Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females

Four experiments were conducted to define a system for the direct transfer of frozen-thawed bovine embryos to recipient females. In Experiment I, nonsurgically recovered embryos were frozen in 1.5 M ethylene glycol (EG), 1.5 M propylene glycol (PG), 1.5 M DMSO or 1.4 M glycerol (GLY), and then thawed and placed directly into holding medium. Viability at 72 hours of post-thaw culture was 70, 11, 25 and 30% for the four groups, respectively. In Experiments II and III, 1.0, 1.5 and 2.0 M concentrations of EG were compared; a concentration of 1.5 M appeared to provide optimal cryopreservation and survival after direct rehydration. In Experiment IV, embryos were packaged in straws containing only 1.5 M EG, in straws containing a column of 1.5 M EG and the embryo and two columns of PB1 in a 1:3 ratio of volumes (EG PB1 ), or were frozen in 1.4 M glycerol. After thawing, embryos in EG and EG PB1 treatments were transferred directly to recipient females, while embryos frozen in GLY were rehydrated using a three-step procedure. In the first trial, pregnancy rates at approximately 60 days of gestation for embryos frozen in EG and GLY groups were 39 and 62%, respectively (P<0.10). In the second trial, the pregnancy rate for embryos frozen in EG PB1 was equal to that of embryos frozen in GLY (50% in both groups). These experiments demonstrate the potential for using ethylene glycol as a cryoprotectant for bovine embryos, thus permitting direct transfer of frozen-thawed embryos to recipient females.     

Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5 degrees C, 5% CO(2)) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0 degrees C, cooled from 0 degrees C to -6 degrees C at -1 degrees C/minute, seeded, held for 10 minutes, and cooled again at -0.3 degrees C or -0.5 degrees C/minute to -30 degrees C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30 degrees C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at -0.3 degrees C vs -0.5 degrees C/minute was not significantly different (P>0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of -0.3 degrees C/minute (64.6%, 31 48 ) than at -0.5 degrees C/minute (22.6%, 12 53 ). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10 21 ); DEG (30%, 3 10 ); EG (74%, 20 27 ); and PG (40%, 4 10 ). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.     

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.     

Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose

The present study was designed to examine the suitability of ethylene glycol as a cryoprotectant for equine embryos. Blastocysts recovered nonsurgically from Day 6 donor mares were cryopreserved by conventional 2-step freezing in the presence of 10% ethylene glycol (EG), 10% glycerol (Gly), or 10% ethylene glycol + 0.1M sucrose (EG + Suc). After thawing, the EG and Gly were removed by a 6-step manner, and the EG + Suc was diluted to one fourth in the freezing straw. The postthaw blastocysts were transferred nonsurgically into the uteri of recipient mares on Days 4 to 7 after ovulation. Pregnancy rates, based on Day 15 ultrasonography, were 25.0% (2/8) and 37.5% (3/8) for the blastocysts frozen in EG and Gly, respectively. Direct transfer following thawing and in-straw dilution of blastocysts frozen in EG + Suc resulted in a pregnancy rate of 63.6% (7/11). In fresh Day 6 blastocysts (control group), the pregnancy rate was 70.0% (7/10). These results indicate that the combined use of ethylene glycol and sucrose in a 2-step freezing regimen allows for the direct transfer of frozen-thawed blastocysts into recipient mares, with an acceptable pregnancy rate.     

Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.     

Methanol as a cryoprotectant for equine embryos

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.     

Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants

Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol (DEG) or 1.3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline. (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0.25-ml plastic straws and were placed directly into a 0 degrees C alcohol bath chamber and held for 2 min. They were cooled from 0 degrees C to -5.5 degrees C at 1 degrees C/min and then seeded, followed by a 10-min holding period at -5.5 degrees C. The straws were then cooled to -30 degrees C at 0.3 degrees C/min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05), between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Cryopreservation of sheep embryos using ethylene glycol

The objective of the study was to evaluate the use of ethylene glycol (EG) for cryopreservation of sheep embryos. A 2 × 2 factorial treatment arrangement examining one-step vs. two-step cryoprotectant addition and removal was used. The one-step cryoprotectant addition involved placement of embryos directly into 1.5 mol EG, whereas the two-step addition utilized an intermediate 10 min exposure to 0.75 mol EG. Similarly, the one-step cryoprotectant removal involved direct placement of thawed embryos into 1.0 mol sucrose, and the two-step procedure included a 10 min exposure to 0.25 mol sucrose before placement in 1.0 mol sucrose. A total of 185 frozen-thawed embryos was placed into in vitro culture for 96 h to determine viability. No differences were observed between cryoprotectant addition or removal techniques, and overall survival was 69%. To validate the results obtained in vitro, a limited number of embryo transfers was performed. Four ewes receiving a total of 11 frozen-thawed embryos produced eight lambs (73% survival) which compared favorably with 74% survival obtained by transferring 19 non-cryopreserved embryos to eight recipients. It is concluded that one-step addition of 1.5 mol ethylene glycol followed by one-step removal in a 1.0 mol sucrose gradient is an appropriate technique for cryopreservation of sheep embryos.     

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.     

Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer

This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; P<0.001) or hatched after 48 h (15% vs. 59%; P<0.001). When blastocysts only were considered, an interaction between VS and additional PVP+trehalose was also observed (P<0.01). Hatching was reduced when they were added to 25% EG+25% DMSO (70% vs. 45%) but was not affected for either 40% EG (44 and 49%) or to 20% EG+20% DMSO+10% BD (72 and 72%). Pregnancy rates (Day 90 ultrasound) of recipients that were transferred either two non-vitrified or two vitrified (20% EG+20% DMSO+10% BD) blastocysts, did not differ (3/6 [50%] and 11/20 [55%]). However, significantly (P<0.02) fewer recipients that received compact morulae maintained pregnancy to Day 90 although this was not affected by vitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.     

应用乙二醇冷冻小鼠胚胎：优化和简化程序的探索

提高解冻胚胎的发育能力和简化冷冻解冻程序是胚胎冷冻研究的两大永恒的主题。尽管乙二醇（EG）广泛用于家畜胚胎冷冻,但很少用于冷冻小鼠和人胚胎。为数很少的以EG慢冻小鼠或人胚胎的研究均采用较为复杂的人胚冷冻程序,未见简化程序和用EG冷冻小鼠桑椹胚的报道。采用简单的牛胚胎冷冻程序研究了发育时期、EG浓度、平衡方法、添加蔗糖以及解冻后脱除EG等对小鼠胚胎冻后发育能力的影响。结果显示：（1）致密晚期桑椹胚冻后体外培养囊胚发育率（81.92%±2.24%）和孵出率（68.56％±2.43%）显著（P＜0.05)高于4-细胞、8-细胞胚胎和致密早期桑椹胚胎;（2）1.8mol/L EG冷冻小鼠致密晚期桑椹胚的囊胚发育和孵出率显著高于其它浓度;（3）在EG中平衡10min的冻后囊胚发育显著好于平衡5、20或30min;（4）两步平衡冷冻胚胎的囊胚发育率和孵出率显著高于一步平衡;（5）用EG冷冻小鼠胚胎无需添加蔗糖;（6）解冻后可不脱除EG;（7）冻后发育的早期囊胚和囊胚细胞数明显少于体内发育胚胎。因此,用EG冷冻小鼠胚胎的最佳方案为：致密晚期桑椹胚用1.8mol/L EG不添加蔗糖、两步平衡15min、以简单的牛胚胎冷冻程序冷冻解冻、解冻后不脱除EG直接培养或移植。     

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from n?turally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.     

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.     

Quick-splitting of bovine embryos

Described is a simplified method of bovine embryo bisection amenable to on-farm embryo transfer. Using a microblade operated by a hand-held micromanipulator, Day 7 bovine embryos were bisected while in the zona pellucida. With a vertical motion, the embryo was pinned between the blade and the bottom of a plastic petri dish and bisected. Demi-embryos were transferred nonsurgically (without zonae pellucidae) into synchronized recipients. Pregnancy rates were normal with 5 13 (38%) and 9 20 (45%) of recipients confirmed pregnant 70 to 80 d after receiving either twin or single half embryos, respectively. This compared to 12 28 (43%) of recipients becoming pregnant from transfer of whole embryos. These data confirm that bovine demi-embryos do not need zonae pellucidae on Day 7 and that simplified field methods of bisection give normal pregnancy results.     

Production efficiency of Japanese black calves by transfer of bovine embryos produced in vitro

The objective of this study was to examine the production efficiency of Japanese Black beef calves after transfer of bovine embryos derived from an in vitro procedure. In vitro-produced (IVP) embryos were obtained from in vitro maturation and fertilization and in vitro development by co-culture with cumulus cells until 7 or 8 days after insemination. In vivo-developed (IVD) embryos from superovulated Japanese Black heifers and cows 7 days after artificial insemination were used as a control group. Bovine embryos were transferred nonsurgically to recipient cows on Day 7 +/- 1 of the estrous cycle. Pregnancy was diagnosed by palpation per rectum at Day 60 to 70 after estrus. Pregnancy, abortion, perinatal accident and birth rates were examined according to the origin of embryos (IVP or IVD), the number of transferred embryos (single or twin) and the storage status (fresh or frozen-thawed). In Experiment 1, production efficiency by twin transfer of fresh IVP embryos was examined. Higher pregnancy rates (52 1% vs 42 9%, P < 0.05) and birth rates (47.0% vs. 33.0%, P < 0.05) were obtained by twin transfer than by single transfer of fresh IVP embryos. Thus, the twin transfer of fresh IVP embryos was effective for production of calves, although the birth rates for single and twin transfers of fresh IVD embryos were still higher (55.5% and 76.1%, P < 0.05). But the abortion and perinatal accident rates for twin transfer of fresh IVP embryos were also significantly greater than those for single and twin transfer of fresh IVD embryos (P < 0.05). In Experiment 2, production efficiency by twin transfer of frozen-thawed IVP embryos was examined. Either single or twin transfer of frozen-thawed IVP embryos resulted in a similar pregnancy rate (41.3% vs. 46.7%, P > 0.05) and birth rate (34.1% vs. 41.1%, P>0.05). Thus, in combination with frozen-thawed IVP embryos, the twin transfer did not enhance production efficiency. In conclusion, Japanese Black beef calves could effectively produce calves by twin transfer to Holstein recipients when using fresh IVP embryos, and by single transfer when using frozen-thawed IVP embryos.     

Pregnancies following transfer of equine embryos cryopreserved by vitrification

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO(2) in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.     

Quick freezing of one-cell mouse embryos using ethylene glycol with sucrose

One-cell mouse embryos were frozen by direct plunging into liquid nitrogen (LN(2)) vapor after equilibration in 3 M ethylene glycol with 0.25 M sucrose (freezing medium) for 5 to 40 minutes. After thawing, the embryos were cultured in vitro and the effects of the equilibration period and dilution method were examined. No significant difference was observed in the in vitro survival of embryos when 0.5 or 1.0 M sucrose was used for the dilution of the cryoprotectant for each equilibration period. The highest survival rate (67.2%) was obtained when the embryos were equilibrated for 10 minutes, and the cryoprotectant diluted with either 0.5 or 1.0 M sucrose after thawing. Shorter (5 minutes) or prolonged (40 minutes) equilibration of embryos in the freezing medium yielded significantly lower survival rates. Dilution by direct transfer of the frozen-thawed embryos into PB1 resulted in lower survival rates than when 0.5 or 1.0 M sucrose was used. The in vitro development to the blastocyst stage of one-cell mouse embryos frozen after 10 minutes equilibration in the freezing medium and diluted after thawing in 0.5 M sucrose was significantly lower than the control (68.0 vs 92.7%). However, transfer of the blastocysts developing from frozen-thawed one-cell mouse embryos into the uterine horns of the recipients resulted in fetal development and implantation rates similar to the control.