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There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.

The efficiency of animal production using cloning technology is still relatively low and research to determine a more efficient nuclear transfer procedure is ongoing. One approach which may be informative in assessing the viability of nuclear transfer embryos is the analysis of embryonic gene expression. Using RT-PCR techniques we have previously detected the aberrant expression of FGF4, FGFr2 and IL6 in a significant proportion of bovine granulosa cell-derived nuclear transfer embryos, which correlated with a limited developmental potential in vivo. In order to analyse the effect of different donor cell nuclei on embryonic gene expression we have now analysed the expression of these genes in nuclear transfer embryos reconstructed with fetal epithelial cell nuclei. In addition, we have compared the expression of these genes in bovine nuclear transfer embryos produced by cell fusion or direct injection with variations in the timing of oocyte activation. In all nuclear transfer embryos analysed, FGFr2 and IL6 transcripts were detected at a similar rate to that in IVF embryos. However, the absence of FGF4 transcripts was again evident in a large proportion of nuclear transfer embryos and most significantly in those embryos whose development was activated almost immediately following the transfer of the donor nucleus. The results demonstrate the effects that different donor cell lines and different nuclear transfer procedures may have on the expression of developmentally important genes in nuclear transfer embryos.

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.

Placental abnormalities and failed implantation are characterized phenotypes that occur in many species as a result of somatic cell cloning. This study examines a number of genes, critical for early placental development and reports aberrant expression patterns in a number of cloned bovine blastocysts, thus implicating a role of these genes in failed implantation. Messenger RNA (mRNA) expression of eight genes critical for early placental and preimplantation development including Acrogranin, Cdx2, Eomes, ErbB3, ERR2, Hand1, MRJ, and Rex1 were analyzed in single, in vivo, in vitro, and cloned bovine blastocysts (produced by hand-made cloning (HMC) and serial hand-made cloning (SHMC)) following complementary DNA (cDNA) amplification with a SMART cDNA synthesis kit. Aberrant expression of Acrogranin, Cdx2, and ERR2 was detected in a number of blastocysts produced by SHMC. Other genes, Eomes and Hand1, were not detectable in, in vivo bovine blastocysts, suggesting a differential expression pattern between bovine and murine embryos. A number of control marker genes including Oct4, IFN-tau, and PolyA were expressed in all single blastocysts analyzed. This is the first study to report that failure of implantation may be due to aberrant expression of genes in the preimplantation cloned embryo, which are crucial for the early regulation and differentiation of the placenta.

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.

Adenosine triphosphate (ATP) plays an important role during fertilisation of the mammalian oocyte through its ability to alter the frequency and duration of calcium oscillations. It has also been shown that higher ATP levels correlate with increased developmental competence in bovine and human oocytes. During somatic cell nuclear transfer (NT), the incoming nucleus is remodelled extensively, undoubtedly using a variety of ATP-dependent enzymes. The aim of the present study was to determine whether additional exogenous ATP influences activation of parthenogenetic (PA), in vitro-fertilised (IVF) or cloned (NT) in vitro-matured bovine oocytes. Blastocyst development and cell numbers in PA embryos were found to increase in a dose-dependent manner following the photorelease of 0, 50, 100, 500 and 1000 μm DMNPE-caged ATP (adenosine 5′-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt). No cleavage was found following release of 2 and 5 mm DMNPE-caged ATP or with DMNPE-caged ATP (not photoreleased). There were also no differences in blastocyst rates or cell numbers between the control group and groups treated with caged, but not photoreleased, ATP. The addition of exogenous ATP before IVF or to NT couplets did not result in a significant increase in blastocyst development or cell number. Embryo transfer is necessary to determine whether exogenous ATP can positively affect reprogramming, resulting in higher cloned pregnancy rates or live-term births.

The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. The technique involves manual bisection of zona-free oocytes, selection of cytoplasts by Hoechst staining and fusion of a single somatic cell and two cytoplasts. In this proof-of-principle experiment, the objective was to examine the developmental competence of HMC embryos following embryo transfer. Modifications to the original method include not selecting of matured oocytes and simultaneous fusion of cytoplasts and karyoplast. Blastocyst rates for embryos cultured in the glass oviduct system as singles (10.5%; 24/228) or in pairs (16.1%; 36/224) did not differ significantly. Fresh and vitrified–thawed blastocysts were transferred to 16 synchronised recipients (three to four embryos per recipient). Ultrasound examination on Days 35–45 showed an initial pregnancy rate of 43.8% (7/16) and a pregnancy rate >8 months of 12.5% (2/16). A male cloned calf (42 kg) derived from a vitrified HMC blastocyst was delivered by Caesarean section on Day 271. The birth and ongoing survival (15 months, 243 kg) of a healthy and apparently normal calf, combining both HMC and vitrification technologies, provides a ‘proof of principle’ of the technology and a promising alternative to traditional nuclear-transfer techniques.

The present study aimed to characterize development of in vivo and somatic cell nuclear transfer (SCNT) Merino ovine embryos on Days 17 and 19 using the mesoderm marker vimentin, the neuroectoderm marker β-tubulin III, and the pluripotency marker OCT4 for primordial germ cells. In vivo embryos were obtained by transferring 10 to 20 zygotes to each of 20 intermediate recipient ewes. On Day 6, 4 final recipients were used to obtain embryos for recovery at Day 17 (n = 2) and at Day 19 (n = 2). SCNT embryos were constructed from in vitro-matured oocytes and adult granulosa cells. On Day 6, 9 embryos (with intact inner cell masses) were transferred to each recipient for collection at Day 17 (n = 2) and Day 19 (n = 2). Ewes were euthanazed with phenobarbitone and excised reproductive tracts flushed with saline. A total of 24 embryos at Day 17 (14 in vivo and 10 SCNT) and 23 embryos at Day 19 (11 in vivo and 12 SCNT) were collected and processed for immunohistochemistry. On Day 17, length of embryo proper for in vivo and SCNT embryos was 6.2 and 5.6 mm, width of allantois was 9.3 and 3.8 mm, and number of somites was 19 and 13, respectively. On Day 19, length of embryo proper for in vivo and SCNT embryos was 11.5 and 7.3 mm, width of allantois was 70.6 and 13.7 mm, and number of somites was 28 and 22, respectively. On both Day 17 and Day 19, in vivo embryos had a much larger allantois, more somites, and were longer in length. Vimentin staining was observed in all in vivo embryos at Day 17 and Day 19; however, in 75% of Day 17 SCNT embryos, differentiation of the somites into dermatome with underlying sclerotome was either delayed or absent. Similarly, at both Day 17 and Day 19, a larger proportion of embryos did not stain for β-tubulin III, and of those that did, only a small amount was found in the neural tube. The intensity of both stains was much weaker in the SCNT embryos compared to in vivo embryos. Oct-4 was initially found in the splanchnic mesoderm lining the endoderm in the wall of the yolk sac and later in the dorsal mesentery and medial aspect of the mesonephros with in vivo embryos having double the number of positively stained cells than SCNT embryos. The delay in mesoderm compartments of SCNT embryos could imply future problems in the musculoskeletal system. However, lack of any neuroectoderm staining in more than half SCNT embryos could point to neurological problems being more of an issue in terms of survival and well-being. Equally, the smaller allantois could induce later placental abnormalities. The current project was funded by the Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Australia.

The Cbx gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation, and chromatin remodeling. In Drosophila embryos, the single family member HP1 localizes to the nuclei from the blastoderm stage, nuclear division cycle 10, suggesting that heterochromatin formation occurs around the time of embryonic transcriptional activation (James et al. 1989 Eur. J. Cell Biol. 50, 170–180). In mice, HP1� is absent in the first cell cycle in both parental nuclei following fertilization (van der Heijden et al. 2005 Mech. Dev. 122, 1008–1022). We report the first study of gene expression, mRNA and protein localization, of the Cbx gene HP1� in in vitro-produced bovine embryos. Expression of the HP1� gene was assessed non-quantitatively in amplified cDNA from 3 individual oocytes or embryos for each stage. HP1� (Cbx5) transcripts were detected in all samples except one oocyte, three 4-cell embryos, and one Day 7 blastocyst. The lack of transcripts at the 4-cell stage may reflect an early degradation of maternal transcripts, or indicate that transcript levels were too low to be detected with this assay. Immuno-localization of HP1� was then performed by using an anti-HP1� monoclonal antibody (Upstate, Auspep Pty., Ltd., Parkville, Victoria, Australia), revealing HP1� to be localized evenly in the cytoplasm of oocytes, and then progressing to both cytoplasmic and nuclear staining at the 2-cell stage. This was followed by nuclear staining from the 4-cell stage onward. Higher power investigation of the subnuclear localization of HP1� showed a diffuse type staining pattern within the nuclei of 2- and 4-cell embryos, followed by punctate staining within the euchromatin in the 8-cell embryos, and within the heterochromatin by the 16-cell stage. At the blastocyst stage, staining appeared more diffuse, but localized to the heterochromatin. This suggests that HP1� is localized to the euchromatin early, followed by subsequent localization to the heterochromatin following the MET. HP1� localization to the euchromatin was recently described (Grigoryev et al. 2004 J. Cell Sci. 117, 6153–6162) during a differentiation event in murine lymphocytes. It is evenly distributed throughout the nucleus in quiescent lymphocytes, but localizes to the vicinity of centromeric chromatin during lymphocyte reactivation. HP1-TIF1α interaction was also recently shown to be required during a short period of time within primitive endoderm-like cells for terminal differentiation (Cammas et al. 2004 Genes Dev. 18, 2147–2160). The results thus indicate that HP1� behaves similarly in embryonic differentiation and lymphocyte reactivation, implying a common mechanism of chromatin remodeling in the 2 cellular systems. Collectively, the results suggest that dynamic changes of the nuclear–cytoplasmic and subnuclear distribution of HP1� may play a key role in the bovine MET.

Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.

Vitrification has been widely developed for the cryopreservation of mammalian embryos. Despite considerable effort, the vitrification of immature bovine oocytes is less successful with poor survival post thawing and low fertilization rates and development to offspring. The MII oocyte is highly sensitive to temperature changes with cryopreservation resulting in high incidence of aneuploidy after fertilization. Recently the concentration of sodium ions has been implicated in vitrification-induced damage. This study investigated the effect of a low-sodium choline-based medium (CJ2) supplemented with either 10% bovine fetal serum (FBS), 0.8% bovine serum albumin (BSA) or 1% polyvinylalcohol (PVA) on the ability of in vitro matured metaphase II (MII) oocytes to survive vitrification. Cumulus removed MII matured oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in CJ2 containing FBS, BSA or PVA for 5 minutes at 37ºC and then transferred into a vitrification solution composed of 20% EG, 20% DMSO and 0.6 M sucrose in CJ2. Twenty MII stage oocytes at a time were aspirated into the Gel Loader tip (GL-tip) with approximately 20 µL of vitrification solution, equilibrated 30 sec before plunging directly into liquid nitrogen and stored for 2 h. Oocytes were thawed rapidly and cryoprotectants removed by step-wise dilution in 0.25 M, 0.15 M, 0 M sucrose in TCM-199, 5 min each. Oocytes were then incubated in TCM-199 for 2 h before being stained with Hoechst-33342 and viewed under epi-florescence to determine survival. Developmental competence was determined by parthenogenetically activating (PA) surviving oocytes using calcium ionophore/6-dimethylaminopurine and cultured for 7 days in mSOF medium supplemented with 0.8% BSA. The recovery from vitrification procedure, survival post thawing and PA rates are summarized in the Table below. Vitrification in GL-tips allows efficient processing of MII oocytes with high rates of recovery. Overall, blastocyst development in this experiment was low in the control, sham groups and treated groups and that results may have been influenced by other factors (i.e. oocyte quality). Nevertheless, results demonstrate that reducing or eliminating sodium ions from the vitrification medium may protect the immature oocytes during vitrification or thawing and allow oocytes to be cryopreserved more effectively.

Advanced reproductive technologies using in vitro production systems in cattle and other livestock species offer significant opportunities for improving the reproductive performance of the female. Impeding the development is the overall inefficiency and significant costs surrounding the high incidence of embryo wastage, particularly during early pregnancy and the occurrence of foetal developmental abnormalities, placentation irregularities and late term losses. Effective molecular interchange and communication between the conceptus and its mother is of utmost importance for the development, growth and continued viability of the foetus. We are using a proteomics approach to determine the protein profile (proteome) of the conceptus fluids (amniotic and allantoic) during the first trimester of normal, IVF and NT pregnancies. Proteomic analysis and comparison of the proteins present in these fluids may lead to the discovery of novel biomarkers which can be used to determine foetal viability status. Collections of foetal fluids from abattoir collections of Day 35 to Day 100 pregnancies were characterised with respect to volume and total protein estimates and subjected to various fractionation procedures. Protein fingerprints of conceptus fluid fractions have been generated by two-dimensional electrophoresis and the identity of proteins determined by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and database searches. Enrichment of the low-abundance and low molecular weight proteins has been achieved by ultrafiltration under denaturing and reducing conditions. We were able to establish the identity of the majority of high-abundance proteins and a significant proportion of low-abundance proteins in both amniotic and allantoic fluids using this method. Searching of MSDB or NCBInr mammalian protein sequence databases coupled with the putative protein database deduced from the first draft assembly of the bovine genome sequence, was achieved using Mascot software.

The generation of cloned animals by somatic cell nuclear transfer has been reported in a number of countries worldwide. However, progress has been impeded by the extremely low efficiency of cloning and health of some of the cloned animals. Surprisingly, little is known of the reproductive performance of viable clones when compared to the original cell donor, given that a major motivation of cloning is dissemination of superior genotypes. The aim of this study was to compare semen collected from three cloned bulls (Clones A1, B1, and B2) to that of the original donor bulls (Donors A and B). Parameters examined included ejaculate volume, sperm concentration and the motility characteristics of frozen/thawed semen using computed-assisted semen analysis (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA). The fertilization ability of each semen sample was examined using in vitro matured oocytes derived from abattoir-source ovaries. Frozen/thawed semen samples from donor and cloned bulls were prepared on a Percoll gradient and diluted with fertilization medium to a concentration of 1 million sperm/mL prior to fertilization (IVF). The number of blastocysts and total cell counts were analyzed on Day 7 of culture. Finally, in vitro-fertilized blastocysts (Day 7) were transfer to synchronized recipients (n = 49) to examine in vivo development. Proportional data for the in vitro development of embryos and subsequent pregnancy rates were analyzed by chi-square test, and embryo cell numbers were analyzed using Student's t-test. Progressive motility percentage between donor and cloned bull did not differ: Donor A (62.25 ± 3.89, n = 12); Donor B (66.69 ± 4.47, n = 13); Clone A1 (71.37 ± 8.57, n = 8); Clone B1 (73.75 ± 2.42, n = 12); Clone B2 (72.41 ± 3.26, n = 12). No obvious differences in kinetic motility parameters were evident between cloned and non-cloned donor animals. However, blastocyst rates were significantly higher in cloned bulls (Clone A1: 30.9%, 81/262; Clone B1: 34.4%, 98/285; and Clone B2: 42.9%, 120/280) compared to donor bulls (Donor A: 20.7%, 54/261; Donor B: 20.9%, 76/364). Total embryo cell numbers did not differ significantly between donor bulls (Donor A: 138.3 ± 5.3, n = 39; Donor B: 133.2 ± 5.2, n = 47) and cloned bulls (Clone A1; 126.3 ± 4.4, n = 45; Clone B1: 134.4 ± 7.1, n = 26; and Cloned B2: 140.1 ± 3.9, n = 46). Initial pregnancy rates on Day 30 were also not different between Donor A (42.3%, 11/26) and Clone A1 (47.8%, 11/23). Preliminary observations from the small data set on postpubertal cloned bulls indicate that semen production, semen morphology, and reproductive performance (in vitro and in vivo) were similar in terms of semen characteristics and reproductive performances when compared to their original donor bulls.

The efficiency of obtaining live calves from somatic cell nuclear transfer remains quite low. One factor implicated in this failure is inadequate chromatin remodelling of the donor nucleus. Five Polycomb group (PcG) genes were investigated as potential remodelling factors in bovine oocytes and preimplantation embryos. These genes (Cbx6, Eed, Edr1, Yy1, and Zfp144) are involved in transcriptional activation and cell cycle regulation. We hypothesize that inadequate expression may cause the developmental abnormalities seen following cloning. This study is aimed at characterizing normal expression, prior to comparative studies with cloned embryos. Three single abattoir-derived in vitro-matured (IVM) oocytes or in vitro-produced (IVP) embryos from each of the following stages: 2-cell, 4-cell, 8-cell, 16–32 cell, morula, Day 7 blastocyst, and Day 8 hatched blastocyst, were studied. Messenger RNA was isolated from individual samples with Dynabeads (Dynal, Inc., Lake Success, NY, USA) and then cDNA was created and amplified with a SMART cDNA synthesis kit (BD Biosciences Clontech, Palo Alto, CA, USA). Products were diluted 1:10 and used to amplify target genes by PCR. PCR products were sequenced for confirmation of identity. All amplified embryo samples expressed the housekeeping genes Poly(A) polymerase and actin. Similarly, all embryos up to the 8-cell stage expressed GDF9, an oocyte-specific gene, while IFN-tau, involved in maternal recognition of pregnancy, was expressed in one morula and all blastocyst samples. Cbx6 was not expressed at any stage. The other four genes were all expressed fairly consistently throughout the pre-implantation period. Eed transcript was detected in all samples, with the exception of one oocyte, one 4-cell embryo, and one 8-cell embryo. Edr1 transcript was detected in all samples except for two oocytes, one 16–32-cell embryo, and one Day 7 blastocyst. Yy1 was expressed in all but one oocyte, one 2-cell embryo, two 4-cell embryos, and one Day 7 blastocyst. Finally, Zfp144 transcript was detected in one oocyte and in all embryos until the 16–32 stage, and then was not detected until seen in one Day 7 and all Day 8 blastocysts. Cbx6, yet to be fully characterized in any species, is also known as the neuronal pentraxin receptor, and is involved in transport and clearance of synaptic debris. It is known to have the characteristic chromobox domain of the Cbx family, of which several family members play a role during pre-implantation development. Eed, Edr1, Yy1, and Zfp144 are expressed throughout the pre-implantation period, although levels of Zfp144 mRNA appear to drop during the embryonic genome activation and then reappear by the late blastocyst stage. This consistent expression may suggest a role for the proteins in chromatin remodelling or modulating patterns of gene expression in early development. Further studies are required to determine if these factors are expressed incorrectly in cloned embryos, potentially providing a link to the abnormalities observed post nuclear transfer.

Two limitations currently restrict the acceptability and adoption of bovine cloning as a commercial reality. The first of these is its low rate of efficiency and the second is that some of the cloned calves are not healthy. Abnormalities in the placenta are thought to contribute to many of the losses in early gestation; however, less is known of the pathology of clone deaths in the perinatal period. To date, the majority of perinatal deaths have been attributed to the “large offspring syndrome” characterized by increased birth weight and a range of morphological abnormalities thought to be associated with in vitro culture and manipulation. This report describes multi-systemic abnormalities in aborted, stillborn, and neonatal genetically modified and unmodified cloned calves weighing less than 60 kg at birth and aged between 6 months gestation and 3 weeks postnatal, generated in various experiments. Three of 14 genetically modified cloned calves had cystic renal dysplasia and osteopetrosis. All three and a fourth had irregular nodular, fibrotic livers with biliary abnormalities. Another two had marked flexion of the fetlock joints. Eleven calves derived from an unmodified cloned cell line by nuclear transfer had nodular, fibrotic livers with biliary anomalies, 9 of 11 had cystic renal dysplasia and cardiomegaly, two had osteopetrosis, and two had contracted tendons. In addition, three calves had polymicrocerebral gyri, two had retinal dysplasia, and one had an aortic aneurysm. Only one calf from a second unmodified cloned cell line produced by nuclear transfer had no significant congenital abnormalities. All calves were negative for bovine virus diarrhea virus (BVDV) by competitive-antigen ELISA, and by virus isolation and no BVDV antibodies were detected by AGID assay. Furthermore, all cell lines and media used were negative for BVDV by virus isolation. Two calves were tested and found to be negative for Akabane virus and Aino virus. There are very few reports of the pathological abnormalities of cloned animals. Similar multi-systemic abnormalities have not been found in non-cloned calves, but several analogous conditions occur in humans, including Simpson-Golabi-Behmel and Zellwegers syndromes. Further ultrastructure studies and genetic analysis are needed to investigate the mechanisms of these multi-systemic disorders, which may ultimately elucidate mechanisms for improved reprogramming and increase the efficiency of generating cloned animals with somatic cells by nuclear transfer.

Abnormalities of the placenta are a major factor contributing to early death in cloned bovine conceptuses. This is primarily due to incomplete chromatin remodeling and reprogramming of the donor nucleus. It is unknown whether genetic aberrations of genes crucial for placental development can be detected in pre-implantation cloned bovine embryos. This study looked at the expression profile of four genes in single bovine blastocysts derived from in vivo, in vitro produced (IVP), or cloning techniques, including handmade cloning (HMC) and serial HMC (SHMC). The genes studied included acrogranin, caudal type homeobox 2 (cdx2), estrogen-receptor-related receptor beta (essrb), and the mammalian relative of DnaJ (MRJ). These genes play a role in trophoblast regulation and placental development. Messenger RNA expression was analyzed by using PCR following cDNA amplification by means of SMART cDNA synthesis (Clontech, Palo alto, CA, USA). Primers were designed from homologous human and mouse sequence. PCR products were sequenced for verification. Five single blastocysts were analyzed from each of the following treatments: in vivo, IVP, HMC, and SHMC. Pooled (n = 10) IVP blastocyst cDNA produced by standard RT was used as a positive control. Grade 1 Day 7 blastocysts were selected for all treatments. Amplified cDNA was tested using control genes polyA, IFN-τ and GDF9. In vitro-produced embryos were matured, fertilized and cultured as published by Ruddock et al. (2004 Biol. Reprod. 70, 1131). Cloned HMC embryos were produced as described by Tecirlioglu et al. (2003 Reprod. Fertil. Dev. 15, 361). Serial HMC embryos were produced as per the HMC embryos, followed by a second round of nuclear transfer at the pronuclear stage. The pooled IVP, in vivo, and IVP blastocysts expressed all four genes of interest. In the HMC-cloned embryos, all four genes were expressed. However, in the SHMC cloned embryos, although MRJ was found to be expressed in all blastocysts, three of the five blastocysts did not express acrogranin. Similarly, two SHMC embryos did not express cdx2, and essrb was weakly expressed in three of the five embryos analyzed. Initial pregnancy rates of HMC and SHMC embryo transfers are similar. Further pregnancy results are pending. These results indicate that aberrations of genes crucial for placental development can be detected in single cloned blastocysts. It also suggests that failed implantation and/or placental defects may stem from patterned genetic defects in the pre-implantation embryo. An increase in the number of embryos analyzed would further strengthen results. These genes could act as markers to identify cloning techniques that produce more embryos with normal genetic profiles. The benefits of developing a screening tool to assess abnormalities in single pre-implantation embryos would be significant.

A major limitation of somatic cell nuclear transfer (NT) for the production of cloned calves is that only 1–5% of cloned embryos produce viable calves. The high rate of mortality is attributed to both pre- and post-natal losses and is primarily due to incomplete reprogramming of donor cells. Almost 50% of the pregnancy losses occur in the first trimester of pregnancy, indicating a major disruption in normal embryo development at NT. The aim of this study was to analyze germ layer formation by stereomicroscopy and immunohistochemical techniques for both NT embryos and their in vivo counterparts on Day 21 and to compare deviations from normal embryonic development as a measure of developmental capacity. Blastocyts derived by IVF (n = 20), conventional NT (n = 20), or hand made cloning (HMC; n = 20) were non surgically transferred to each synchronized recipient cow (n = 3). Each group of twenty embryos was transferred to one recipient. Cows were slaughtered on Day 21 and uterine tracts recovered and flushed with phosphate-buffer solution with 10% serum. Recovered Day 21 embryos were fixed in 4% paraformaldehyde, and embedded in paraffin; serial sections were stained with hematoxylin and eosin and evaluated by light microscopy. Immunohistochemical localization of cytokeratin 8 was used as a marker for potential ectoderm, alphafetoprotein for potential endoderm, and vimentin for potential mesoderm. Four IVF (20%; 4/20) embryos, six NT (30%; 6/20) embryos, and ten HMC (50%; 10/20) embryos were recovered following flushing. No obvious morphological differences were seen in the formation of a neural tube, differentiation of mesoderm, and number of somites among IVF (25%; 1/4), NT (33.3%; 2/6), and HMC (20%; 2/10) embryos. Delayed development with respect to the formation of neural groove and mesoderm differentiation was observed in 25% (1/4) of IVF, 16.7% (1/6) of NT, and 30% (3/10) of HMC embryos. In addition, 25% (1/4) of IVF, 16.7% (1/6) of NT, and 33.3% (2/6) of HMC embryos had not initiated gastrulation (i.e. displayed hypoblast and epiblast), suggesting a more substantial developmental delay. The remaining embryos showed gross abnormalities compared to their in vivo counterparts, including degenetration of epiblast and hypoblast cells. Cytokeratin was detected in the trophoblast, ectoderm, hypoblast, and endoderm. Alpha-fetoprotein was detected in the hypoblast while vimentin was seen in the mesoderm. In conclusion, although localization of staining in IVF and cloned embryos was consistent with that of in vivo embryos, the intensity was weaker, suggesting compromised or delayed development. It is also possible that the differences observed were due to the recipient and not to the treatment group. This work was supported by Genetics Australia Cooperative Limited, Victoria, Australia.

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

Culture medium supplemented with sera is commonly used for the in vitro production (IVP) of livestock embryos. However, serum induced complications including batch variation, the potential risk of virus and mycoplasma contamination and the implication in the large offspring syndrome in domestic animals impels the development of a serum-free culture system. In this study, we investigated whether replacement of fetal bovine serum (FBS) with bovine serum albumin (BSA) in three maturation media, tissue culture medium-199 (TCM-199), a modified synthetic oviduct fluid (mSOF) routinely used in our laboratories and a commercially available SOF-VC (Vitro Cleave, Cook Australia). Harvested oocytes were matured, parthenogenetically activated and in vitro cultured (Day 7) to measure maturation efficiency, embryo development and quality with the aim of developing a simplified and defined culture medium for the in vitro production of bovine embryos. Abattoir derived cumulus oocyte complexes were matured in TCM-199, mSOF and SOF-VC media supplemented with LH and beta-estradiol in the presence of 15% FBS or 0.08% BSA at 39ºC in 5% CO2 in air. Polar body extrusion was assessed twenty-two hour post maturation and selected MII occytes were activated using calcium ionophore/6-dimethylaminopurine and cultured for seven days in SOF medium supplemented with 0.8% BSA. On day seven, blastocyst development was assessed and randomly selected blastocysts were stained to determine inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN). Supplementation with either BSA or FCS did not significantly affect the maturation efficiency, blastocyst rates or differential cell numbers within each maturation media tested. However, maturation efficiency and blastocyst rates were significantly lower (P < 0.01) when oocytes were matured in either mSOF or SOF-VC regardless of FBS or BSA supplementation. From this study, we conclude that BSA effectively replaces FCS and TCM-199 is superior to SOF (mSOF or SOF-VC) in terms of oocyte maturation regardless of protein source. Once matured SOF and TCM-199 parthenogenetically blastocysts were equivalent in terms of embryo development and quality.

The establishment of viable embryonic development following in vitro production (IVP) or nuclear transfer by SUZI (subzonal injection) and handmade cloning (HMC) methods is pivotal for practical implementation of embryo technology. The development of cloned embryos is currently impeded by the high rate of pre- and postnatal losses. This project is aimed at developing screening methods using both ultra-structural and molecular markers of development of post-hatching/pre-implantation stage embryos to enable selection of embryos with greater developmental competence. Initially twenty embryos derived by IVP or nuclear transfer (SUZI or HMC) were transferred to six recipient cows on Day 7 (n=120). Embryos were collected on Days 14 and 21 by flushing the uterine tracts of slaughtered cows. Embryos were fixed in 4% paraformaldehyde and processed for paraffin embedding. Serial sections were stained with haematoxylin and eosin and evaluated by light microscopy. On each day of collection, certain characteristics were expected to be observed according to normal in vivo development (Maddox-Hyttel et al., 2003, Reproduction 125, 607–623). On Day 14, IVP embryos (n=6) were ovoid to filamentous in shape. Three embryos had extensive foldings of trophoblast in the process of forming an amniotic cavity. NT-SUZI embryos (n=4) had a mainly round uneven shape. No NT-HMC embryos were recovered. On Day 21, IVP embryos (n=4) more closely resembled in vivo embryos than any other group and formed either a neural groove or a neural groove with somites. NT-SUZI embryos (n=6) were mainly filamentous, with one embryo showing complete development of the neural tube and 5 somite pairs. NT-HMC (n=17) development ranged from ovoid to multi-protrusions of the yolk sac. One embryo displayed the formation of a neural tube. Embryos, irrespective of production method, which had an abnormal morphology, presented only an incomplete hypoblast and severe degeneration of the epiblast. Those embryos that were morphologically comparable to in vivo embryos also displayed developmental stages which ranged from presentation of a primitive streak and formation of a neural tube to having a neural tube, differentiation of mesoderm and somites. At present, the low number of embryos recovered as well as their abnormal development clearly reflect the low success rate to term of live calves. It is possible that no Day 14 NT-HMC embryos were recovered due to collection efficiency not being optimal. It is apparent that IVP and cloned embryos have different developmental time lines as compared to that of in vivo embryos.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g. pig organs) used for human transplantation are normally destroyed by the host within minutes by hyperacute xenograft rejection. An improved understanding of the immune recognition and rejection of xenografts has resulted in new therapies that can partially overcome hyperacute rejection (HAR), delayed xenograft rejection (DXR) or acute vascular xenograft rejection. Strategies to diminish immunogenicity following xenotransplantation can be divided into two approaches: those directed at the recipient (e.g. antibodies or complement depletion or inhibition and tolerance induction) and those directed at the donor (e.g. transgenic modifications to express human complement-regulatory proteins or removal or displacement of alphaGal epitopes). DXR is likely to be controlled by transgenic inhibition of endothelial cell activation (e.g. inhibition of NF-kappaB). Transgenic pigs required for xenotransplantation will soon be generated at a greater efficiency and precision using nuclear transfer and cloning when compared to pronuclear injection. Of greater significance is that nuclear transfer offers the ability to target gene insertion selectively to specific gene loci and to delete specific genes in the pig. Experimental pig-to-primate organ xenotransplantation is currently under way, and results show increased transplant function from minutes to days and weeks. The final therapeutic regimen that allows survival of a discordant xenograft is likely to involve a combination of 'modified' functional genes in the donor organ, the development of immunological tolerance to pig antigens and administration of novel therapeutic agents, including immunosuppressants, that can control natural killer (NK) cell and monocyte mediated responses.