Tracing recombinant bovine somatotropin ab(use) through transcriptomics: the potential of bovine somatic cells in a multi-dose longitudinal study

Abstract

In the European Union, the use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden. Monitoring rbST (ab)use by its direct detection in animal matrices still remains a challenging task. New monitoring methods based on indirect detection of the substance are necessary. A new transcriptomic system based on the use of high-throughput real-time PCR in combination with somatic cells was developed to control rbST administration in dairy animals. A total of nine cows, separated into control and rbST-treated groups, were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group every 14 days, up to a total of 12 doses. Milk somatic cells (MSCs) were sampled from each animal at different time points throughout 8 months of study. It was possible to obtain the transcriptomic profile of 18 genes in MSCs of rbST-treated and control groups, and using univariate and multivariate statistical analysis control and treated animals were discriminated. The transcription of CCND1, IGF-1R, TNF and IL-1β genes resulted strongly influenced by rbST treatment. The combination of MSCs, transcriptomic tools and statistical analysis has allowed the selection of four genes as potential biomarkers that could be used in a transcriptomic panel for monitoring rbST administration in cows.

Figure 1

Overview of the rbST animal experiment, showing the days of milk sample collection (marked with a tube with a blue cap), and the days of animal treatment (days highlighted in bold numbers).

Figure 2

Relative abundance of IGF-1R and CCND-1 in the first and second cycles of rbST administration in control group (N = 3) and treated group (N = 6). The nonparametric Mann–Whitney U test was used to compare the milk yield in each cycle between treated and control group. Asterisks represent statistically significant differences between treated group and both control group. *(p < 0.05), **(p < 0.01), ***(p < 0.001).

Figure 3

Relative abundance of CCND1, IGF-1R, IL1β and TNF transcription of rbST treated group (N = 6) and control group (N = 3) on days -10, 1, 9, 23, 35, 84. Samples for a single day of outside control cows (N = 3) were added. A one-way ANOVA approach was applied for comparisons of more than two groups. Asterisks represent statistically significant differences between treated group and both control group. *(p < 0.05), **(p < 0.01), *** (p < 0.001).

Figure 4

Evolution of the relative abundance of some target genes during all the sample points of the study in rbST treated group (N = 6) and control group (N = 3).

Figure 5

PCA plot built using full transcriptomic profiles of milk somatic cells (MSCs), in which a discrimination between cows treated with rbST (N = 6, red circles) and control animals (N = 3; black boxes) can be glimpsed. MSC samples are labelled according to day of experiment, being day 0 the day of the first rbST dose in treated animals.

Figure 6

OPLS-DA scatter plot (a) constructed using trasncriptomic profiles from milk somatic cells, showing a discrimination between control (N = 3; black circles) and rbST (N = 6; red dots) animals, labelled according to the experimental day (being day 0 the day of 1st dose in rbST group). S-plot (b) associated to the OPLS discriminant analysis, highlighting the genes more affected by rbST on the upper right corner of the plot, and hence with a higher discriminative power.