Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification

The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.     

Morphological changes of in-vitro-produced bovine blastocysts after vitrification,in-straw direct rehydration,and culture

Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In-vitro-produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two-step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in-straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re-assembled in a central area forming a small blastocoele, cell adhesion structures were re-established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure. Mol. Reprod. Dev. 48:9–17, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

Successful vitrification of bovine blastocysts on paper container

Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P < 0.05) than those obtained from non-vitrified blastocysts (38.0 and 32.7%) but were comparable (P > 0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos.     

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.     

Successful vitrification of bovine blastocysts,derived by in vitro maturation and fertilization

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74–77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7–0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts. © 1993 Wiley-Liss, Inc.     

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).     

The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and warmed using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199 + 20% CS with PBS + 20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinylalcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification.     

Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing

Factors affecting viability of IVF-derived bovine blastocysts after freezing and thawing were investigated. A total of 1,101 ova matured and fertilized in vitro were cultured under 2 different conditions, 1) in TCM-199 on granulosa cell monolayers at 5% CO(2) in air and 2) in synthetic oviduct fluid (SOF) medium without somatic cell support at 5% CO(2), 5% O(2), 90% N(2). All blastocysts that developed from the 2 different culture systems were individually classified into 4 grades of embryo quality and were then frozen by conventional slow freezing. Developmental rates of the IVF-derived ova to blastocysts and the survival rates of the frozen-thawed blastocysts were not different between the SOF medium (16 and 49%) and the co-culture system (13 and 61%, respectively). Survival of frozen-thawed blastocysts was affected by embryo quality in both the SOF and co-culture systems (P<0.001). Blastocysts produced in vitro were also individually classified into 3 developmental stages and were then cultured for 3 d in the co-culture system with granulosa cells after freezing and thawing. There was a difference in the survival rate of frozen-thawed embryos between blastocyst developmental stages (early vs mid, P<0.05; mid vs expanded, P<0.01; early vs expanded, P<0.001). The post-thawing survival rate of blastocysts frozen at Day 7 (62%) of culture was higher compared with that of Day 8 (45%), but there was no difference in survival rate between Day 7 and 8 of culture. The results indicate that the quality and developmental stage of blastocysts are important factors influencing their survival after freezing and thawing.     

TUNEL analyses of bovine blastocysts after culture with EGF and IGF-I

Experiments were carried out to investigate the beneficial effects of IGF-I or EGF on bovine embryo development in chemically defined embryo culture media and resultant incidences of nuclear DNA fragmentation as an indication of embryo quality. Presumptive IVF zygotes were randomly cultured in either control (with no added growth factor) or treatment groups, i.e., with 50 ng/ml IGF-I (experiment 1) or 5 ng/ml EGF (experiment 2). IGF-I supplemented to culture media significantly improved proportions of blastocysts from oocytes inseminated compared to untreated controls (38.0% vs. 28.5%). Only embryos reaching the blastocyst stage on day 8 showed significant effects of IGF-I treatment by resulting in higher blastocyst cell numbers (162 vs. 141) and lower percentages of TUNEL positive nuclei (2.1% vs. 3.3%) when compared to controls. Blastocyst development from oocytes was also improved by EGF supplementation compared to untreated controls (38.5% vs. 30.7%). Cell numbers of either day 7 or day 8 blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei when compared with controls. Similar proportions of parthenogenetically activated oocytes developed to blastocysts as for inseminated oocytes (28.8%). Parthenogenetic blastocysts contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than were found for IVF embryos.     

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.     

Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro

We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.     

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.     

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.     

Effect of lecithin on in vitro and in vivo survival of in vitro produced bovine blastocysts after cryopreservation

The use of soybean lecithin in an glycerol-based solution for slow freezing of in vitro matured, fertilized and cultured (IVMFC) bovine embryos was examined. Embryos were developed in vitro in INRA Menezo's B2 medium supplemented with 10% fetal calf serum (FCS) on Vero cells monolayers. Day 7 blastocysts were frozen in a two-step protocol consisting of exposure to 5% glycerol and 9% glycerol containing 0.2 M sucrose in F1 medium + 20% FCS. Soybean lecithin was either added or not to the freezing solutions at a final concentration of 0.1% (w/v). In Experiment 1, blastocysts were equilibrated in cryoprotectant solutions without cooling. Cryoprotectant was diluted from embryos with 0.5 M and 0.2 M sucrose. The percentages of fully expanded and hatched blastocysts treated with or without lecithin after 24 and 48 h in culture were not significantly different (100 versus 100% and 93.3 versus 100%, respectively). In Experiment 2, the in vitro survival of frozen-thawed IVMFC blastocysts was compared when cryoprotectant solutions were either supplemented or not with lecithin. No significant effect of lecithin was found on the ability of frozen-thawed blastocysts to re-expand after 48 h in culture (65.6 and 54.2%, respectively). However, the post-thaw hatching rate of embryos cryopreserved in the presence of 0.1% lecithin was significantly higher after 72 h in culture (52 and 31.8%, respectively). In Experiment 3, the ability of frozen-thawed IVMFC blastocysts to establish pregnancy following single embryo transfer was determined. Transfers of 58 and 66 frozen-thawed embryos cryopreserved with or without lecithin resulted in 6 and 10 (10.3 and 15.1%, respectively) confirmed pregnancies at Day 60. Addition of lecithin to cryoprotectants did not improve the in vivo development rate of cryopreserved IVMFC bovine blastocysts.     

Postthaw survival of in vitro-produced bovine blastocysts loaded onto the inner surface of a plastic vitrification straw

In this study, we investigated whether vitrification of an embryo by attachment to the inner surface of a plastic straw, which requires a small volume of vitrification solution, improves the survival of thawed embryos. In vitro-produced Korean native cattle blastocysts were randomly assigned into four groups: (1) blastocysts attached to the inner surface of a plastic straw (aV); (2) blastocysts loaded into the column of a plastic straw (cV); (3) blastocysts directly dropped into liquid nitrogen (dV); and (4) nonvitrified blastocysts (control). The postthaw recovery rate did not significantly differ among the aV, dV, and cV groups (98.3% vs. 81.5% vs. 91.4%). The postthaw survival rate was greater in the control, aV, and dV groups than in the cV group (100%, 87.7%, and 81.8% vs. 26.4%, P < 0.05), but did not significantly differ among the control, aV, and dV groups. The total number of cells per blastocyst did not significantly differ among the groups (134.4 ± 38.9 in control vs. 114 ± 48.1 in aV, 105.6 ± 33.9 in dV, and 102 ± 35.1 in cV group). However, the number of apoptotic cells per blastocyst was higher in the dV and cV groups than in the control group (10.9 ± 9.6 and 14.5 ± 9.5 vs. 0.4 ± 1.4; P < 0.05), but did not significantly differ between the control and aV groups (0.4 ± 1.4 vs. 6.6 ± 9.5). In addition, the blastocoel of each blastocyst was left intact or was mechanically punctured to reduce its volume, and the blastocysts were then vitrified using the aV method. At 12 hours after thawing, the re-expansion rate did not significantly differ among the control, punctured aV, and nonpunctured aV groups (93.3% vs. 85.2% vs. 82.8%). However, at 24 hours after thawing, the hatching rate was greater in the control and punctured aV groups than in the nonpunctured aV group (75% and 62.9% vs. 37.1%; P < 0.05). The total number of cells per blastocyst was greater in the control group than in the nonpunctured aV group (143 ± 37.2 vs. 94.5 ± 18.6; P < 0.05), but did not significantly differ between the control and punctured aV groups (143 ± 37.2 vs. 119.4 ± 19.7). The number of apoptotic cells per blastocyst was greater in the nonpunctured aV group than in the control and punctured aV groups (11.3 ± 6.1 vs. 5.9 ± 5.8 and 6.3 ± 4.4; P < 0.05). Taken together, the data show that the aV method improved the survival and quality of vitrified-thawed blastocysts. Furthermore, puncture of the blastocoel increased the hatching rate and reduced the number of apoptotic cells in vitrified-thawed blastocysts generated using the aV method.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.