Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08-73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83-70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x(2) = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.

Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA.

As one of the transport systems on the sperm plasma membrane, CD4 molecule plays a distinct role in the process of sperm/DNA interaction. This makes it possible to explain the mechanism of sperm-mediated gene transfer (SMGT), which at present is still a mystery in this area. In this study, seminal samples of 60 individuals from seven breed bucks were collected to detect the ability of sperm in internalizing exogenous DNA, and genomic DNA from 147 individual blood samples (including 60 bucks referred ahead) were extracted to test the polymorphisms of CD4 genes by using PCR-SSCP technique. Then the correlation between them was evaluated. The results showed that: (1) it was a novel finding that breed-dependence of exogenous DNA binding to goat spermatozoa. There was the most significant difference among the buck breeds of sperm in binding exogenous DNA (F((6, 53)) = 4.811, P = 0.001) and in internalizing them into nuclei (F((6, 53)) = 4.587, P = 0.001). The ability of Lezhi Black goat was the lowest (P < 0.01) among the seven breeds. (2) There was no significant correlation between the ability of sperm in internalizing exogenous DNA and each semen quality parameter (P > 0.05). (3) In particular, three single-nucleotide polymorphisms (SNP) were described and there was one SNP (G/A(700)) of CD4 gene that made G234R substitution in the amino acid sequence of CD4 molecule. Nanjiang Yellow goat and Lezhi Black goat had higher hereditary variation compared with other breeds. (4) CD4 polymorphisms were highly associated with the ability of sperm in internalizing exogenous DNA. The SNP of Caprine CD4 gene exon 6 might be an important molecular marker of the ability to internalize exogenous DNA into sperm.

The role of periampullary diverticulum on the incidence of pancreaticobiliary diseases and the outcome of endoscopic retrograde cholangiopancreatography.

INTRODUCTION: Periampullary diverticulum (PAD), although commonly discovered in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP), remains controversial regarding its role in pancreaticobiliary diseases and the failure rate of cannulation. The aim of this study was to evaluate the association of PAD with pancreaticobiliary diseases and its impact on the outcome of ERCP. MATERIAL AND METHODS: A retrospective analysis was carried out on 1455 patients who underwent an ERCP. Patients were divided into a PAD group and a control group without PAD, and propensity score matching was performed to adjust for clinical differences. The comparison was focused on pancreaticobiliary diseases, technical success, and complications of ERCP. RESULTS: The occurrence of PAD is associated significantly with increasing age (p < 0.001). Incidences of acute pancreatitis (AP), suppurative cholangitis, and pancreatic head cancer were significantly higher in the PAD group (p < 0.05). After propensity score matching, the PAD group exhibited a higher rate of post-ERCP complications including haemorrhage, post-ERCP pancreatitis (PEP), and hyperamylasaemia (p < 0.05). However, the prevalence of perforation and the success rate of ERCP did not differ between groups (p > 0.05). CONCLUSIONS: Periampullary diverticulum develops with aging and seems to be associated with an increase in pancreaticobiliary diseases and post-ERCP complications except for perforation. Additionally, the presence of PAD does not affect the technical success of ERCP.

Efficiency of methods applied for goat estrous synchronization in subtropical monsoonal climate zone of Southwest China.

Two experiments were undertaken to select the efficient method applied for goat estrous synchronization. In experiment 1, a total of 120 does (Capra hircus) were divided into five groups with a randomized block design, and the does of treatment 1 were synchronized by intravaginal sponges impregnated with 30 mg Levonorgestrel inserted for 10 days. Does of treatments 2, 3, and 4 were treated with further injection of 25 IU follicle-stimulating hormone (FSH), 0.05 mg prostaglandin F2 alpha (PGF(2alpha)), and 25 IU FSH + 0.05 mg PGF(2alpha) at sponge withdrawal, respectively. The does in the control group (n = 40) without estrous synchronization treatment and natural estrous does were observed. In experiment 2, a total of 140 does in five goat farms in breeding and non-breeding seasons were treated with the selective efficient procedure. The results presented that all the employed treatments were capable of inducing and synchronizing estrous goats. According to estrous response and economy, the use of intravaginal sponges impregnated with 30 mg Levonorgestrel and 0.05 mg PGF(2alpha) (treatment 3) is the first choice for estrous synchronization, and 95.0% of synchronized does demonstrated estrus, which was significantly higher than that of treatment 1 (P < 0.05) and control group (P < 0.01). The percentages of ovulating of treatments 3 and 4 were the same (95.0%), which were significantly higher than that of treatment 1 (P < 0.01). The ovulation rates among different groups were not significant (P > 0.05). When the selective procedure was applied to five goat farms, 85.7% (120/140) of does demonstrated estrus, and the kidding percentage, litter size, and prolificacy rate were 53.6%, 0.95, and 177%, respectively.

Efficacy and safety of endoscopic submucosal dissection for large gastric stromal tumors.

BACKGROUND AND AIM: Endoscopic submucosal dissection (ESD) of gastric stromal tumors is becoming increasingly common. However, there have been few studies analyzing the therapeutic efficacy and safety of this technique on large (≥ 3 cm) gastric stromal tumors (LGSTs). The aim of this study was to determine the feasibility of ESD for the removal of LGSTs and to investigate the clinical safety and efficacy of ESD for this indication. METHODS: A retrospective analysis was carried out on 82 patients with LGSTs who underwent an ESD. Data on therapeutic outcomes and follow-up were collected for an analysis of the rates of en block resection and complete resection. A logistic regression model was used to identify potential risk factors for ESD-related complications, and a receiver operating characteristic (ROC) curve was generated for qualifying independent risk factors. RESULTS: En bloc resection was achieved in 81 lesions (98.8%), and complete resection was achieved in 80 lesions (97.6%). The rates of intraoperative and postoperative bleeding were 6.1% and 3.7%, respectively. The accidental perforation rate was 12.2%, the postoperative perforation rate was 3.7%, the intentional perforation rate was 28.0%, and the postoperative infection rate was 12.2%. There was no postoperative mortality. LGSTs originating from the deep muscularis propria (MP) layer (OR = 4.905, 95% CI: 1.362-17.658, P = 0.015), located at the gastric fundus (OR = 4.927, 95% CI:1.308-18.558, P = 0.018) and with an irregular shape (OR = 4.842, 95% CI: 1.242-18.870, P = 0.023) increased the rate of complications. The prediction model that incorporated these factors demonstrated an area under the ROC curve of 0.77 (95% CI: 0.66-0.89). No tumor recurrence or distant metastasis was observed during the follow-up period, which ranged from 6-36 months. CONCLUSIONS: ESD is a feasible, safe, effective and minimally invasive approach for the resection of LGSTs. Tumors originating from the deep MP layer, located at the fundus and with an irregular shape were identified as risk factors for the development of complications.

Outcomes of endoscopic sphincterotomy vs open choledochotomy for common bile duct stones.

BACKGROUND: Endoscopic sphincterotomy (EST) for the management of common bile duct stones (CBDS) is used increasingly widely because it is a minimally invasive procedure. However, some clinical practitioners argued that EST may be complicated by post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) and accompanied by a higher recurrence of CBDS than open choledochotomy (OCT). Whether any differences in outcomes exist between these two approaches for treating CBDS has not been thoroughly elucidated to date. AIM: To compare the outcomes of EST vs OCT for the management of CBDS and to clarify the risk factors associated with stone recurrence. METHODS: Patients who underwent EST or OCT for CBDS between January 2010 and December 2012 were enrolled in this retrospective study. Follow-up data were obtained through telephone or by searching the medical records. Statistical analysis was carried out for 302 patients who had a follow-up period of at least 5 years or had a recurrence. Propensity score matching (1:1) was performed to adjust for clinical differences. A logistic regression model was used to identify potential risk factors for recurrence, and a receiver operating characteristic (ROC) curve was generated for qualifying independent risk factors. RESULTS: In total, 302 patients undergoing successful EST (n = 168) or OCT (n = 134) were enrolled in the study and were followed for a median of 6.3 years. After propensity score matching, 176 patients remained, and all covariates were balanced. EST was associated with significantly shorter time to relieving biliary obstruction, anesthetic duration, procedure time, and hospital stay than OCT (P < 0.001). The number of complete stone clearance sessions increased significantly in the EST group (P = 0.009). The overall incidence of complications and mortality did not differ significantly between the two groups. Recurrent CBDS occurred in 18.8% (33/176) of the patients overall, but no difference was found between the EST (20.5%, 18/88) and OCT (17.0%, 15/88) groups. Factors associated with CBDS recurrence included common bile duct (CBD) diameter > 15 mm (OR = 2.72; 95%CI: 1.26-5.87; P = 0.011), multiple CBDS (OR = 5.09; 95%CI: 2.58-10.07; P < 0.001), and distal CBD angle ≤ 145° (OR = 2.92; 95%CI: 1.54-5.55; P = 0.001). The prediction model incorporating these factors demonstrated an area under the receiver operating characteristic curve of 0.81 (95%CI: 0.76-0.87). CONCLUSION: EST is superior to OCT with regard to time to biliary obstruction relief, anesthetic duration, procedure time, and hospital stay and is not associated with an increased recurrence rate or mortality compared with OCT in the management of CBDS.