Combination of hCG and Deslorelin Acetate on the Induction of Ovulation in Mares: Changes in Follicular Fluid Protein Profile

The composition of follicular fluid (FF) is essential for the proliferation and differentiation of granulosa cells in addition to processes related to rupture of the follicular wall, maturation and fertilization of the oocyte and luteinization. In the mare, there are few studies evaluating FF proteome (FAHIMINIYA, Prot Sci, 9:1, 2011; PETRUCCI, J Eq Vet Sci, 34:115, 2014). The ability to induce ovulation in a reliable way is important in equine reproductive management in different situations the main ovulation-inducing agents used are hCG and deslorelin. The aim of this study was to compare the protein profile of FF on induced ovulation of mares with hCG or with the combination of hCG and deslorelin acetate. Fourteen mares were used (3-12 years). Following the observation of follicles ≥ 35 mm and with endometrial edema, the mare was submitted to the induction protocols: Group H 1000 UI, IV, of hCG or Group HG 1000 UI hCG, IV, + 1,5 mg of Deslorelin acetate, IM. In the subsequent cycle mares were submitted to a protocol different from the previous cycle. Samples were collected by transvaginal aspiration 32 h after induction and submitted to the quantification of proteins by the Bradford method. Two-dimensional electrophoresis was performed in 12.5% polyacrylamide gel and stained with Coomassie G250, scanned and analyzed using PDQuest v.8.0.1. Spots with significant differences in relative abundance between group H and HG were cut out submitted to trypsin digestion and mass spectrometry. In this study the total protein concentration in the mare FF from Group HG were higher (73.07 ± 6.42 mg/ml) than those induced with hCG alone (63.97 ± 6.97 mg/ml). Comparative analysis showed a significant difference in the abundance of five spots between groups. Two Alpha-1-antiproteinase 2 (A1AT2), the Serotransferrin (TF) and Antithrombin III (ATIII) had lower relative expression in group HG and the Haptoglobin (HP) showed greater abundance in the same group. The lowest expression of A1AT2 at the final moment of follicular maturation prior to ovulation is likely related to the need for lower inhibitory action on the proteolytic activity allowing fine adjustment that controls the ECM degradation, inflammation and the coagulation cascade (BIANCH, J Prot, 90:61, 2013). ATIII is also serine-type endopeptidase inhibitor activity. The last protein less expressed in the group HG was TF. Increase in cellular iron levels stimulates the expression of some MMPs that degrade the ECM. There are reports of increased transferrin in granulosa cells and oocyte follicles in more advanced stages of maturation, which could explain the reduction in FF transferrin. Haptoglobulin showed increased abundance in the group HG and exerts anti-inflammatory action due to inhibition of oxidative damage. These proteins are probably related to the final events of oocyte/follicle maturation that trigger ovulation and subsequent luteinization.