Rabbit Morulae Research Articles

The harmful effect of removing the extracellular vitrification medium during embryo cryopreservation using a nylon mesh device in rabbit

During the last decades, many techniques have been developed to reduce sample volume and improve cooling and warming rates during embryo vitrification. The vast majority are based on the “minimum drop size” concept, in which the vitrification solution around embryos is reduced by aspiration, leaving a tiny part of volume surrounding embryos. However, novel cryodevices were aimed to remove the entire vitrification solution. This study was designed to compare the “minimum drop size” technique using Cryotop® with the nylon mesh as cryodevice on rabbit morula embryos. The outcomes assessed were the in vitro development rates (experiment 1) and the offspring rates at birth (experiment 2). Embryos were vitrified in a two-step procedure; equilibrium (10% EG + 10% Me2SO) for 2 min and vitrification (20% EG + 20% Me2SO) for 1 min. In experiment 1, embryos (n = 323) were warmed and subsequently in vitro cultured for 48 h to assess the embryo developmental capability to reach the hatching-hatched blastocyst stage. In experiment 2, embryos were transferred using the laparoscopic technique (n = 369) to assess the offspring rate at birth. In this context, rates of in vitro embryo development were similar between vitrified groups (0.73 ± 0.042% and 0.66 ± 0.047% for Cryotop® and nylon mesh device, respectively), but lower than in the fresh group (0.97 ± 0.016%, p < 0.05). In experiment 2, there were no significant differences in survival rates (offspring born/total embryos transferred) among the Cryotop® device group and fresh group (0.41 ± 0.049% and 0.49 ± 0.050%, respectively). But significantly lower value was obtained in the nylon mesh device group (0.18 ± 0.030%). These results indicate that nylon mesh is not suitable as cryodevice for rabbit morula vitrification, remaining those using the “minimum drop size” methodology as the best option.

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment. The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species. Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Rapid cooling of rabbit embryos in a synthetic medium

Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. We aimed to evaluate the effect of replacing BSA in rabbit embryo rapid cooling “freezing” and warming media with a chemically defined medium with no animal-derived products: STEM ALPHA. Cryo3 (“Cryo3”).A total of 1540 rabbit morulae were divided into three cryopreservation groups (group 1: BSA, group 2: 20% Cryo3 and group 3: 100% Cryo3) and a fresh controls group. After rapid cooling, embryos were cultured (in vitro approach), or transferred into synchronized does (in vivo approach). In the in vitro approach, post-warm survival rates obtained with 100% Cryo3 (94.9%) were superior to BSA (90.8%) and 20% Cryo3 (85.6%). The blastocyst formation rate was similar between BSA, 20% Cryo3 and 100% Cryo3 groups (85.1, 77.9 and 83.3%, respectively), as was the expansion/hatching rate (63.1, 63.4 and 58.0%, respectively) and embryo mitochondrial activity. In the in vivo approach, pregnancy (80.0, 68.0 and 95.2%, respectively), implantation (40.5, 45.9 and 44.8%, respectively), and live-foetus rates (35.6, 35.5 and 38.1%, respectively) were similar between the three groups. To conclude, Cryo3 can replace BSA in rabbit embryo rapid cooling “freezing” and warming media.

Does vitrification alter the methylation pattern of OCT4 promoter in rabbit late blastocyst?

Vitrification is replacing slow freezing as the most popular method for oocyte and embryo cryopreservation. However, very little information is available on alterations in epigenetic regulation. Previous studies reported post-implantation effects of vitrification on fetal development and gene expression. This study was conducted to determine if vitrification procedure induce alterations in OCT4 promoter methylation profile which could determine the set point of fetal losses and transcriptomic alterations observed after implantation. Rabbit morulae were recovered at Day 3 of development and vitrified and transferred, or directly transfer, to recipient till Day 6. A conserved regulation region of OCT4 promoter was examined in control and vitrified embryos by bisulfite sequencing and quantitative PCR was used to measure the gene expression. No significant differences were observed in methylation levels or gene expression of OCT4. This work was the first approach in rabbit to the study of possible epigenetic alterations associated with vitrification procedure.

Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.

Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation

Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10–14days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen–thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term.Further studies in subsequent embryo–foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects.

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in ∼1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO 2 in air at 39 °C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.

Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60 s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO−S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG−20DMSO+S) and two-step (20EG−20DMSO−S) groups (0–6% versus 36–50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham’s F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG−20DMSO+S) group in s-PBS (35–45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO−S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG−20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG−20DMSO−S), regardless of percentage of FCS in the PBS-based media (81% versus 41–45%; P<0.05). In conclusion, we propose either the one step (20EG−20DMSO+S) or two steps (20EG−20DMSO−S) prepared in PBS plus 20% serum for use in future works.

Induction of Arylhydrocarbon Receptor Expression in Embryoblast Cells of Rabbit Preimplantation Blastocysts upon Degeneration of Rauber's Polar Trophoblast

The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor and mediates carcinogenic, teratogenic, and toxic effects of xenobiotics such as dioxin and coplanar polychlorinated biphenyls. The AhR nuclear translocator (ARNT) is involved in AhR signal transduction. We have analyzed the expression of AhR and ARNT mRNA and AhR protein in Day 3 pc (postcoitum) rabbit morulae and Days 4 and 6 pc blastocysts using RT–PCR, nested PCR, whole mount in situ hybridization, and whole mount immunohistochemistry with subsequent confocal laser scanning analysis. AhR and ARNT transcripts were detected in all stages investigated, indicating coexpression of both transcription factors. AhR protein was localized in the cytoplasm. It was detected in Day 3 pc morulae and in blastocysts. In Day 4 pc blastocysts, only trophoblast cells but not embryoblast cells were immunopositive. However, at Day 6 pc, the embryoblast cells also expressed AhR protein and this expression was correlated with the degeneration of Rauber's trophoblast layer.

Effect of 3- and 4-day FSH superovulation treatments on the in vivo development of vitrified rabbit morulae

Effect of 3- and 4-day FSH superovulation treatments on the in vivo development of vitrified rabbit morulae

In vivo survival rate of rabbit morulae and blastocysts after vitrification in ethylene glycol-devethyl sulfoxide solution

To research developmental competence of transgenic somatic cell by serial nuclear transplantation, goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including human finger-domain lacking t-PA gene was microinjected into the cytoplasm of the MII oocytes. Goat embryos (G0) were cloned by this procedure. A single blastomere from 16-cell to 64-cell goat cloned embryos (G0) was microinjected into the Intracytoplasm of the MII oocytes. Goat embryos (G1) were cloned by this procedure. Goat embryos (G2, G3) were recloned using 16-cell to 64-cell recloned embryos. The developmental time of donor embryo affected the developmental rate of recloned embryos (G1, G2). The result indicates: the cleavage rate of cloned embryos (G0) (76.45±1.17%) was no difference significantly with recloned embryos (G1 G2 G3) (72.18%±1.97%, 76.05%±2.38%, 75.99%±2.84%). The developmental rate of morulae and blastocysts of cloned embryos (47.20%±2.93%, 11.00%±1.42%) were higher than of recloned embryos (34.99%±2.66%, 28.23%±2.00%, 23.34%±1.99%) (3.87%±0.67%, 2.08%±1.66%, 0); The morulae rate (29.57%±1.53%, 24.43%±1.87%), blastocysts rate (1.96%±1.31%, 2.01%±1.34%) of recloned embryos (G1 G2) from 16-cell recloned embryos was lower than that (34.32%±1.31%, 29.76%±1.66%), (3.86%±1.03%, 3.48%±0.34%) from 32–64-cell recloned embryos (P>0.05). In conclusion, nuclear transfer embryos were not recloned mostly; Embryos recloned using 32-cell to 64-cell embryos achieved higher developmental ability compared with using 16-cell embryos.

Embryotoxicity of polychlorinated biphenyls (PCBS) for preimplantation embryos

In a previous study oral administration of a commercial PCB mixture (Aroclor 1260) to female rabbits (4 mg/kg b.wt. for 14 weeks) resulted in a significant accumulation of PCBs in 6-d-old blastocysts and increased preimplantation embryo mortality (1). In the present study, the direct toxicity of PCBs on preimplantation rabbit embryos was investigated during in vitro culture. One-day-old cleavage stages and 3-d-old rabbit morulae were cultured in BSM II medium supplemented with 1.5% BSA in a reduced oxygen concentration of 5% O 2. They were exposed to 50, 5, or 0.5 μg Aroclor 1260/mL culture medium for 24 h. PCB (50 μg) led to a complete degeneration of the exposed embryos. Following exposure to 5 μg only 16% of the morulae developed into blastocysts. The others were either arrested in the morulae stage or were degenerated. Cell proliferation of the nondegenerated embryos was approximately 20% of that of corresponding control embryos. Compared with nonexposed controls, addition of 0.5 μg PCB/mL showed either no or only a slight impairment of development. Preliminary embryo transfer experiments showed that morulae exposed to 5 μg PCB with clear signs of degeneration after 24 h in vitro culture were able to implant. Aroclor 1260 is embryotoxic in a dose-dependent manner in cultured rabbit preimplantation embryos.

Direct transfer of vitrified rabbit embryos

In this study we looked at the feasibility of transfering vitrified rabbit embryos directly into recipient does. Compacted morulae were vitrified in a solution of 20% ethylene glycol and 20% dimethyl sulfoxide. After thawing, and without step-wise diluted solution, the vitrified embryos were transferred into the recipient's uterine horns. Survival rate at birth differed from fresh rabbit embryos (40% vs 55%, P < 0.05). However, the percentage of does that delivered (94%) and the survival rate suggested this method is suitable for both storage and simple transfer of rabbit morulae.

Effects of lithium chloride on rabbit blastocyst expansion, and accumulation of phosphoinositides and inositol phosphates

The effects of lithium, an inhibitor of the recycling of inositol in the phosphatidylinositol cycle, on rabbit blastocyst growth and metabolism of phosphoinositides were investigated. Day 2 rabbit morulae were first cultured for 2 days in basic culture medium and then transferred to medium containing myo-[2-3H]inositol for culture for a further 3 days. At the end of culture, the resulting blastocysts were incubated with LiCl (0, 1, 5, 10, 20 mmol l-1) for 1 h. The blastocysts were then lysed and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin layer chromatographic separation of the lipid portion indicated that lithium had no significant effect on formation of radiolabelled phosphoinositides. However, high performance anion exchange chromatography indicated that lithium significantly stimulated accumulation of radiolabelled inositol monophosphate and inositol 1,4,5-trisphosphate. This result indicates that the phosphatidylinositol cycle is turning over in rabbit blastocysts. Continuous culture of rabbit embryos for 5 days in media containing LiCl (5, 10, 15 and 20 mmol l-1) significantly decreased blastocyst growth as measured by blastocyst expansion and incorporation of [3H]thymidine. However, supplementing the medium with excess inositol (up to 9375 mumol l-1), in an attempt to increase the intracellular uptake of inositol and thus compensate for the inhibitory effect of lithium on inositol recycling, did not reverse the inhibitory effect of lithium on blastocyst growth.

Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae

Comparisons were made of the osmotic and cryoprotective effects on rabbit embryos preserved by vitrification with 2 solutions and by conventional freezing. Embryos obtained from rabbits killed 70 to 72 h after mating were used in the study (n = 948). Initially, toxicity of the 3 cryoprotectants was studied in fresh (unfrozen) embryos (n = 135). Subsequently, embryos placed in ethylene glycol (EG, 40% v/v; n = 88) and ethylene glycol with dimethyl sulfoxide (EG+DMSO, 20% v/v each, respectively; n = 344) were loaded into straws and plunged directly into liquid nitrogen. Embryos placed in 1.5 M DMSO and 20% heat inactivated rabbit serum were subjected to conventional freezing in a programmable freezer (control group, n = 363). The osmotic effect was estimated by measuring the changes in the embryonic and interzonal volume (crossectional area) and in the thickness of the mucin coat (n = 18). Cryoprotective effectivity was determined by development to the blastocyst stage in vitro, or birth of normal pups after transfer into synchronized recipients.Osmotic effects of cryoprotective solutions on embryonic and interzonal volume and mucin coat thickness were variable and overall not significant. Survival rate of cryopreserved embryos in vitro and development to blastocysts, was worst in the EG-treated embryos. Survival rate at birth was higher in vitrified vs frozen embryos. We conclude that rabbit morulae can be vitrified successfully in EG+DMSO medium.

Influence of the mucin coat on the survival of rabbit and mouse embryos stored at 0°c

To examine the factors affecting the survival of refrigerated embryos, rabbit and mouse morulae were stored at 0 degrees C in modified phosphate-buffered saline (PB1) or in PB1 containing 0.75 M sucrose. Survival was defined as the ability to develop into an expanded blastocyst in culture. The data was analyzed with special reference to the presence of a mucin coat around the embryos. When rabbit morulae were stored in isotonic PB1 for 2, 4, 6, and 8 days, survival rates were 98%, 88%, 85%, and 50%, respectively. However, if the mucin coat had been removed before storage, the rates were lower (95%, 75%, 36%, and 3%, respectively). Sucrose impaired the survival of rabbit morulae irrespective of the presence of the mucin coat. Only 11% of mouse morulae survived 2 days of storage in PB1 medium, but if the medium contained sucrose, survival rates after storage for 2, 3, 4, and 5 days were higher (83%, 55%, 31%, and 7%, respectively). To provide them with a mucin coat around the zona pellucida, mouse embryos were incubated in a rabbit oviduct. Survival rates of these embryos after storage in the presence of sucrose did not decrease over 4 days of refrigeration (98-92%), and the rates after storage for 5, 6, and 7 days were 65%, 40%, and 30%, respectively; embryos that had been stored for 5 days were transferred to recipient mice, and live young were born. Agar embedding of mouse morulae did not have the same effect as the mucin coat.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an aromatase inhibitor, 4-androsten-4-ol-3,17 dione acetate, on in vitro blastocyst formation of rabbit embryos

The role of estradiol-17β (E 2) in early embryonic development was investigated using an estrogen synthesis blocking aromatase inhibitor, acetoxy-androstene-dione (AAD). A total of 281 morphologically intact rabbit morulae (32- to 64-cell stage) from superovulated (100 IU pregnant mare's serum gonadotropin (PMSG) and 100 IU human chorionic gonadotropin (hCG)) New Zealand White does were cultured in vitro in B 2 medium and the blastocyst formation rate (BFR) was recorded after 24 and 48 h. Differences between groups were compared using Kruskal-Wallis one-way analysis of variance. The addition of AAD (25.0 μg ml −1) to the culture medium significantly ( P<0.001) reduced the BFR when compared with the controls (20.8±3.5% vs. 90.4±3.9%). To overcome the detrimental effects of AAD (25.0 μg ml −1), the medium was supplemented with E 2 (1.0 μg ml −1). This treatment significantly ( P<0.001) increased the BFR and partly reversed the effect of the AAD (62.6±5.0% vs. 20.8±3.5%). The addition of E 2 (1.0 μg ml −1) only to the culture medium did not influence the BFR when compared with the controls (88.8±2.9% vs. 90.4±3.9%). It is concluded that E 2 is not only an essential factor for the transformation of morulae to blastocysts but it is presumably produced by the early rabbit embryo itself.

Effect of the Composition of Vitrification Media on Survival of Rabbit Embryos

A total of 448 rabbit morula stage embryos were used to examine the effect of modified vitrification procedure and vitrification media containing 10% (1.36 M) glycerol, 20% (2.72 M) 1,2-propanediol, and 1 M sucrose. The best result, 89.5% embryos developed in vitro, was obtained after equilibration, vitrification, and dilution of embryos using media in which the salt concentration was maintained constant on a molar basis. Media prepared on a molal basis were more toxic and/or less efficient.

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.