When was recombinant bovine somatotropin discovered

In the specific case of rbST, it is essential to monitor its misuse during the period of lactation, before milk reaches the market, and therefore the collection of samples after slaughtering animals is not a real option in terms of food safety.

All this means that target samples must be easy to collect, the method of sample collection must cause minimal or no pain to the animal, and it has to be economically viable. One of the main target tissues of rbST in dairy cattle is the mammary gland, where this peptide hormone exerts an important galactopoietic effect.

Due to this direct relationship, recent studies have used post-mortem mammary tissue to evaluate the effect of rbST on its transcriptional profile However, as mentioned before, it is important to detect the use of this substance during lactation to avoid the entry of milk produced using rbST into the market.

At this point, milk somatic cells appear as a good alternative to carry out in vivo transcriptomic assays to detect the use of rbST. A comparative study, in which five different RNA sources were evaluated to examine the lactating bovine mammary gland transcriptome, concluded that detecting MSCs released into milk during lactation is one of the simplest methods to isolate RNA.

Also, the MSC transcriptome is representative of mammary gland tissue and can be used as an effective alternative to study mammary gland tissue gene expression without the need for a tissue biopsy In a practical way, Toral et al. The aim of this study is to evaluate the potential of high-throughput real-time PCR to obtain a gene expression profile in MSCs collected from cows treated with rbST.

MSCs were collected from six treated cows and three control animals at 36 different time points to analyse the expression of selected genes and seek transcriptomic differences between groups. Additional control samples from rbST-free farms have been also included. As described in a previous work carried out by this research group 30 , nine Holstein cows in first or second lactation and in an age range from 1.

Feed was distributed to the cows twice a day, and they had ad libitum access to fresh water. Milk production in volume of each individual animal was recorded every day during eight consecutive months days by using individual milk meters, starting collecting data eighteen days before the first rbST dose pre-dose or conditioning period and finalizing 1.

The nine cows were divided into two groups: control group composed of three cows and rbST-treated group composed of six cows. A total of 12 rbST doses were administered to the treated group during the study, and milk samples of the first milking of the day were collected from both control ant treated animals at different time points during the whole experiment Fig.

Between the fifth and sixth rbST treatment, there was a day gap with no rbST dose. Also, since the last rbST dose was administered, milk production was recorded by two months. 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 Monthly somatic cell counts, as a measure of mastitis, were required.

The sites where bST was injected were monitored for any signs of adverse reactions. To evaluate safety, companies had to use one, three, and five times the expected dosage level of bST for two consecutive lactations in one of their test herds.

Heifers born to treated cows were raised through breeding age and monitored for abnormalities. Companies seeking approval for bST were also required to prove that its use was not harmful to the environment. The Monsanto Company of St. Louis, Missouri, developed the drug. However, the drug could not be used immediately due to a day moratorium imposed by Congress during the summer of The moratorium was designed to give the White House Office of Management and Budget time to study possible consumer reaction and the drug's impact on the dairy industry.

The FDA approval also carried with it some provisions to deal with antibiotic residue concerns. In September , the General Accounting Office reported that the FDA had found evidence in submitted clinical trials that bST-treated cows have a slightly increased incidence of mastitis. This report raised concerns that antibiotic treatments for mastitis could lead to increased antibiotic residues in milk.

States require milk to be tested for drug residues. Milk found to have unsafe levels of residues must be discarded. Although an FDA advisory committee concluded in March that adequate safeguards exist to prevent unsafe levels of antibiotic residues from entering the milk supply, additional steps were taken to ensure that any unsafe residues in the milk of bST-treated cows are detected before the milk or its products are marketed.

According to a news release issued by the U. The most intense controversy surrounding approval of bST for use in dairy cows has occurred in major dairy producing states in the Great Lakes and New England areas. Representatives of the dairy industry are concerned about the ultimate economic effect on producers. Consumer and environmental advocacy groups have expressed opposition based on concerns about milk quality and the use of biotechnology in general.

Dairy producers Some producers are afraid that they will not be able to keep up with new technologies and they will suffer economically as a result. Others feel that a product such as bST will work to the disadvantage of producers in the Great Lakes States and the Northeast. Natural resistance to new technology adoption and a fear of genetic engineering techniques cause some producers to resist the approval and use of bST.

Special interest groups Activist groups with a variety of agendas and motives have addressed the bST issue. Some have stated that milk from treated cows may not be safe after all, and more testing is needed. Others see this as a scare tactic to delay or block the use of bST and undermine consumer confidence in milk from bST-treated cows.

Some animal rights groups see the use of animals for food, under any circumstances, as inhumane or a violation of those animals' "rights". S Others have stated that cows have a right not to be injected with bST. Others Other opposing arguments state that the FDA does no independent testing of its own, but only monitors the studies of the companies seeking approval.

The persistent oversupply of milk and dairy products has also been cited as a reason to block the use of bST. Some dairy farmers oppose the use of bST but feel they would have no choice but to use the product in their own herd in order to stay competitive if bST came into general use McDermott. Table 2 summarizes arguments for and against the use of bST in the categories of food safety and its effect on the number and size of farms.

What Lies Ahead? Use of bST will have a significant effect on the research and development investment in agricultural biotechnology by commercial firms.

Universities will be expected to provide unbiased scientific information. Patience, tolerance, and understanding will be required by educators, extension workers, and other professionals in agriculture who work with groups that either support or oppose implementation of technology such as bST.

Safety is not an issue; bST is naturally present in milk and would be broken down in the digestive process. Can we really be sure that milk from bST-treated cows is safe? Do we really know we need to know? Consumers will be reassured when they get the complete story of safety of milk from treated cows. We won't see lower milk prices. Retail food prices increase even when farm prices decline. Dairy product consumption at best will be unaffected, and more likely will be hurt by approval of bST.

Some consumers will prefer RorganicS non-bST-treated milk. Marketers could sell such an RorganicS product. Consumers wonUt believe the chemical companies and FDA when they refute negative campaigns.

The dairy industry canUt afford to stake everything on what some people say is scientific truth. Increased feed and forage for bST-treated cows is more difficult to produce on smaller farms. Small farms will be pushed out faster. Farm buildings and other assets will be worth less as more producers are forced out of dairy production. Farm size has been increasing and number of dairy farms has been decreasing for years.

This will occur with or without approval of bST. In the Northeast and Midwest, fewer farms will result in decreased service and supplier support for remaining farms. There is an adverse effect on the environment and quality of life when large farms are favored.

University of Illinois 69 Mumford Hall W. Gregroy Drive Urbana, IL University of Minnesota 3 Coffey Hall St. Paul, MN Purdue University S. Second St. Lafayette, IN University of Missouri S. Fifth St. Columbia, MO Ames, IA University of Nebraska Dept. Communications Lincoln, NE North Dakota State University Ag. Michigan State University 10B Ag.

East Lansing, MI University of Wisconsin Ag. Bulletin, Rm. Murray St. Madison, WI South Dakota State University Ag. Center Box Brooking, SD The FDA is understaffed and over-worked. Their credibility is not high. But this same finding has also been reported in people who drink soy milk. This suggests that the increase in IGF-1 may not be specific to cow's milk, and may be caused by protein, minerals, or some other factors in milk unrelated to rBGH.

There have been no direct comparisons of IGF-1 levels in people who drink ordinary cow's milk vs. At this time, it is not clear that drinking milk, produced with or without rBGH treatment, increases blood IGF-1 levels into a range that might be of concern regarding cancer risk or other health effects.

These were:. At least 8 other national and international review committees have evaluated the evidence concerning potential health effects of rBGH on humans and dairy cows. These reviews and the most recent year they convened are listed below.

Several of these reports document adverse effects on cows, including higher rates of mastitis, foot problems, and injection site reactions. Although the use of rBGH is still approved in the United States, demand for the product has decreased in recent years. Many large grocery store chains no longer carry milk from cows treated with rBGH.

The available evidence shows that the use of rBGH can cause adverse health effects in cows.