Can supplementation of leonurine to in vitro culture medium affect the cryosurvival of bovine embryos?

The aim of this thesis study was to determine whether blastocysts developed in a culture medium supplemented with leonurin during in vitro embryo production (IVEP) would remain viable after being thawed following vitrification and stored for different periods. A total of 1,717 Grade-A oocytes were collected by aspiration from 368 abattoir-derived ovaries. During the in vitro culture phase, two groups were established: a control group (no supplementation) and a leonurine group (20 µM). Blastocysts obtained during culture were vitrified using the open-pulled straw (OPS) method. Following storage for 1 week, 1 month, or 6 months, thawed embryos were evaluated for re-expansion and hatching rates. The embryos were thawed after storage periods of one week, one month and six months, after which their re-expansion and hatching rates were evaluated. Re-expanded embryos were classified to determine the inner cell mass (ICM), trophectoderm (TE) and total cell counts (TCC) using differential staining; to determine the apoptotic cell (AHC) and TCC and apoptotic index using TUNEL staining; and to measure the levels of reactive oxygen species (ROS) and glutathione (GSH) using different staining methods. The blastocyst formation rate was 35.85% in the leonurine group and 28.04% in the control group (p < 0.001). Re-expansion rates after vitrification were significantly higher in the leonurine group across all storage durations (p < 0.001), whereas hatching rates did not differ between groups (p > 0.05). After vitrification, embryos thawed and re-expanded after storage periods of 1 week, 1 month, and 6 months showed THS values in the leonurin group of 186.70±31.07; 146.50±13.5 and 123.80±20.63, respectively; in the control group, they were determined as 146.30±24.81; 130.70±17.58 and 115.20±26.56 (p<0.001). Concurrently, AHS was found to be 6.70±2.45, 7.30±3.02, and 7.50±3.02 in the leonurin group, respectively, and 9.80±2.25, 11.40±1.26, and 17.60±5.35 in the control group. Differential staining revealed higher cell counts in the leonurin group (p<0.001). The ROS levels of the embryos on the specified days were 22.02±5.3; 42.07±4.05 and 79.46±9.80 in the leonurin group, and 35.14±0.05; 63.45±7.01 and 125.66±25.62, respectively (p<0.001). Concurrently, it was ascertained that the GSH level was elevated in the leonurin group (p<0.001). However, this level remained constant in both groups due to the extended storage period (p>0.05). Consequently, it was determined that leonurin, recognized for its antioxidant properties, could be administered as a supplement to the culture stage of IVEP in cattle. This approach has the potential to enhance the blastocyst rate and preserve the viability of embryos undergoing vitrification.