Estradiol receptors and IGF-1 signaling components in the mammary gland of prepubertal dairy heifers are altered by gastrointestinal nematode infection.

MEJIA, ME; PERRI, AF; BARAVALLE, C.; LICOFF, N; BECU-VILLALOBOS, D; FORMIA, N; DALLARD, B.; ISABEL MARIA LACAU

Cairns

Congreso; XXVIII World Buiatrics Congress; 2013

Objectives (100 words) Mammary growth during prepuberty occurs mainly in response to stimulation by IGF-1 and estradiol. We previously demonstrated that dairy heifers infected with gastrointestinal parasites had reduced IGF-1 circulating levels. This reduction resulted in delayed puberty and reduced proportion of the mammary parenchyma, as well as parenchymal cell division rate.  We therefore wish to investigate some components of the regulatory patterns within the mammary gland, comparing antiparasitic treated with untreated heifers reared on pastures naturally infected with gastrointestinal nematode larvae.   Method (250 words) Forty four female Holstein calves were randomly assigned to an untreated or to a treated group at birth. Heifers in the treated group received monthly, from birth to 18 months of age, different anthelminthic drugs, in order to minimize parasite burden avoiding drug resistance generation. The drugs (and doses) used were ivermectin (0.63 mg/Kg), fenbendazol (7.5 mg/Kg) and levamisol (10 mg/Kg). At 20, 30, 40 and 70 weeks of age, blood samples for IGF-1 determination by RIA and feces for EPG counting (adapted McMaster method) were taken to all the animals. Mammary biopsies were taken from 6 heifers in each group with a Tru-Core-I fully automatic biopsy gun provided with a Tru-Core 14 G X 20 cm needle, at the same times. Samples were processed for immunohistochemical studies. Primary antibodies for alpha estradiol receptors (ESR1), IGFBP2 and IGFBP3 were used. The reactions were then developed with the DAB-peroxidase method and counterstained with hematoxylin.  Images were obtained by a color camera mounted on a optical microscope (40X) and digitalized for the analyses with the Image-Pro Plus 3.0.11 software. The area labeled by each antibody reaction (for ESR1, IGFBP-2 and IGFBP-3) was quantified as a percentage of total explored area, using a segmentation color method with mask generation. Treatment effect on EPG and IGF-1 values was analyzed by two-way ANOVA for repeated samples (treatment x age). Non parametric tests (Friedman and Mann-Whitney) were used to compare percentages of immunolabeled areas. Statistical significance was fixed at 0.05 and a tendency to significance was considered for P between 0.05 and 0.1.   Results (250 words) EPG in feces were lower and IGF-1 levels were higher in the treated compared to the untreated heifers, as expected. Immunolabeling for ESR1 in the mammary slices was evidenced in stained nuclei, at all studied ages, only in the parenchyma. No staining was evidenced in the stroma. Immunolabeled area was higher in treated than in untreated heifers (P = 0.02). In the treated group age effect was evidenced by a peak of immulnolabeled area at 30 weeks (P = 0.04).   Specific staining for IGFBP-2 and IGFBP-3 was cytoplasmic and was present in stroma and parenchyma. IGFBP-2 immunolabeled area was lower in treated than in untreated heifers (P = 0.04), and there was a tendency to temporal variation in the treated group (P = 0.06) with a maximal expression of the protein at 40 weeks of age. No differences were observed for IGFBP-3 immunolabeled area between treated and untreated groups or during development.     Conclusions (100 words) These results demonstrate that parasite burden in young heifers influence the components of the regulatory system of mammary gland development during prepuberty and peripuberty and add new evidence about the importance of parasite control.  The fact that parasite presence during prepuberty alters mammary developmental features may indicate that the future productive capacity of the gland would be affected.