Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C.

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE: Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.

Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites.

Compared to INRA 401 lambs reared in France, Black Belly (BB) lambs reared in Guadeloupe (F.W.I.) were highly resistant to both primary and secondary experimental infection with Haemonchus contortus. To investigate this huge inter-breed difference, a nucleus flock of BB was constituted, and experiments were conducted to: (i) confirm this difference in lambs born in France, (ii) check whether it was similar for Trichostrongylus colubriformis and Teladorsagia circumcincta, and (iii) find out whether this difference was age-related. Forty BB lambs, 84 F1 lambs (BB siresxINRA 401 ewes) and 88 INRA 401 lambs born in two cohorts were used in an experimental design involving three host breeds, both genders and two age-groups (3.5- and 7-month-old when first infected). The limited availability of BB lambs made the study incomplete. Infection consisted of the administration of two doses of 10,000 infective larvae of one of the nematode species, separated by an anthelmintic treatment and an interval of 1 week before the second dose was administered. Fecal egg counts (FECs) were done on Days 28 and 35 after each infection; ewe lambs of the INRA 401 and F1 breeds were necropsied, the worm burden was established, the length of the female worms measured and the eggs in utero counted. For H. contortus and T. colubriformis, the FEC was lower in the BB than in the INRA 401 lambs, and the FEC found for the F1 lambs was intermediate. For T. circumcincta, only data for the F1 and INRA 401 lambs were available, and the FECs for these breeds were the same. In all three breeds, the FEC determined after the second dose was significantly lower than that found after the first dose in the ewe lambs, but not in the ram lambs. Infecting the lambs when they were 7- instead of 3.5-month-old significantly lowered egg excretion of both parasites in the F1 (P<0.0001), but not the INRA 401 lambs. Worm numbers in the F1 and in the INRA 401 ewe lambs confirmed the FEC data, many F1 lambs being free of T. colubriformis. The H. contortus female worms were shorter and had fewer eggs in utero in the F1 than in the INRA 401 lambs. A higher proportion of T. circumcincta was at the fourth larval stage in the F1 lambs. In conclusion, the BB breed is much more resistant than INRA 401 to H. contortus, with the F1 lambs being more like the BBs after the second dose. This difference was also found for T. colubriformis and, to a lesser extent, for T. circumcincta.

Hepatitis C virus infection and assisted reproduction.

BACKGROUND: In assisted reproduction, hepatitis C virus (HCV) transmission may pose a risk for the baby, technicians, and gametes or embryos from non-contaminated parents. This study aimed at determining the prevalence and risk factors for HCV infection in a group of infertile couples. METHODS: HCV infection was investigated in 409 patients attending the infertility clinic at Hospital de Clínicas de Porto Alegre, Brazil, between 1997 and 1998. Serum was screened for anti-HCV using ELISA and for hepatitis B surface antigen (HBsAg) using an enzyme-linked fluorescent assay (ELFA). HCV infection and semen viraemia was also investigated using HCV RNA detection. RESULTS: The overall prevalence of anti-HCV was 3.2% (8/248) among women and 3.7% (6/161) among men. All subjects were negative for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). From the 14 HCV-positive patients, two were lost, and serum was collected from the remaining 12 patients for assessment of HCV RNA, resulting in five HCV-positive cases (one woman and four men). Only one of the HCV-positive men had viraemia levels >500 000 RNA copies/ml. There was a significant risk associated with being HCV-positive in women with HCV-positive male partners (P < 0.001). In male patients, the correlation between use of intravenous drugs and HCV-positivity was also significant (P < 0.001). CONCLUSIONS: Since the risk for vertical and laboratory HCV infection is not well determined, and HCV prevalence is not negligible in this group, we recommend that infertile patients be screened before assisted reproductive techniques.

Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.

The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.

Conservative management for an extensive type A aortic dissection complicating coronary angioplasty.

Aortic dissection is a recognized, though rare complication of percutaneous revascularization procedures. We report a case of an extensive type A dissection that occurred during an attempt to recanalize a chronic total occlusion of a right coronary artery. The patient was treated conservatively and was followed for 36 months, during which he remained well. We conclude that, even though surgery remains the preferred option, conservative management could also be considered in certain patients.

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins.

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.

Urinary protein/creatinine ratio in hypertensive pregnant women.

OBJECTIVES: To determine the correlation between the protein/creatinine ratio and 24-h proteinuria; to estimate the sensitivity and specificity of this ratio for the diagnosis of significant proteinuria; to establish its cutoff point with the best predictive value for the diagnosis of significant proteinuria in patients with systemic arterial hypertension. STUDY DESIGN: A cross-sectional study of 47 hypertensive patients who had been pregnant for 20 weeks or more seen at the Maternity of the University Hospital of Porto Alegre. The studied factor was the protein/creatinine ratio measured in a single random urine sample and the outcome was protein determination in 24-h urine. The level of significance was set at 0.05. RESULTS: The correlation coefficient between the protein/creatinine ratio and 24-h proteinuria was 0.94 when urine was properly collected. A receiver-operator characteristic curve was constructed to determine the sensitivity and specificity of the ratio for the diagnosis of significant proteinuria (> or = 300 mg in 24 h). Specificity and predictive positive value were 100% for a ratio > or = 0.8. The best values for sensitivity, specificity, positive predictive value, and negative predictive value in the diagnosis of proteinuria > or = 300 mg in 24 h were obtained when the protein/creatinine ratio was 0.5 (0.96, 0.96, 0.96, and 0.96, respectively). CONCLUSION: The protein/creatinine ratio measured in a single urine sample taken at random from hypertensive pregnant women showed good sensitivity and specificity for the diagnosis of 24-h proteinuria > or = 300 mg and was strongly correlated with 24-h proteinuria. A ratio of 0.5 mg/mg is predictive of significant proteinuria and can be used for the diagnosis and follow-up of hypertensive pregnant women.

Stiff hydrophilic glidewire: a useful tool for percutaneous insertion of intraaortic balloon counterpulsation catheters.

Intra-aortic balloon catheters have a central lumen able to accommodate dedicated 0.030" guidewires but not conventional 0.035" guidewires. Due to its extreme lubricity, the 0.035" hydrophilic "glidewire" fits into these catheters and is particularly useful in its stiff, angled version for femoral percutaneous insertion in patients with severe calcific iliac disease.

A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE.

SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.

Clinical experience with a new biocompatible phosphorylcholine-coated coronary stent.

AIMS: The BiodivYsio stent is a new stent coated with phosphorylcholine, a biocompatible molecule designed to reduce the formation of thrombus and potentially the risk of restenosis. The feasibility, safety, and efficacy of elective and urgent implantation of this coated coronary stent were prospectively studied. METHODS AND RESULTS: We studied 224 patients who underwent elective (67%) or bail-out implantation of 303 BiodivYsio stents in 286 lesions. Most lesions (62%) had unfavorable characteristics (type B2 or C) and half of them (50%) had thrombus and/or chronic total occlusion. Clinical follow-up was obtained in all patients at one month and in the first 132 patients at six months. Repeat angiography was undertaken in all patients with recurrent ischemia. Successful stent deployment was achieved in 284 lesions (99.3%). One emergency coronary artery bypass graft (CABG) was required. Angiographic success rate was 98.3% (281/286). There was 1 (0.4%) subacute stent thrombosis associated with Q-wave myocardial infarction and two (0.9%) in-hospital deaths. Reference vessel diameter was 2.82 +/- 0.32 mm. Minimum luminal diameter (MLD) increased from 0.38 +/- 0.25 mm to 2.97 +/- 0.35 mm and diameter stenosis decreased from 83.8 +/- 12.1% to 5.8 +/- 9.7%. Clinical restenosis rate was 6.1% (8/132 patients) at 6-month follow-up. Target vessel revascularization rate at 6-month follow-up was 5.4%. CONCLUSIONS: This initial clinical experience indicates that the implantation of stents coated with phosphorylcholine appears to be safe and efficacious in the treatment of complex coronary lesions and is associated with an extremely low target vessel revascularization rate.

Preliminary clinical experience with the Bard XT coronary stent.

BACKGROUND: The Bard XT stent is a balloon expandable stent with a new design, consisting of discrete zigzag modules welded onto a flexible longitudinal spine. METHODS: To assess the safety and efficacy of this recently introduced coronary stent, we studied 81 patients (107 lesions) who underwent implantation of 108 such stents (66% elective). RESULTS: Primary success in stent delivery was 97.1% and angiographic success was achieved in all cases. Reference segment diameter was 2.69+/-1.4 mm and average lesion length 10.9 mm. Mean minimal luminal diameter before the procedure was 0.79+/-1.11 mm and increased to 2.55+/-1.09 mm after stenting. Diameter stenosis decreased from 76%+/-24% to 9%+/-12%. One Q wave and 1 non-Q-wave myocardial infarction occurred but no other significant complications were observed. At 1-month follow-up, angiographically documented subacute thrombosis occurred in 2 cases at days 1 and 3. Conclusions These preliminary data suggest that the Bard XT stent has a promising design for the safe and effective treatment of coronary lesions, even in the presence of high-risk clinical and unfavorable angiographic characteristics.

Bilateral internal mammary angiography through a right radial approach: a case report.

This case report demonstrates that bilateral internal mammary angiography through a single right-radial approach is feasible and convenient. It avoids double-brachial or radial artery puncture, is adapted to the variable characteristics of right internal mammary artery origin, and may be considered when the femoral approach is contraindicated.

Recanalization of chronic coronary occlusions using a new hydrophilic guidewire.

Chronic total occlusion remains a relative contraindication and the main cause of failure of coronary angioplasty. Previously available hydrophilic guidewires had numerous limitations. The Crosswire is a new 0.014" hydrophilic nitinol guidewire which can be accommodated by very-low-profile balloon catheters and has a shapeable and highly radiopaque platinum-iridium coiled tip. This guidewire was used in 55 patients with 56 chronic coronary occlusions in which recanalization by conventional guidewires had failed. Clinical success was 79%. Coronary perforation occurred in two cases, one of them requiring pericardiocentesis for tamponade. These results illustrate the usefulness of this new guidewire in the treatment of chronic total occlusions.

Preliminary experience with the NIR coronary stent.

We prospectively studied 223 patients (288 lesions) who underwent elective or bail out implantation of 309 NIR stents (Scimed, Boston Scientific Corporation, Galway, Ireland). Most lesions (68.4%) had unfavorable characteristics (type B2 or C). Primary success in stent deployment was achieved in 305 (98.6%). There was no Q-wave myocardial infarction. Emergency coronary artery bypass grafting (CABG) was required in 1 patient and 1 death occurred. Subacute thrombosis rate was 0.4%. Reference diameter was 2.65+/-0.67 mm. Minimum luminal diameter (MLD) increased from 0.62+/-0.45 to 2.69+/-0.57 mm and diameter stenosis decreased from 78.3+/-13.4% to 12.7+/-5.9%. Clinical follow-up was performed in the first 135 patients for 5.3+/-1.6 months and repeat angiography was undertaken in 35 (16%) with recurrence of symptoms at 4.6+/-1.3 months. Clinical restenosis rate was 9.6%. We conclude that the NIR coronary stent exhibits favorable performance characteristics and appears to be safe and efficacious in the treatment of coronary lesions even in the presence of high-risk characteristics.