How does rbst affect cows

It was made to determine the existence of some effect of this lack of dose on gene expression of rbST group in comparison to control group. All procedures were performed respecting animal welfare and causing no more pain, suffering, distress or lasting harm than the equivalent to that caused by the introduction of a needle in accordance with good veterinary practice. 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.

The collection of milk somatic cells MSCs was carried out as previously described by our research group Briefly, a total of two litres of homogenized milk from the morning milking of each cow were collected at the milking parlour in sterile bottles Deltalab, Spain and immediately transported in refrigerated conditions to the laboratory.

Before milking, udder was cleaned and first contaminated milk streams were dismissed. The remaining milk that was not collected for experimental assays was discarded. This step was repeated five times for each sample in the same conical plastic tube to concentrate the pellet.

Following the same procedure, a total of three MSCs samples from three different cows belonging to an external dairy farm unrelated to the farm where the cows of the study were housed were collected. The selection of those genes was mainly based in previous transcriptomic studies of cows treated with rbST and anabolic agents 26 , 27 , 31 , Of these 18 genes, three were used as endogenous controls to calculate the relative expression of the other 15 candidate genes.

A plate design of 18 assays in triplicate and 56 samples was chosen. Briefly, in a well plate, 1. LinRegPCR imports non-baseline-corrected data and performs a baseline correction on each sample. Then, a window of linearity is determined, and linear regression analysis is used to determine the PCR efficiency per sample from the slope of the regression line.

The mean PCR efficiency of each amplicon tested and the Cq value per sample were used to calculate a starting concentration N 0 per sample, expressed in arbitrary fluorescence units.

After that, Factor Correction qPCR software was used to remove multiplicative between-session variation in experiments A session factor is used to correct the observed data and it can be calculated from a matrix of between-session ratios or estimated using a maximum likelihood approach. Corrected values are obtained by dividing the observed values by the session factor. Finally, the gene expression ratio was calculated by dividing the N 0 of the target gene by the N 0 of the geometric mean of the three reference genes.

Test T was used to determine significant differences between groups. The nonparametric Mann—Whitney U test was used when data was no normally distributed.

A supervised method, i. Lastly, to determine the variables genes more affected by rbST administration, and hence more appropriated to discriminate treated animals, the S-plot of the OPLS-DA model was used.

From cycle 0 to cycle 3, milk production seems to increase in both groups, possibly indicating that animals had not yet reached their peak of lactation. Although the first dose of rbST was administered around 2 months post-partum, as recommended by the manufacturer, it is important to note that this recommendation is based on mathematical models that locate the milk production peak at 60—90 days postpartum.

However, these models are only theoretical approaches and days in milk DIM at the milk peak have a wide variation between herds and cows, being conditioned, amongst other factors, by welfare and feeding At this point, the production differences observed between groups returned to levels very close to those observed at cycle 0. On the basis of the biweekly administration pattern of rbST recommended by the manufacturer, on day 70 the group of treated animals should have received a new dose of recombinant growth hormone.

However, that dose was not applied so as to be able to evaluate if the rbST group could recover the pre-dose transcriptional profile after 28 days without hormonal administration Fig. Possibly due to this lack of hormone, the statistically significant differences existing in milk production between the two groups disappeared, highlighting the effects of rbST on milk production rates Table 2.

Curiously, these significant differences were maintained until the end of the study, even almost two months after the last dose 12 th dose, day was administered Table 2. MSCs are easy-to-collect source of RNA for gene expression studies, as these cells can be isolated from raw milk following a very simple protocol.

Besides, sample collection is cheap and non-invasive, since milk can be collected in the milking room without direct contact with the animal and therefore without causing stress to it which could alter the results obtained.

Although the inclusion of this step increases the sample price in routine analysis, it allows obtaining samples with higher purity. The mean RIN value observed for the samples analysed was 6. The samples used in this study were bovine somatic cells isolated from milk.

Milk is characterized by a complex microbiota and RNases derived from that microbiota could be responsible of a lower RNA integrity. In addition, some of somatic cells present in milk could be partially degraded. These facts were also observed in a study that used bovine vaginal smear for transcriptomics studies with the aim to find biomarkers to trace the misuse of anabolic agents Also, the process of milking, milk collection and transportation to the lab could, despite being carried out with the as swiftly as possible, affect to the RNA integrity.

This selection was based on the potential of these genes to obtain a characteristic rbST transcriptomic signature that could be used as a standard to control the ab use of rbST in dairy cattle. Previous gene expression studies with rbST were designed as single-dose and single sampling studies in post-mortem mammary tissue 27 or multi-dose five doses studies with blood and muscle sampling in vivo Those methods are considered invasive, relatively expensive and therefore impractical for control purposes on dairy farms.

In the case of somatotropin, the control must be performed in vivo , since the ultimate goal is to avoid the entry of rbST-milk into the dairy market. In this study, the expression of IGF-1 was only detected at all the sample points in one cow cow 7 treated with rbST and it was not detectable in the MSCs of control group.

The fact that it was only possible to detect the IGF-1 target at all the sample points in one cow may indicate the existence of different local mammary gland responses to exogenous rbST among individuals.

This result could be due to MSCs being composed mainly of leukocytes 40 in which the repressive effect of IGFBP may not be as important as in mammary tissue. On day 1 the rbST group 0. Different studies that evaluated the concentration of rbST in blood after administration observed that the higher concentrations of this recombinant hormone were found after rbST administration 14 , Therefore, the significant differences observed in treated group could be due as a response to the IGF-1 synthesized as response the higher concentrations of rbST in treated cows after first dose administration.

In Fig. This day coincided with the sixth administration of rbST, and between this dose and the fifth dose there was a gap of 28 days instead of 14 days. These results indicate that IGF-1R is a possible good candidate for inclusion in a panel of genes to detect the use of rbST in dairy farms.

Although other authors did not find a significant effect of rbST on IGF-1R levels in skeletal muscle 31 , in this study a clear response was observed in the levels of this receptor transcript in MSCs.

The difference between the two studies could be due to the different matrices used. MSCs could possibly respond better to the higher levels of circulating IGF-1 and increase the number of receptors for this molecule in their membrane.

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. Therefore, it is possible that the transcriptomic changes caused by rbST in these two cytokines are more evident in the second week of an rbST cycle. Curiously enough, on day 84 28 days after the 5 th rbST administration , there was no significant differences between groups Fig.

In this context, other previous studies have used powerful transcriptomic technologies to detect the use of anabolic agents in cattle 26 , However, the previously mentioned studies did not find an effect of anabolic agents on TNF, while the present research found a strong influence of rbST administration on the relative abundance of TNF. With this regard, it has been reported that the exogenous administration of rbST during lactation can enhance the immune response in cows Actually, milk somatic cell counts increased earlier and faster in cows suffering from coliforme mastitis when rbST was administered Growth hormone and its recombinant version show the ability to modulate the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland in health and diseased cows In screening studies by using transcriptomics it is very difficult to find a specific gene that could be used as very trusty gene.

Different studies carried out in the United States have observed that large farms are more likely to adopt rbST, suggesting not only a potential farm-size component of rbST use and profitability but also an operator age and education component 48 , Recombinant somatotropin has been frequently reported as a management-intensive technology, associated with the use of other productivity-oriented technologies and management practices that are characteristic of larger farms, being less frequent among grazers 49 , In this sense, subclinical mastitis causes a reduction in milk production in affected cows Therefore, it should be interesting to combine the data of transcriptomic assays with the milk production data.

Also it could be accompanied by a milk microbiological assay of suspected cows to detect the principal pathogens associated with subclinical mastitis. But what really increases the potential for discrimination is the inclusion of more genes in the panel. The cell cycle is controlled by cyclins and cyclin-dependent kinases. Of those, cyclin D endoded by the CCND1 gene coordinates cell cycle progression through extracellular stimulation e.

However, most adult cells are maintained in a quiescent state known as G0 phase, a resting state, and they can re-enter the cell cycle in G1 phase under appropriate mitogenic stimuli Before the first dose of rbST, there were no significant differences between the control and rbST groups Fig.

However, after the first dose, it was possible to observe significant differences between the two groups at different sample points. Thus, on days 9, 23 and 35 of the study, the relative abundance of CCND1 was significantly higher in the rbST group than in the control group Fig. Finally, on day 84 28 days after the 5 th rbST administration , there were no significant differences between groups Fig. This can result in the activation of cell in G0 phase, and in the particular case of mammary tissue, an increase in milk production through increasing the number of alveolar cells of mammary gland The circulation of this peptide hormone in the organism would result in the activation of the cell cycle in the target cells.

However, in the other rbST cycles, the relationship between dose administration and changes in the relative abundance of SIRT2 transcripts was not as evident as in the second dose. In this study, the relative abundance of EEF1G transcripts did not follow an obvious tendency related to rbST administration. However, on days 17 and 30 3 and 2 days after the second and third rbST doses, respectively , the relative abundance of EEF1G transcripts was significantly higher in the rbST group.

A previous study 27 concluded that rbST treatment increases the levels of EEF1G transcripts in mammary tissue but it only used one sample point 6 days after somatotropin administration. Therefore, it is not possible to directly compare those results with the results obtained in this study, in which 36 sample points were used for transcriptomic assays over the course of 8 months.

In particular, McCoard et al. However, as mentioned before, that study included only one data point after a single rbST dose. The present long-term multi-dose experiment has demonstrated that transcription patterns in bovine animals treated with rbST have great variability over time.

For example, data obtained for CCND1 showed that the effect of rbST on the transcription of some genes increases with the number of doses.

Although it is possible that rbST influences the transcription of the MFGE8 gene, obtained results showed both up- and down-regulation. Therefore, MFGE8 cannot be suggested as an ideal candidate for tracing rbST ab use in cattle, as it was not possible to observe a clear tendency in its transcription.

However, it was not possible to find significant differences in its transcription as a result of rbST administration. Lactoferrin LTF is an iron-binding glycoprotein belonging to the transferrin family Figure 4 shows the evolution of LTF gene relative abundances at all sample points evaluated in this study.

Although upregulation of LTF was observed in previous studies with anabolic substances in cattle 38 , it was not possible to establish a clear tendency for LTF in relation to rbST treatment. For example, on days 9 and 53, the relative abundance of LTF considerable lower in rbST-treated animals. However, on day 91, the relative abundance was considerable higher in that group. A previous study concluded that treatments with rbST in dairy cattle cause upregulation of the COL3A1 gene in mammary tissue 6 days after rbST administration However, in the present work it was not possible to detect transcripts of the COL3A1 gene.

Also ESR2 transcription was not detected in this study. The final aim of this work was to propose a routine panel of genes whose combined transcription pattern would allow the development of a screening method to control the misuse of rbST in dairy farms via MSCs.

Unlike other previous studies 27 , 31 , 32 , this work analysed the transcription patterns of rbST-related genes in a 8-month real-conditions experiment including 12 cycles of rbST administration and control animals. This approach permitted more accurate data to be obtained in order to differentiate between rbST-treated cows and control cows. Multivariate statistical analysis was run to elucidate modifications in MSCs transcription patterns as a consequence of rbST administration.

The main goal of PCA analysis is to identify global patterns in data, detecting the correlation between different variables, i. Projection of the samples into the new multidimensional space of the principal components PCs would potentially allow a differentiation between rbST and control groups, highlighting also those genes with a greater ability as biomarkers of the treatment.

In the score plot of the PCA analysis shown in Fig. Various samples of rbST group appeared mixed with controls on the left side of the plot but, curiously enough, the majority of them correspond to a day of somatotropin administration days 14, 28, 57 or 84 or to the first 2—3 days after a dose days 2, 3, 17, 44 or , in which an evident transcriptomic disturbance was not detected. MSC samples are labelled according to day of experiment, being day 0 the day of the first rbST dose in treated animals.

The transcriptomic profiles were also subjected to an OPLS-DA, from which potential discriminative genes were also pointed out. In a similar way to PCA, several rbST samples are projected among control samples on the left side of the ellipse.

However, these samples were collected when the transcriptomic disturbance caused by rbST had disappeared or had not appeared yet , so strictly, they could be classified as controls in that specific moment.

Conversely, very few control samples were misclassified as rbST. When no test set is available as is this case here, the cross-validation method is the main strategy to assess the quality of a model. Results of the cross-validation procedure are summarized by the value of different quality parameters. These values indicate a relatively good description of the data by the model and average predictability The fact that this model was built with a panel of genes with different discriminating ability should not be ignored, as it affects the discriminating power of the OPLS.

Some rBGH products on the market differ chemically from a cow's natural somatotropin by one amino acid. Both the natural and recombinant forms of the hormone stimulate a cow's milk production by increasing levels of another hormone known as insulin-like growth factor IGF Concerns about possible health effects on humans from milk produced using rBGH have focused on 2 main issues.

If it does, would this be expected to have any health effects in people, including increasing the risk of cancer? Several scientific reviews have looked at these issues and are the main focus of this document. Second, cows treated with rBGH tend to develop more udder infections mastitis.

These cows are given more antibiotics than cows not given rBGH. Does this increased use of antibiotics lead to more antibiotic-resistant bacteria, and is this a health concern for people? This remains a concern, but it has not been fully examined in humans. Bovine growth hormone levels are not significantly higher in milk from rBGH-treated cows. On top of this, BGH is not active in humans, so even if it were absorbed from drinking milk, it wouldn't be expected to cause health effects.

This drug is based on the somatotropin naturally produced in cattle. Somatotropin is a protein hormone produced in the pituitary gland of animals, including humans, and is essential for normal growth, development, and health maintenance.

Early research in the s and s in Russia and England found that milk production in cows could be increased by injecting cattle pituitary extracts, specifically bST. English scientists attempted to increase milk production in cows during World War II with pituitary-derived bST to alleviate food shortages. This process is also advantageous for producing a more consistent and purified source of bST.

The drug also must be effective, meaning that it works as intended.