In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.     

Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.     

Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival

Two experiments were conducted to examine the effect of membrane stabilization through the modification of in vitro culture medium or freezing medium on post-thaw survival of in vitro-produced bovine embryos. In Experiment 1, Day 7 (Day 0 = day of IVF) late morulae and blastocysts that developed following culture in SOF/aa/BSA (IVC medium) were frozen slowly to -35 degrees C in the presence of 1.5 M ethylene glycol prepared in ovum culture medium (OCM) or in OCM supplemented with 10, 25 or 50% fetal calf serum (FCS) or 5, 10 or 25 mg/mL BSA. Post-thaw survival was assessed by re-expansion and/or hatching following 48 h of culture in IVC medium + 10% FCS. Overall, survival was significantly (P < 0.01) affected by embryo stage, with more hatched blastocysts surviving (71%) than blastocysts (59%) or late morulae (51%). Addition of FCS significantly (P < 0.01) reduced survival compared with control embryos or those frozen in BSA-supplemented medium (50.48 vs 68.01 vs 63.53%, respectively). There was also a significant interaction between embryo stage and protein type (P < 0.05). The survival of late morulae/early blastocysts following freezing was improved in the presence of additional BSA but not FCS. In Experiment 2, the IVC medium was supplemented with liposomes containing lecithin, sphingomyelin and cholesterol. Sphingomyelin and cholesterol at ratios of 1:1, 1:4 and 4:1 were added to 50, 100 or 150 micrograms/mL lecithin to yield a final lipid concentration of 200 micrograms/mL. A further group contained 200 micrograms/mL lecithin only. Blastocysts were frozen in 1.5 M ethylene glycol in OCM, then thawed and assessed as in Experiment 1. The presence of liposomes during IVC did not affect the proportion of cleaved embryos that developed to blastocysts or survival following freezing. However, the survival of blastocysts that developed in the presence of 200 micrograms/mL lecithin only was significantly lower than in any other treatment (6%; P < 0.03). These studies demonstrate that the protein composition of the freezing medium can significantly affect survival after thawing and that the survival of late morulae can be improved with additional BSA. The presence of lecithin only in the liposome preparation did not affect embryo development, but significantly reduced survival after freezing, suggesting it can affect post-thaw embryo survival, perhaps by altering embryonic membrane composition.     

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.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

The objective of this study was to determine the effect of fetal calf serum (FCS) on the quality of in vitro produced bovine embryos. Cumulus oocyte-complexes (COCs, n = 2 449) recovered by ovum pick-up from Bos taurus indicus donors were randomly assigned to experimental groups. Sperm selected by Percoll gradient was used for in vitro fertilization (insemination = Day 0). In Experiment 1 (n = 1 745 COCs), zygotes were cultured in vitro in Synthetic Oviduct Fluid + 4 mg/mL of bovine serum albumin (BSA), or BSA + 2% FCS (BSA+FCS). In Experiment 2 (n = 704 COCs), the COCs were cultured in SOF + BSA, BSA + 2% FCS, or BSA + 2% FCS on D4 (BSA + FCSD4). In Experiment 1, blastocyst yield (51%) and Quality I blastocysts (41%) at Day 7 were higher (P < 0.05) in the BSA + FCS treatment than in BSA (42 and 30%, respectively). In Experiment 2, blastocyst yield was higher (P < 0.05) in the BSA+FCS (47%) treatment. Quality I blastocyst yield was higher (P < 0.05) for BSA + FCS (34%) and BSA+FCSD4 (32%) compared to the BSA treatment (20%). A total of 820 embryos were transferred, with no significant differences among groups in pregnancy rates. In conclusion, in vitro culture in SOFaaci + BSA + FCS enhanced blastocyst yield and Quality I blastocysts; adding FCS to the culture medium increased the efficiency of IVP of bovine embryos.     

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition—Control; after 60 h—PES Day 2.5 of embryo culture; and after 96 h—PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification (Control: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts (Control: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Freezability of porcine blastocysts at different peri-hatching stages

The freezability of porcine peri-hatching stage blastocysts was investigated by the cryopreservation of embryos at -196 degrees C with 1.5 M glycerol and by thawing, followed by in vitro culture. Of 66 expanded blastocysts frozen, 34 (51.5%) developed in vitro after thawing, while only 2 (6.7%, P<0.05) of 30 earlier stage blastocysts survived freezing. After freezing of 85 hatched blastocysts with an embryonic diameter of 150 to 300 mum, 59 (69.4%) surviving embryos were obtained, whereas none of the 78 advanced staged hatched blastocysts (>300 mum) survived the cryopreservation. High post-thaw survival (32 39 , 82..1%) was obtained with in vitro-hatched blastocysts precultured in Whittingham's M-16 medium containing 12mg/ml bovine serum albumin (BSA). In contrast, none of the 14 in vitro-hatched blastocysts precultured in the M-16 medium supplemented with 15% fetal calf serum (FCS) survived freezing. Similarly 51 of 56 hatced blastocysts (diameter = 150 to 300 mum) precultured in the M-16 medium supplemented with BSA survived cryopreservation, compared with 3 of 26 embryos precultured in the medium supplemented with FCS (P<0.001). Because both groups of the embryos precultured with BSA or FCS possessed normal ability to develop after transfer (developmental rate = 61.1 and 93.3%), the improved freezability of the embryos precultured with BSA may relate to a favorable change of embryonic cell membranes during the culture period. It was concluded that in vitro-hatched blastocysts precultured in medium containing BSA and in vivo-hatched blastocysts at the appropriate stage of development could both tolerate deep freezing to -196 degrees C; however, a differece in the freezability of embryos between breeds of pig was suggested from a further experiment performed with German Landrace embryos.     

Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage

Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post-warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or 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.     

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.     

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.     

Effect of lipid polarization by centrifugation at different developmental stages on post-thaw survival of bovine in vitro produced 16-cell embryos

The developmental rate to the blastocyst stage of frozen-thawed bovine in vitro produced embryos at stages earlier than Day 6 morula is not sufficiently high for practical utilization. The present study was undertaken to determine the effect of polarization of lipid droplets in the cytoplasm of bovine in vitro produced embryos from zygotes to the 8-cell stage, by centrifugation without following micromanipulation, on the survival rate of Day 4 16-cell embryos. After centrifugation at 15,500 x g in medium containing cytochalasin D, embryos were cultured to the 16-cell stage, classified as either mostly or partially delipidated by degree of lipid droplet removal, and then frozen. Embryos centrifuged at the 2-cell stage developed to the 16-cell stage similarly to those centrifuged at the 8-cell stage. The developmental rate to blastocysts after freezing of the mostly delipidated 16-cell embryos centrifuged at the 2-cell stage was higher than that of those centrifuged at the zygote stage, those that were partially delipidated at the 2-cell stage, and those that were not centrifuged. The results demonstrate that polarization of lipid droplets at the 2-cell stage by centrifugation without micromanipulation improved the survival rate of mostly delipidated 16-cell embryos after freezing.     

Development and quality of sheep embryos cultured in commercial G1.3/G2.3 sequential media

Present study assesses the developmental ability and quality of ovine IVP embryos derived from culture in sequential media G1.3/G2.3. A total of 1474 cumulus-oocyte complexes were matured in M199 supplemented with EGF and FCS for 24h in 5% CO2 in humidified air at 39 degrees C. Oocytes were co-incubated in SOF medium with 1 x 10(6) spermatozoa/ml at the same temperature and gas conditions (Day 0 p.i.). Presumptive zygotes at 20 h p.i. were denuded, washed and placed in culture in SOF (control; n=742) or G1.3 media supplemented with 3mg/ml of BSA (n=732) under mineral oil in a humidified and controlled atmosphere at 39 degrees C. Embryos in the treated group were changed to G2.3 medium on Day 3 of culture. A group of blastocysts in each group were frozen by conventional method (SOF, n=55; G1.3/G2.3, n=48). In vivo embryos (n=72) were recovered at Day 7 from the uterus of progestagen+eCG treated females and they were cultured in defined medium (n=38) or frozen (n=34) directly after recovery. Cleavage rate of IVP embryos recorded at 48 h p.i. was similar for control and treated embryos (49.8 versus 47.5%). There were no significant differences in blastocyst development from the two groups on Day 6 (26.0 versus 25.6%), 7 (42.1 versus 38.6%) or 8 (50.8 versus 43.2%). Blastocyst development rates from total oocytes cultured were comparable (24.1 versus 21.5%). However, the proportion of hatched blastocysts was significantly higher for control embryos (86.6 versus 44.3%, P<0.0001). In addition, embryos cultured in SOF had higher re-expansion rates post-thawing at 24h (38.2 versus 6.2%), 48 h (36.4 versus 4.1%) and 72 h (34.5 versus 4.1%) and hatching rate (32.8 versus 2.0%) than embryos cultured in sequential media (P<0.0001). In vivo embryos showed higher hatching rate (61.7%) than IVP groups (SOF, P<0.01; G1.3/G2.3, P<0.0001) but lower than their fresh cultured counterparts (86.8%; P=0.01). In conclusion, G1.3/G2.3 media supported high developmental rates of embryos in vitro but the quality of the embryos was impaired.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells

Human "spare" embryos, judged unsuitable for freezing because of their poor quality, were cocultured for 5 days on a "Vero" cell layer. These epithelial cells were selected because kidney and genital tract have a common embryologic origin and "Vero" cells are a safe and highly controlled cellular support used for vaccine production. In the control group, the embryos were cultured in culture medium alone (B2 + 15% serum). At the end of the culture, the number of blastocysts was significantly higher in the coculture group: 61% vs. 3%. Moreover, at least half of the blastocysts were expanding and hatching (13/25), with a chronologically normal development. These observations suggest that (1) the coculture system improves human embryonic development; (2) it can rescue early degenerating embryos; (3) beneficial effects of coculture are not strictly genital-tract specific, but rather epithelium dependent. This coculture system could be used for in vitro fertilization to prolong in vitro culture and thus make it possible to transfer embryos at a more appropriate time, to eliminate early-blocked eggs, and to freeze embryos at the blastocyst stage, when freezing procedures are most successful.     

Progesterone enhances in vitro development of bovine embryos

Increased pregnancy rates in cattle given progesterone (P₄) prior to 5 d after breeding have recently been reported. The objective was to determine if this increase in pregnancy rate could be attributed to a direct positive effect of P₄ on the developing embryo. In Experiment 1, 280 bovine oocytes were inseminated in vitro and at Day 3 (insemination = Day 0), good quality 8 cell embryos (n = 206) were randomly allocated to be cultured in either CR1aa+serum with 0 or ∼15 ng/mL (n = 102 and n = 104, respectively). In Experiment 2, 881 bovine oocytes were used; on Day 3, good quality 8 cell embryos (n = 511) were randomly allocated to either the control (CR1aa+FCS, n = 168), vehicle (CR1aa + FCS + ethanol, n = 170), or P₄ treatment (CR1aa + FCS + ∼15 ng/mL P₄ in ethanol, n = 173). On Day 7, in both experiments, there were increased numbers of blastocysts developing in the P₄ group (Experiment 1, 59% and Experiment 2, 71%) compared to the vehicle (Experiment 2, 53%) or control (40 and 62% in Experiments 1 and 2, respectively). The addition of P₄ (8%) stimulated the rate of embryo development (early blastocysts or more advanced stages on Day 6) compared to vehicle (3%) and control (0%) and the P₄ group had more hatched or hatching blastocysts (33%) on Day 9 compared to the control or vehicle group (21 or 22%). Additionally, the P₄ group had greater embryo diameter and significantly more Grade 1 blastocysts on Day 7. In conclusion, P₄ had a direct, positive effect on developing bovine embryos cultured in vitro.