Search

Atraumatic chest pain is a common emergency department (ED) presentation and the American College of Cardiology and American Heart Association recommends stress testing within 72 hours. The HEART score predicts major adverse cardiac events (MACE) in ED populations and does not require universal stress testing. An evaluation based solely on history, electrocardiography, and biomarkers, therefore, is an attractive approach to risk stratification in resource-limited settings. The HEART score has not been previously evaluated in a safety net hospital setting. We therefore implemented an interdisciplinary clinical care guideline utilizing the HEART score to stratify patients presenting to our inner-city hospital. During a 6-month study period, 1170 patients were evaluated (521 before and 649 after implementation). Among the 998 patients with confirmed follow-up 6-weeks after the index ED encounter, the prevalence of MACE (all-cause mortality, acute myocardial infarction, or coronary revascularization) was 0% [95% confidence interval (CI), 0%-1%] for low, 9% (95% CI, 7%-12%) for moderate, and 52% (95% CI, 39%-65%) for high-risk groups. Guideline implementation significantly increased admissions (+12%, 95% CI, 7%-17%) primarily in the moderate risk group (+38%, 95% CI, 29%-47%), but significantly decreased median ED length of stay (-37 minutes, 95% CI, 17-58). It also led to an increase in stress testing among moderate and high-risk patients (+10%, 95% CI, 0%-19%). In conclusion, the HEART score effectively stratified risk of MACE in a safety net population, improved evaluation consistency, and decreased ED length of stay. However, implementation was associated with an increase in hospitalizations and stress testing. Although the American Heart Association/American College of Cardiology guideline regarding atraumatic chest pain in the ED recommends universal noninvasive testing, the value of this approach, particularly in conjunction with the HEART score is uncertain in safety net hospitals. Further evaluation of the costs and clinical advantages of this approach are warranted.

Background High-intensity glucocorticoid regimens are commonly used to induce and maintain remission in Juvenile Dermatomyositis but are associated with several adverse side-effects. Evidence-based treatment guidelines from North American and European pediatric rheumatology research societies both advocate induction with intravenous pulse steroids followed by high dose oral steroids (2 mg/kg/day), which are then tapered. This study reports the time to disease control with reduced glucocorticoid dosing. Methods We retrospectively reviewed the records at a single tertiary-care children’s hospital of patients diagnosed with Juvenile Dermatomyositis between 2000 and 2014 who had a minimum of 2 years of follow-up. The primary outcome measure was time to control of muscle and skin disease. Additional outcome measures included glucocorticoid dosing, effect of treatment on height, frequency of calcinosis, and complications from treatment. Results Of the 69 patients followed during the study period, 31 fulfilled inclusion criteria. Median length of follow-up was 4.58 years, (IQR 3-7.5). Myositis control was achieved in a median of 7.1 months (IQR 0.9-63.4). Cutaneous disease control was achieved in a median of 16.7 months (IQR 4.3-89.5). The median starting dose of glucocorticoids was 0.85 mg/kg/day, (IQR 0.5-1.74). The median duration of steroid treatment was 9.1 months, (IQR 4.7-17.4), while the median duration of any pharmacotherapy was 29.2 months (IQR 10.4 to 121.3). Sustained disease control off medications was achieved in 21/31 (68%) patients by the end of review. Persistent calcinosis was identified in only one patient (3%). Conclusion Current accepted treatment paradigms for Juvenile Dermatomyositis include oral glucocorticoids beginning at 2 mg/kg/day and reduced over a prolonged time period. However, our results suggest that treatment using reduced doses and duration with early use of steroid-sparing agents is comparably effective in achieving favorable outcomes in Juvenile Dermatomyositis.

Objective To validate an ocular phantom as a realistic educational tool utilizing in vivo and phantom optic nerve sheath (ONS) images obtained by ultrasound. Methods This prospective study enrolled 51 resident physicians from the Denver Health Residency in Emergency Medicine (EM) and 10 ultrasound fellowship-trained EM attending physicians. Participants performed optic nerve sheath diameter (ONSD) measurements on five in vivo and five phantom ocular ultrasound images and rated the realism of each image on a 5-point Likert scale. Chi-square analysis was performed to evaluate the subjective “realness” of in vivo and phantom images. Results Sixty-one participants performed ONSD measurements. Mean Likert scale values were 3.43 (95% confidence interval: 3.31–3.55) for in vivo images and 3.41 (95% confidence interval: 3.28–3.54) for phantom images. There was no statistical difference in subjective “realness” between in vivo and phantom ONSD ultrasound images among EM residents. Ultrasound fellowship-trained EM attending physicians aptly differentiated between in vivo (p < 0.01) and phantom (p < 0.01) images, as compared with EM residents. Conclusion Our ocular phantom simulates in vivo posterior ocular anatomy. EM resident physicians found the phantom indistinguishable from in vivo images. Our ONS model provides an inexpensive and realistic educational tool to teach bedside ONSD sonography.

Objectives To compare the degree of agreement of sonographic optic nerve sheath diameter (ONSD) measurements by ultrasound fellowship–trained and resident emergency medicine (EM) physicians. Methods Ten ultrasound fellowship–trained EM attending physicians from multiple institutions and 51 resident EM physicians at a single residency were enrolled to measure the ONSD using a computerized ruler on five separate still-frame sonograms of adult eyes that included the retrobulbar optic nerve. Descriptive and bivariate statistics for each ultrasound image were calculated to compare median ONSD measurements among groups. Intraclass correlation coefficients (ICCs) were calculated to estimate the inter-rater reliability. Results Although the median ONSD measurement of each image did not significantly differ between resident and ultrasound fellowship–trained EM physicians, we found greater variation in agreement of ONSD measurements by resident EM physicians (ICC 0.50; 95% confidence interval 0.25–0.89) than ultrasound fellowship–trained EM physicians (ICC 0.73; 95% confidence interval 0.44–0.96). Conclusions Although both resident and ultrasound fellowship–trained EM physicians showed good inter-rater reliability of ONSD measurements, variation in agreement among ultrasound fellowship–trained EM physicians may be as wide as 0.44, suggesting that even in skilled hands, ultrasound measurement of the ONSD may not be as dependable a screening tool for elevated intracranial pressure as previously suggested in the literature.

Background Chest CT is more sensitive than a chest X-ray (CXR) in diagnosing rib fractures; however, the clinical significance of these fractures remains unclear. Objectives The purpose of this study was to determine the added diagnostic use of chest CT performed after CXR in patients with either known or suspected rib fractures secondary to blunt trauma. Methods Retrospective cohort study of blunt trauma patients with rib fractures at a level I trauma center that had both a CXR and a CT chest. The CT finding of ≥ 3 additional fractures in patients with ≤ 3 rib fractures on CXR was considered clinically meaningful. Student’s t-test and chi-square analysis were used for comparison. Results We identified 499 patients with rib fractures: 93 (18.6%) had CXR only, 7 (1.4%) had chest CT only, and 399 (79.9%) had both CXR and chest CT. Among these 399 patients, a total of 1,969 rib fractures were identified: 1,467 (74.5%) were missed by CXR. The median number of additional fractures identified by CT was 3 (range, 4 - 15). Of 212 (53.1%) patients with a clinically meaningful increase in the number of fractures, 68 patients underwent one or more clinical interventions: 36 SICU admissions, 20 pain catheter placements, 23 epidural placements, and 3 SSRF. Additionally, 70 patients had a chest tube placed for retained hemothorax or occult pneumothorax. Overall, 138 patients (34.5%) had a change in clinical management based upon CT chest. Conclusions The chest X-ray missed ~75% of rib fractures seen on chest CT. Although patients with a clinical meaningful increase in the number of rib fractures were more likely to be admitted to the intensive care unit, there was no associated improvement in pulmonary outcomes.

Contents The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre-clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre-puberal miniature pigs were poor responders to oestrous synchronization treatments, while post-puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non-cycling post-puberal miniature pig group (p

Preimplantation embryo development typically involves sequential morphological events connecting embryonic cleavage, morula compaction and blastocyst formation, and occurs in parallel with transcriptional regulation, specifically, the maternal to embryonic transition. The underlying homeostatic and metabolic mechanisms governing embryo development are influenced by both genetic and epigenetic factors that respond to environmental stimuli and may impact development during later gestational and fetal growth. There is a renewed interest in the identification and characterization of developmentally important genes during embryonic and fetal development. Perturbations in gene expression, resulting from environmental conditions, can have serious consequences on further embryonic development, homeostasis and disease pathogenesis. The bovine embryo is, however, capable of tolerating and adapting to a wide range of conditions, although little is known of the molecular fingerprint required for oocyte maturation, fertilization and development to term. The genomic revolution united with promising new technologies offer greater opportunity to elucidate the mechanisms behind this well-orchestrated biological process. This paper reviews the current literature on gene expression in the bovine embryo with reference to environmental interference and the development of new technologies to observe this biological process. Defining the difference in molecular signalling between in vivo and in vitro systems will undoubtedly improve the safety and efficiency of assisted reproductive technologies. The future challenge is to devise culture conditions that mimic the changing environment required by developing embryos to allow the correct temporal and spatial expression of a cohort of developmental genes in a manner similar to that seen in

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 efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.

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.

While the expression and epigenetic differences of imprinted genes have been extensively characterized in the mouse and human, little is known about imprinted genes in livestock species. In the current study, eight genes that are imprinted in the human or mouse were investigated in preimplantation bovine embryos. Amplified cDNA was created from three single metaphase II (MII) oocytes or embryos throughout preimplantation development. The imprinted genes Dlk1 and Mest (isoform 1) had no detectable transcripts during preimplantation development. Gnas and Grb10 were expressed in most embryos from the 2-cell to blastocyst stages of development. Mest (isoform 2) was expressed in all oocytes and embryos, except for one blastocyst sample. Ndn and Xist were expressed from the 8‐16-cell stage (maternal-to-zygotic transition, MZT) onwards. Sgce was expressed until the MZT, and Nnat in both early (a form) and late (b form) stage embryos. The paternally imprinted genes Gnas, Grb10, and Xist were expressed in both in vitro-fertilized (IVF) and parthenogenetically activated (PA) blastocysts as expected. Of the four maternally imprinted genes expressed in the blastocyst (Mest, Ndn, Nnat, and Sgce), Nnat alone showed differential mRNA expression between IVF and PA blastocysts, suggesting imprinting by this stage of development. In conclusion, seven of the eight genes investigated showed mRNA expression during preimplantation development, indicating a potential role during early development. Also significant is the observation that Nnat is imprinted by the blastocyst stage of development although the other genes are not, indicating a temporal imprinting program. early development, embryo, developmental biology, gene regulation, in vitro fertilization

Objectives To determine whether the addition of ultrasound to traditional physical examination instruction improves junior medical students' abilities to locate the femoral pulse. Methods Initially, 150 second-year medical students were taught the femoral pulse examination using traditional bedside teaching on standardized patients and online didactic videos. Students were then randomized into 2 groups: group 1 received ultrasound training first and then completed the standardized examination; and group 2 performed the standardized examination first and then received ultrasound training. On the standardized patients, the femoral artery was marked with invisible ink before the sessions using ultrasound. Compared to these markers, students were then evaluated on the accuracy of femoral artery pulse palpation and the estimated location of the femoral vein. All students completed a self-assessment survey after the ultrasound sessions. Results Ultrasound training improved the students' ability to palpate the femoral pulse (P= .02). However, ultrasound did not facilitate correct estimation of the femoral vein's anatomic location (P = .09). Confidence levels in localizing the femoral artery and vein were equal between groups at baseline, and both increased after the ultrasound sessions. Conclusions The addition of ultrasound teaching to traditional physical examination instruction enhanced medical student competency and confidence with the femoral vascular examination. However, understanding of anatomy may require emphasis on precourse didactic material, but further study is required.

The integration of bedside ultrasound into medical school curricula is limited by the availability of skilled faculty instructors. Peer mentors have been utilized successfully to teach clinical and procedural skills and may serve as a valuable resource for potential ultrasound instructors. We describe a method to train senior medical students as peer instructors for a combined ultrasound/physical exam curriculum and assessed junior medical students' perceptions of peer instruction relative to faculty.The University of Colorado has incorporated ultrasound into ocular, abdominal, musculoskeletal, cardiac, vascular, and pulmonary physical exam instruction for 1st-year (n=155) and 2nd-year (n=155) medical students. Fourth-year medical students who completed a 2- or 4-week bedside ultrasound elective were recruited as peer instructors. Both peer and faculty instructors received similar session training and were assigned to random groups of junior medical students. Instructor evaluation scores completed by students were collected after every session.Twenty students and 29 faculty served as instructors for the curriculum. Comparisons of evaluation scores between faculty and student teachers were equivalent (α.05) in 5 out of 6 sessions. In addition, students who taught more than 1 session showed improvement in their instructor scores and had higher average scores than students who taught only 1 session. Student instructors who completed the 4-week elective had higher average scores than students who completed the 2-week elective.Students' perception of peer instructors' teaching competency was equivalent to faculty instructors for the majority of sessions. Senior students who have completed an elective ultrasound rotation may serve as a useful resource for circumstances where the availability of skilled instructors is limited. However, further research is required to evaluate their effectiveness.

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.

Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20–24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer. Disclosure of potential conflicts of interest is found at the end of this article.

Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more divergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

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.

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

In order to determine the variability inherent in conceptus-related measurements in first trimester bovine pregnancies, conceptus and fetometric parameters from beef cattle pregnancies (n=103) estimated to be between Days 36 and 103 of gestation were examined. During this period, the protein concentration of amniotic fluid ranged between 0.181 and 0.501mg/mL. The amniotic fluid volume gradually increased from

In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.

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.

Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement-mediated rejection, as shown here using non-immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high-level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement-mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti-Galalpha(1,3)Gal epitope (anti-GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre-sensitized with antibody were highly resistant to human complement-mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non-immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at approximately 24-h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti-GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced approximately 8-fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.

While the expression and epigenetic differences of imprinted genes have been extensively characterized in the mouse and human, little is known about imprinted genes in livestock species. In the current study, eight genes that are imprinted in the human or mouse were investigated in preimplantation bovine embryos. Amplified cDNA was created from three single metaphase II (MII) oocytes or embryos throughout preimplantation development. The imprinted genes Dlk1 and Mest (isoform 1) had no detectable transcripts during preimplantation development. Gnas and Grb10 were expressed in most embryos from the 2-cell to blastocyst stages of development. Mest (isoform 2) was expressed in all oocytes and embryos, except for one blastocyst sample. Ndn and Xist were expressed from the 8-16-cell stage (maternal-to-zygotic transition, MZT) onwards. Sgce was expressed until the MZT, and Nnat in both early (alpha form) and late (beta form) stage embryos. The paternally imprinted genes Gnas, Grb10, and Xist were expressed in both in vitro-fertilized (IVF) and parthenogenetically activated (PA) blastocysts as expected. Of the four maternally imprinted genes expressed in the blastocyst (Mest, Ndn, Nnat, and Sgce), Nnat alone showed differential mRNA expression between IVF and PA blastocysts, suggesting imprinting by this stage of development. In conclusion, seven of the eight genes investigated showed mRNA expression during preimplantation development, indicating a potential role during early development. Also significant is the observation that Nnat is imprinted by the blastocyst stage of development although the other genes are not, indicating a temporal imprinting program.

beta 2 microglobulin knockout (beta2M-/-) mice lack CD8+ T and natural killer T cells. We hypothesized that beta 2M-/- mice are resistant to lethal intraabdominal sepsis. To test this hypothesis, mortality, cytokine production, and physiologic function were assessed in beta 2M-/- mice during sepsis caused by cecal ligation and puncture (CLP). beta 2M-/- mice survived significantly longer than wild-type mice after CLP but ultimately exhibited 100% mortality. Treatment of beta 2M-/- mice with anti-asialoGM1 to deplete natural killer cells conferred greater than 70% long-term survival. Compared with wild-type mice, beta 2M-/- mice treated with anti-asialoGM1 produced decreased amounts of proinflammatory cytokines and did not exhibit hypothermia or metabolic acidosis after CLP. Adoptive transfer of CD8+ T and natural killer cells into beta 2M-/- mice treated with anti-asialoGM1 re-established CLP-induced mortality. CD8 knockout mice treated with anti-asialoGM1, which are specifically deficient in CD8+ T and natural killer cells, exhibited 40% long-term survival after CLP. Furthermore, treatment of wild-type mice with antibodies to CD8 and asialoGM1 conferred a significant survival benefit compared with wild-type mice treated with nonspecific IgG. These findings demonstrate that beta 2M-/- mice treated with anti-asialoGM1 are resistant to CLP-induced mortality and that depletion of CD8+ T and natural killer cells largely accounts for the survival benefit observed in these mice.

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.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

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.