Retzius-sparing robot-assisted laparoscopic radical prostatectomy: combining the best of retropubic and perineal approaches.

OBJECTIVE: To compare the early peri-operative, oncological and continence outcomes of Retzius-sparing robot-assisted laparoscopic radical prostatectomy (RALP) with those of conventional RALP. MATERIALS AND METHODS: Data from 50 patients who underwent Retzius-sparing RALP and who had at least 6 months of follow-up were prospectively collected and compared with a database of patients who underwent conventional RALP. Propensity-score matching was performed using seven preoperative variables, and postoperative variables were compared between the groups. RESULTS: A total of 581 patients who had undergone RALP were evaluated in the present study. Although preoperative characteristics were different before propensity-score matching, these differences were resolved after matching. There were no significant differences in mean length of hospital stay, estimated blood loss, intra- and postoperative complication rates, pathological stage of disease, Gleason scores, tumour volumes and positive surgical margins between the conventional RALP and Retzius-sparing RALP groups. Console time was shorter for Retzius-sparing RALP. Recovery of early continence (defined as 0 pads used) at 4 weeks after RALP was significantly better in the Retzius-sparing RALP group than in the conventional RALP group. CONCLUSIONS: The present results suggest that Retzius-sparing RALP, although technically more demanding, was as feasible and effective as conventional RALP, and also led to a shorter operating time and faster recovery of early continence. Retzius-sparing RALP was also reproducible and achievable in all cases.

Laparoendoscopic single-site (LESS) robot-assisted nephroureterectomy: comparison with conventional multiport technique in the management of upper urinary tract urothelial carcinoma.

OBJECTIVE: To compare the peri-operative, pathological and oncological outcomes of laparoendoscopic single-site (LESS) robot-assisted nephroureterectomy (LESS-RALNU) with those of multiport robot-asssisted nephroureterectomy (M-RALNU). PATIENTS AND METHODS: A total of 38 patients with upper urinary tract urothelial carcinoma underwent LESS-RALNU (n = 17) or M-RALNU (n = 21) by a single surgeon at a tertiary institution. Data were obtained from a prospectively maintained database. RESULTS: Patients' demographics and tumour characteristics were similar between the M-RALNU and LESS-RALNU groups. The mean follow-up was 48.4 months for M-RALNU and 30.9 months for LESS-RALNU (P = 0). The mean operating time, estimated blood loss and length of hospitalization for M-RALNU and LESS-RALNU were 251 min, 192 mL, 6.5 days and 247 min, 376 mL and 5.4 days, respectively (P > 0.05). Overall, there were no significant differences in complication rates, although three patients in the LESS-RALNU group required blood transfusion, whereas no patient in the M-RALNU group did (P = 0.081). The proportion of patients with bladder recurrence, local recurrence and distant metastases was similar between the two groups. There were no significant differences in the recurrence-free survival, cancer-specific survival and overall survival rates between the two groups. CONCLUSIONS: Although the oncological and peri-operative outcomes of patients who underwent LESS-RALNU compared well with those who underwent M-RALNU and with series of other surgical approaches, LESS-RALNU might result in greater intra-operative blood loss. We suggest careful selection of patient for this technique.

Tumor volume adds prognostic value in patients with organ-confined prostate cancer.

PURPOSE: This study was designed to assess the independent prognostic value of tumor volume (TV) and whether adding TV provides additional prognostic information for predicting biochemical recurrence (BCR) after radical prostatectomy. METHODS: We reviewed the medical records of 1,129 patients who underwent radical prostatectomy between July 2005 and July 2011. TV was categorized as minimal (≤1.0 ml), moderate (1.1-5.0 ml), or extensive (>5.0 ml). Cox regression analysis was performed to identify independent predictors of BCR. The predictive accuracies of Cox's proportional hazard regression models with and without TV were quantified and compared using time-dependent receiver operating characteristic curve analysis. RESULTS: Increasing TV was associated with higher prostate specific antigen, pathological Gleason score, and pathologic tumor stage. TV was an independent predictor of BCR in multivariate analysis (p<0.001). When patients were stratified by organ-confined and nonorgan-confined tumor groups, TV remained an independent predictor of BCR in organ-confined tumors (p<0.001). In the nonorgan-confined tumor group, a significant difference was found only between extensive versus minimal TV (p=0.023). The predictive accuracy of the Cox regression model increased significantly by adding TV in organ-confined tumor group (0.748 vs. 0.704, p<0.05) but not in nonorgan-confined group (0.742 vs. 0.734, p>0.05). CONCLUSIONS: TV was an independent prognostic predictor of BCR in organ-confined prostate cancers and provided additional prognostic information with increased predictive accuracy. In contrast, TV did not increase the predictive accuracy in nonorgan-confined tumor. TV should be considered as a prognosticator in organ-confined tumors.

Gleason 5+4 has worse oncological and pathological outcomes compared with Gleason 4+5: significance of Gleason 5 pattern.

BACKGROUND: The purpose of this study was to evaluate the pathological and oncological significance of Gleason (G) 5 pattern in high-grade PCa after robotic-assisted radical prostatectomy (RARP). MATERIALS AND METHODS: From a cohort of 1,046 men, 159 post-RARP patients by a single surgeon with pathological G8 (N=79) and G9 (N=80) met our inclusion criteria. G9 cancers were sub-stratified into G4+5 (N=58) and G5+4 (N=22). Clinical and pathological outcomes were evaluated with the t test or Mann-Whitney U test for continuous variables and the Pearson χ2 test for categorical variables. The Kaplan-Meier method was used to estimate the biochemical recurrence-free survival (BCRFS), and survival curves were compared using the log-rank test. Multivariate analysis was performed with Cox regression analysis. RESULTS: Baseline characteristics across all subgroups were similar except for number of positive cores on biopsy. There was a trend toward worse pathological and oncological outcomes in G9 cancers when compared with G8, although only tumor volume (TV), extracapsular extension (ECE) of tumor and lymph nodes involvement (LNI) achieved statistical significance. G4+5 PCa were statistically more likely to have ECE and a higher TV than G4+4 although the BCRFS were not significantly different. G5+4 cancers were associated with a significantly higher proportion of patients with LNI and had a statistically significant poorer BCRFS compared with G4+5 patients. CONCLUSIONS: Oncological and pathological outcomes of G8 were significantly better than G9 and merited distinction between them. G5+4 harbors a much poorer BCRFS compared with G4+5, and hence we suggest considerations for immediate adjuvant treatments.

Upgrading of Gleason score and prostate volume: a clinicopathological analysis.

OBJECTIVE: To more clearly elucidate the association between prostate volume and Gleason score (GS) upgrading. PATIENT AND METHODS: We reviewed 451 patients with prostate cancer with a GS of 6 on biopsy, who underwent radical prostatectomy without neoadjuvant treatment. As a preoperative variable, we assessed the independent effect of prostate volume on GS upgrading. To evaluate the association between prostate volume and GS upgrading, we developed multivariate models with volumetric pathological variables, including postoperative tumour volume and percent tumour volume (tumour volume as a percentage of prostate volume). RESULTS: GS upgrading was observed in 194 patients (43.0%). As a preoperative variable, smaller prostate volume was an independent predictor of GS upgrading. In regression analysis, prostate volume and postoperative tumour volume were inversely correlated. On multivariate analysis including volumetric pathological variables, tumour volume was a strong independent factor influencing GS upgrading, and prostate volume lost statistical significance after adjusting for tumour volume. Percent tumour volume was inversely correlated with GS upgrading after adjusting for tumour volume. CONCLUSIONS: Smaller prostate volume was an independent predictor of GS upgrading as a preoperative variable. The inverse relationship between prostate volume and GS upgrading seems to be attributable to cancer biology, which was represented by tumour volume in our study. Percent tumour volume was also inversely associated with GS upgrading. These results suggest that biological factors and sampling error both play important roles in GS upgrading.

Extended vs standard lymph node dissection in robot-assisted radical prostatectomy for intermediate- or high-risk prostate cancer: a propensity-score-matching analysis.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Although lymph node dissection (LND) is known as the most accurate method of nodal staging, the therapeutic role of LND remains undetermined. This is mainly because of the lack of randomized prospective studies and the fact that retrospective analyses often result in bias and misinterpretation. To overcome the limitation of retrospective analysis, we matched preoperative variables using propensity scores and compared the outcomes between patients treated with robot-assisted eLND and sLND. In the matched cohort, robot-asssited eLND achieved an increased detection rate of lymph node metastases; however, the therapeutic benefit was not statistically significant between the two groups on short-term follow-up. OBJECTIVE: To compare the pathological and biochemical outcomes between extended lymph node dissection (eLND) and standard lymph node dissection (sLND) in patients undergoing robot-assisted radical prostatectomy for intermediate- or high-risk prostate cancer. PATIENTS AND METHODS: A total of 905 patients underwent robot-assisted radical prostatectomy and lymph node dissection (LND) by a single surgeon between June 2006 and January 2011. Of these, 170 patients who underwent robot-assisted eLND and 294 patients who underwent robot-assisted sLND for intermediate- or high-risk prostate cancer were included in the study. Propensity-score matching was performed using the preoperative variables which included age, body mass index, prostate-specific antigen, clinical stage, biopsy Gleason score 1 and 2, total number of biopsied cores, number of positive cores and prostate volumes. Pathological and biochemical outcomes were assessed according to the extent of LND. RESULTS: The median (range) follow-up period was 36 (12-77) months and the median number of lymph nodes removed was 21 and 12 in the eLND and sLND groups, respectively. Propensity-score matching resulted in 141 patients in each group. Although patients who underwent eLND had a higher clinical stage, biopsy Gleason score and number of positive cores than those treated with sLND in the entire cohort, there were no preoperative between-group differences in the matched cohort. In the matched cohort, lymph node metastases were detected at a significantly higher rate in the eLND than in the sLND group (12.1 vs. 5.0%, P = 0.033). In the matched cohort, the 3-year biochemical recurrence-free survival rates were 77.8 and 73.5% in the eLND and sLND groups, respectively, which was not significant (hazard ratio 0.85, P = 0.497). CONCLUSION: Robot-assisted eLND achieved an increased lymph node yield and higher detection rate of lymph node metastases; however, robotic eLND did not alter biochemical outcomes in a short-term follow-up.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

Current status of robot assisted laparoscopic radical nephroureterectomy for management of upper tract urothelial carcinoma.

Upper tract urothelial carcinomas (UUT-UC) are usually aggressive tumours and require radical treatments. The standard of care for localised UUT-UC is radical nephroureterectomy (RNU). Robot-assisted laparoscopic surgeries are currently employed in various urological procedures, including RNU. We conducted a literature search on medical databases (PubMed/ MEDLINE) using free text keywords nephroureterectomy, distal ureter, bladder cuff, urothelial carcinoma and/or robotic. In this review, we aim to provide an up-to-date status on robot-assisted laparoscopic nephroureterectomy (RAL-NU) for the management of UUT-UC. The various surgical techniques and approaches for RAL-NU and retroperitoneal lymph node dissection (RPLND) will be discussed and their perioperative and early oncological outcomes reported. The feasibility and safety of RAL-NU has been demonstrated in a number of studies but intermediate and long term clinical and oncological outcomes are still lacking.

Current status of robot-assisted laparoscopic radical prostatectomy: how does it compare with other surgical approaches?

The aim was to review the current status and evaluate the outcomes of robot-assisted laparoscopic radical prostatectomy in comparison with open radical prostatectomy and laparoscopic radical prostatectomy. Between January 2008 and June 2012, published English language comparative studies comparing robot-assisted laparoscopic radical prostatectomy with either open radical prostatectomy and/or laparoscopic radical prostatectomy were reviewed. End-points for this review include oncological, functional and perioperative outcomes, and complications. Compared with laparoscopic radical prostatectomy and/or open radical prostatectomy, robot-assisted laparoscopic radical prostatectomy offered at least equivalent oncological control. Current evidence seems to suggest a superiority of robot-assisted laparoscopic radical prostatectomy over open radical prostatectomy and laparoscopic radical prostatectomy in terms of functional outcomes, such as urinary continence and potency. Risks of perioperative complications were also low after robot-assisted laparoscopic radical prostatectomy. Robot-assisted laparoscopic radical prostatectomy offers at least equivalent oncological and functional outcomes with low risks of complications when compared with open radical prostatectomy and laparoscopic radical prostatectomy. However, there is a paucity of high-level evidence available in current literature.

Effects of Trichostatin A on In vitro Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer.

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

Predicting Future Incidences of Cardiac Arrhythmias Using Discrete Heartbeats from Normal Sinus Rhythm ECG Signals via Deep Learning Methods.

This study aims to compare the effectiveness of using discrete heartbeats versus an entire 12-lead electrocardiogram (ECG) as the input for predicting future occurrences of arrhythmia and atrial fibrillation using deep learning models. Experiments were conducted using two types of inputs: a combination of discrete heartbeats extracted from 12-lead ECG and an entire 12-lead ECG signal of 10 s. This study utilized 326,904 ECG signals from 134,447 patients and categorized them into three groups: true-normal sinus rhythm (T-NSR), atrial fibrillation-normal sinus rhythm (AF-NSR), and clinically important arrhythmia-normal sinus rhythm (CIA-NSR). The T-NSR group comprised patients with at least three normal rhythms in a year and no atrial fibrillation or arrhythmias history. Clinically important arrhythmia included atrial fibrillation, atrial flutter, atrial premature contraction, atrial tachycardia, ventricular premature contraction, ventricular tachycardia, right and left bundle branch block, and atrioventricular block over the second degree. The AF-NSR group included normal sinus rhythm paired with atrial fibrillation or atrial flutter within 14 days, and the CIA-NSR group comprised normal sinus rhythm paired with CIA occurring within 14 days. Three deep learning models, ResNet-18, LSTM, and Transformer-based models, were utilized to distinguish T-NSR from AF-NSR and T-NSR from CIA-NSR. The experiments demonstrated the potential of using discrete heartbeats in predicting future arrhythmia and atrial fibrillation incidences extracted from 12-lead electrocardiogram (ECG) signals alone, without any additional patient information. The analysis reveals that these discrete heartbeats contain subtle patterns that deep learning models can identify. Focusing on discrete heartbeats may lead to more timely and accurate diagnoses of these conditions, improving patient outcomes and enabling automated diagnosis using ECG signals as a biomarker.

Effects of repetitive ionomycin treatment on in vitro development of bovine somatic cell nuclear transfer embryos.

To artificially activate embryos in somatic cell nuclear transfer (SCNT), chemical treatment with ionomycin has been used to induce transient levels of Ca(2+) and initiate reprogramming of embryos. Ca(2+) oscillation occurs naturally several times after fertilization (several times with 15- to 30-min intervals). This indicates how essential additional Ca(2+) influx is for successful reprogramming of embryos. Hence, in this report, the experimental design was aimed at improving the developmental efficiency of cloned embryos by repetitive Ca(2+) transients rather than the commonly used ionomycin treatment (4 min). To determine optimal Ca(2+) inflow conditions, we performed three different repetitive ionomycin (10 µM) treatments in reconstructed embryos: Group 1 (4-min ionomycin treatment, once), Group 2 (30-sec treatment, 4 times, 15-min intervals) and Group 3 (1-min treatment, 4 times, 15-min intervals). Pronuclear formation rates were checked to assess the effects of repetitive ionomycin treatment on reprogramming of cloned embryos. Cleavage rates were investigated on day 2, and the formation rates of blastocysts (BLs) were examined on day 7 to demonstrate the positive effect of repeated ionomycin treatment. In Group 3, a significant increase in BL formation was observed [47/200 (23.50%), 44/197 (22.33%) and 69/195 (35.38%) in Groups 1, 2 and 3, respectively]. Culturing embryos with different ionomycin treatments caused no significant difference among the groups in terms of the total cell number of BLs (164.3, 158.5 and 145.1, respectively). Additionally, expression of the anti-apoptotic Bcl-2 gene and MnSOD increased significantly in Group 3, whereas the expression of the pro-apoptotic Bax decreased statistically. In conclusion, the present study demonstrated that repeated ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.

Morphological abnormalities, impaired fetal development and decrease in myostatin expression following somatic cell nuclear transfer in dogs.

Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT-cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down-regulated at the mRNA level in tongues and skeletal muscles of SCNT-cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT-cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates.

Increased cavernous expression of transforming growth factor-ß1 and activation of the Smad signaling pathway affects erectile dysfunction in men with spinal cord injury.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) is implicated in bladder fibrosis after spinal cord injury (SCI) and in the fibrosis in the corpus cavernosum tissue after cavernous nerve injury. AIM: We investigated the differential expression of TGF-ß1 and the Smad transcription factor, the key molecule for the initiation of TGF-ß-mediated fibrosis, in cavernous tissue from SCI patients. METHODS: After obtaining informed consent and approval from the patients and our institutional review board, we enrolled 5 patients with psychogenic erectile dysfunction (ED) (mean age 36.8 years; range 20-50 years) and 10 patients with neurogenic ED from SCI (mean age 38.8 years; range 18-50 years). Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), or antibodies to TGF-ß1 and phospho-Smad2. MAIN OUTCOME MEASURES: Semi-quantitative analysis of TGF-ß1 and phospho-Smad2 was performed, and the numbers of apoptotic cells were counted. We also quantified the cavernous collagen area with the use of an image analyzer system. RESULTS: The expression of TGF-ß1 and phospho-Smad2 protein was significantly higher in the SCI group than in the psychogenic group. The TUNEL assay revealed a higher apoptotic index in the SCI group than in the psychogenic group. Higher TGF-ß1 and phospho-Smad2 expression and more apoptotic cells were noted mainly in endothelial cells, smooth muscle cells, and fibroblasts of the SCI group. Double labeling of cavernous tissue with TUNEL and antibody to phospho-Smad2 revealed that most TUNEL-positive cells showed immunoreactivity to phospho-Smad2 staining. Cavernous collagen content was significantly greater in the SCI group than in the psychogenic group. CONCLUSION: Upregulation of TGF-ß1 and activation of the Smad signaling pathway may play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which warrants early pharmacological intervention to protect erectile tissue from irreversible damage.

Transcriptome Analysis of the Japanese Pine Sawyer Beetle, Monochamus alternatus, Infected with the Entomopathogenic Fungus Metarhizium anisopliae JEF-197.

The Japanese pine sawyer (JPS) beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), damages pine trees and transmits the pine wilt nematode, Bursaphelenchus xylophilus Nickle. Chemical agents have been used to control JPS beetle, but due to various issues, efforts are being made to replace these chemical agents with entomopathogenic fungi. We investigated the expression of immune-related genes in JPS beetle in response to infection with JEF-197, a Metarhizium anisopliae isolate, using RNA-seq. RNA samples were obtained from JEF-197, JPS adults treated with JEF-197, and non-treated JPS adults on the 8th day after fungal treatment, and RNA-seq was performed using Illumina sequencing. JPS beetle transcriptome was assembled de novo and differentially expressed gene (DEG) analysis was performed. There were 719 and 1953 up- and downregulated unigenes upon JEF-197 infection, respectively. Upregulated contigs included genes involved in RNA transport, ribosome biogenesis in eukaryotes, spliceosome-related genes, and genes involved in immune-related signaling pathways such as the Toll and Imd pathways. Forty-two fungal DEGs related to energy and protein metabolism were upregulated, and genes involved in the stress response were also upregulated in the infected JPS beetles. Together, our results indicate that infection of JPS beetles by JEF-197 induces the expression of immune-related genes.

Post-activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei.

The objective of this study was to examine the effect of cytochalasin B (CB) and/or demecolcine (Dc) on the remodeling of donor nuclei, nuclear ploidy, and development of somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) pig embryos. SCNT and PA oocytes were either untreated (control), or treated with CB, Dc, or both CB and Dc after electric activation, and then cultured or transferred to surrogates. In SCNT, blastocyst formation was higher after treatment with CB and/or Dc (26-28%) than in the controls (16%). The number of oocytes that formed a single pronucleus (PN) was higher after treatment with Dc (86%) and CB + Dc (86%) than under control conditions (44%) or after treatment with CB (63%). In PA, blastocyst formation was higher after CB treatment (47%) than under control conditions (28%), while the formation of a single PN was higher after treatment with Dc (88%) and CB + Dc (84%) compared to controls (34%). The rate of formation of diploid embryos was higher after treatment with Dc and CB + Dc than under control conditions. Dc treatment resulted in a farrowing rate of 50% with 1.1% production efficiency, while controls showed a farrowing rate of 37.5% and a production efficiency of 0.7%. The results of our study demonstrate that post-activation treatment with Dc improves preimplantation development and supports normal in vivo development of SCNT pig embryos, probably because Dc induces formation of a single PN and this leads to normal nuclear ploidy.

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.

Entomopathogenic Fungi as Dual Control Agents against Both the Pest Myzus persicae and Phytopathogen Botrytis cinerea.

The green peach aphid (Myzus persicae), a plant pest, and gray mold disease, caused by Botrytis cinerea, affect vegetables and fruit crops all over the world. To control this aphid and mold, farmers typically rely on the use of chemical insecticides or fungicides. However, intensive use of these chemicals over many years has led to the development of resistance. To overcome this problem, there is a need to develop alternative control methods to suppress populations of this plant pest and pathogen. Recently, potential roles have been demonstrated for entomopathogenic fungi in endophytism, phytopathogen antagonism, plant growth promotion, and rhizosphere colonization. Here, the antifungal activities of selected fungi with high virulence against green peach aphids were tested to explore their potential for the dual control of B. cinerea and M. persicae. Antifungal activities against B. cinerea were evaluated by dual culture assays using both aerial conidia and cultural filtrates of entomopathogenic fungi. Two fungal isolates, Beauveria bassiana SD15 and Metarhizium anisopliae SD3, were identified as having both virulence against aphids and antifungal activity. The virulence of these isolates against aphids was further tested using cultural filtrates, blastospores, and aerial conidia. The most virulence was observed in the simultaneous treatment with blastospores and cultural filtrate. These results suggest that the two fungal isolates selected in this study could be used effectively for the dual control of green peach aphids and gray mold for crop protection.

Cloning of exotic/endangered species: desert bighorn sheep.

Cloning using somatic cell nuclear transfer (SCNT) may be a useful tool for conserving genetic diversity and for propagating exotic and/or endangered animal species. Somatic cells can be obtained easily, expanded in culture, cryopreserved, and thawed at a later date for use in NT. Significant challenges relevant to using SCNT for cloning wild and endangered animal species include the need for using interspecies NT and interspecies embryo transfer. Animal care and welfare issues raised that are unique to exotic and endangered species also are raised. In this chapter, the methods used in attempts to clone the wild animal species of Desert Bighorn Sheep are described.

A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes.

To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.