feeding of nurslings, even when feeding an infant under six months.

Keywords: breastfeeding; human milk; infant feeding; lactation; metabolome; microbiome; tandem feeding.

Figure 1

Fat Percentage, Volume, and Estimated Bacterial Load of Human Milk. (a) Total fat percentage of different groupings; (b) total volume of different groupings; (c) 16S rRNA gene copy qPCR for bacterial load for different groupings; (d–f) fat percentage, volume, and 16S rRNA gene copy for preferential side feeding. X axis labels indicate grouping: 03to06 = three to six months nursling age of exclusive breastfeeding dyads; Y < 6 = youngest tandem feeding nursling younger than six months; Y < 12 = youngest tandem feeding nursling younger than 12 months; Y < 24 = youngest tandem feeding nursling younger than 24 months; O < 36 = oldest tandem feeding nursling younger than 36 months; O > 36 = oldest tandem feeding nursling older than 36 months.

Figure 2

REIMS Fingerprinting of Human Milk of Sole Tandem Feeding Nurslings. (a) REIMS negative analysis of all tandem feeders against 3 to 6 month group; (b) REIMS negative univariate analysis showing no significant differences; (c) REIMS positive analysis of all tandem feeder against 3 to 6 month group; (d) REIMS positive univariate analysis showing no significant differences.

Figure 3

REIMS Fingerprinting of Human Milk Based on Age and Preference of Tandem Feeding Nursling. Metabolic fingerprinting of human milk based on negative ion detection mode data for (a) age of oldest child, (b) age of youngest child, and (c) milk from breast that is used solely to feed the youngest or oldest nursling. Metabolic fingerprinting of human milk based on positive ion detection mode data for (d) age of oldest child, (e) age of youngest child, and (f) milk from breast that is used solely to feed the youngest or oldest nursling.

Figure 4

Tentative Metabolite Identifications of Detected Features in REIMS Spectra. Extracted features from REIMS mass spectra in both (a) negative ion detection mode and (b) positive ion detection mode were tentatively identified against the Human Metabolome Database using a cut-off of <10 ppm. Due to number of features in spectra, metabolites are classified according to the superclass level of chemical taxonomy in the HMDB collection to allow visual presentation.

Figure 5

Metataxonomic Profiling of Human Milk Based on Age of Tandem Feeding Nursling. Metataxonomic analysis of human milk for 3 to 6 month nursling and tandem feeder nurslings using (a) Chao1 alpha diversity index (p value = 0.141) and (b) beta diversity (R² = 0.021, p value = 0.556). Metataxonomic analysis of human milk based on age of oldest nursling using (c) Chao1 alpha diversity index (p value = 0.134) and (d) beta diversity (R² = 0.197, p value = 0.005). Metataxonomic analysis of human milk based on age of youngest nursling using (e) Chao1 alpha diversity index (p = 0.137) and (f) beta diversity (R2 = 0.112, p Value = 0.085).