201 Oocyte maturation is the key for bovine embryo developmental pace

Transvaginal ultrasound-guided follicle aspiration (OPU) allows for the production of a large number of embryos irrespective of the reproductive status of the donor. However, its efficiency largely depends on the use of ovarian stimulation and the optimisation of the associated in vitro embryo production. The aims of the study were to determine the developmental ability of oocytes matured in vivo or in vitro following OPU, and to compare blastocyst developmental rates in the presence or absence of serum during maturation and in vitro culture. 21 Holstein heifers were included in the study and randomly allocated to three experimental groups: In vivo matured (M) or in vitro matured in either serum-free (SF) or serum-supplemented (S) conditions. Repeated OPU for immature oocytes (SF + S; n = 47) was performed in 3–7 day intervals. Superovulation consisted in a total of 500 IU of FSH (Pluset) administered twice a day in decreasing doses for four days, followed by gonadotrophin-releasing hormone (GnRH) injection 22 h later. OPU (n = 9) was performed 21 h after GnRH, while ovulation was expected 26–32 h after GnRH. Immature OPU-collected oocytes (n = 230) were matured in vitro per donor in TCM199 supplemented with either 15% FBS (S) or 20 ng/mL EGF (SF) for 22 h. Zygotes from in vitro-matured oocytes were cultured in SOFaa supplemented with either 5% FBS (S) or 0.4% BSA 5 µg/mL, insulin 5 µg/mL, transferrin 5 ng/mL, selenium (SF). Zygotes from in vivo-matured oocytes (M; n = 75) were cultured in serum-free medium. Total and freezable blastocysts according to the IETS guidelines were recorded and vitrified 6, 7, and 8 days post-insemination. Blastocyst rates were analysed using a binary logistic regression model, while data concerning number of oocytes and blastocyst per OPU were analysed using Kruskal-Wallis test. Differences at P < 0.05 were considered significant. More oocytes were obtained per OPU of M (11.56 ± 3.62) than of immature oocytes (5.56 ± 0.68) P = 0.035. However, there was no difference in the number of viable oocytes (M 8.33 ± 2.88, immature 4.79 ± 0.55). Regarding total blastocysts rates (day 6+7+8), no differences were found between M (36%) and SF (25.8%), but both were significantly higher than S (13.1%). Considering only total freezable blastocysts (Day 6 + 7 + 8), M (34.7%) was higher than S (13.1%) P = 0.003, but not higher than SF (19.6%); no differences were found between SF and S. The same significant differences were observed considering total freezable blastocysts per OPU (M 2.89 ± 1.01^a; SF 0.83 ± 0.16^ab; S 0.58 ± 0.2^b). Superscript letters indicate: in vivo matured (a), serum-free (ab) and serum (b). Interestingly, the development of SF was slower, with 52.6% of the total freezable blastocyst developing only at Day 8, compared to 15.4% of M (P = 0.018) and 14.3% of S (P = 0.044). In conclusion, in vivo maturation did not yield higher blastocyst rates or blastocyst per OPU than in vitro maturation in the same embryo culture conditions. However, the development of the blastocyst originated from oocytes matured in vitro was slower than their in vivo counterparts. Serum supplementation of maturation and culture media hampered blastocyst development.

This work was funded by IJC2019-039404-I and Moving Minds 2022.