Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes

Abstract Background: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. A study was conducted to examine the effects of adding melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 hours at 38.5°C in 5% CO2. The matured oocytes were fertilized in vitro and cultured in SOF media for 7 days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability, mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.20%), cleavage (89.20%), and transferable embryo (48.20%) rates compared to the group without melatonin (69.20%, 75.00%, and 35.80% respectively). Additionally, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in both fresh with melatonin (TCM+Mel) and Vitrified with melatonin (VS2+Mel groups ) (80% and 76.70%, respectively), while the mitochondrial intensity was higher in the TCM+Mel group (1698.60) among vitrified groups. In conclusion, Melatonin supplementation improves developmental competence and mitochondrial distribution in buffalo oocytes during in vitro maturation and vitrification.