| Since the first successful cryopreservation of mammalian embryos, cryopreservation of bovine embryos has been widely used commercially. The equipment required for traditional slow freezing is relatively expensive, the process is time-consuming, and survival rates depend on the quality and developmental stage of embryos produced in vitro, but they are generally low. Vitrification seems to be a promising way to resolve most of these problems as it can be done in a simple container of liquid nitrogen, the time required for incubation is short and the reported in vitro and in vivo survival rates are reasonable. However, in the past30years, at least20different combinations of cryoprotectants have been published for vitrification of mammalian oocytes and embryos. The number of variations regarding the concentrations, incubation times and other conditions is almost infinite. There are opportunities for simplifying these vitrification methods for production. This study have analysed some factors which affectted the vitrification efficiency of bovine embryos. The first part focused on protocols of cryoprotecctants(CPAs) adding and diluting that might affect vitrification; the second part focused on discussing relationship between incubation time and the concentration of intracellular cryoprotectants(ICP); Using the conclusions from the former two experiments we optimized the two-step vitrification and in-straw dilution method in bovine blastocysts vitrification. Results show that:(1) MIl stage bovine oocytes equilibrated by EG20for5min and then transferred to ESI (40%EG,0.3M sucrose in DPBS) or ES3(40%EG,0.8M sucrose in DPBS) for30s. After this two-step treatment, oocytes were diluted by DPBS,0.25M sucrose or0.5M. Oocytes’survival rates of ESI groups was3.33%,56.67%and76.67%respectively for diluting by DPBS,0.25M sucrose or0.5M sucrose; Was56.67%,83.33%and96.67%respectively for ES3group. These results demonstrate that raise the sucrose concentration in vitrification solutions could yield better oocytes survival rate after diluting with lower concentration dilution medium.(2) Bovine blastocysts after EG20equilibrated for5min, transferred to EG40, ESI, ES2or ES3(with40%EG in each solutions, but sucrose concentration was0,0.3,0.5and0.8M) for30s. After diluted by DPBS, the survival rates were90.32%,93.33%,86.60%,75.00%respectively. The EG40group (without sucrose), also got a high survival rate after DPBS directly diluting(90.32%). Which means even without sucrose in the vitrification solution, could also dilute bovine blastocysts by DPBS directly.(3) After oocytes incubated in EG20for5min, EG40for30s, transferred them to Gly30, Gly40or Gly50. Oocytes expansion can be observed in Gly30and Gly40but not in Gly50. Which suggests that, after the two-step EG treatment, the intracellular oosmotic pressure was higher than Gly30and Gly40, ranged between Gly40to Gly50.(4) After oocytes treated by EG20for5min and EG40for30s, the oocytes’volume was about half of its initial volume, at this point transferred oocytes to a solution with6.9M (38.3%v/v) EG in2×DPBS. In this solution oocytes hold their volume at this level. This result suggests at this point the intracellular osmotic pressure was equilibrated with the extracellular. The ionic concentration and EG concentration was equal, means the intracellular EG concentration was6.9M at this point. As calculated above, after oocytes equilibrated with EG20for5min, when transferred to ES3, the intracellular EG could reach a concentration about7.55M.(5) Since oocytes in EG40just for a short period(30s) and at4℃, we hypothesis this step’s main function was to make dehydration. We omitted the EG40step, oocytes were pretreated in EG20for5min, exposed to pre-cooled (4℃) Gly50, for30sec, and then dipped into liquid nitrogen. After warming,81.08%of the oocytes survived, and the surviving oocytes developed into embryos at cleavage (63.49%) and blastocyst (19.96%) stages after parthenogenetic activation. Nevertheless glycerol was considered to have minimal permeability to oocytes, these data demonstrated that in a two-step vitrification procedure, the permeability of the second step is not necessary; it is possible that the second step is only required to provide a high enough osmotic pressure to condense the intracellular CPA concentrated to a level necessary for successful vitrification.(6) Bovine blastocysts equilibrated with EG20for5min or3min, then in EFS1for30s for vitrification. After warming, EG20treating for3min group got a blastocysts re-expansion rate of79.41%, higher than EG20treating5min group71.42%(P<0.05), which indicate a better permeability of EG to blastocysts, preatreat3min in EG20could be better equilibrated already. Prolong the pre-treatment time will introduce extra toxicity; Raising sucrose concentration from0.3to0.5M in the EFS40solution, could slightly improve the survival rate after blastocyst freezing and warming (83.33%vs.80.65%). But, when the concentration of sucrose raised to0.8M, the survival rate decreased sharply (19.44%), indicates a higher toxicity was introduced. While we reduced the EG concentration to35%in EFS4group, we got better blastocysts re-expansion rate after vitrification and warming.Conclusions:During two-step vitrification, if proper pre-treatment was taken, the second step’s main function was to provide a high osmotic pressure to concentrate the intracellular CPA to a level facilitates vitrification. The second step did not need any more CPA permeate into cells; It is better that when cells shrunk to the minimum volume in the vitrification solution put them into liquid nitrogen for cryopreservation, that needs no more than30s to complete this procedure; There are cases that in a two-step vitrification protocol, the second step’s ICP could higher than cryoprotectant concentration of extracellular. These conclusions could help researchers to understand vitrification more simply and may conduct them yield better result in practice. |
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