Osmotic characteristics of sheep and cattle embryos

100

Abstract.To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development. Key words: Mouse, Embryo, Oocyte, Permeability, Cryoprotectant (J. Reprod. Dev. 51: 235-246 , 2005) he survival of mammalian oocytes and embryos after cryopreservation varies with the stage of maturation and development. In general, oocytes/embryos at earlier stages appear to be more sensitive to cryopreservation. In the mouse, the rate of survival after cryopreservation is lower for unfertilized oocytes than embryos and increases as development proceeds up to the 8-cell or morula stage, although it may decrease as the blastocoel enlarges Cryoprotectants for mammalian oocytes and embryos are virtually limited to these five agents both in slow freezing and in vitrification [8].Although the protective action of cryoprotectants is considered colligative [9], each agent has its own specific properties. Among them, the permeating p r o p e r t y i s o f g r e a t i m p o r t a n c e , b e c a u s e permeation of the cell with a cryoprotectant is critical for the successful cryopreservation of

mentioning

confidence: 99%

Permeability of Mouse Oocytes and Embryos at Various Developmental Stages to Five Cryoprotectants

Pedro

Yokoyama

Zhu

et al. 2005

100

“…Széll et al [15] and Somgsasen et al [16] found an enhanced permeability for EG in ovine embryos than other cryoprotectant. In addition, they found an improvement in the post-thaw survival rate of embryos frozen with EG.…”

Section: Discussionmentioning

confidence: 96%

“…However, the survival rates of ET and IVC of frozen-thawed molurae were higher than those of blastocysts (Tables 2 and 3). Some workers reported a higher conception rate by ET of morulae frozen with EG rather than blastocysts [5,15]. The differences in the survival rate between treatments for morula have been explained by differences in the permeation rates associated with cryoprotectants in early developmental stage of the embryos [15].…”

Section: Discussionmentioning

confidence: 99%

Non-Surgical Transfer of Fresh or Frozen-Thawed Ovine Embryos by Laparoscopy.

Ishida

Jung

Itagaki

et al. 1999

Abstract. The present study was conducted to examine survival of fresh and frozen-thawed ovine embryos after embryo transfer (ET) using laparoscopy or in vitro culture. Embryos were collected on Day 7.5 (Day 0 =sponge removal) from donor ewes superovulated by pre-treatment of progestogen-impregnated intravaginal sponges (FGA) for 12 days and a single injection of follicle stimulating hormone (FSH) and equine chorionic gonadotrophin (eCG), at 2 days and 1 day before sponge removal. The donor ewes showed estrus between 12 h and 30 h (the mean time: 15.6 h) after sponge removal. Embryo transfer of fresh or frozen-thawed embryos was carried out on Day 7.5 in 37 recipient ewes using laparoscopic technique. Frozen-thawed morulae or blastocysts were cultured to examine their viability in a modified synthetic oviduct fluid medium (SOFM) for 24 h. The lambing rates after ET with fresh and frozen-thawed embryos were 33.3% (6/18) and 26.3% (5/19), respectively. The developmental stage (morula or blastocyst) affected the survival rate of frozenthawed embryos transferred (54.5% and 0%, respectively). The survival rate (development to blastocyst or hatched blastocyst) of frozen-thawed embryos after 24 h in culture was 40.9% (9/22). The present results indicate that ovine ET using laparoscopy is a useful technique. But, the lambing and survival rates of both fresh and frozen-thawed embryos were low, and remained to be improved.

“…It has been shown that bovine embryos are more permeable to EG than to GLY, but the former is more toxic [3,18]. Therefore, the combination of EG and GLY is assumed to reduce not only the toxicity of each cryoprotectant but also the osmotic damage at warming because EG is more likely to diffuse out of the cell rapidly.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown that bovine blastocysts are less permeable to GLY than to EG and that the permeating speed of the former is slower than that of the latter [18]. Permeation of a certain amount of cryoprotectant into the embryo, which increases as the duration of exposure increases, has been suggested to be an essential factor in cryosurvival [3].…”

Section: Vitrification Of Ivf and Scnt Embryosmentioning

confidence: 99%

Effect of Cryoprotectant Composition on In Vitro Viability of In Vitro Fertilized and Cloned Bovine Embryos Following Vitrification and In-Straw Dilution

Taniguchi

Ikeda

Arikawa

et al. 2007

Abstract. In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.