Effect of slow freezing on morphology and developmental competence of buffalo (Bubalus bubalis) immature oocytes.

The present study was conducted to evaluate the effects of three cryoprotectants, dimethyl sulphoxide (DMSO), ethylene glycol (EG) and 1,2-propanediol (PROH), each used at two concentrations (1.0 and 1.5 M) on the morphology, maturation rate and developmental capacity of usable quality immature buffalo oocytes subjected to slow freezing. The addition of the cryoprotectant before freezing and its dilution after thawing were carried out in a two- (for 1.0 M) or three-step manner (for 1.5 M). The incidence of damage was found to be significantly higher (P<0.05) with the lower concentration of 1.0 M, compared to that with 1.5 M for all the three cryoprotectants examined. The proportion of immature oocytes recovered in a morphologically normal state was significantly higher (P<0.05) for DMSO than those for EG or PROH at both 1.0 and 1.5 M concentrations. Among the six combinations evaluated, that of DMSO at 1.5 M concentration was found to be superior to others. Irrespective of the type or concentration of the cryoprotectant, partial or complete loss of the cumulus mass was the most prevalent damage. Following in vitro maturation, the nuclear maturation rate was significantly higher (P<0.05) for DMSO than those for EG or PROH at both 1.0 and 1.5 M concentrations. When the in vitro matured oocytes were subjected to in vitro fertilization after slow freezing, using 1.5 M DMSO as cryoprotectant, 4.5% and 0.6% of them were able to develop to morulae and blastocysts, respectively, on Day 9 post insemination, compared to 19.2% and 10.6%, respectively, for the controls. In conclusion, DMSO was more effective than EG or PROH for the slow freezing of immature buffalo oocytes and blastocysts could be produced from immature buffalo oocytes subjected to slow freezing in 1.5 M DMSO.

Gautam SK ，Verma V ，Singh B ，Palta P ，Singla SK ，Chauhan MS ，Manik RS ... -  《ANIMAL REPRODUCTION SCIENCE》

Effect of type of cryoprotectant on morphology and developmental competence of in vitro-matured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or vitrification.

Gautam SK ，Verma V ，Palta P ，Chauhan MS ，Manik RS ... -  《REPRODUCTION FERTILITY AND DEVELOPMENT》

Maturation rates of vitrified-thawed immature buffalo (Bubalus bubalis) oocytes: effect of different types of cryoprotectants.

Cryopreservation of oocytes collected from slaughtered animals of high genetic value, their subsequent utilisation for production of embryos for transfer may provide an opportunity to replenish the valuable germplasm lost. Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH) and glycerol at different concentrations (3.5, 4, 5, 6 and 7 M each with 0.5M sucrose and 0.4% BSA in DPBS) on morphological survival and in vitro maturation of vitrified-thawed immature buffalo oocytes. The cumulus oocyte complexes were harvested from the ovaries obtained from a local slaughterhouse by aspirating the visible follicles. Less number of oocytes reached metaphase-II stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation (40.3, 42.5, 40.4 and 23.5%) was obtained in 7 M DMSO, EG, PROH and glycerol, respectively. Oocytes reaching to M-II stage from the oocytes cryopreserved in 7 M glycerol were significantly lower than that of the oocytes vitrified in 7 M DMSO, EG and PROH. It can be concluded that 7 M solutions of DMSO, EG and PROH can be used for vitrification of immature buffalo oocytes for subsequent utilisation of these oocytes in IVM/IVF and embryo production for transfer.

Wani NA ，Misra AK ，Maurya SN 《ANIMAL REPRODUCTION SCIENCE》

Effect of different combinations of cryoprotectants on in vitro maturation of immature buffalo (Bubalus bubalis) oocytes vitrified by straw and open-pulled straw methods.

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open-pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 +/- 1.9% in EG, 47.5 +/- 3.4% in EG + DMSO, 36.8 +/- 1.2% in EG + glycerol and 29.9 +/- 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 +/- 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase-I or metaphase-II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 +/- 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 +/- 0.6% for EG + glycerol and 17.0 +/- 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.

Mahmoud KG ，Scholkamy TH ，Ahmed YF ，Seidel GE Jr ，Nawito MF ... -  《-》

Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants.

Lim JM ，Ko JJ ，Hwang WS ，Chung HM ，Niwa K ... -  《THERIOGENOLOGY》