UFPA - Universidade Federal do Pará - R. Augusto Corrêa, Belém - PA, Brazil.
UFRA - Universidade Federal Rural da Amazônia - Estr, Belém - PA, Brazil.
Reprod Domest Anim. 2024 Mar;59(3):e14548. doi: 10.1111/rda.14548.
The in vivo fertilization process occurs in the presence of follicular fluid (FF). The aim of this study was to evaluate the effect of in vitro fertilization medium supplementation with 5% or 10% bovine follicular fluid (BFF) on the production of in vitro bovine embryos. FF was collected from ovarian follicles with a diameter of 8-10 mm, and cumulus-oocyte complexes (COCs) were co-incubated with sperm for 24 h in the commercial medium BotuFIV® (BotuPharma©), being distributed among the experimental groups: oocytes fertilized in a control medium; oocytes fertilized in a medium supplemented with 5% BFF; and oocytes fertilized in a medium supplemented with 10% BFF. After fertilization, the zygotes were cultured in vitro for 8 days. Embryo development was assessed through cleavage rates (day 2) and blastocyst formation rates (day 8). The relative expression of the genes OCT4, IFNT2, BAX, HSP70 and SOD2 was measured using the real-time polymerase chain reaction method. There was no difference (p > .05) among the different experimental groups in terms of cleavage rates and blastocyst formation rates. Regarding the gene expression results, only the blastocysts from oocytes fertilized with 10% BFF showed significantly lower expression of IFNT2 (p = .003) and SOD2 (p = .01) genes compared to blastocysts from oocytes fertilized in control medium alone, while there was no difference between blastocyst from oocytes fertilized in control medium and the ones from oocytes fertilized with 5% BFF. In addition to this, the blastocysts from oocytes fertilized with 5% BFF showed significantly reduced levels of expression of the heat shock protein HSP70 (p < .001) and the pro-apoptotic protein BAX (p = .015) compared to blastocysts from oocytes fertilized with control medium. This may indicate that lower supplementation of BFF to the IVF medium creates a more suitable environment for fertilization and is less stressful for the zygote.
体内受精过程发生在卵泡液（FF）的存在下。本研究的目的是评估在体外受精培养基中添加 5%或 10%牛卵泡液（BFF）对体外牛胚胎生产的影响。FF 是从直径为 8-10mm 的卵巢卵泡中收集的，将卵丘-卵母细胞复合物（COCs）与精子共同孵育 24 小时，在商业培养基 BotuFIV®（BotuPharma©）中，分为以下实验组：在对照培养基中受精的卵母细胞；在添加 5% BFF 的培养基中受精的卵母细胞；和在添加 10% BFF 的培养基中受精的卵母细胞。受精后，将受精卵在体外培养 8 天。通过卵裂率（第 2 天）和囊胚形成率（第 8 天）评估胚胎发育情况。使用实时聚合酶链反应法测量 OCT4、IFNT2、BAX、HSP70 和 SOD2 基因的相对表达。在卵裂率和囊胚形成率方面，不同实验组之间没有差异（p>.05）。关于基因表达结果，只有用 10% BFF 受精的卵母细胞产生的囊胚中 IFNT2（p=.003）和 SOD2（p=.01）基因的表达显著降低，与单独用对照培养基受精的卵母细胞产生的囊胚相比，而用对照培养基受精的卵母细胞和用 5% BFF 受精的卵母细胞产生的囊胚之间没有差异。此外，与用对照培养基受精的卵母细胞产生的囊胚相比，用 5% BFF 受精的卵母细胞产生的囊胚中热休克蛋白 HSP70（p<.001）和促凋亡蛋白 BAX（p=.015）的表达水平显著降低。这可能表明，向 IVF 培养基中添加较低水平的 BFF 为受精创造了更适宜的环境，对受精卵的压力较小。
