1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development

Abstract

In vitro maturation is an important process in the in vitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and in vivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21 h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5 µg mL⁻¹ FSH, 5 µg mL⁻¹ LH, 100 U mL⁻¹ penicillin/streptomycin] or maturation medium supplemented with 20 ng mL⁻¹ human LIF, 20 ng mL⁻¹ IGF1, and 40 ng mL⁻¹ FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28 M sorbitol fusion medium (0.1 mM calcium, 0.5 mM magnesium, 0.5 mM HEPES, 1 g mL⁻¹ bovine serum albumin) by a single pulse of 1.75 kV/cm for 22 ms. Reconstructed embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by 4 h of incubation in 2 mM 6-(dimethylamino)purine and 10 μg mL⁻¹ cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean ± s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2 ± 2.33%, n = 885 vs. 66.8 ± 1.82%, n = 822; P < 0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2 ± 1.34%, n = 259 vs. 90.9 ± 1.22%, n = 208; P > 0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6 ± 5.1%, n = 446 vs. 24.3 ± 2.9%, n = 300; P < 0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3 ± 20.9%, n = 48 vs. 29.0 ± 20.6%, n = 31; P < 0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined.

This research was supported by UAES project (1343) and RFBR (18-29-07089).