Effect of culture system on survival rate of vitrified bovine embryos produced in vitro

This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups.In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.     

Varied approach of using MSCs for bovine embryo in vitro culture

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p?<?0.0001), and between SOF and SOF conditioned (monolayer and drop) (p?<?0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.     

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.     

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.     

Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing

Vitrification is a novel cryopreservation method for mammalian blastocysts. This study was designed to compare different vitrification methods and slow freezing for their effects on survival rate and DNA integrity in mouse and human blastocysts. In Experiment 1, embryo survival and DNA integrity were compared between mouse blastocysts with collapsed and non‐collapsed blastoceles. In Experiment 2, embryo survival and DNA integrity were compared between vitrified and slow‐frozen mouse blastocysts. In Experiment 3, embryo survival and DNA integrity were compared between vitrified and slow‐frozen human blastocysts. Fresh blastocysts were used as controls in all experiments. Higher (P < 0.05) blastocyst survival rates were obtained in mouse blastocysts vitrified with collapsed versus intact blastoceles, although DNA‐integrity indices in the surviving blastocysts were the same among vitrified and fresh blastocysts. More mouse blastocysts (P < 0.05) survived after vitrification (100%) as compared to slow freezing (82.5%). DNA‐integrity indices examined in the surviving blastocysts were also higher (P < 0.001) in fresh (93.6%) and vitrified/warmed (93.7%) blastocysts than in slow‐frozen/thawed (75.8%) ones. More human blastocysts survived with a higher DNA‐integrity index after vitrification/warming than after slow freezing/thawing. These results indicate that higher survival rates can be obtained by vitrification of blastocele‐collapsed blastocysts, and that vitrification causes less cell apoptosis in both mouse and human blastocysts compared to slow freezing. Vitrification of blastocysts after blastocele collapse by single laser pulse supports a higher survival rate and less DNA apoptosis, suggesting that laser blastocele collapse is a safe procedure for blastocyst vitrification. Mol. Reprod. Dev. 79: 229–236, 2012. © 2011 Wiley Periodicals, Inc.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Piglets born after vitrification of embryos using the open pulled straw method

Morulae and unhatched blastocysts from Large White hyperprolific (LWh) and Meishan (MS) gilts were selected to test an ultrarapid open pulled straw (OPS) vitrification method with two media. The viability of vitrified/warmed embryos was estimated by the percentage of embryos that developed to the hatched blastocyst stage in vitro or by birth after transfer. In Experiment 1, two cryoprotectant dilution media were compared for cryopreservation of MS and LWh blastocysts: TCM was a standard Hepes-buffered TCM199 + 20% NBCS medium and PBS was a PBS + 20% NBCS medium. After a two-step equilibration in ethylene glycol, dimethyl sulfoxide, and sucrose, 2-5 blastocysts were loaded into OPS and plunged into liquid nitrogen. Embryos were warmed; a four-step dilution with decreasing concentrations of sucrose was applied. In PBS, LWh blastocysts (27%) had a lower viability in vitro than MS blastocysts (67%; P = 0.001). In TCM, no significant difference was observed between genotypes (41% for LWh and 43% for MS blastocysts) and both viability rates were lower than that of the control groups. In Experiment 2, morula-stage LWh and MS embryos were vitrified and warmed using PBS. The viability rate was low and did not differ between LWh (11%) and MS (14%). In Experiment 3, 200 MS and 200 LWh blastocysts were vitrified/warmed as described in Experiment 1 (PBS). In each of 20 MS recipients, 20 embryos were transferred. The farrowing rate was 55% and recipients farrowed four and five piglets (median) for MS and LWh blastocysts, respectively. The OPS method is therefore appropriate for cryopreservation of unhatched porcine blastocysts.     

Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification

The aim of this study was to assess the effect of a bovine in vitro culture system on blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were cultured in either synthetic oviduct fluid (SOF) in 5% CO2, 5% O2, 90% N2; or TCM199-granulosa cells (TCM199-GCM) in 5% CO2 in air. In vivo blastocysts were used as a control. Culture in SOF resulted in a significantly higher blastocyst yield on both Day 7 (31.3 vs 13.2%, P < 0.001) and 8 (36.8 vs 23.7%, P < 0.001) than did culture in TCM199-GCM. After vitrification, survival at 72 h of in vivo blastocysts was significantly higher than both in vitro groups, while significantly more blastocysts produced in TCM199-GCM survived compared to those produced in SOF (0, 43.5, 78.3% for SOF, TCM199-GCM and in vivo, respectively P < 0.01). In Experiment 2, SOF-GCM proved to be the best post-warming culture system of those tested and was adopted as the post-warming medium for all subsequent experiments. In Experiment 3, zygotes were cultured in SOF or SOF-GCM, in either 5% CO2 in air, or 5% CO2, 5% O2, 90% N2. In agreement with Experiment 1, culture in SOF in 5% O2 resulted in significantly more blastocysts at Day 7 (26.4 vs 17.3%, P < 0.01) and Day 8 (31.5 vs 23.2%, P < 0.01) than did culture in SOF-GCM. However, survival at 72 h post vitrification was significantly higher for SOF-GCM (44 vs 8.3%, P < 0.001). Increasing the O2 concentration to 20% significantly reduced the blastocyst eld from SOF (31.5 vs 17.3%, P < 0.001). In addition, the quality of blastocyst produced was reduced in terms of survival post vitrification (8.3 vs 0%, P < 0.05). In contrast, there was no difference in blastocyst yield (23.2 vs 25.2%) or survival (44.0 vs 36.9%) in SOF-GCM, irrespective of O2 concentration. Experiment 4 examined the duration of exposure to GCM necessary to acquire improved blastocyst quality. Zygotes were cultured in SOF; SOF until Day 3, followed by SOF-GCM for the remainder of the culture; SOF until Day 5, followed by SOF-GCM for the remainder of the culture; or SOF-GCM for the entire culture. Survival at 72 h post vitrification was significantly higher (P < 0.05) in Groups 2 (50.0%, 13/26) and 4 (55.3%, 26/47) than in Groups 1 (21.7%, 10/46) and 3 (10.8%, 4/37). In conclusion, culture system can affect blastocyst yield and quality and crytolerance is a useful indicator of blastocyst quality.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

Effects of culturing bovine oocytes either singly or in groups on development to blastocysts

In vitro maturation, fertilization and culture (IVM/IVF/IVC) of cattle oocytes from individual cows requires adapting existing culture protocols so that small numbers of oocytes can be cultured. The culture of single oocytes is desirable for correlating the relationship between follicular properties with oocyte developmental competence or for facilitating ovum pick-up procedures. In Experiment 1 we compared group and single culture under cell-free conditions on embryo development; significantly higher (P<0.001) rates of cleavage (66.4 vs 47.6%) and blastocyst formation (7.5 vs 0.5%) were observed in the group cultured oocytes. In Experiment 2 we compared group and single oocyte co-culture with granulosa cells. Although there was no effect of oocyte number on the percentage cleaving (73.1 vs 66.6%), there were significantly higher blastocyst yields (37.4 vs 10.1%) and blastocyst cell numbers (91.6 vs 66.2) in group-cultured oocytes. In Experiment 3 we examined the effect of group size (1, 5, 10, 20 and 40 oocytes) in a co-culture system using granulosa cell monolayers. The results show a difference in cleavage rates between the single cultured oocytes (66.8%) and each group of cultured oocytes, with the highest cleavage rate (81.5%) obtained in the 20-oocyte group. The blastocyst yield from both cleaved and total oocytes showed that group culture of 20 or 40 oocytes resulted in the highest number of blastocysts (32.5%), with smaller group sizes yielding significantly (P<0.05) fewer blastocysts. In Experiment 4 we examined the effects of co-culture on the development of single vs group-cultured oocytes. The results showed no significant difference (P>0.05) in the cleavage rate between single and group culture systems. No blastocysts were formed with single oocytes cultured without monolayers, while the blastocyst formation rate for those co-cultured with granulosa cells was 12.4%. Blastocyst formation was significantly higher (P < 0.006) in group co-culture on monolayers (24.2 vs 8.5%). These data indicate that oocytes cultured in groups are developmentally more competent and suggest that for optimum development oocytes need some undefined paracrine activity that is absent from the culture medium in addition to coculture with granulosa cells, which enhances development to the blastocyst stage of both group and singly cultured oocytes.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media

Treatment of in vitro matured bovine oocytes with colcemid results in a membrane protrusion that contains maternal chromosomes, which can be easily removed by aspiration. Four experiments were designed to evaluate the overall and temporal effects of conditioned medium (CM) by bovine cumulus cells on development of nuclear transfer (NT) bovine embryos and to examine the chromosomal composition and allocation of inner cell mass (ICM) and trophectoderm (TE) of the subsequent blastocysts. The nuclear transfer embryos were cultured in various CR1aa media conditioned by preculture with bovine cumulus cells. Development to the blastocyst stage in BSA-containing CM (BCM) and serum-containing CM (SCM) were similar to co-culture group (24-30%). The 24 hr-conditioned BCM yielded higher blastocyst development than 48 and 72 hr-conditioned BCM. Temporary exposure of embryos to BCM and SCM followed by CR1aa was also studied. Morula and blastocyst development were not different among the groups cultured in BCM for 72, 96, and 168 hr, but were significantly higher (P < 0.01) than groups exposed to BCM for 24 and 48 hr, respectively. Blastocyst development in SCM for 24 hr (29%), 96 hr (25%), and 168 hr (27%) were much higher (P < 0.05) than those in SCM for 48 hr (12%) and 72 hr (10%). The analyses of chromosomal composition of the resulting blastocysts indicate approximately 80% of the blastocysts cultured in CR1aa with co-culture or groups initially exposed to BCM for 24 hr followed by culture in CR1aa were diploid. However, the incidence of diploidy were only 36-60% in SCM-cultured groups and groups cultured in BCM beyond 48 hr. Conditioned media did not affect the allocation of ICM and TE in the blastocyst. No difference was found in the ratio of inner cell mass to total cells in co-culture, BCM or SCM groups (0.424, 0.441, and 0.473, respectively). In conclusion, bovine cumulus cell-CM and CR1aa with co-culture supported comparable development and blastocyst ICM:total cell ratio of bovine NT embryos. However, CM affected the blastocyst chromosomal composition and induced higher mixploidy.     

Effect of energy source during culture on in vitro embryo development, resistance to cryopreservation and sex ratio

The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.