Differences In Penetration Rates Research Articles

Quercetin supplementation to the thawing and incubation media of boar sperm improves post-thaw sperm characteristics and the in vitro production of pig embryos

Elevated levels of reactive oxygen species can cause oxidative stress, which could lead to membrane damage, decreased fertility, and spermatozoan morphological deformities. Antioxidants can be supplemented to reduce the impacts of oxidative stress. The objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75 mM) during the thawing and incubation of frozen-thawed boar semen on spermatozoan characteristics, IVF kinetics (n = 400) and subsequent embryonic development (n = 1340). Spermatozoa were evaluated for motility, viability, and membrane lipid peroxidation levels at 0, 2, 4, 6, 8, and 10 h after thawing. Embryos were evaluated for IVF kinetics 12 h after IVF (penetration, polyspermy, male pronucleus formation, IVF efficiency) and cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. Spermatozoa supplemented with 0.25 mM quercetin had significantly higher (P < 0.05) motility (51.67±8.50 %) and percent of viable cells (61.21 ± 2.44 %) compared to all other treatments at 10 h after thawing, in addition to having significantly (P < 0.05) lower levels of hydroperoxide (3.38 ± 0.88 μM/107cells). There were no differences in penetration rates and male pronucleus formation between treatment groups. Supplementation of quercetin significantly decreased (P < 0.05) polyspermy and significantly increased (P < 0.05) the percentage of embryos reaching blastocyst stage of development by 144 h after IVF compared to no supplementation. Results indicated that supplementing frozen-thawed boar semen with 0.25 mM quercetin improves sperm characteristics up to 10 h after thawing and decreases polyspermy while improving early embryonic development in pigs.

146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P&amp;lt;0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P&amp;lt;0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P&amp;lt;0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

Pig sperm preincubation and gamete coincubation with glutamate enhance sperm-oocyte binding and in vitro fertilization

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, in vitro fertilization (IVF) and sperm parameters during in vitro induced capacitation.For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1 h and then coincubated for 1 h with denuded in vitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). MSG 1 and 10 mM significantly (P < 0.05) increased the mean number of sperm bound to ZP compared with control (12.3 ± 9.0, 17.8 ± 11.3, 17.6 ± 10.8, MSG 0, 1 and 10 mM respectively).For in vitro fertilization trials, both sperm preicubation (1 h) and gamete coincubation (1 h) were performed in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). After 19 h of culture in fresh IVF medium, oocytes were fixed. MSG 1 mM significantly (P < 0.05) increased the penetration rate compared with control (53.7 ± 20.4 vs. 36.8 ± 16.2).The addition of MSG during in vitro induced capacitation of boar spermatozoa did not cause any significant difference, compared with control, on the percentage of viable cells, spermatozoa with intact acrosome and the percentage of spermatozoa displaying tyrosine-phosphorylation of sperm tail proteins.In order to evaluate whether the effect elicited by MSG could be due to glutamate uptake in boar spermatozoa, fertilization trials were performed in presence of either 1 mM MSG or 1 mM MSG + 100 μM DL-threo-beta-hydroxyaspartic acid (THA), a non selective inhibitor of glutamate uptake. A significant increase (P < 0.05) in the penetration rate in both MSG and MSG + THA groups compared to control was recorded (39.8 ± 15.7, 53.7 ± 22.1, 52.2 ± 23.7, Control, MSG and MSG + THA respectively) while no difference in penetration rate between MSG and MSG + THA treatment was observed suggesting that sperm glutamate transporters are not involved in the pathway mediating this effect.Our study demonstrates for the first time that glutamate exerts a positive effect on sperm-oocyte binding and fertilization. Further studies are needed to clarify the mechanism by which glutamate exert his effect.

Dysphagia in infants with single ventricle anatomy following stage 1 palliation: Physiologic correlates and response to treatment.

Deficits in swallowing physiology are a leading morbidity for infants with functional single ventricles and systemic outflow tract obstruction following stage 1 palliation. Despite the high prevalence of this condition, the underlying deficits that cause this post-operative impairment remain poorly understood. Identify the physiologic correlates of dysphagia in infants with functional single ventricles and systemic outflow tract obstruction following stage 1 palliative surgery. Postoperative fiberoptic laryngoscopies and videofluoroscopic swallow studies (VFSS) were conducted sequentially on infants with functional single ventricles following stage 1 palliative surgery. Infants were dichotomized as having normal or impaired laryngeal function based on laryngoscopy findings. VFSS were evaluated frame-by-frame using a scale that quantifies performance within 11 components of swallowing physiology. Physiologic attributes within each component were categorized as high functioning or low functioning based on their ability to support milk ingestion without bolus airway entry. Thirty-six infants (25 male) were included in the investigation. Twenty-four underwent the Norwood procedure and twelve underwent the Hybrid procedure. Low function physiologic patterns were observed within multiple swallowing components during the ingestion of thin barium as characterized by ≥4 sucks per swallow (36%), initiation of pharyngeal swallow below the level of the valleculae (83%), and incomplete late laryngeal vestibular closure (56%) at the height of the swallow. Swallowing deficits contributed to aspiration in 50% of infants. Although nectar thick liquids reduced the rate of aspiration (P = .006), aspiration rates remained high (27%). No differences in rates of penetration or aspiration were observed between infants with normal and impaired laryngeal function. Deficits in swallowing physiology contribute to penetration and aspiration following stage 1 palliation among infants with normal and impaired laryngeal function. Although thickened liquids may improve airway protection for select infants, they may inhibit their ability to extract the bolus and meet nutritional needs.

175 EFFECTS OF GLUCURONIC ACID AND N-ACETYL-D-GLUCOSAMINE SUPPLEMENTATION ON THE PERIVITELLINE SPACE DURING THE IN VITRO MATURATION OF PORCINE OOCYTES

Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced perivitelline space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P &lt; 0.05) with oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (3.20 ± 0.29) and all of maturation (2.78 ± 0.21) compared with no supplementation (2.22 ± 0.13) and supplementation during only the second half of maturation (2.02 ± 0.16). The amount of hyaluronic acid present at 24 h of maturation was significantly greater (P &lt; 0.05) in oocytes supplemented with the PVS components (2.03 ± 0.07 pg/oocyte) compared with no supplementation (0.21 ± 0.02 pg/oocyte). At the end of maturation, oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the entire maturation had significantly greater (P &lt; 0.05) amounts of hyaluronic acid present (4.16 ± 0.19 pg/oocyte) compared with all other groups. There was no significant difference in penetration rates between the groups. Polyspermic penetration was significantly less (P &lt; 0.05) in oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation compared with no supplementation or supplementation during only the second half of maturation. Oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (87.36 ± 4.01) compared with no supplementation (76.47 ± 5.67) or supplementation during only the second half of maturation (80.23 ± 3.21) had significantly higher percentages (P &lt; 0.05) of male pronuclear formation by 12 h after IVF. Supplementing 0.01 mM GA and 0.01 mM GlcNAc during maturation significantly increased (P &lt; 0.05) the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF compared with those that were not supplemented during maturation (55.00 ± 6.43, 20.00 ± 6.16). There were no significant differences between the supplementation treatment groups at 48 or 144 h after IVF. These results indicate that supplementing GA and GlcNAc to the media during maturation, specifically during the first 24 h, decreases polyspermic penetration by increasing PVS thickness, hyaluronic acid amount, and male pronuclear formation, which improves subsequent embryonic development.

Polyethylene Wear Associated With 26- and 32-mm Heads in Total Hip Arthroplasty: A Multicenter, Prospective Study

BackgroundAlthough there were many clinical studies of highly cross-linked polyethylene (XLPE) wear among different femoral head diameters, few referred to thickness of XLPE in case larger femoral heads were used because smaller sockets were frequently used for Asian population. MethodsThis prospective study included 240 hips that underwent primary total hip arthroplasty using XLPE combined with 26-mm (group S) or 32-mm (group L) cobalt–chromium head with maximum follow-up of 10 years. We measured 3-dimensional (3-D) linear penetration rate of XLPE among same implant design groups except head diameter and estimated the validity of thinner XLPE. ResultsOur study demonstrated comparable 3-D linear penetration rates, which were 0.06 ± 0.07 mm/y for group S and 0.03 ± 0.02 mm/y for group L at 10 years after surgery and penetration rates seemed to be almost constant with no significant difference after 3 years. Minimum liner thickness (5.3 mm for 48-mm socket in combination with 32-mm femoral head) and the second thinnest XLPE (6.3 mm in case of socket from 50 mm to 54 mm combined with 32-mm femoral head) was distributed in 25% and 72% with group L, respectively, and there were no significant differences in penetration rates between 5.3-mm– and 6.3-mm–thickness groups. ConclusionOur study suggested that whether to select 26- or 32-mm diameters of femoral head does not affect XLPE wear in combination with this type of articulation.

Metals Leak from Tilled Soil in a Century – A Review

&lt;p&gt;Toxic metals are mobilized on a large scale in modern society. Many of those metals end up in sewage sludge. The objective of this review was to elucidate the threat to groundwater due to a few metals lost from tilled sludge amended soils. It is sometimes suggested that these metals are immobilized in the topsoil and do not move downward. In contrast, dozens of long term field studies around the world indicate that penetration depths for metals increase with time since deposition.&lt;/p&gt;&lt;p&gt;Such studies were examined in depth in the current analysis. An equation was developed for calculation of long term mean metal penetration rates into the topsoil for copper and silver. The equation is valid for about a century but not much longer. The mean depths of a basic set of 11 cases from studies over 4 years to 100 years were predicted with a standard deviation of 11%. A typical penetration rate was 3 mm per year. There was no significant difference in penetration rate between several cations. Extremely large amendments were associated with larger penetration rates.&lt;/p&gt;&lt;p&gt;When metals have traversed the topsoil, the groundwater will be contaminated. The European Groundwater Pollution Directive stipulates that pesticide levels should be kept below 0.1 µg/l. When sludge is applied to agricultural soil, this level may by far be exceeded for many metals, even if strict limitations are applied to the metal contents of the sludge. This calls for careful assessment of the groundwater consequences of sludge amendment.&lt;/p&gt;&lt;p&gt;Extensive supplementary material provides many detailed tables, texts and references.&lt;/p&gt;

Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability

The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.

Using Gray‐Based Taguchi Method to Construct Multi‐Objective Optimal Model in Super‐Resolution Near‐Field Photolithography

This study integrated thermally induced super-resolution into near-field photolithography and conducted simulation and analysis on line segment fabrication. This technique involves passing a laser beam through an aluminum-plated optical fiber probe onto a thin film of indium (approximately 10 nm thick). The indium film opens a melted aperture narrower than the width of the laser beam, creating a melted region and a crystalline region. The difference in penetration rate between the two regions leads to the generation of thermally induced super-resolution. This paper proposes a combination of Taguchi method with gray relational analysis, in which S/N ratios obtained using the Taguchi method are converted into gray relational grades to identify an optimal combination of parameters capable of meeting multiple quality objectives. This optimal combination includes a probe aperture of 100 nm (A1), exposure energy/μm of 0.002nJ/μm (B2), development time of 60 s (C3), and indium film with a thickness of 7 nm (D1). The optimal parameters were (A1B2C3D1) for the gray relational analysis and (A1B1C1D1) for the Taguchi method. Results showed a negative improvement of -14.3% in line width from 126.2 (Taguchi method) to 144.2 nm (gray relational analysis). Working depth, however, showed a significantly improvement of 140.4% from 5.7 (Taguchi method) to 13.7 nm (gray relational analysis). The proposed approach resolves the conflicts that commonly occur among factor levels in Taguchi analysis under the requirements of multiple quality requirements.

In Vitro Dermal Penetration of 4-Chloro-3-Methylphenol from Commercial Metal Working Fluid and Aqueous Vehicles

The biocide 4-chloro-3-methylphenol (CMP, CAS number 59-50-7) is a common additive to metal-working fluids (MWF) and building materials. National Institute for Occupational Safety and Health (NIOSH) researchers previously identified and quantified CMP in a commercial water-soluble MWF, TRIM VX, and demonstrated irritancy and sensitization potential of both TRIM VX and CMP alone after dermal exposure in a murine model. In the current study, the in vitro human epidermal permeability of CMP contained in a working dilution of TRIM VX (20% in water) was evaluated and, for comparison, permeability from an aqueous buffer was also assessed. CMP penetration was also measured from transient exposures to 20% TRIM VX. To address differences in penetration rates from 20% TRIM VX and from buffer, the role of thermodynamic activity of CMP in the 2 vehicles on dermal penetration was investigated. Static headspace gas chromatography was used to measure vapor pressures and infer fractional thermodynamic activities of CMP in the mixtures. Permeability coefficient (kp ) of CMP from 20% TRIM VX was (4.1 ± 0.8) × 10−3 cm/h (mean ± SD, n = 5), and CMP was found at a concentration of 3555 ± 191 μg/ml in this donor. In contrast, kp was 0.18 ± 0.03 cm/h (n = 5) at a similar concentration (3919 ± 240) from buffer donor. Steady-state fluxes from 20% TRIM VX and buffer were comparable when expressed as functions of thermodynamic activity of CMP in the donor, rather than as concentrations. Transient (20 or 40 min) exposures of epidermal membranes to 20% TRIM VX (n = 4) resulted in total penetration of 4.2 ± 1.2 and 7.3 ± 0.8 μg/cm2, respectively; these amounts are comparable to amounts predicted using a simple algebraic equation.

Periapical Inflammation and Bacterial Penetration After Coronal Inoculation of Dog Roots Filled With RealSeal 1 or Thermafil

Introduction The purpose of this study was to subject 2 carrier-based root filling products to a 4-month microbial challenge in a dog model with histologic markers to assess periapical inflammation and bacterial penetration of the 2 filling materials. Histologic evidence of bacterial penetration and periapical inflammation were the outcome parameters used to compare the products. Methods Teeth were aseptically prepared and then filled with carrier-based Resilon (RealSeal 1 [RS-1], n = 25) or with carrier-based gutta-percha (Thermafil, n = 25) and were left exposed for 4 months. The first control group received a coronal seal over either RS-1 or Thermafil root fillings (n = 8). A second control group was instrumented and left completely empty (n = 8). Results Histologic evidence of periapical inflammation was observed in 29% of the Thermafil group and in 9% of the RS-1 group. This difference was only significant when controlling for a possible tooth position effect on inflammation presence ( P < .05). Histologic evidence of bacterial penetration was present in 9% of the RS-1 group and in 70% of the Thermafil group. The difference in penetration rates between RS-1 and Thermafil was statistically significant when controlling for any dog or tooth position effects on bacterial penetration ( P < .001). Furthermore, there was a statistically significant correlation between histologic evidence of inflammation and histologic evidence of infection ( P = .002). Conclusions RS-1 appeared to resist bacterial penetration more effectively than Thermafil under the conditions of this study.

Galantamine Delivery on Buccal Mucosa: Permeation Enhancement and Design of Matrix Tablets

The most important feature in transbuccal drug delivery is the low drug passage through the buccal mucosa. In our previous work we demonstrated the aptitude of Galantamine to penetrate the buccal tissue. The collected data suggested that Galantamine passively crosses the membrane, but the calculated Js and Kp values showed that the drug amount that crosses the membrane wasn’t sufficient to assure blood therapeutic level. So, in this study, ex vivo permeation tests, using porcine buccal mucosa, were performed in presence of physical or chemical enhancers. No significant differences in penetration rate were observed using chemical enhancers as sodium dehydrocholate, EDTA disodium salt and trisodium citrate dihydrate; while, Js and Kp were extensively affected by application of electric fields. Tablets, designed for Galantamine administration on buccal mucosa, were prepared by direct compression of drug loaded Eudragit® RS 100 matrices. When the tablets were coated with lipophilic material, Galantamine is slowly discharged from buccal tablets, following the Higuchian kinetic. Buccal tablets containing Galantamine may represent a potential alternative dosage form in Alzheimer management.

Release of naltrexone on buccal mucosa: Permeation studies, histological aspects and matrix system design

Transbuccal drug delivery has got several well-known advantages especially with respect to peroral way. Since a major limitation in buccal drug delivery could be the low permeability of the epithelium, the aptitude of NLX to penetrate the mucosal barrier was assessed. Ex vivo permeation across porcine buccal mucosa 800 μm thick was investigated using Franz type diffusion cells and compared with in vitro data previously obtained by reconstituted human oral epithelium 100 μm thick. Both fluxes (Js) and permeability coefficients ( K p) are in accordance, using either buffer solution simulating saliva or natural human saliva. Permeation was evaluated also in presence of chemical enhancers or iontophoresis. No significant differences in penetration rate were observed using chemical enhancers; in contrast, Js and K p were extensively affected by application of electric fields. Tablets, designed for Naltrexone hydrochloride (NLX) administration on buccal mucosa, were developed and prepared by direct compression of drug loaded (56%) poly-octylcyanocrylate (poly-OCA) matrices. NLX is slowly discharged from buccal tablets following Higuchian kinetic. Histologically, no signs of flogosis ascribable to NLX and/or poly-OCA were observed, while cytoarchitectural changes due to iontophoresis were detected. Buccal tablets containing NLX may represent a potential alternative dosage form in addiction management.

Reduced Penetration Rate of Pyraclofos into Housefly, Musca domestica, as a Resistance Mechanism

Ethylacetate was found to be suitable for washing pyraclofos from the insect body in experiments to determine the role of the cuticle in the penetration of pyraclofos into the body, and was recommended as a favorable solvent to study housefly resistance to pyraclofos. Three ml of ethylacetate washed off 99% of pyraclofos from the housefly body surface. The penetration through integument played an important role in resistance mechanism of the housefly to pyraclofos. There were considerable differences in penetration rate, excretion rate, and metabolism of the insect body between the resistant (YBOL) and susceptible (SRS) strains of housefly. In penetration study, the YBOL (resistant) strain showed a slightly greater inhibition of penetration than the SRS strain during 1 h after treatment at both the 8, 000-dpm and 4, 000-dpm treatments. Therefore, the reduced penetration rate of pyraclofos is one of the resistance mechanisms in the YBOL strain of housefly even though there were no significant differences of radioactivity between the YBOL and SRS strains after 2 h. For both 8, 000-dpm and 4, 000-dpm treatments, radioactivity in the YBOL surface extract was somewhat higher than in that of SRS, while radioactivity in the housefly body of SRS was higher than that of YBOL. Excretion rate was faster in the resistant housefly. This suggests that metabolic process might be also one of the resistance mechanisms of housefly to pyraclofos.

Liquid Gallium Penetration along Grain Boundaries in Pure Al and Al-Based Alloys

The kinetics of liquid Ga penetration along grain boundaries in pure Al, Al-Mn (50 ppm Mn) and Al-Ga alloys (with concentration 0.7, 1 and 3% mass. Ga) was studied. It was shown that crack-like channels filled by liquid Ga were formed along all grain boundaries. Their width was about 1-3 µm. Investigation of quenched state showed that in all samples, channels had grown with linear kinetics, and that the propagation rate depended on impurity concentration in alloys: 35µm/s for Al-Mn alloy, 14 µm/s for pure Al and 9±1 µm/s for Al with (0.7-3) % Ga alloys. The difference in penetration rates of pure Al and Al based alloys are discussed in terms of internal stress and mechanical properties.

89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P &lt; 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P &lt; 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.

Birth of piglets by in vitro fertilization of zona-free porcine oocytes

The present experiments were conducted to optimize in vitro fertilization conditions for zona pellucida-free (ZP-free) oocytes and their subsequent development. The results demonstrated that: (1) maximal fertilization efficiency was achieved at 200 spermatozoa per ZP-free oocyte. At this sperm dose, there were no significant differences in penetration rates and polyspermy rates from controls (zona-intact oocytes with 1000 spermatozoa/oocyte), indicating that ZPs of in vitro matured pig oocytes failed to block polyspermy during in vitro fertilization. (2) In vitro development of zygotes from ZP-free oocytes showed that there was no difference in cleavage rates. The blastocyst rate was slightly lower in the ZP-free group than the control. However, there was no difference in cell number per blastocyst between the control and the ZP-free group. (3) Examination of acrosome status by a specific fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining procedure revealed that frozen–thawed pig spermatozoa could undergo acrosome reaction and penetrate oocytes without induction by ZP. These data suggested that there are alternative mechanistic pathways for acrosome reaction induction during the fertilization process than the widely accepted sperm–zona receptor models. Finally, the viability of ZP-free derived embryos was demonstrated by full-term development and the delivery of healthy piglets following embryo transfer. In conclusion, the present experiments showed for the first time in farm animals, that normal embryos could be produced by in vitro fertilization of ZP-free oocytes in optimized conditions and that they could develop normally to full-term.

Numerical modelling of the petroleum oil penetration into sandy beach sediments

A numerical model describing the penetration of petroleum oil into sandy beach sediments was developed to assess the behavior of stranded oil at tidal zone as a result of tanker accidents, and so on. To understand the penetration behavior, penetration rate of three species of petroleum oil (two species of fuel oil C and one species of crude oil) was observed experimentally with artificial tidal zone equipment. As a consequence, two types of oil were distinguished from the viewpoint of penetration rate. One (fuel oil C-1) kept its homogeneity in composition and showed relatively rapid penetration, the other (fuel oil C-2 and crude oil) became heterogeneous and slow in penetration. Considering this aspect as the adsorption of polar compounds (i.e. asphaltenes) on the sediment surface, a numerical model that described oil penetration into sandy beach was developed. As a result, the difference in penetration rate between these two types of oil could not be replicated sufficiently only by consideration of the adsorption. However, the change of a parameter value which represents the apparent viscosity of oil led to good agreement with observations. Simulation results indicated that when fuel oil C or crude oil used in this study was stranded at a sandy beach located in Hiroshima Bay, Japan, 2 to 39% of total stranded oil might penetrate into the deeper zone (> 3 cm in depth) over 50 days.

Development of in vitro embryo production systems for red deer ( Cervus elaphus) : Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes

In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4–12 h on COEM or for 4 h in TALP medium supplemented with 20 μg of heparin. COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm ( P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24 h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4 h and no motile sperm were observed by 8 h. Overall, the penetration rates of in vitro red deer oocytes were low (5–28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin ( P<0.001). Penetration rates were similar for 4–12 h pre-incubation of sperm on COEM ( P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24 h compared to 12 h ( P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24 h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine.

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, beta-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 microM BME, 0.5 microgram/ml LH, 0.5 microgram/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5-6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78-89%). The mean differences in penetration rate (69-77%), polyspermy rate (31-40%), male pronuclear formation rate (93-96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32-39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13-15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.