Abstract

Background and Aim: In in vitro embryo production (IVEP) systems, the efficiency of oocyte maturation and subsequent embryo development is often limited by oxidative stress and suboptimal mitochondrial function. Supplementation of maturation media with growth factors and antioxidants has been proposed as a strategy to enhance oocyte developmental competence. Insulin-like growth factor-1 (IGF-1) promotes cell survival and proliferation, while antioxidants such as lycopene and α-tocopherol reduce intracellular reactive oxygen species (ROS) and protect cellular structures from oxidative damage. Although these supplements have individually demonstrated beneficial effects in various species, comparative studies evaluating their influence under identical conditions in buffalo (Bubalus bubalis) oocytes are limited. Therefore, this study aimed to evaluate and compare the effects of IGF-1, lycopene, and α-tocopherol supplementation during in vitro maturation on nuclear maturation, embryo developmental competence, and mitochondrial dynamics in buffalo oocytes.

Materials and Methods: A total of 1,485 high-quality buffalo oocytes were subjected to in vitro maturation (IVM) in four experimental groups: control (Tissue culture medium [TCM]-199), TCM-199 supplemented with 100 ng/mL IGF-1, TCM-199 supplemented with 0.2 µM lycopene, and TCM-199 supplemented with 100 µM α-tocopherol. Oocytes were incubated for 22 h at 38.5°C under 5% CO₂. Mature oocytes (n = 1,149) were then fertilized in vitro using Fert-TALP medium and cultured in modified synthetic oviductal fluid (mSOF) for 7 days to evaluate cleavage, morula, and blastocyst formation rates. Mitochondrial activity and distribution were assessed in 120 mature oocytes using MitoTracker Red FM staining followed by confocal laser scanning microscopy. Mitochondrial patterns were classified as diffuse, semi-diffuse, semi-peripheral, or peripheral. Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc test or chi-square analysis, with significance set at p ≤ 0.05.

Results: The nuclear maturation rate (metaphase II stage) was significantly higher (p < 0.01) in the IGF-1 and lycopene groups (85.2% and 87.3%, respectively) compared with the control (73.3%) and α-tocopherol groups (76.2%). Cleavage, morula, and blastocyst formation rates were also significantly higher (p < 0.01) in the IGF-1 (89.3%, 28.5%, and 20.6%) and lycopene (84.2%, 30.8%, and 32.7%) groups than in the control (75.1%, 20.3%, and 12.2%) and α-tocopherol (76.7%, 23.2%, and 14.4%) groups. Lycopene produced the highest blastocyst yield. Mitochondrial fluorescence intensity was significantly greater (p < 0.01) in all supplemented groups compared with the control. Diffuse mitochondrial distribution predominated in IGF-1- and lycopene-treated oocytes, indicating improved cytoplasmic competence and metabolic activity, whereas α-tocopherol treatment was associated with increased peripheral mitochondrial localization.

Conclusion: Supplementation of IVM medium with 100 ng/mL IGF-1 or 0.2 µM lycopene significantly enhances nuclear maturation, mitochondrial activity, and embryo developmental competence of buffalo oocytes. Lycopene demonstrated the most pronounced improvement in blastocyst formation, suggesting superior antioxidant protection during maturation. These findings highlight the importance of optimizing mitochondrial function and oxidative balance in buffalo IVEP systems and provide a potential strategy to improve reproductive biotechnology outcomes in buffalo.

Keywords: buffalo oocytes, embryo development, insulin-like growth factor-1, in vitro maturation, lycopene, mitochondrial dynamics, mitochondrial function, α-tocopherol.