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SummarySomatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

We injected mouse zygotes with combinations of Cas9 protein, Cas9 mRNA, and two gRNAs targeting a single exon of type I interferon receptor (IFNAR1) to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than Cas9 mRNA when each was used with a single gRNA, regardless of which gRNA was used. When Cas9 mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

Methyl-CpG-binding domain 3 (Mbd3) is a core repressor complex component. Although Mbd3 is required for the pluripotency of embryonic stem cells (ES), the role of Mbd3 in mouse ES (mES) cell apoptosis remains undefined. In this study naïve-state mES were derived and maintained in the presence of a selective protein kinase C pathway inhibitor (PKCi; Gӧ6983) to study the function of Mbd3 during mES apoptosis. Mbd3 overexpression in mES decreased the total cell number and viability, and it also dramatically increased the rate of apoptosis. Further investigation of Mbd3 overexpression revealed a 3-fold increase in the proapoptotic/prosurvival protein ratio (Bax/Bcl-2) and elevated RNA expression levels of apoptosis-related genes, including Bim, Trail, Fasl, and caspase 3, with reduced Bcl-2 RNA expression levels. Removal of PKCi from the mES cell culture resulted in upregulated Mbd3 expression and apoptosis, similar to the effects of Mbd3 overexpression. Furthermore, specific knockdown of endogenous Mbd3 partially rescued the mES apoptosis induced by the removal of PKCi, thus increasing the total cell number and viability while decreasing the rate of apoptosis. Additionally, Bax, Bim, Trail, and caspase 3 RNA expression levels were partially reduced, and that of Bcl-2 was partially increased. Our findings support Mbd3 as a pivotal regulator of apoptosis in mES.

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0-48.0%; P 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.

The guinea pig is an excellent but underused animal model due to its reproductive biology, which poses difficulties in inducing superovulation, embryo manipulation

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2–2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo-derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo-derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle-stimulating hormone (FSH) sources, Folltropin(®) Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well-established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo-derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.

Epigenetic modification and expression of key pluripotent factors are critical for development, cell fate determination, and differentiation in early embryos. In this study, we systematically examined the dynamic patterns of histone modifications (H3K4me3 and H3K27me3) and Nanog expression during the development of preimplantation rabbit embryos. Rabbit oocytes, 1-, 2-, 4-, 8-, and 16-cell embryos, morulae, and blastocysts were collected at specific time points following superovulation and assessed for nuclear H3K4me3, H3K27me3, and Nanog expression by immunofluorescence microscopy. The frequency of H3K4me3-positive nuclear staining was highest in oocytes through 4-cell embryos (100%), decreased in 8-cell (97.2%) and 16-cell (94.4%) embryos (P > 0.05), declined dramatically in morulae (86.7%) (1- through 8-cell embryos vs morulae, P < 0.05), and was the lowest in blastocysts (76.2%) (P < 0.05). Nuclear staining of H3K27me3 was negative in oocytes and embryos through the 16-cell stage but was positive in 25.9% of morulae and 34.2% of blastocyst (P < 0.05). Similarly, rabbit oocytes and embryos through the 16-cell stage did not express Nanog, but Nanog was expressed in 24.9% of morulae and 36.5% of blastocysts (P < 0.05). The observed decrease in H3K4me3 and increase in H3K27me3 as development progressed in preimplantation rabbit embryos, together with late Nanog expression, indicates a correlation of these factors with early embryonic cell fate determination and differentiation. Our study provides a specific and dynamic profile of histone modifications and gene expression that will be important for the derivation of rabbit embryonic stem cells and improving rabbit cloning by somatic cell nuclear transfer.

Exogenous genes can be site-specifically integrated into the genomic DNA of animals by homologous recombination, generating transgenic animals. These animals have a clear hereditary background, although position effects of the exogenous genes and potential functional disruption of host genes can be caused by the genetic inserts. Therefore, the generation of mammary gland bioreactors via gene-targeting methods is a great asset for producing recombinant proteins in milk. Here, we describe a method of generating gene-targeted goats with the human alpha-lactalbumin gene (hα-LA) integrated into the beta-lactoglobulin gene (BLG) locus. The milk from these goats will be less allergenic and will be enriched with components of human milk protein.

Exogenous genes can be site-specifically integrated into the genomic DNA of animals by homologous recombination, generating transgenic animals. These animals have a clear hereditary background, although position effects of the exogenous genes and potential functional disruption of host genes can be caused by the genetic inserts. Therefore, the generation of mammary gland bioreactors via gene-targeting methods is a great asset for producing recombinant proteins in milk. Here, we describe a method of generating gene-targeted goats with the human alpha-lactalbumin gene (hα-LA) integrated into the beta-lactoglobulin gene (BLG) locus. The milk from these goats will be less allergenic and will be enriched with components of human milk protein.

Hair follicle stem cells (HFSCs) are an important source for skin tissue engineering studies and clinical applications. Here, we describe a differential enrichment approach to derive HFSCs from hair follicles of vibrissae and ear skin using the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In the presence of Y-27632, primary cultured hair follicle cells grew in clustered colonies surrounded by keratinocyte-like cells and simultaneously expressed three HFSC markers: CD34, K15, and ITGB1. HFSCs cultured in medium containing Y-27632 were presented at a stable ratio of 30.7%, 34.1%, and 32.9% after passages 5, 10, and 15, respectively. By contrast, in medium containing epidermal growth factor, clustered HFSC colonies disappeared after 6 passages and lacked HFSC marker expression. After withdrawal of Y-27632 from the medium, HFSCs rapidly differentiated into keratinocyte-like cells. Furthermore, HFSCs derived with Y-27632 formed spherical clusters in collagen matrix

The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model.

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.

The first successful rabbit SCNT was achieved more than one decade ago, yet rabbits remain one of the most difficult species to clone. The present study was designed to evaluate the effects of two histone deacetylase inhibitors (HDACis), namely trichostatin A (TSA) and scriptaid (SCP), on cloning efficiency in rabbits. The in vitro development, acetylation levels of histone H4 lysine 5 (H4K5), and octamer-binding transcription factor 4 (Oct-4) expression patterns of cloned embryos were systemically examined after various HDACi treatments. Supplementation of TSA (50 nM) or SCP (250 nM) in the culture medium for 6 hours improved blastocyst development rates of cloned embryos compared with the treatment without HDACi. The combined treatment with TSA (50 nM) and SCP (250 nM) further enhanced morula (58.6%) and blastocyst (49.4%) rates in vitro. More importantly, compared with single HDACi treatments, embryos with the combined treatment had a higher level of H4K5 and an increased total cell number (203.7 ± 14.4 vs. 158.9 ± 9.0 or 162.1 ± 8.2; P < 0.05) with a better Oct-4 expression pattern in hatching blastocysts, indicating substantially improved embryo quality. This was apparently the first report regarding Oct-4 expression in cloned rabbit embryos. We inferred that most cloned rabbit embryos had an aberrant inner cell mass (ICM) structure accompanied with abnormal spatial distribution of Oct-4 signals. This study demonstrated a synergistic effect of TSA and SCP treatments on cloned rabbit embryos, which might be useful to improve cloning efficiency in rabbits.

We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

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.

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.

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, 𝑃 = . 0 0 1 ). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, 𝑃 = . 0 0 1 ). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

The average number of oocytes collected by ovum pick up (OPU) from Bos taurus cattle is8 per live donor. The objective was to determine whether development of small numbers of cattle embryos (produced by OPU and IVF), was enhanced by including "helper" embryos, produced from abbatoir-derived oocytes and embedded in agarose. Oocytes were from abbatoir-derived ovaries (Experiments 1 and 2) or OPU of elite donors (Experiment 3). In Experiment 1, cleaved embryos (2-8 cells), were randomly allocated. Controls were groups of 1, 3, 5, 10, and 20 cleaved embryos cultured in 50 μL serum-free SOF, whereas treatments were groups of 1, 3, and 5 embryos freely cultured along with helpers in groups of either 9, 7 or 5 embedded in agarose per droplet. Therefore, there were 10 cleaved embryos per droplet in combinations of 1 + 9, 3 + 7 or 5 + 5 (free + helper), respectively. There was an increase in the progression to blastocyst for 1-5 embryos per droplet, compared to 10 and 20 (6.6-24.2% vs. 39.2-43.3%, P0.05). For the tested free embryos, those cultured with helpers had increased blastocyst development over their control counterparts (39.3-49.5% vs. 6.6-24.2%, P0.05). When the number of embryos per droplet was 10 or 20, blastocyst percentage was similar (39.2-49.5%, P0.05). In Experiment 2, addition of an agarose chip to the culture medium did not significantly affect development to the blastocyst stage. In Experiment 3, after fertilizing OPU oocytes with sorted X-sperm, a group of three cleaved embryos were cultured in a droplet with either seven helpers (3 + 7) or alone (3 + 0). Blastocyst development of OPU oocytes in the 3 + 7 group was 37.1%, higher than that in the 3 + 0 group (11.8%, P0.05). In conclusion, limited numbers of OPU/IVF oocytes had competent development when cultured with helpers (embedded in agarose to provide physical separation).

Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3'UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied.

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4–23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8–54.5%) was also achieved with oocytes collected at 10–12 hpi than from those collected 14–16 hpi (8.3–14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2–42.8 %) were significantly higher than from those collected at 16 hpi (6.8–8.4 %) (p

International audience; In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer versus in vivo-derived embryos. Nuclear transfer (NT) was conducted using cumulus cells collected from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification. Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n=6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (NT: n=65 recipients; Vivo: n=20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45 and 75 and a sample of animals were slaughtered at each time point to recover the fetus/placenta for further analyses. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 versus 67.7%, P0.05), Day 45 (92.8 versus 49.1%, P0.01) and Day 75 (70.0 versus 20.8%, P0.0). There was no significant difference (P=0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14+/-0.23 g, n=8) versus in vivo transfers (0.75+/-0.19 g, n=8). On Day 45, there was no significant difference in the weight of either fetus (P=0.393) or membranes (P=0.167) between NT embryos (fetus: 2.76+/-0.40, n=12; membranes: 59.0+/-10.0, n=11) or in vivo-derived embryos (fetus: 2.60+/-0.15, n=6; membranes: 41.8+/-5.2, n=4). However, on Day 75 the weight of the fetus and several of the major organs were heavier from NT embryos. These data suggest that morphological abnormalities involving the fetus and the placenta of cloned pregnancies are manifested after Day 45.

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 μM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 μg/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1–4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

Canine retina expressed two forms of Type II procollagen mRNA, IIA and IIB, due to alternate splicing of exon 2 at the N-propeptide region. The N-propeptide region of Type II procollagen showed a high degree of homology with human, approximately 96% both at the nucleotide and the amino acid levels. Expression of IIA and IIB forms of Type II procollagen from early stage of development to adult life suggests the important role of both forms for the development as well as maintenance of the ocular structure.

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315 bp (-134/+181), 446 bp (-265/+181), and 1,100 bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos.

Stem cells are found in all multicellular organisms, including two broadly defined cell types: embryonic stem cells (ESC) (C. Y. Cheong and T. Lufkin; and N. Lifantseva et al.) that are derived from the inner cell mass of blastocyst-stage embryos and adult stem cells that are present in adult tissues (C. M. Teven et al., R. Chung et al., and A. C. Wilber et al.). Nuclear reprogramming refers to the erasure and remodeling of epigenetic marks, which is a part of normal mammalian development. This reprogramming is likely required for totipotency of the newly formed embryo and erasure of acquired epigenetic changes (Felici). Advances in stem cells including induced pluripotent stem (IPS) cells (D. Dey and G. R. D. Evans; and P. Noisa and R. Parnpai) and nuclear reprogramming (C. M. Teven et al.) will provide new insights into the mechanisms of cellular differentiation, during embryonic development (N. Lifantsevaa et al.) as well as in adult tissues (C. M. Teven et al.), and their pluripotency (A. C. Wilber et al.), which may lead to cell-based therapies (R. Eggenschwiler et al.) for several human diseases (R. Chung et al.). Fuliang Du Mark G. Carter Giorgio A. Presicce Shinn-Chih Wu Perng-Chih Shen

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

The present study was conducted to compare bovine embryo developmental quality, after culture in different defined culture media, up to blastocyst stage, and subsequently cultured in media supplemented with beta-mercaptoethanol (beta-ME) following blastocyst vitrification and thawing. In part one of this study, presumptive zygotes were randomly allocated into the following media: (1) CR1, (2) KSOM, (3) SOF, and (4) sequential KSOM-SOF. In the second part of the study, blastocysts derived from four different culture media were subjected to a solid surface vitrification (35% (v/v) ethylene glycol+0.5M Sucrose+5% (w/v) Polyvinylpyrrolidone (PVP), and tested for the effect of beta-ME on their post-vitrification survival. Following thawing, blastocysts were cultured with or without beta-ME. Culture medium had no effect on cleavage rates; however, a significantly greater number of zygotes cultured in KSOM, KSOM-SOF, or SOF developed to the 8-cell stage, compared with those cultured in CR1. A greater proportion of the zygotes cultured in SOF or KSOM-SOF reached blastocysts, than did those cultured in CR1 or KSOM. The use of sequential KSOM-SOF significantly increased total cell numbers of Day 7 expanded-blastocysts when compared to those cultured in CR1, KSOM, or SOF. Addition of beta-ME into culture media after vitrification and thawing improved blastocyst survival, hatching rates, and total cell numbers of blastocysts. In conclusion, supplementation of beta-ME into culture medium after vitrification and thawing significantly increased blastocyst survival, hatching rates, and their total cell numbers. These results suggest that vitrified IVF embryos should be thawed and briefly cultured in beta-ME medium prior to embryo transfer.

The Japanese Black cattle (Wagyu) plays a significant part in the Japanese beef industry because it is numerically the largest breed group and comprises ~93% of the national purebred beef cow herd. Japanese Black cattle are genetically predisposed to intense marbling and to a high percentage of unsaturated fat resulting in a meat characterised by both high quality and price on the market. Like other breeds, genetic improvement and production traits in Wagyu can be fostered by the implementation of traditional reproductive strategies such as multiple-ovulation embryo transfer. A multiple-ovulation embryo transfer case study was performed in a leading production centre in Hainan island, South China. Donors (n = 40) were split into 2 groups receiving either a total dose of 400 mg of FSH Folltropin (FSH USA; Folltropin Bioniche Inc., USA; n = 24), or 10 mg of an FSH formulation produced from the Institute of Zoology of the Chinese Academy of Sciences (FSH CHN; n = 16). In both cases, dosages were equally distributed over a 4-day administration schedule. Both donors and recipients (198 heifers) were synchronized for fixed-time embryo transfer (ET) and AI, respectively, by adopting the Ovsynch protocol. Such protocol consists of GnRH administration at Day 0, followed by prostaglandin administration at Day 7 and a second administration of GnRH at Day 9. Artificial insemination was performed on donor animals at 12 and 24 h from the last GnRH administration, whereas for recipient synchronization receiving fresh embryos, the second GnRH administration was given at the time of second AI on donors, and ET was performed 7 days following the first AI. Final synchronization at the time of ET, judged by the ultrasonic presence of a functional corpus luteum, was 53% (105/198). The following parameters for FSH USA and FSH CHN were found to be not significantly different (Student's t; mean ± s.e.): i) ovulations (10.5 ± 1.2 v. 8.5 ± 1.2; P = 0.2); ii) embryos (6.3 ± 1.2 v. 5.1 ± 1.1; P = 0.5), and iii) embryos from ovulated donors (6.8 ± 1.3 v. 5.8 ± 1.1; P = 0.6). Recovered embryos from the 2 groups were also not different: i) degenerated embryos (1.0 ± 0.4 v. 0.8 ± 0.2; P = 0.6); ii) morula (4.0 ± 0.8 v. 2.6 ± 0.5; P = 0.1), and iii) early blastocysts (4.0 ± 1.2 v. 3.3 ± 0.5; P = 0.6). Blastocysts were recovered only from donors treated with FSH USA. Out of 233 recovered embryos, 34 were transferred as fresh and 71 as frozen/thawed. Pregnancy rate at 60 days following ET for fresh and frozen/thawed embryos was 47.1 and 35.2%, respectively (P = 0.2). Within frozen embryos, pregnancy rates derived from transferred morulas and blastocysts were 25 and 45.5% (P = 0.07). When considering the two sources of hormones, overall pregnancy rates were similar between the two groups (28/71, 39.4% v. 13/34, 38.2%; P = 0.9). Finally, pregnancy rates from the transfer of fresh embryos (9/21, 42.8% v. 7/13, 53.8%; P = 0.6) and frozen/thawed embryos (19/50, 38% v. 6/21, 23.8%; P = 0.6) were also not different. In conclusion, all parameters in this study did not differ between the 2 sources of FSH; however, a lower incidence of degenerated embryos and a higher pregnancy rate following transfer of frozen/thawed embryos occurred when FSH USA was used in donor animals.

The disease phenotype of oculo-skeletal dysplasia (OSD) detected in Labrador retrievers and Samoyeds shows a large degree of similarity with human Stickler and Kniest dysplasia. Type II collagen (COL2A1) mRNA, which is defective in a larger number of Stickler and Kniest patients, has been cloned and characterized from normal dog. The amino acid sequence of the canine type II procollagen is predicted to contain 1487 residues, with high degree of homology with its human homologue, and maintains all the characteristic structural domains. In addition to cartilage, expression of COL2A1 has also been detected in canine retina and testes. In testes, the N-propeptide region of COL2A1 displayed differential splicing and expressed both splice variants, IIA (with exon 2) and IIB (without exon 2), suggesting the importance of both forms in testis maturation and maintenance. Despite a severe decrease of type II collagen protein in the vitreous of OSD affected Labrador retrievers, COL2A1 gene has been excluded from having any causal association with the disease locus by linkage analysis. Using an intragenic RFLP marker, COL2A1 gene has also been tested as a candidate gene for the non-allelic form of the other canine OSD identified in Samoyeds, and excluded by linkage analysis. Oculo-skeletal dysplastic Labrador retriever and Samoyed provide two animal models for chondrodysplasia with genetic heterogeneity.

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

The main scope of this study was to evaluate the likelihood of a helper effect of agar-embedded cleaved embryos on a low number of free embryos at a similar stage of development within the same culture droplet. Such an improved system could be beneficial within ovum pickup/in vitro embryo production (OPU/IVEP) combined protocols whenever a low number of OPU-derived cleaved embryos are produced per donor. Oocytes were recovered from abattoir ovaries, and after in vitro maturation (IVM) and in vitro fertilization (IVF), presumptive zygotes were deprived of cumulus investment and allocated into culture droplets for 24 h. At 48 h from IVF, 4- to 8-cell cleaved embryos were randomly allocated into a control and a treatment group. Control groups consisted of 1, 3, 5, and 10 embryos, respectively, in 50-μL droplets. Treatment groups consisted of 1, 3, and 5 free embryos with the addition of 9, 7, and 5 embryos, respectively, at a similar stage of development embedded in agar chips, so as to reach a total number of 10 cleaved embryos in each culture droplet. Culture was performed for both the control and treatment groups in SOF medium droplets covered with mineral oil, with the supplementation of essential and nonessential amino acids in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2 at 39°C. Final embryo output was checked at Day 7 from IVF. When considering only free embryos, the difference in progression to blastocyst development was highly significant between the control and treatment groups: 1) group 1 v. 1 + 9: 6.6 v. 84.3% (P = 0.00000); 2) group 3 v. 3 + 7: 11.1 v. 41.3% (P = 0.00001); 3) group 5 v. 5 + 5: 24.4 v. 42.2% (P = 0.00001). Rate of blastocyst development in the control group containing 10 cleaved embryos was not significantly different from free cleaved embryos in the 3 + 7 (39.2 v. 41.3%, P = 0.71) and 5 + 5 treatment groups (39.2 v. 42.2%, P = 0.54), but was significantly lower when compared with the 1 + 9 treatment group (39.2 v. 84.3%, P = 0.000). For 1, 3, 5, and 10 control group embryos, the numbers of replicates and total cleaved embryos used (n) were 30 (n = 30), 27, (n = 81), 27 (n = 135), and 39 (n = 390), respectively. For the 1 + 9, 3 + 7, and 5 + 5 treatment group embryos, the numbers of replicates and total cleaved embryos used were 32 (n = 32), 29 (n = 87), and 27 (n = 135), respectively. In conclusion, a beneficial effect of agar-embedded embryos on the development of free embryos within the same culture droplet was shown. A striking improvement in late-stage embryo development was particularly evident when considering the 1 v. 1 + 9 control and treatment groups. These results may foster a different strategic approach in in vitro culture to enhance embryo development from highly valuable donors.

The objective of this study was to examine the effect of heparin on bovine IVF and to improve the efficiency of IVF production by using sex-sorted sperm. The fertility performance of sex-sorted and unsorted semen from 4 bulls was compared to determine the optimal heparin concentration during preimplantational embryo development. A total of 7615 matured bovine oocytes were randomly allocated among different heparin concentrations (0, 2.5, 5, 10, 20, 40, 60, 80, and 100 μg mL–1) in Brackett-Oliphant medium and coincubated with either sex-sorted or unsorted sperm for 6 h. Presumptive zygotes were cultured in CR1aa+ 6 mg mL–1 of BSA in 5% O2 , 5% CO2 and 90% N2 at 39°C until Day 8 (Day 0, culture post-IVF). Cleavage rates at Day 2 and embryo development to blastocyst (BL) at Day 8 were recorded. Data (4 replicates) were analyzed by a general linear model (SPSS 11.0, SPSS Inc., Chicago, IL). The optimal heparin concentration for each treatment was determined as the lowest value from those groups that resulted in the highest BL rates. The results (Table 1) demonstrated that a differential requirement of heparin concentration was important for the highest preimplantational BL development between sexed sperm and unsorted control within each bull. By optimizing heparin concentration, in 3 out of 4 (75%) bulls, the in vitro BL development with sex-sorted sperm could be increased to a level that was comparable to the highest BL rate from unsorted sperm (bulls A, B, and C, P > 0.05). A higher heparin concentration was required for optimal BL development in bulls A and C; however, a lower concentration was desirable for bulls B and D, indicating that a partial capacitation to the sperm may have taken place in bulls B and D during the sorting process, as reported by Lu and Seidel (2004 Theriogenology 62, 819–830). The fertility of sorted sperm from bull D (1 out of 4, 25%) was adversely affected, even after heparin optimization for BL development (P < 0.05). This result suggests that sperm sorting could affect the IVF fertility of sorted sperm in a bull-specific manner, but it was not significant for all bulls. Table 1. Blastocyst (BL) development in bovine IVF after heparin optimization using sorted and unsorted sperm This project was supported by the SBIR program under a USDA Cooperative State Research, Education, and Extension Service (CSREES) grant to F. Du (USDA #2006-03069).

Nuclear reprogramming is dependent upon a number of factors, including chromatin organization and modification. Trychostatin A (TSA), a histone deacetylase inhibitor, was used to increase histone acetylation and to improve reprogrammability in both cattle and mice. The objective of the study was to determine whether TSA could improve the pre-implantational development potential of rabbit nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of FSH and hCG. Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in 10% FBS M199 and confirmed under fluorescence microscopy. Cumulus cells were prepared as nuclear donors for NT; a donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte, and subsequently fused with the oocyte recipient by application of 3 direct current pulses at 3.2 kV cm−1 for a duration of 20 µs/pulse. Fused embryos were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg mL−1 cycloheximide for 1 h. Rabbit NT embryos were cultured in 5 nM TSA-2.5% FBS-B2 medium for 10 h before being transferred into regular medium (FBS-B2). The TSA-treated embryos (5 nM vs. 0 nM) were cultured in 400 µL FBS-B2 medium for 5 days in 5% CO2 in a humidified atmosphere at 38.5°C (initiation of activation = Day 0). NT embryo development to cleaved (2 to 4 cell), morula, and blastocyst stages was evaluated on Day 2, Day 3, and Day 5, respectively. The selected NT blastocysts were counted for cell numbers following Hoechst 33342 epifluorescenin staining. The results (Table 1) showed that there was no difference on pre-implantational development of cloned embryos between TSA-added and control groups (P > 0.05). However, a significantly higher cell number per NT blastocyst was found in the TSA-added group (357 vs. 113; P < 0.05). This indicated that the blastocyst quality in NT embryos was improved with the addition of TSA by increasing histone acetylation activity. The developmental potential of TSA-treated NT embryos to term is under investigation. Table 1.Effects of TSA on the pre-implantational development of cloned rabbit embryos This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.

The developmental potential of oocytes fertilized in vitro by sexed–sorted frozen–thawed sperm remains low. The aim of this study was to find an optimal concentration of heparin during IVF using frozen–thawed sex-sorted sperm to improve the percentages of fertilization and blastocyst formation. A total of 1708 matured bovine oocytes were randomly allocated among different concentrations of heparin (0, 2.5, 5, 10, 20, and 40 µg mL−1) in Bracket-Olifant medium and co-incubated with non-sexed or sex-sorted sperm for 6 h (Day 0). The sperm (sorted or not) used for IVF was from one bull of proven fertility. Presumptive zygotes then were cultured in CR1aa + 6 mg mL−1 of BSA in 5% O2, 5% CO2, and 90% N2 at 39°C until Day 8. Percentages of cleavage were recorded on Day 2. Data (3 replicates) were analyzed by ANOVA. The results (Table 1) demonstrated that addition of different concentrations of heparin in IVF medium for non-sexed sperm groups did not alter cleavage and blastocyst formation. However, the lowest percentages of cleavage were observed with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups, demonstrating that high concentrations of heparin (20 and 40 µg mL−1) positively affected the percentages of cleavage and blastocyst formation. A greater proportion of zygotes in 20 and 40 µg mL−1 sexed sperm groups developed into grade 1 blastocysts on Day 7, and subsequently formed more blastocysts on Day 8 compared with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups. In conclusion, addition of heparin was not necessary when frozen–thawed non-sexed sperm were used for IVF with this particlar bull. However, addition of higher concentrations of heparin during IVF improved fertilization and blastocyst formation in vitro (40 µg mL−1 of heparin could be used as an alternative concentration for frozen–thawed sex-sorted sperm with this particular bull). The incidence of polyspermy as well as polyploidy in blastocysts is currently being studied. Table 1.Effect of heparin concentrations during IVF on cleavage and blastocyst formation

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

The aim of this experiment was to determine the effect of the sucrose concentration (0 to 0.33 M) in the dilution medium on the viability, fertilizability, and development of vitrified bovine oocytes. Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries and in vitro-matured as reported previously. After 24-h maturation in TCM199-based medium under 5% CO2 humidified air at 39°C, these were exposed to hyaluronidase and carefully pipetted to remove all except the 3–5 innermost layers of cumulus. Oocytes were put into the pre-equilibration medium for 3 min and then into vitrification solution containing HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol, and dimethylsulphoxide for 25–30 s; they were then vitrified by modified solid surface vitrification (Dinnyes et al. 2000 Biol. Reprod. 63, 513–518).The oocytes were warmed at 39°C by placing them in holding medium with 0, 0.08, 0.17, 0.25, or 0.33 M sucrose. Non-vitrified oocytes were used as controls. Oocytes were inseminated 30 min after warming, and the presumptive zygotes were cultured in CR1-aa medium supplemented with 6 mg/mL BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for eight days. Data were analyzed by one-way ANOVA. As shown in Table 1, there was no significant difference in survival rate (P > 0.05) of the vitrified oocytes that were placed in dilution solution containing 0.17, 0.25, or 0.33 M sucrose and the non-treated controls. On Day 2 (fertilized on Day 0), cleavage to the 8-cell stage was similar for the 0.17, 0.25, and 0.33 M dilution groups, but the rates for all three were significantly lower (P < 0.05) than for the control group. The blastocyst rate on Day 8 was significantly higher for the 0.25 M group than for any other experimental group but still significantly lower than for the control. In conclusion, this study suggests that with this vitrification/warming procedure the optimum concentration of sucrose in the dilution solution is 0.25 M. Table 1. Oocyte survival after vitrification/warming and subsequent embryo development The authors would like to thank Ms Colleen Shaffer for the preparation of bovine oocytes.