Klaus-Gerd Hadeler

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows.

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.

Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes.

Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig‐to‐non‐human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3‐galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1‐KO pigs have successfully been produced by transfecting somatic cells with zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT.

Clip closure versus endoscopic suturing versus thoracoscopic repair of an iatrogenic esophageal perforation: a randomized, comparative, long-term survival study in a porcine model (with videos)

BACKGROUND Esophageal full-thickness wall repair is an important but unsolved issue in endoscopy. It is unknown how well endoscopic clip closure (ECC) and endoscopic closure with suturing (ECS) perform compared with the criterion standard of thoracoscopic closure (TC). OBJECTIVE Comparison of technical success, feasibility, long-term patency, complications, and histological quality of the different closure techniques (ECC, ECS, TC) for esophageal perforations. DESIGN Comparative animal study. SETTING Approved animal facility. SUBJECTS Eighteen pigs. INTERVENTIONS Eighteen pigs were randomized, 6 each into 3 groups (ECC, ECS, TC). After endoscopic wall incision and mediastinoscopy, closure was performed by using 1 of the 3 techniques. After 8 to 12 weeks, pre-euthanasia endoscopic, necropsy, histological, and morphometric analyses were performed. MAIN OUTCOME MEASUREMENT Long-term survival and histological quality of the repair. RESULTS The closure of the esophageal incisions was successful in all pigs. On days 2 and 6, 1 animal died of mediastinitis, 1 in the ECS group because of reflux of gastric contents into the mediastinum before the repair and 1 in the TC group because of leakage of the sutured closure (P = 1.0). No strictures were seen on prenecropsy endoscopy. At necropsy, 1 mediastinal abscess was found in an ECS animal (P = 1.0). Minor complications included periesophageal adhesions and reactive lymph nodes in 3 of 6 (ECC group) and 5 of 6 (TC and ECS groups). Histology showed muscle layer defects up to 12 mm in width and 21 mm in length, with a trend toward smaller defect size of width and length in the ECS group of animals. LIMITATIONS Animal study of limited size. CONCLUSIONS Overall, ECS and ECC performed similarly to TC. ECS showed the smallest histological defects in the long-term repair.

Laparoscopical intrauterine insemination with different doses of fresh, conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.

In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 μg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

In vivo oocyte IGF-1 priming increases inner cell mass proliferation of in vitro-formed bovine blastocysts

Studies addressing the effects of supraphysiological levels of IGF-1 on oocyte developmental competence are relevant for unravelling conditions resulting in high bioavailability of IGF-1, such as the polycystic ovary syndrome (PCOS). This study investigated the effects of supraphysiological levels of IGF-1 during in vivo folliculogenesis on the morula-blastocyst transition in bovine embryos. Compacted morulae were non-surgically collected and frozen for subsequent mRNA expression analysis (IGF1R, IGBP3, TP53, AKT1, SLC2A1, SLC2A3, and SLC2A8), or underwent confocal microscopy analysis for protein localization (IGF1R and TP53), or were cultured in vitro for 24 h. In vitro-formed blastocysts were subjected to differential cell staining. The mRNA expression of SLC2A8 was higher in morulae collected from cows treated with IGF-1. Both IGF1R and TP53 protein were present in the plasma membrane and cytoplasm. IGF-1 treatment did not affect protein localization of both IGF1R and TP53. In vitro-formed blastocysts derived from morulae recovered from IGF-1-treated cows displayed a higher number of cells in the inner cell mass (ICM). Total cell number (TCN) of in vitro-formed blastocysts was not affected. A higher mean ICM/TCN proportion was observed in in vitro-formed blastocysts derived from morulae collected from cows treated with IGF-1. The percentage of in vitro-formed blastocysts displaying a low ICM/TCN proportion was decreased by IGF-1 treatment. In vitro-formed blastocysts with a high ICM/TCN proportion were only detected in IGF-1 treated cows. Results show that even a short in vivo exposure of oocytes to a supraphysiological IGF-1 microenvironment can increase ICM cell proliferation in vitro during the morula to blastocyst transition.

Endoscopic ultrasound-guided lymph node ablation with a novel radiofrequency ablation probe: feasibility study in an acute porcine model.

BACKGROUND AND STUDY AIMS Radiofrequency ablation (RFA) is an accepted method of tissue destruction for solid organ tumors. Endoscopic ultrasound (EUS)-guided RFA has been used for lesions in the pancreas and liver, but there is limited experience of lymph node ablation using EUS-guided RFA. The aim of this study was to determine feasibility and safety of prototype EUS-guided RFA of mediastinal lymph nodes. METHODS This was an endoscopic experimental feasibility study in a porcine model. After EUS-guided puncture of targeted lymph nodes, the stylet of a 19-G needle was replaced by a prototype RFA probe. RFA was performed by ERBE generator (bipolar settings: 10 watts, effect 2, 2 minutes). The animals were euthanized, and the targeted lymph nodes were identified and removed for histology and measurement of the effect achieved. RESULTS A total of 18 mediastinal lymph nodes were ablated (mean size 20.8 ± 6.6  mm in the long axis). The average length of exposed probe was 10.0 ± 3.0 mm. The mean length and diameter of necrosis was 9.8 ± 3.6  mm and 5.5 ± 1.6  mm, respectively. Linear regression comparing needle length with necrosis diameter revealed a coefficient gradient of r = 0.92 (P = 0.0001). With EUS-RFA a mean of 17.6 ± 10.3 % (range 8.0 % - 53.2 %) of the respective lymph node area was ablated. No complications (i. e. hemodynamic instability, local bleeding, tissue damage) occurred during the procedure. Technical problems included stripping of the probe by the EUS needle and bending of the tip of the probe. CONCLUSIONS EUS-RFA of lymph nodes was performed safely and successfully using a prototype EUS-compatible probe. This method may have the potential for future use in patient care.

Endoscopic transesophageal vs. thoracoscopic removal of mediastinal lymph nodes: a prospective randomized trial in a long term animal survival model.

BACKGROUND AND STUDY AIMS In cases where biopsies remain inconclusive, removal of mediastinal lymph nodes for further analysis requires surgical means. Natural orifice transluminal endoscopic surgery (NOTES) procedures allow incision/closure of the gut wall, which might enable endoscopic excision of pre-marked nodes. The aims of the current study were to investigate the feasibility, safety, and reproducibility of lymph node generation in an animal model to enable endoscopic ultrasound-guided (EUS) lymph node removal (ELR) using transesophageal NOTES access/closure and to compare this procedure with thoracoscopic lymph node removal (TLR) in a randomized long term survival animal study. PATIENTS AND METHODS Lymph node creation using graphite injection was performed in 12 pigs. After randomization into ELR and TLR groups, lymph nodes were marked with newly developed anchors under EUS guidance and removed using either ELR or TLR. ELR included incision of the esophageal wall and closure after lymph node removal. The main outcome measures were success in lymph node generation, technical success of lymph node removal, complications, and comparability of ELR and TLR. RESULTS Generation of lymph nodes proved successful in all animals in 46/48 sites injected (96 %). Anchors were placed through the selected nodes in a mean of 9.4 minutes. TLR and ELR were successful in all cases. One bleeding occurred during esophageal incision in ELR, which was stopped endoscopically. After lymph node removal, endoscopic suturing of the incision took a mean of 18 minutes. Procedure time was longer for ELR than TLR (mean 48 vs. 42 minutes). All animals survived the procedures. Autopsy after 4 weeks showed two thoracic wall abscesses in the TLR group and none in the ELR group.  Microscopic analysis revealed well healed esophageal scars. CONCLUSION ELR proved to be feasible in this limited sample size and complications were not observed more frequently in this group than in the TLR group.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

A novel endoscopic prototype device for gastric full-thickness biopsy for the histopathologic diagnosis of GI neuromuscular pathology: in vivo porcine long-term survival study (with videos)

BACKGROUND Many GI motility disorders are associated with underlying GI neuromuscular pathology, which requires full-thickness biopsies (FTB) for histopathologic diagnosis. Currently, none of the endoscopy-based attempts to obtain FTB specimens have proven suitable for routine use. This study evaluated a novel endoscopic prototype device (ED) for this purpose. OBJECTIVE To determine (1) the ability of the ED to obtain suitable FTB specimens, (2) associated complications, (3) feasibility of reliable defect closure, and (4) ability to evaluate intramural neuromuscular components. DESIGN Preclinical proof-of-concept study in 30 pigs. SETTING Animal laboratory. INTERVENTION Gastric FTB specimens were obtained with a circular cutter and anchor. The defect was closed by over-the-scope clips/T-tags. The resection site was inspected via laparoscopy. After 2 to 4 weeks, necropsy was carried out to evaluate late complications. MAIN OUTCOME MEASUREMENTS Feasibility, safety, and closure rate of the procedure. FTB specimens were assessed by histology/immunohistochemistry to visualize enteric neuromusculature. RESULTS A total of 29 of 30 procedures were successfully performed; one hemorrhage required endoscopic treatment. A total of 29 of 30 FTB specimens (mean diameter 9.1 mm) were retrieved in 7.1 ± 0.4 minutes (range 3.0-12.5 minutes), displaying optimal tissue quality. Defect closure took 10.8 ± 0.9 minutes (range 7.2-32 minutes). Laparoscopy did not reveal damage to adjacent organs. Necropsy showed well-healed scars at the resection site and no complications, peritonitis, or abscess formation. Histology showed smooth muscle layers and submucosal and myenteric ganglia. LIMITATIONS Survival animal pilot study, no patients. CONCLUSION The novel ED enabled safe harvesting of well-preserved FTB specimens. Defect closure proved to be reliable. All neuromuscular structures relevant for histopathologic evaluation of GI neuromuscular pathology were demonstrated. Further studies are needed to verify the efficacy of this prototype device in the entire gut and in humans.