Nuclear maturation of immature bovine oocytes after vitrification using open pulled straw and cryotop methods

To date, at least two well known methods have been widely used for vitrification of oocytes and embryos at different stages in a variety of species. However, there is no reported data regarding the comparative effectiveness of these two methods for vitrification of immature bovine oocytes. The objective of this study is to compare the nuclear maturation of immature bovine oocytes vitrified using open pulled straw (OPS) and cryotop methods. Two experiments were conducted in this study. In the first experiment, cytotoxicity of vitrification solutions (VS) from both methods was studied. After removal of cryoprotectants, cumulus oocyte complexes (COCs) was cultured in vitro and cleavage rate was monitored on Day 2 post-insemination (pi), whereas, morulae and blastocyst yields on Days 5 and 8 pi, respectively. The VS solutions significantly reduced zygotic cleavage rate, morulae and blastocyst rates compared with the control group (P < 0.05). The lowest cleavage rate resulted from prolonged exposure time to OPS-VS solutions (35.1%; P < 0.05). However, the morulae and blastocyst rates were significantly higher (P < 0.05) for embryos derived from oocytes exposed to cryotop solutions (40.5 and 22.4%, respectively). In the second experiment, effectiveness of both vitrification methods was compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop solution and device showed higher percentages of PB+ (36%) and MII (51%) rates. In addition, the lowest percentage of degenerated oocytes resulted from cryotop solution. The highest degenerated oocytes obtained by equilibration in OPS solution and vitrified using OPS device (40%; P < 0.05). In conclusion, our data demonstrated that cryotop solution was less toxic to the immature bovine oocytes and vitrification with the cryotop method resulted in higher survival and nuclear maturation rates.Key words: Immature oocyte, bovine, vitrification, cryotop, open pulled straw (OPS)

Effects of exposure to dmso in vitrification solution on cytotoxicity and in vitro viability of immature bovine oocyte.

Based on previous studies for vitrification of oocytes, it has been shown that short term exposure to DMSO during vitrification could improve the maturation rate and cause not spontaneous parthenogenesis (Isachenko et al., 2006). In addition, it was reported that DMSO in freezing media caused disassembly of microfilaments and chromosomal abnormalities in mouse oocytes (Vincent et al., 1990). On the other hand, DMSO is categorized as a potent glass former and its existence in vitrification solution seems necessary. The aim of this study was to determine the in vitro viability of immature bovine oocytes vitrified by short or long time exposure to DMSO. Materials and Methods: Cumulus oocytes complexes (COCs) with homogenous ooplasm were recovered from slaughterhouse ovaries and used in this study. The vitrification protocol was adapted from Kuwayama et al (2005) with minor modifications. Briefly, oocytes were washed twice in holding solution (HS, Hepes-buffered TCM medium supplemented with 20% fetal calf serum, FCS) and kept there for about 15 min. Group of four COCs were incubated in the first vitrification solution (VS1; 7.5% DMSO and 7.5% EG in HS) for 12 min. Equilibration in VS1 was performed in three steps of increasing concentration. First (F) and second (S) steps contained 1/3 and 2/3 of VS1 diluted in HS, and the third (T) step contained only pure VS1. Based on removal of DMSO from each step, five treatment groups were designed: (G1) control, (G2) VS1, (G3) F w/o DMSO, (G4) F+S w/o DMSO, and (G5) F+S+T w/o DMSO. For G3, G4 and G5, similar concentration of EG was added to replace DMSO in VS1. All treatment groups were equilibrated into the second vitrification solution (VS2; 15% DMSO, 15% EG and 0.5M sucrose in HS) for a further 60 sec. Two experiments were performed: (a) cytotoxicity after only exposure, and (b) in vitro viability after vitrification processes. In cytotoxicity test, immature oocytes were directly transferred to the warming solution (WS). In vitrification experiment, oocytes were instantly loaded on a Cryotop device and submerged into liquid nitrogen (LN2) for storage. The time of exposure from VS2 to LN2 was not longer than 90 s. Vitrified samples were maintained in LN2 for at least 10 days. Immediately after removing the Cryotop from LN2, thin strip of Cryotop was submerged in 3 ml HS plus 1M sucrose (WS; 39°C) and smoothly tried to detach oocytes from Cryotop device. Immature oocytes were left in WS for one minute and then transferred to HS plus 0.5M and 0.15M sucrose solution for 3 and 5 min, respectively. Finally, the immature oocytes were washed twice in HS for 5 min each and processed for in vitro maturation. Significant differences among treatments used in the experiment were revealed by one-way analysis of variance and followed by Duncan's multiple range test for mean comparisons (P < 0.05) using SAS software (ver. 9.1)

Cryotop and development of vitrified immature bovine oocytes

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10% DMSO + 10% ethylene glycol (EG) for 30-45sec and 2: 20% DMSO + 20% EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58% respectively. Vitrified oocytes using OPS and EMG showed 26 and 32%; and 35 and 46% of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocyte