• bST
• buffalo
• gene expression
• milk
• recombinant growth hormone
• somatotropin

Recombinant bovine growth hormone (rbGH) is a synthetic peptide hormone used in slow-release
formulations to increase the milk yield in bovines. With the Decision 1999/879/CE the European Union banned its use within the Member States, based on the negative effects on the animal welfare.
Furthermore, at present, there are no unequivocal proofs on the safety for human consumption of milk and dairy products coming from treated animals. Despite the ban, an illegal use of the hormone has been strongly indicated by the findings of pharmaceuticals in dairy farms of both bovines and buffalos.
From here, the need to develop analytical methods detects rbGH treatments, in order to counteract or prevent rbGH misuse. Unfortunately, the very high similarity of the recombinant forms with the natural hormone makes it extremely difficult to develop analytical methods to directly reveal the presence of rbGH in biological fluids. Therefore, indirect analytical approaches, which involve the exploitation of biomarkers, could represent a possible alternative. Even though a number of studies on this matter have already been carried out on bovines, there are still no data on buffalos, which represent a very important resource for dairy production in many Countries, Italy included.
For this reason, and with the aim of searching biomarkers for rbGH treatment, we treated eight
lactating buffalos with biweekly injections of a slow-release formulation of rbGH, according to the protocol suggested by the producer, for ten weeks and analyzed a number of parameters to assess their power as potential indirect indicators of the treatment.
In particular, the investigated parameters were:
- Serum growth hormone (GH) and Insulin-like growth factor 1 (IGF-1) levels and milk IGF-1
levels, by means of immunoassays developed in this study;
- expression levels, in leukocytes, by means of Real-time PCR, of ten genes belonging to the
somatotropic axis (GHR, IGF-1, IGF-2, IGFBP-1, -2, -3, -4, -5, -6, IGF-1R), of five GHR
5’-UTR variants (1B, 1C1, 1C3, 1D, 1I) and of ten other genes identified by a preliminary
microarray analysis as being the most relevant in terms of difference in expression levels
between treated animals and controls (ABCG1, DQA1, DQA5, DQB, DLC, Ferritin-like heavy chain, Integrin β-fragment, KKCC, TKDP3, SUCLG).
Collection of the samples was performed on days 2, 5, 9, 14 of each cycle of rbGH administration
for blood; on days 2, 7, 14 of the second and last cycle for milk; on days 2 and 14 of each cycle for leukocytes.
Results showed an extraordinary increase of GH serum levels (tens of times) on day 2 of each cycle in treated animals (p<0.001), followed by a rapid decrease in the subsequent days, even though significant differences with the controls were observed until day 5 (p<0.001). At the same time, IGF-1 concentrations promptly raised in serum (2~3 times, p<0.001) until day 9, despite, on an average, they were still significantly higher in the treated animals even on day 14 (p<0.01). Similarly, IGF-1 concentrations in milk were found to be significantly higher during the whole cycle, with peaks (more than 2 times the levels of the controls) on days 2 (p<0.001) and 7 (p<0.01).
These results promote serum GH levels as a good and reliable indicator of treatment, but the rapid decrease after the peak limits the exploitable period for sample collection within each cycle of rbGH administration. On the other hand, IGF-1 concentration in serum would allow to rely on a longer period for inspection. However, the moderate increase of IGF-1 levels may be diriment for a legal purpose, when single animals are taken into consideration. Nevertheless, an analysis plan involving a number of animals belonging to the same farm would make the finding of high levels of serum IGF-1 a strong indicator for treatment. Higher IGF-1 concentrations in milk of treated buffalos, rather than to be considered an inspection indicator, should be regarded as a potential concern in term of food safety, as suggested by a number of scientists with regard of the IGF-1 increase in milk of treated bovines.
Finally, biomolecular analysis revealed that exogenous GH significantly influenced the expression of some of the investigated genes (IGFBP-1, p<0.001; DQA5, p<0.01; DQB p<0.05; DLC p<0.001; Ferritin-like heavy chain, p<0.05; Integrin β-fragment, p<0.05). However, only DQA5 expression level seemed to be strongly triggered and sustained by the hormonal treatment, but the findings of high values in one non-treated buffalo indicated the possibility of false positives. For this reason, the gene expression analysis should be used with caution, even though it could be intended as a further support to the serum indicators.