133 Effect of cytokines on the quality bovine oocytes matured invitro

Abstract

As has been shown previously, fibroblast growth factor 2 (FGF2), leukaemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination play a positive role in maintaining the quality of mammalian oocytes maturing invitro. In the present work, we studied the effects of these cytokines in optimal concentrations when added in combination to IVM medium on the nuclear status and development competence of bovine oocytes. Slaughterhouse-derived cumulus–oocyte complexes (COC) (n=1107 COC) were cultured for 22h in either IVM medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH] (Control) or the same IVM medium supplemented with FGF2, LIF, and IGF1. The eight combinations of cytokines tested during maturation were (1) 20ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 1); (2) 20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 2); (3) 20ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 3); (4) 20ngmL−1 LIF/20ngmL−1 IGF1/40ngmL−1 FGF2 (Group 4); (5) 5ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 5); (6) 5ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 6); (7) 5ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 7); and (8) 5ngmL−1 LIF/20ngmL− 1 IGF1/40ngmL−1 FGF2 (Group 8). After IVM, matured and denuded oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, the cleavage and blastocyst rates were determined. The data from 6 to 7 replicates (122–181 oocytes per treatment) were analysed by ANOVA. The rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and reached 67.1 to 84.5%. Cleavage rate was higher (84.5±2.9%, P<0.05) in Group 1 compared with Control (68.9±2.7%), Group 4 (67.1±4.9%) and Group 7 (66.6±2.0%), but not compared with Group 2 (76.5±4.8%), Group 3 (76.6±4.8%), Group 5 (78.2±3.1%), or Group 8 (79.9±3.1%). The percentage of blastocyst formation relative to the total number of MII oocytes in the control group was 19.6±2.4%. The culture of COC in Group 1 (20 ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2) and Group 2 (20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2) caused the blastocyst yield to increase to 33.0±3.8 and 31.2±4.3%, respectively (P<0.05), whereas the culture COC in other cytokine-treated groups had no effect. In Groups 3 to 8, the blastocyst rates were 22.9±4.6, 19.2±3.0, 24.2±3.2, 27.8±1.7, 21.6±1.8, and 25.5±9.0%, respectively, and did not differ (except Group 4) compared with those of Group 1 and Group 2. In conclusion, LIF, FGF2, and IGF1 in optimal combinations can maintain competence for parthenogenetic development of bovine COC during their maturation invitro. This research was supported by RFBR (projects No. 18-29-07089) and the Ministry of Science and Higher Education of Russia.