290 CYTOKINE PROFILE IN THE AMNIOTIC FLUID OF NELORE CALVES CONCEIVED BY IN VITRO PRODUCTION AND EMBRYO TRANSFER

Abstract

Cytokines have several functions in many cells and their appropriate balance in the maternal-fetal connections is necessary to maintain a successful pregnancy. The incidence of fetal problems reported at the delivery moment in cows with conceptus conceived by in vitro production (IVP) and lack of literature in the immunology gestation area in bovine prompted this study. The aim of the present study was to determine the levels of cytokines such as tumor necrosis factor a (TNF-α), interferon-y (IFN-γ), interleukin-6 (IL-6), and IL-8 in amniotic fluid of calves from IVP and conventional embryo transfer at the moment of delivery. Forty animals were used and divided into 2 groups: 1 - Twenty Nelore cross-breed cows pregnant with Nelore calves conceived by conventional embryos transfer method (ET group); 2 - Twenty Nelore cross-breed cows pregnant with Nelore calves obtained by IVP after follicular aspiration (IVP group). Around labor, cows were transferred to a maternal paddock to permit observation of delivery. During the expulsion phase the amnion was punctured and 15 mL of fluid was collected in a plastic tube and stored in a freezer for later analysis. The cytokine levels were measured by immunoenzymatic assay (ELISA). The commercial kits used were ESS0011 (Bovine TNF Alpha), ESS0026 (Bovine IFN-gamma), and ESS0029 (Bovine IL-6) from Pierce Biotechnology™ (Rockford, IL, USA) and kit D8000C (Human IL-8) from R&D Systems™ (Minneapolis, MN, USA). The protocol was performed according to the manufacturer’s recommendations. The densities were evaluated by using an automatic optical reader ELISA. The well plates were read at 450 nm. The ANOVA on ranks was used for statistical analysis because of nonparametric distribution of data, with 5% of significance. No calves in this experiment showed signs of sepsis or death during the peripartum period. There was no significant difference in the concentration of TNF-α between the animals of the ET (250.40 pg mL^-1) and IVP (398.70 pg mL^-1) groups (P > 0.05). There was no difference statistically for IFN-γ; the medians of this cytokine were 35.7 pg mL^-1 for the ET group and 48.22 pg mL^-1 for the IVP group (P > 0.05). Only samples 15 and 18 of the IVP group had detectable concentrations of IL-6 (2227.47 and 2686.30 pg mL^-1, respectively) and for IL-8 only 15 of the samples in the same group (577.6 pg mL^-1). The results for TNF-α and INF-γ in this experiment can be adopted as standards for normal pregnancy in cattle, because there are no reports of such data in the literature. However, additional research in the area of gestation immunology area is necessary to describe the role of cytokines in the maintenance of pregnancy and at the moment of delivery in cattle.

Supported by FAPESP, Brazil.