The sustained decline in fertility of dairy cows considerably affects agriculture in New Zealand and worldwide. One of its causes is the metabolic burden of increased milk production in lactating cows. Investigating differences in the metabolome of the uterine fluid between viable and non-viable pregnancies may help in finding a biomarker for pregnancy success. To understand the effect of the uterine environment in differences in fertility, we will use metabolomics to measure the uterine fluid metabolome. In a pilot study we have used a GC-MS/MS targeted metabolomics method on a Shimadzu 8040 TQGCMS to identify compounds present in uterine luminal fluid (ULF) and optimise the extraction method. ULF was collected by flushing the uterine horn using a standard method (Ledgard et al., 2012). We tested the effect of single phase or biphasic extraction and the use of a 10 kDa cut-off filter, with five replicates for each sample preparation permutation. Samples were analysed in a randomised order.
We were able to detect around 200 metabolite peaks out of 450 metabolites included in the targeted GC-MS/MS method, irrespective of extraction method. We found that biphasic extraction on filtered ULF wash gave the best reproducibility with 96 peaks having a relative standard deviation <10 %. The use of protein filters, however, led to contamination with glycerol. The metabolites detected were intermediates in 33 pathways mapped in the KEGG metabolic pathway database, including central carbon amino acid and fatty acid metabolic pathways.
A wide range of metabolic pathways in bovine ULF can be assessed using targeted GC-MS/MS metabolomics, with a combination of protein cut-off filters and biphasic extraction giving the best reproducibility.