Ghrelin antagonist: possible solution for in vitro oocyte maturation from cows in negative energy balance

CARRANZA A; NIKOLOFF N; ANCHORDOQUY J MATEO; PASCUA AM; ANCHORDOQUY J PATRICIO; RELLING AE; FURNUS C

Foz do Iguazu

Simposio; 10th International Ruminant Reproduction Symposium; 2018

International Ruminant Reproduction Symposium Team

Ghrelin is a gut hormone released when cows are in negative energy balance (NEB) with adverse effects on oocyte in vitro maturation (IVM) (Sirini et al., 2017. Zygote, 25:601-611). The aim of this study was to investigate the effect of ghrelin antagonist (Lys3-GHRP-6). For this purpose, cumulus oocytecomplexes(COC) were obtained from slaughterhouse ovaries; and then maturated for 24h in TCM199 with 0.4% BSA, FSH (1μg/mL) and estradiol 17-beta (1μg/mL). The COC were matured in IVM medium: a) alone (Control: C); or supplemented with b) 60pm/mL ghrelin (G); c) 60pm/mL ghrelin+20pM/mL Lys3-GHRP-6 (ghrelin antagonist 1: GA1); d) 60pm/mL ghrelin+60pM/mL Lys3- GHRP-6 (ghrelin antagonist 2: GA2); e) 60pm/mL of ghrelin+100pM/mL Lys3-GHRP-6 (ghrelin antagonist 3: GA3). After IVM oocyte nuclear maturation was analyzed by Hoechst 33342, cumulus expansion area (CEA) was calculated with microscopic photographs by ImageJ, cumulus cells (CC)viability was evaluated by FDA, CC apoptosis and necrosis was classified by Anexina V and propidium iodide (Pläsier et al., 1999. J Immunol Methods, 229:81-95) and CC DNA damage was estimated by comet assay. Results of oocyte nuclear maturation, CC viability and rates of apoptosis and necrosis were analyzed by logistic regression using GENMOD procedure, and CEA and genetic damage index were analyzed by mixed model (SAS Institute, Cary, NC, USA). There were not differences among groups in CEA and nuclear maturation rates at any IVM condition analyzed (P>0.1). On the other hand, CC viability was significantly higher in C than G or ghrelin plus high ghrelin antagonist supplementation (C137.39±9.07 vs G 95.5±9.07, GA2 101.52±9.3 and GA3 96.68±9.04; P0.1; 124.34±9.07 vs. 137.39±9.07, respectively). Apoptosis was not significant different between treatments. However, there was a significant difference in CC necrosisbetween C and G (P0.1; 15.5%, 13.5%, 17%, and 14.3%, respectively). Comet assay data demonstrated that genetic damage index (GDI) was significantly higher in G (P