Vitrification of immature bubaline cumulus oocyte complexes by the open-pulled straw and conventional straw methods and their subsequent in vitro fertilization

Abstract

The <i>in vitro</i> maturation (IVM), fertilization (IVF) and morphological changes in buffalo cumulus oocyte complexes (COCs) cryopreserved by ultra rapid freezing using conventional (CON) and open-pulled straw (OPS) methods were tested. COCs were cryopreserved using a vitrification solution comprising of DPBS + 0.5M sucrose + 0.4% BSA and two concentrations (4.5 or 5.5M) of each cryoprotectant ethylene glycol (EG) and dimethylsulfoxide (DMSO) and cryopreserved by either CON or OPS method. Vitrified COCs were stored in LN<sub>2</sub> for seven days and then thawed, and morphologically normal COCs were used for IVM (<i>n</i> = 864) and IVF (<i>n</i> = 933) in two separate experiments to record (1) morphological damage of COCs due to vitrification, (2) nuclear maturation 24 h after culture (nine replicates) and (3) fertilization 24 h after insemination (10 replicates). The COCs were matured <i>in vitro</i> in TCM-199 medium using hormone supplements and fertilized using TALP-BSA. Freshly collected COCs were separately used for IVM (<i>n</i> = 110) and IVF (<i>n</i> = 130) and kept as control. The arcsin transformed data of the proportion of COCs matured or fertilized was compared by DNMR test. The highest proportion of morphologically normal COCs were seen in 5.5M EG with CON method (94.5%) and the lowest were seen in 4.5M DMSO with OPS method (82.4%). At the end of Experiment 1, it was revealed that IVM in all vitrification groups was significantly lower (<i>P</i> < 0.05) compared to control (66.4%). Amongst the various vitrification treatments the highest IVM was seen in 5.5M EG with OPS method (39.2%) and the lowest in 4.5M DMSO with CON method (19.3%). Comparison of both concentrations of EG and DMSO showed that the proportion of COCs attaining metaphase-II (M-II) increased with increasing concentration of both the cryoprotectants. However, at equal concentration of EG and DMSO the proportion of COCs attaining M-II were significantly higher in OPS method compared to CON method. In Experiment 2, a significantly higher (<i>P</i> < 0.05) IVF was seen for fresh COCs (45.4%) compared to vitrified COCs. Amongst the vitrification treatments the highest fertilization was seen for 5.5M EG with the OPS method (33.6 %) and the lowest for the 4.5M DMSO with CON method (15.17%). A dose dependant increase in the proportion of oocytes fertilized was seen with increasing concentration of both EG and DMSO [CON: 4.5M (15.2%), 5.5M (25.6%), OPS: 4.5M (21.3%) and 5.5M (27.5%)] in both CON and OPS methods. Comparison of the two cryoprotectants revealed that EG was better compared to DMSO. At equal concentrations of EG or DMSO a significantly higher (<i>P</i> < 0.05) proportion of fertilized oocytes were seen in OPS method compared to the CON method. It was concluded that developmental capacity of vitrified buffalo COCs could be improved by using OPS in comparison to conventional straws.