46 Evaluation of viability, hatching, and apoptosis of slow frozen and vitrified in vitro produced buffalo embryos

The study was performed with objectives to (1) measure the cleavage rate of cumulus–oocyte complexes (COCs) recovered from ovaries procured from slaughterhouses; (2) study the effect of slow freezing and vitrification on the re-expansion rate and hatching rate of in vitro-produced buffalo embryos; and (3) evaluate the apoptosis rate of frozen–thawed, and vitrified-warmed embryos. The COCs were recovered by aspiration method from the collected ovaries. The IVM, IVF, and in vitro culture (IVC) were performed using commercially available media (Vitrogen). Presumptive zygotes were cultured for 6 days in IVC medium in a benchtop incubator at 38.5°C, 5% O₂, 5% CO₂ and balance N₂ with 90% humidity for development of blastocysts (BLs). After 72 h of IVC, 63.1% (1015/1640) cleavage rate was observed, and on Day 6 after IVC, 20% (295/1640) blastocyst rate was observed. The BLs of grades I and II were randomly assigned to the fresh (n = 34), frozen–thawed (n = 47), and vitrified-warmed (n = 50) groups. In slow freezing, allotted BLs were exposed to ethylene glycol + sucrose (IMV France) for 8 min at room temperature (RT) and frozen using a programmable bio-freezer. Similarly, in the vitrified-warmed group, BLs were vitrified using a vitrification kit (Vitrogen) at RT. Blastocysts were exposed to V1 media for 8 min and subsequently, 40 s in a microdrop of V2 media. After that, BLs were loaded into the vitrification device (VitriFit^TM), and plunged into LN₂. The BLs in the frozen–thawed group were thawed in a water bath at 32°C for 30 s, while warming of vitrified BLs was performed using warming media (Vitrogen) at RT. Specifically, vitrification devices were opened and BLs were exposed to D1 medium (devitrification medium; Vitrogen) for 1 min. Subsequently, the BLs were transferred and exposed in D2 for 3 min and D3 for 5 min. The re-expansion rates of frozen–thawed (n = 47) and vitrified-warmed BLs (n = 50) were observed at 0, 2, 12, 24, and 48 h. Re-expansion and hatching rate data were analysed used nonparametric chi-squared test, while terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay data were assessed using a one-way ANOVA in SPSS 25.0 software. The re-expansion rate at 2 h for frozen–thawed BLs was 74% (35/47), which was significantly lower than that of vitrified-warmed BLs (41/50 = 82%). Further, the total re-expansion rate of frozen–thawed BLs was 91.5% (43/47), which was significantly lower than for the vitrified-warmed BLs (48/50 = 96%). To study hatching rate, a fresh group was included and hatching was observed at 2, 12, and 24 h. The hatching rate for the fresh group (79.4%; 27/34) was greater than for the vitrified-warmed (76%; 38/50) and frozen–thawed (68.1%; 32/47) embryo groups. In addition, a significantly higher hatching rate was observed for BLs in the vitrified-warmed group as compared to BLs in the frozen–thawed group. The apoptosis rate was assessed using the TUNEL assay. The apoptosis rate for frozen–thawed BLs was greater than for vitrified-warmed BLs (13.7 ± 0.7 vs 9.0 ± 0.7); however, BLs in the vitrified-warmed groups also had a greater apoptosis rate compared to fresh control BLs (6.6 ± 0.5). Results of the study indicate that BLs exposed to vitrification-warming had greater re-expansion and hatching rates and less DNA fragmentation as compared to BLs subjected to slow freezing. Moreover, the observed results suggest that vitrification is a more effective method than slow-freezing for cryopreservation of in vitro-produced buffalo embryos.