The establishment of an in vitro culture system for complete oocyte maturation from the early stages of ovarian follicles is still a challenge. The aim of the present study was to assess the effect of different matrix with different culture media on the developmental growth of ovarian follicles in vitro. An ovarian histoarchitectural study was carried out to identify the primordial (0.027–0.039 mm), primary (0.041–0.079 mm), small preantral (0.085–0.131 mm), large preantral (0.132–0.294 mm), small antral (0.387–0.589 mm), and large antral (1.188–1.366 mm) follicles. Thus, large preantral follicles (0.2–0.3 mm) were mechanically isolated and cultured subsequently in different microconditions such as Dulbecco's modified Eagle's medium, Tissue Culture Medium-199 (TCM-199) and Opti-minimum essential medium, with same supplements where control (without matrix) was compared with matrix (coculture and encapsulation), which includes (1) buffalo fetal fibroblast cells, (2) cumulus cells, (3) ovarian mesenchymal cells, (4) collagen, (5) gelatin, and (6) Matrigel, cultured for 7 days in CO₂ incubator at 38.5°C (5% CO₂ in air). Cultured follicles were evaluated for growth rate (107.88% ± 10.24%), maturation rate (51.06% ± 6.53%), survivability rate (56.52% ± 3.42%), and antioxidant (catalase; CAT [1.58 ± 0.04 U/mg], superoxide dismutase; SOD [4.63 ± 0.05 U/mg], lactate dehydrogenase; LDH [1.48 ± 0.01 U/mg]) enzymatic activities, which showed significantly (p < 0.05) positive results in growth model with media TCM-199 than other studied groups. Furthermore, the development of large preantral follicles augmented significantly (p < 0.05) for growth rate (248.54% ± 9.51%), maturation rate (75.81% ± 7.07%), survivability rate (81.82% ± 3.02%), antioxidant (CAT [2.05 ± 0.03 U/mg], SOD [3.13 ± 0.12 U/mg], LDH [2.55 ± 0.51 U/mg]), and estradiol (175.83 ± 5.92 pg/mL) activities when they were encapsulated in Matrigel with nutritional requirements fulfilled by media TCM-199. These results provide better insight for the optimization of culture conditions for in vitro follicular development in the water buffalo, which will eventually assist in resolving the limitation of obtaining fewer competent oocytes for the embryo production in the species.

中文翻译：

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不同培养基3D培养条件的优化和比较，共培养和包封系统在水牛体外卵泡发育中的作用

建立从卵泡早期完全成熟的卵母细胞体外培养系统仍然是一个挑战。本研究的目的是评估不同基质和不同培养基对体外卵泡发育生长的影响。. 进行了卵巢组织结构研究以识别原始（0.027-0.039 mm）、初级（0.041-0.079 mm）、小窦前（0.085-0.131 mm）、大窦前（0.132-0.294 mm）、小窦（0.387-0.589）毫米）和大窦卵泡（1.188-1.366 毫米）。因此，大的窦前卵泡（0.2-0.3 mm）被机械分离并随后在不同的微量条件下培养，例如 Dulbecco 改良的 Eagle 培养基、组织培养基 - 199（TCM-199）和 Opti-minimum 基本培养基，与对照相同的补充剂（无基质）与基质（共培养和包封）进行比较，其中包括（1）水牛胎儿成纤维细胞，（2）卵丘细胞，（3）卵巢间充质细胞，（4）胶原蛋白，（5）明胶，和（6） Matrigel，在 38.5°C（5% CO ）的 CO _{2培养箱中培养 7 天2}在空气中）。评估培养的卵泡的生长率（107.88% ± 10.24%）、成熟率（51.06% ± 6.53%）、存活率（56.52% ± 3.42%）和抗氧化剂（过氧化氢酶；CAT [1.58 ± 0.04 U/mg]，超氧化物歧化酶；SOD [4.63 ± 0.05 U/mg]，乳酸脱氢酶；LDH [1.48 ± 0.01 U/mg]) 酶活性，在 TCM-199 培养基的生长模型中显示出显着 ( p  < 0.05) 的阳性结果。团体。此外，大窦前卵泡的发育显着增强（p < 0.05) 生长率 (248.54% ± 9.51%)、成熟率 (75.81% ± 7.07%)、存活率 (81.82% ± 3.02%)、抗氧化剂 (CAT [2.05 ± 0.03 U/mg]、SOD [3.13 ± 0.12 U/mg]、LDH [2.55 ± 0.51 U/mg]) 和雌二醇 (175.83 ± 5.92 pg/mL) 的活性，当它们被封装在 Matrigel 中时，其营养需求由培养基 TCM-199 满足。这些结果为优化水牛体外卵泡发育的培养条件提供了更好的见解，这最终将有助于解决获得较少合格卵母细胞用于该物种胚胎生产的限制。