Development Of Rabbit Embryos Research Articles

Asynchrony between in vivo and in vitro rabbit embryos

Background: Comparative features of embryos developed under in vitro and in vivo conditions are particularly important in designing embryo transfer procedures that fulfil embryo-recipient synchronization requirements. Objective: To determine the degree of asynchrony in rabbit embryo development between cultured and in vivo embryos. Methods: A total of 55 non-lactating multiparous female rabbits were used. Embryos were classified as 16-cells or early morulae at 48 hours post-coitum (hpc). Embryos were cultured during 30 or 32 h and embryo development was compared with in vivo embryos of 72 hpc. In vitro and in vivo embryos at 72 hpc were classified as early or compacted morulae. Bayesian statistics was used. Difference between in vivo and in vitro embryos and the actual probability of the difference between the in vivo and in vitro embryo higher than zero (P) was estimated. Results: The percentage of compacted morulae was higher in in vivo embryos than in in vitro embryos with +6 h of asynchrony (73.5 and 32.8%, P=1.00). But the percentage of compacted morulae was similar with +8 h asynchrony. Conclusions: In vitro embryos delay their development by + 8 hours compared to in vivo embryos.

Whole transcriptome sequencing reveals that non-coding RNAs are related to embryo morphogenesis and development in rabbits

The roles of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in embryonic development remain unclear. We performed a comprehensive analysis of lncRNA and circRNA profiles in rabbit embryos at different stages by whole transcriptome sequencing. We identified 719 lncRNAs and 744 circRNAs that were differentially expressed between stages S1, S2 and S3. A total of 241 differentially expressed lncRNAs and 166 differentially expressed circRNAs were significantly involved in embryonic morphogenesis and development. An RNA network was established and of the embryonic development-associated RNAs, the lncRNAs TCONS_00009253 and TCONS_00010436 were persistently downregulated, while circRNA_07129, circRNA_15209, and circRNA_12526 were persistently upregulated, and their co-expressed mRNAs TBX1, WNT3 and FGFR2 were persistently downregulated during embryonic development. These candidate RNAs were mainly involved in the Wnt, PI3K-Akt, and calcium signaling pathways. This study reports candidate lncRNAs and circRNAs that may be indispensable for the morphogenesis and development of rabbit embryos.

Effect of PPARGC1A on the development and metabolism of early rabbit embryos in vitro.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.

The effect of unilateral ovariectomy on early embryonic survival and embryo development in rabbits

Unilateral ovariectomy can be used to study uterine capacity in rabbits because an overcrowding of the functional uterine horn is produced. Due to the uterus duplex, the rabbit is the ideal model for such studies. However, this technique may affect embryo survival. The aim of this work is to study the effect of unilateral ovariectomy on early embryo survival and development in rabbit. A total of 101 unilateral ovariectomised females and 52 intact females were compared after slaughter at 30 h post-mating. Early embryo survival was estimated as the ratio between number of embryo recovered and ovulation rate. No differences were found between intact and unilaterally ovariectomised females in this trait. Unilateral ovariectomy did not change embryo development, measured as the number of embryo cells. Variability of embryo development was not affected either. At 30 h post-mating, the majority of embryos (86.2%) were 4-cell stage. Embryo quality was evaluated according to morphological criteria. No difference in embryo quality between intact and unilaterally ovariectomised females was found. Therefore, unilateral ovariectomy performed before puberty in rabbit does not modify early embryo survival and development.

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.

The Oct4 promoter-EGFP transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.

Dynamic profiles of Oct-4, Cdx-2 and acetylated H4K5 in in-vivo-derived rabbit embryos

This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.There are very limited studies on key transcription factors and epigenetic programming events in preimplantation-stage rabbit embryos. Previously we reported the spatial and temporal distribution of Oct-4 and acetylated H4K5 (H4K5ac) in-vitro-cultured rabbit embryos. In the present study, we report the similar distribution patterns of Oct-4 and H4K5ac using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where such signal was similarly weak in both the ICM and the TE cells. A consistent strong H4K5ac signal was observed in the TE cells in all stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Importantly, we also report, as far as is known for the first time, the distribution pattern of Cdx-2 in rabbit embryos. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts. It became evident in the TE cells exclusively afterwards. The present study provides novel information on key transcription factors and epigenetic events during rabbit embryo development, and demonstrates that a combination of Oct-4, Cdx-2 and H4K5ac could be used as biomarkers to identify different lineage cells in rabbit blastocysts.

Spatial and temporal distribution of Oct-4 and acetylated H4K5 in rabbit embryos

Rabbit is a unique species to study human embryology; however, there are limited reports on the key transcription factors and epigenetic events of rabbit embryos. This study examined the Oct-4 and acetylated H4K5 (H4K5ac) patterns in rabbit embryos using immunochemistry staining. The average intensity of the Oct-4 signal in the nuclei of the whole embryo spiked upon fertilization, then decreased until the 8-cell stage and increased afterwards until the compact morula (CM) stage. It decreased thereafter from the CM stage to the early blastocyst (EB) stage, with a minimum at the expanded blastocyst (EXPB) stage and came back to a level similar to that of the CM-stage embryos in the hatching blastocysts (HB). The Oct-4 signal was observed in both the inner cell mass (ICM) and the trophectoderm (TE) cells of blastocysts. The average H4K5ac signal intensity of the whole embryo increased upon fertilization, started to decrease at the 4-cell stage, reached a minimum at the 8-cell stage, increased again at the EXPB stage and peaked at the HB stage. While TE cells maintained similar levels of H4K5ac throughout the blastocyst stages, ICM cells of HB showed higher levels of H4K5ac than those of EB and EXPB.Understanding key genetic and epigenetic events during early embryo development will help to identify factors contributing to embryo losses and consequently improve embryo survival rates. As a preferred laboratory species for many human disease studies such as atherosclerosis, rabbit is also a pioneer species in the development of several embryo biotechnologies, such as IVF, transgenesis, animal cloning, embryo cryopreservation and embryonic stem cells. However, there are limited reports on key transcription factors and epigenetic events of rabbit embryos. In the present study, we documented the temporal and spatial distribution of Oct-4 protein and H4K5 acetylation during early embryo development using the immunostaining approach. We also compared the patterns of these two important biomarkers between the inner cell mass (ICM) and the trophectoderm (TE) cells in blastocyst-stage embryos. Our findings suggest that a combination of Oct-4, H4K5ac and possibly other biomarkers such as Cdx-2 is needed to accurately identify different lineages of cells in morula and blastocyst stage rabbit embryos. Importantly, we revealed a novel wave of Oct-4 intensity change in the ICM cells of rabbit blastocysts. The signal was high at the early blastocyst stage, reached a minimum at the expanded blastocyst stage and returned to a high level at the hatching blastocyst stage. We hypothesize that the signal may have reflected the regulation of Oct-4 through enhancer switching and therefore may be related to cell lineage formation in rabbit embryos. These findings enrich our understanding on key genetic and epigenetic programming events during early embryo development in rabbits.

Differential mRNA Expression in Rabbit In vivo Pre‐implantatory Embryos

To study genes involved in embryo developmental competence and implantation in rabbits, the expression of a panel of genes related to pluripotency, angiogenesis, proliferation, apoptosis and differentiation were evaluated in late rabbit blastocysts. Thirty nulliparous does were used to obtain a total of 184 in vivo-derived blastocysts on days 4, 5 and 6 of development. The relative transcript abundance of vascular endothelial growth factor (VEGF), epidermal growth factor receptor 3 (erbB3), transforming growth factor β2 (TGF β2) and transcription factor OCT-4 were analysed from eight pools of each stage of development, using quantitative real-time reverse transcriptase-polymerase chain reaction (qrtRT-PCR). mRNA expression was detected for all genes in 4-, 5- and 6-day-old blastocysts, according to blastocyst growth and implantation proximity. Significant differences in OCT-4, VEGF and TGF β2 expression were observed between days of development. Results show a down-regulation of OCT-4 from the 4th day, contrasting with the up-regulation of VEGF and TGF β2 at 6-day-old blastocyst. These findings corroborate the importance of VEGF and TGF β2 in rabbit embryo development and implantation and suggest a possible regulator role of OCT-4 in embryonic angiogenetic factors. On the other hand, no differences were found for erbB3 expression. Therefore, the study of specific gene transcripts in rabbit blastocyst could provide novel embryo developmental competence markers and might be used as a new tool for further studies of embryo quality and in vitro development.

Dextran vitrification media prevents mucin coat and zona pellucida damage in rabbit embryo

Vitrification of embryos is being increasingly important for cryopreservation in mammals. However, damage and toxicity has to be reduced even more. The composition of cryoprotective medium used to immerse the embryos affects viability and developmental potential. The aim of this work was to assess the effect of the Polyvinylalcohol-PVA- and Dextran addition to vitrification media on the in vitro development of rabbit embryos from superovulated and non-superovulated females. Superovulation group were treated intramuscularly with 25 IU rhFSH. The vitrification media contained the same permeable cryoprotectans (Ethylene Glycol-ET- and Dimethyl Sulfoxide-Me 2SO-) and different macromolecules (PVA and Dextran) in different combinations. There was a significantly higher proportion of embryos without damages in mucin coat or zona pellucida after warming (undamaged embryos) in the control than in the superovulation group (95.8% vs. 83.2%, respectively). The proportion of undamaged embryos was significantly affected by the vitrification solution composition. The rate of undamaged embryos after warming in media containing 20% Me 2SO was significantly lower in media supplemented with PVA than in media with dextran (67.3 vs. 93.8, respectively). However, the proportion of undamaged embryos for the medium supplemented with dextran was similar for media with 15 or 20% Me 2SO. In conclusion, the addition of dextran to the vitrification media improve the preservation of rabbit embryos and permits to reduce the amount of Me 2SO for vitrification. Additionally, in vitro developmental ability of undamaged embryos were not affected by superovulation treatment nor vitrification media.

Full-Term Development of Rabbit Embryos Produced by ICSI with Sperm Frozen in Liquid Nitrogen without Cryoprotectants

The aim of the present study was to establish the technology of intracytoplasmic sperm injection (ICSI) in rabbit by using the sperm frozen without cryoprotectants. Observation under an electron microscope revealed that the rabbit spermatozoa frozen without cryoprotectants had severe damage especially in the plasma membrane and junction between head and tail. However, after being injected into the oocytes, the sperm frozen without cryoprotectants retained the capability of supporting the cleavage and development of the ICSI oocytes, with no significant difference from that of fresh sperm, although the development of ICSI embryos derived from either frozen sperm or fresh sperm is much lower than that of in vivo-fertilized zygotes. When additional artificial activation was applied following ICSI, the rates of cleavage and blastocyst formation of ICSI oocytes were significantly increased when compared with the oocytes without additional activation. Yet, the cell numbers in blastocysts were not significantly different between the activation and non-activation group. After embryo transfer, four offspring were obtained from the oocytes microinjected with the sperm frozen without cryoprotectants. The technology established by this study may facilitate exploring the ICSI-based transgenic method in rabbit and broaden the application of ICSI technique in related field.

Effect of β-mercaptoethanol supplementation on in vitro development of rabbit embryos derived from parthenogenesis and somatic cell nuclear transfer

To investigate effects of β-mercaptoethanol (β-ME) supplementation in in vitro culture (IVC) medium on developmental competence of embryos and total cell numbers (TCN) of blastocysts derived from parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) in rabbits.

Refinement of a morphological scoring system for postimplantation rabbit conceptuses

The rabbit is used extensively in developmental toxicity testing, yet basic information on rabbit embryo development is lacking. The goals of this study were to refine a rabbit embryo morphology scoring system, and use it to evaluate rabbit whole embryo cultures (WEC). A total of 265 conceptuses were harvested between GD 8.0 and 12.0 (coitus = GD 0) at 6-hr intervals and examined in detail. Discreet developmental landmarks were then established for 18 morphological features and assigned scores ranging from 0 up to 6. The scoring system was then validated on a subset of randomly selected in vivo conceptuses, and was used to evaluate conceptuses grown for 12, 24, 36, or 48 hr in WEC beginning from GD 9.0 or 10.0. A few embryos also were examined using microscopic computed tomography (microCT)-based virtual histologytrade mark to assess the utility of this technology. Morphology scores of in vivo developed conceptuses increased linearly (r2 = 0.98) with advancing gestational age, from means of 0.0 on GD 8.0 to 67.9 on GD 12.0. Application of the scoring system, supplemented with evidence from Virtual histologytrade mark, indicated that the WEC system supported normal morphological development of rabbit conceptuses. However, when explanted at GD 9, the rate of development was about 20% slower than in vivo, whereas the rate of development in WEC from GD 10 was indistinguishable from in vivo. This work enhances the evaluation tools available to study mechanisms of normal and abnormal development in this widely used animal testing species.

Effect of atosiban on rabbit embryo development and human sperm motility

To investigate embryotoxic potential and effects on human sperm motility of the mixed vasopressin V(1a)/oxytocin receptor antagonist atosiban considered for novel indication of improvement of uterine receptivity in embryo-transfer recipients. One-cell rabbit embryo bioassay and human sperm motility bioassay were performed in control media or in media containing atosiban. Private center of reproductive medicine and academic research institute of reproduction biotechnology. Rabbit females (New Zealand and California, N = 15) aged 4.5-6.5 months. In vitro exposure of one-cell rabbit embryos and human sperm to atosiban in the range of therapeutic concentrations clinically occurring in human beings. Embryo development and sperm motility. Preimplantation development of one-cell rabbit embryos was not affected by atosiban in the concentrations < or =15,000 nM, which was 50-fold higher than the mean plasma concentration reached during regular therapy (300 nM). Atosiban did not affect human sperm motility in concentrations of < or =3,000 nM, in other words, 10 times the human mean plasma concentration. Clinical application of atosiban in the proposed indication may be safe for embryos because it is compatible with preimplantation rabbit embryo development and human sperm motility.

Differential development of rabbit embryos following microinsemination with sperm and spermatids

Microinsemination is the technique of delivering male germ cells directly into oocytes. The efficiency of fertilization after microinsemination and subsequent embryo development may vary with the animal species and male germ cells used. The present study was undertaken to observe the in vitro and in vivo developmental ability of rabbit embryos following microinsemination with male germ cells at different stages. First, we assessed their oocyte-activating capacity by injecting them into mouse and rabbit oocytes. The majority of mouse oocytes were activated irrespective of the type of rabbit male germ cell injected (61-77%), whereas rabbit oocytes were activated differently according to the type of male germ cells (89%, 75%, and 29% were activated by spermatozoa, elongated spermatids, and round spermatids, respectively; P < 0.05). After 120 hr in culture, 66%, 45%, and 13%, respectively, of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts (P < 0.05). Additional electric pulse stimulation of round spermatid-injected oocytes increased the blastocyst rate to 43%. After 24 hr in culture, some four to eight cell embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from spermatozoa and elongated spermatids, but not from round spermatids. Karyotypic analysis at the morula/blastocyst stage revealed that the majority of round spermatid-derived embryos had abnormal ploidy (8 out of 12 embryos). Our study indicates that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. As these differential developmental patterns are similar to those reported for humans in vitro and in vivo, rabbits may provide an alternative small animal model for studying the biological nature and molecular basis of human microinsemination techniques, especially those using immature male germ cells.

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.

The DNA methylation events in normal and cloned rabbit embryos

To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite sequence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and precociously de novo methylated, while the satellite sequence maintained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellite sequences may have aberrant methylation patterns in cloned embryos.

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.

Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer.

Parthenogenetic development (PA) is often used as a model to investigate activation protocols for nuclear transfer (NT) embryos. The objective of this study was to compare the development, as well as the dynamics of the nuclear materials and microtubules of PA and NT embryos following similar activation treatment. Our results demonstrate that, during parthenogenesis, activation through either electrical pulses or chemical stimulation alone resulted in low cleavage rates and compromised development. A combination of two sets of electrical pulses and a 2-h-exposure to chemical activation medium (5 microg/ml cycloheximide (CHX) and 2 mM 6-dimethylaminopurine (6-DMAP) in KSOM+0.1% BSA) could effectively activate rabbit oocytes, and resulted in a 99% (n = 73) cleavage rate with greater than 60% (n = 73) developing to blastocysts at day 4. However, the same activation protocol following NT resulted in only 65-72% of oocytes cleaved (depending on donor cell type), with less than 20% developing to the blastocyst stage. The differences observed between NT and PA embryos subjected to the same activation protocol were also evident in terms of the time required for their development to the blastocyst stage, as well as the cell numbers present in blastocysts at day 6. Furthermore, laser confocal microscopy revealed that pronuclear formation in the NT embryos was delayed by comparison to that in the parthenotes. In conclusion, our study suggests that an effective protocol for parthenogenesis cannot promise a comparable outcome for NT embryos.

Developmental Rate of Rabbit Parthenogenetic Embryos Derived Using Different Activating Protocols

The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated atl8 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+5 min culture in the presence of 5 mM lonomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3xEP schemes, comparing with EP+lonomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment EP+6-DMAP, which may be potentially used for nuclear transfer protocol.