Safety assessment of poly(N-vinylcaprolactam) as a potential drug carrier in extenders for boar sperm cryopreservation.

Polymers may be used to deliver compounds in freezing extenders to minimize injuries in spermatozoa during cryopreservation, although their activity and toxicity for boar sperm are unknown. This study investigated the effects of the polymer (N-vinylcaprolactam) (PNVCL), when included in extenders for boar sperm cryopreservation. In Experiment 1, sperm was exposed to PNVCL at: 0 (control); 39.1; 78.1; 156.3; and 312.5 µg/mL. Spermatozoa structure, kinetics and biochemical functions were unaltered in contact with PNVCL at 38 °C (P > .05) but declined with prolonged exposure (10, 60 and 120 min) in all treatments (P > .05). In Experiment 2, after inclusion of PNVCL in the freezing extender at the same concentrations, post-thawing sperm quality did not differ compared to the control (P > .05). Lipid peroxidation and the production of reactive oxygen species were the only parameters of sperm quality that were unaffected in both experiments, even after contact with PNVCL for 120 min (P > .05). As no negative effects were observed in post-thawing boar sperm quality, PNVCL did not incur in cytotoxicity and may be a potential carrier for antioxidants in freezing extenders.

Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles.

PURPOSE: Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles. METHODS: Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham's F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5% CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h. RESULTS: Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 µg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture. CONCLUSIONS: In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 µg/ml for 30 min, being perfectly compatible with PAF vitrification.

[Genotoxicity risk assessment and oocytes: basis of genetic toxicology and application in reproductive science]. / Risque génotoxique et ovocytes : principes de toxicologie génétique et applications.

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line and on mouse oocytes.

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender.

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.

In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

[Pharmacological and cost effectiveness bases of the use of categel and categel S [correction of F] in urological practice]. / Farmakoékonomicheskoe obosnovanie primeneniia preparatov katedzhel' i katedzhel' S v urologicheskoi praktike.

Preparations catedgel and catedgel S made in Austria (Montavit) was tried in Moscow hospital N 50. Categel is a sterile gel of methylcellulose with 2% lidocain and 0.05% chlorhexidine, catedgel S contains the same components but lidocain. Categel significantly reduces the risk of infectious-inflammatory complications after endourological manipulations, improves endoscopic diagnosis and makes some manipulations less painful. Comparative pharmacological cost-effect assessment of categel S and glycerine effects in prostatic transurethral resection. Categel was found 2.11 times more effective. It also improves quality of life of the patients. Categel can be recommended for wide use in urology.

Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation.

The aims of the present study were: (i) to validate the accuracy of flow cytometry for assessment of viability and acrosomal status of canine spermatozoa; and (ii) to evaluate the cryopreservation protocols currently used for dog spermatozoa using flow cytometry. Data obtained by flow cytometry analysis of fresh dog spermatozoa stained with carboxyfluorescein diacetate (CFDA) and propidium iodide, or with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide, were compared with those obtained by microscopic evaluation. The results demonstrated that flow cytometry is a precise method for evaluating the viability and acrosomal status of fresh samples of dog semen. A new triple staining procedure, using carboxy-SNARF-1, propidium iodide and FITC-PSA, was developed and was an efficient method for evaluating the following aspects of cryopreservation protocols for dog spermatozoa: (i) addition of 0.5% (v/v) Equex STM paste to a Tris-egg yolk-based extender; (ii) dilution of the semen in one or two steps; (iii) freezing semen by placing 0.5 ml straws horizontally above liquid nitrogen in a styrofoam box or lowering them vertically into a liquid nitrogen tank; (iv) thawing semen at two different rates; (v) packaging semen at different sperm concentrations; and (vi) diluting semen at different rates after thawing. The highest sperm survival and longevity was obtained when Equex was present in the semen extender, the semen dilution was performed in two steps to obtain a concentration of 2.0 x 10(8) spermatozoa ml-1, the freezing was carried out using the styrofoam box, the straws were thawed at 70 degrees C for 8 s and the semen was diluted 1:4 after thawing.