Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes

ABSTRACT

The study aimed to determine the effects of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes. The vitrification solution [VS] consisted of Dulbecco's phosphate buffered saline [DPBS] supplemented with 0.5 M sucrose, 0.4% bovine serum albumin [BSA] and different types of molar [M] concentrations of the cryoprotectants which composed of either glycerol [G], ethylene glycol [EG] or dimesthyl sulfoxide [DMSO] in order to determine the best type of vitrification cryoprotectants. The concentrations tested were 4 M, 7 M and 7M concentration of G, EG and DMSO, respectively. Cumulus oocyte complexes [COCs] were obtained from slaughterhouse ovaries. Oocytes were vitrified immediately after collection .The COCs were pre-equilibrated in 50% of the VS for 3-5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 ml straws for 7-10 days of storage in liquid nitrogen. The straws were thawed in warm water at 37°C for 10 seconds and COCs were evaluated for morphological damage. Morphologically normal COCs were cultured in vitro and evaluated for maturation. Oocytes were fertilized with frozen-thawed semen capacitated in Brackett and Oliphant [BO] medium contained heparin and caffeine and evaluated for cleavage and embryonic development. The results revealed that the proportion of buffalo oocytes found to be morphologically normal was significantly [p<0.05] higher in EG and DMSO than those obtained in G [85.0 and 83.33 vs 65.0%, respectively]. Among the damaged oocytes, cracking of zona pellucida was the most frequent abnormality observed. A significantly higher [p<0.05] percentage of maturation and cleavage rates derived from vitrified -thawed immature oocyte in EG and DMSO than those obtained in G [47.05, 46.67%; 28.57, 25.71 vs 30.76% and 10.0%, respectively]. A similar trend was observed in blastocyst stage produced in vitro. However, in vitro developmental competence was higher for vitrified-thawed fresh oocytes [control] than those obtained from all groups of cryoprotectants. that 7M solution of EG or DMSO could be used for vitrification of immature buffalo oocytes for their subsequent utilization in the in vitro maturation, fertilization and embryo production