L-carnitine supplementation in conventional slow and ultra-rapid freezing media improves motility, membrane integrity, and fertilizing ability of dog epididymal sperm.

This study aimed to assess the impact of L-carnitine (LC) supplementation in conventional-slow (CS) and ultra-rapid (UR) freezing media on post-thaw quality and fertilizing ability of dog epididymal spermatozoa. Sperm samples were collected from 60 epididymides obtained from 30 adult orchiectomized dogs via retrograde flushing. Twenty pooled sperm samples were then created (3 epididymal samples/pool). Four treatments were established according to the freezing method (CS and UR) and LC supplementation (5 and 0 mM [control, Co]): CS-LC5, CS-Co, UR-LC5, and UR-Co. The CS freezing involved exposing 0.25 mL straw to liquid nitrogen vapors (LN2), while UR freezing submerged 30-µL drops of sperm samples directly into LN2. Sperm kinematics, membrane integrity, and fertilizing ability (by heterologous in vitro fertilization using bovine oocytes) were evaluated for all treatments. Post-thaw results revealed that the CS freezing treatments resulted in significantly higher values (P < 0.05) of curvilinear and average-path velocities, and beat-cross frequency compared to the UR freezing treatments, regardless of LC supplementation. The CS-LC5 and UR-LC5 treatments cryoprotected the sperm by increasing (P < 0.05) the percentage of 'live-sperm/intact-acrosome' compared to their controls treatments CS-Co and UR-Co. Regarding fertilizing ability, the CS-LC5 treatment yielded a higher percentage (P < 0.05) of pronuclei formation compared to both UR treatments. The UR-LC5 treatment, however, obtained greater percentage (P < 0.05) than their control UR-Co. In conclusion, supplementation with L-carnitine in conventional-slow and ultra-rapid freezing improved sperm motility, plasma, and acrosome membranes integrity and fertilizing ability of dog epididymal spermatozoa.

BoviPure® density-gradient centrifugation procedure enhances the quality of fresh and cryopreserved dog epididymal spermatozoa.

BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non-selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm motilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat-cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of individual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the individual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactness.

Development of a real-time RT-PCR assay for the detection of potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a quarantine pathogen in the European Union and causes damaging diseases of solanaceous crops. Under the EU Plant Health directive 2000/29/EC, countries must have the ability to detect and identify accurately and rapidly the introduction of harmful organisms in plants or plant products; furthermore, if the quarantine pathogen is found, be able to survey extensively for it. In this respect, PSTVd poses an interesting technical problem, since its RNA does not code for any proteins and thus any diagnostic method must be based on the detection of the RNA and be suitable for scaling up to testing large sample numbers. With this in mind a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed. Investigations were carried out into various aspects of the assay relevant to the efficient amplification of targets that have a significant amount of secondary structure such as viroids. Thus comparisons were made of reverse transcription temperature, concentration and type of reverse transcriptase, RNA denaturation, sample purity and single versus two-tube reaction format. The assay developed was shown to be able to detect a wide range of isolates of PSTVd and in comparison with a chemi-luminescent hybridisation system was shown to be 1000-fold more sensitive. A further significant advantage of this assay format compared with hybridisation is that it is suitable for scaling up to large sample numbers using robotic liquid handling systems.

[Congenital atresia of the vagina]. / Atresia congénita de vagina.

The articles includes eight cases of congenital atresia of the vagina seen and studied at the Pediatric Surgery Department. All cases were congenital five of them corresponded to the Mayer-Rokitansky-Kuster-Hauser Syndrome an the remaining three to congenital adrenal hyperplasia. No other malformations were associated to any of the cases. The karyotype, hormones and secondary sexual characteristics were normal in those cases of Mayer-Rokitansky-Kuster-Hauser Syndrome. In the other three cases of congenital adrenal hyperplasia the karyotype was found to be 46XX, the 17-ketosteroids and pregnanediol were high and the patients were found to have hirsutism and were virilized. The ultrasonography, CAT and magnetic resonance scans showed excellent anatomical information on the congenital vaginal atresia and the intrapelvic organs. No other invasive study was needed to confirm its existence. Six of the patients were operated using the McIndoe and Bannister technique while on the other two the Tobin and Day technique was used. Post-surgical complications included vaginal stenosis in two cases within the first three months. The article includes an analysis of the complications and both the anatomical and functional results obtained.

Potentiation by caffeine of the analgesic effect of aspirin in the pain-induced functional impairment model in the rat.

The ability of caffeine to potentiate the analgesic effect of aspirin was studied in the pain-induced functional impairment model in the rat. Female Wistar rats received an intra-articular injection of 30% uric acid in the right hind limb, inducing its dysfunction. Once the dysfunction was complete, animals received aspirin oral doses of 0, 0.55, 0.98, and 1.74 mmol/kg with and without 0.17 mmol/kg of caffeine, and the recovery of functionality over time was considered as an expression of analgesia. Blood samples were drawn simultaneously with hind limb functionality determinations, and plasma concentrations of aspirin, salicylic acid, and gentisic acid were measured by high-performance liquid chromatography. Aspirin induced a dose-dependent analgesic effect. Caffeine alone was ineffective. However, caffeine significantly increased the analgesic effect of aspirin at all doses, without modifying aspirin, salicylic acid, or gentisic acid plasma levels. It is concluded that caffeine potentiates the analgesic effect of aspirin by a pharmacodynamic, but not by a pharmacokinetic mechanism.