Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants.

A total of 498 porcine embryos at various stages of development collected from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blastocysts (HB) were used to determine the effect of exposure to concentrated solutions of ethylene glycol as cryoprotective additives (CPAs) on embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethylene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solution of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. To vitrify the embryos, they were exposed to 2.0 M EG for 5 min and then were pipetted directly into short columns of 8.0 M EG-PVP contained within (1.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, the embryos were immediately mixed with galactose within the straws by shaking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method 1: Embryos in the EG-PVP-galactose mixture were expelled from the straws and rinsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cultured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. After 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method II, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB vitrified embryos diluted by Method III and transferred into a recipient, four developed into live piglets; two other recipients failed to litter although one had been pregnant for 65 days. These results demonstrate that porcine embryos can be successfully cryopreserved by rapid cooling in EG-PVP and by careful dilution of the CPA after warming.

Kobayashi S ，Takei M ，Kano M ，Tomita M ，Leibo SP ... -  《CRYOBIOLOGY》

The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts.

A total of 678 bovine blastocysts, which had been produced by in vitro maturation, fertilization, and culture, were placed into plastic straws and were vitrified in various solutions of ethylene glycol (EG) + polyvinylpyrrolidone (PVP). Part of the straw was loaded with TCM199 medium + 0.3 M trehalose as a diluent; the diluent portions of the straw were prefrozen to either -30 or -196 degrees C. Then, the embryos suspended in the vitrification solution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degrees C and then agitated to mix the diluent and cryoprotectant solution for 5 min followed by culture in TCM199 + 10% FCS + 5 + microg/ml insulin + 50 microg/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitrification, the PVP concentration, and the temperature of exposure to EG + PVP prior to vitrification. Survival and hatching rates of the blastocysts exposed to 40% EG in four steps at 4 degrees C were higher than those of embryos exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% and 71.5 +/- 4.7%, respectively; P < 0.05). The same indices were superior following vitrification-thawing of the blastocysts in 40% EG + 20% PVP than it was in 40% EG + 10% PVP (76.1 +/- 5.5% vs 63.7 +/- 1.8%; P < 0.05; and 61.6 +/- 6.0% vs 70.5 +/- 4.7%; P < 0.01, respectively). Exposure to the vitrification solution (40% EG + 20% PVP) at higher temperatures (37.5 degrees C vs 4 degrees C) reduced both survival and hatching rates (45.8 +/- 6.9% vs 83.9 +/- 4.4% and 41.5 +/- 1.8% vs 64.0 +/- 4.7%, respectively; P < 0.001). These results indicate that blastocysts vitrified after prefreezing the diluent portions of the straws do favor developmental competence of in vitro produced embryos.

Mtango NR ，Varisanga MD ，Dong YJ ，Otoi T ，Suzuki T ... -  《CRYOBIOLOGY》

In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

Campos-Chillòn LF ，Walker DJ ，de la Torre-Sanchez JF ，Seidel GE Jr ... -  《THERIOGENOLOGY》

Cryopreservation of porcine blastocysts by vitrification.

The objective of this study was to assess the possibility of cryopreservation of porcine expanded and hatched blastocysts by vitrification. The following four types of vitrification solutions were applied in this study: (i) EFT, a mixture of 7.2 M ethylene glycol, 0.003 M ficoll, and 0.3 M trehalose; (ii) DAP213, 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol; (iii) DAP213-T, DAP213 supplemented with 0.3 M trehalose; and (iv) EPT, 4 M ethylene glycol, 3.1 M propylene glycol and 0.3 M trehalose. The embryos collected on Days 5 to 7 (Day 0 = onset of estrus) were allocated to eight experimental groups according to the types of vitrification solution and the developmental stage. In the toxicity test, the embryos were equilibrated in the respective vitrification solutions either in a single step or in four steps and then transferred to 1 M sucrose in a single step at 22 degrees C without cooling. As a whole, the viabilities of embryos equilibrated in the solutions were lower than those of control embryos. The stepwise equilibration was superior to a single-step equilibration in the in vitro survival of, especially, expanded blastocysts after dilution. No significant difference was observed between the vitrification solutions for the four-step method. In the vitrification test, the embryos were equilibrated in the solutions by the four-step method, loaded into a 0.25-ml plastic straw, and plunged into liquid nitrogen. Although viable embryos were obtained after warming from all of the combinations except the hatched blastocyst-EPT group, viabilities were further reduced by cooling (range of reduction rates: 60 to 100%). The possible cause of low survival after warming is also discussed concerning the cryophysical properties of vitrification solutions.

Yoshino J ，Kojima T ，Shimizu M ，Tomizuka T ... -  《CRYOBIOLOGY》

Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification.

In this study, the cryoprotectant ethylene glycol (EG) was tested for its ability to improve and facilitate the cryopreservation of in vitro produced (IVP) bovine embryos. Embryos were cryopreserved in EG solutions supplemented with either newborn calf serum (NBCS) or polyvinyl alcohol (PVA). To assess EG toxicity, the embryos were equilibrated in EG concentrations from 1.8 to 8.9 M at room temperature for 10 min and then cultured for 72 h on a cumulus cell monolayer. The hatching rate was highest for day 7 blastocysts frozen in 3.6 M EG (98%) and was not different from the control group (85%). The controlled freezing (0.3 degrees C/min to -35 degrees C) of expanded day 7 blastocysts resulted in a hatching rate of 81%, which was similar to that of the nonfrozen controls (76%). Differential staining revealed only very few degenerate blastomeres attributed to freezing and thawing. Upon direct nonsurgical transfer of day 7 expanded blastocysts frozen in 3.6 M EG, a pregnancy rate of 43% was achieved, while the pregnancy rate after transfer of other developmental stages was significantly lower (22% with expanded day 8 blastocysts). When bovine IVP embryos were incubated at room temperature in 7.2 M EG preceded by preequilibration in 3.6 M EG, the hatching rate of day 7 expanded blastocysts reached 93%. Upon vitrification of IVP day 7 and day 8 blastocysts and expanded blastocysts in 7.2 M EG, the latter showed a higher hatching rate (42%) than blastocysts (12%). Overall, PVA as supplement to the basic freezing solution instead of NBCS had deleterious effects on survival after controlled freezing or vitrification. The simple cryopreservation protocol employed in this study and the low toxicity of ethylene glycol highlight the usefulness of this approach for controlled freezing of IVP embryos. However, further experiments are needed to improve the pregnancy rate following embryo transfer and to enhance survival after vitrification.

Sommerfeld V ，Niemann H 《CRYOBIOLOGY》