Evaluation of the in vitro performance of generic and original adapalene gel.

OBJECTIVE: The aim was to evaluate the difference of the in vitro behavior between the commercially available generic adapalene gel and original product with Topical Classification System (TCS), and to analyze the effect of changes of excipients on the release behavior. SIGNIFICANCE: Establishing in vitro performance assays to understand the impact of formulation variables on the critical quality attributes (CQA) is critical for the quality assessment of semi-solid generic drug. METHODS: In vitro release (IVR), in vitro permeation (IVP), viscosity, and pH measurement methods for adapalene gels were established and validated. The differences between generic adapalene gel from 7 companies and original products were evaluated by correlation analysis (CA) and principal component analysis (PCA), and the relationship among 4 parameters was elucidated. The effect of excipients on the above variables was examined by univariate tests. RESULTS: There were some differences between the gels of 5 of the 7 imitation enterprises and reference listed drug (RLD). There were varying degrees of correlation between viscosity, pH, the adapalene amount retained in skin and release rate. The result validated the key role of IVR, and identified that pH value, type of suspending agent, the amount of carbomer, etc. had certain effects on the release rate. CONCLUSIONS: The factors mentioned above should be considered when developing and manufacturing generic adapalene gels, and the application of TCS in the evaluation of generic topical drugs was advanced. Additionally, our research revealed some discrepancies from USP<1724>, which could be valuable information for the revision.

ß-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

Influence of temperature-humidity index on conception rate of Nelore embryos produced in vitro in northern Brazil.

Considering the high temperatures that the tropical climate provides to most of Brazil and the effects of thermal stress on reproductive processes, the objective of the present study was to analyze, in the warmer months of 2016, conception rates of Nelore bovine embryos in Acre state. For this purpose, oocytes were aspirated (ultrasound-guided follicular aspiration), matured, fertilized with Nelore bull semen, cultured for 6 days, and then the embryos were transferred to crossbred recipients. Pregnancy diagnosis was performed 30 and 60 days after embryo transfer. Meteorological data were obtained at www.inmet.gov.br to generate temperature-humidity index (THI). The data from the conception rates and periods of the year were submitted to the chi-square test at 5% probability to verify independence. Regression analysis was used to verify the relationship between THI and gestation rate. There was a strong relationship between conception rates and THI values, verified by an increase in conception rates as THI values were reduced and a decrease when THI reached the highest value. Our findings demonstrated a negative effect of heat stress in conception rates of crossbred cows in northern Brazil.

Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos.

The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 µM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 µM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 µM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 µM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4-8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.

Gene expression analysis and in vitro production procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects.

Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved and in particular bovine IVP is now widely applied under field conditions. This in vitro technique provides new opportunities for cattle producers, particularly in the dairy industry, to overcome infertility and to increase dissemination of animals with high genetic merit. Improvements in OPU/IVP resulted in large-scale international commercialization. More than half a million IVP embryos are generated on the yearly basis demonstrating the enormous potential of this technology. These advances and the fact that bovine and human early development is remarkably similar have also prompted the use of bovine embryos as a model system to study early mammalian embryogenesis including humans. Despite all the improvements, embryos generated in vitro still differ from their in vivo derived counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo. The successful clinical application of the techniques in reproductive biotechnology requires both species-specific clinical skills and extensive laboratory experience. The recent advances in transcriptome profiling have also provided deeper insight into RNA expression and regulation at an unprecedented resolution. The development of these high-throughput DNA sequencing methods has resulted in new approaches for both mapping and quantifying transcriptomes. The aim of this review is therefore to summarize the available data related to gene expression analyses as well as in vitro embryo production procedures and to provide ideas about future concepts.

Framework for evaluating external and internal parameters associated with Sea Based Container Culture (SBCC): Towards understanding rearing success in European lobsters (Homarus gammarus).

Sea Based Container Culture (SBCC) is a mariculture technique that relies on the natural maintenance of environmental conditions, such as Dissolved Oxygen (DO) concentration and feed availability. This paper discusses a framework to evaluate the rearing success of European Lobsters (Homarus gammarus) in SBCC based on temporal and spatial variations of external parameters, including current velocity, wave velocity, turbulent fluctuations and dissolved oxygen concentrations. The temporal variations considered annual changes to the environment and the effect of biofouling growth, and the spatial variations considered the geographical location (case study of Falmouth bay, Cornwall) and vertical position in the water column. The internal parameters of the containers were modelled using transfer functions derived from previous experimental data. The internal parameters were compared to rearing limitations selected from available literature, which included foraging and mobility behaviours, and DO consumption. The time that internal parameters exceeded the rearing limitations was quantified, allowing rearing success to be predicted. This paper uses a case study of external parameters measured in Cornish waters, UK, to demonstrate the framework methodology. The framework showed that in situ measurements of current, wave and turbulence could be used to predict the internal parameters of SBCC containers, which can be used to predict theoretical rearing success based on rearing limitations. The framework indicated that DO concentrations within the containers should not affect rearing success; however, the foraging and mobility limits were exceeded by 0 to 30% of the time (depending on vertical position in the water column and assessment method). The paper aims to demonstrate the generic framework methodology and understands its limitations in predicting rearing success. The framework provides a tool to optimise the SBCC design for spatial and temporal varying conditions related to a geographical location or (vice versa) identify suitable mariculture sites based on SBCC design and environmental conditions. Additionally, the framework can optimise the vertical position of the SBCC in the water column and identify, from parameters considered, those that are most likely to affect rearing success.

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos.

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.

Short communication: Genetic correlations between number of embryos produced using in vivo and in vitro techniques in heifer and cow donors.

Multiple embryos can be produced from a heifer or cow donors using an in vivo or an in vitro technique. Comparisons of the number of embryos produced by the same donors as heifers and cows and using different techniques are limited. The main objectives of this study were to assess the genetic correlation between the number of embryos produced by Holstein donors using an in vivo and in vitro technique as a heifer and as a cow. The data set used was recorded by Holstein Canada and included all successful superovulations or ovum pickup and in vitro fertilization procedures performed on Holstein donors for more than 20yr. The type of technique used was known for all records and the status of the donor at recovery was retrieved from calving records. Bivariate repeatability animal model analyses were performed for both the total number of embryos (NE) and the number of viable embryos (VE) recovered per procedure. Logarithmic transformation was performed on the traits to normalize the data. Heritability estimates for the donor varied between 0.14 (0.02) and 0.19 (0.03) over all analyses, indicating that the number of embryos produced by a donor is influenced by the genetic potential of the donor. Genetic correlations between records produced in vivo and in vitro were moderately high and positive (NE=0.85±0.07; VE=0.63±0.09), suggesting that donors with high genetic potential for in vivo superovulation tend also to have high potential to produce multiple embryos in vitro. Similarly, the moderately high genetic correlations (NE=0.79±0.05; VE=0.72±0.05) found between heifer and cow records indicate that a donor tends to produce a comparable number of embryos as a heifer or as a cow. The estimated repeatabilities (0.23 to 0.35) indicated that the number of embryos recovered should be somewhat repeatable in the same donor over time. On the other hand, the service sires seem not to play an important role on the total number of embryos produced by a donor no matter the technique used or the status of the donor at recovery.

Effect of sperm preparation on development of bovine blastocyst in vitro.

Sperm preparation is an important step in the in vitro production of embryos. Centrifugation through colloids has been used to select normal sperm for assisted reproduction in several species. Animal models can sometimes be used as a preliminary step to investigate sperm preparation methods that are potentially of use for human fertility treatments. In this study bovine semen was prepared using three variants of the single-layer centrifugation sperm selection technique (Small, Mini, Mini-EP) with Bovicoll (Androcoll-B). Computer-assisted sperm motility analysis, the hypo-osmotic swelling test, and the sperm chromatin structure assay were performed on unselected (control) and SLC-selected sperm samples. Mini and Mini-EP gave the highest yield of motile spermatozoa, progressive motility and membrane integrity. In vitro fertilization trials were performed to investigate the fertilizing ability of the frozen-thawed bovine spermatozoa selected with Bovicoll. Mini-SLC (single-layer centrifugation) and swim-up (Control) were performed and cleavage rate and blastocyst rate did not differ significantly between groups. As there was a trend to an increased number of cells in blastocysts in the SLC group, the Mini-SLC method is at least as good as swim-up for selecting frozen-thawed bull spermatozoa for in vitro fertilization (IVF). This method could potentially be used to prepare human sperm for assisted reproduction.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods.

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification-warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts.

Effect of body condition and season on yield and quality of in vitro produced bovine embryos.

The aim of our study was to examine the effects of cow's body condition score (BCS; scale 1-5) and season on the quality of bovine in vitro produced embryos. The proportion of good quality oocytes (Q1 and Q2) was higher (P < 0.05) in the BCS 2 (57.60%) and BCS 3 (60.90%) groups compared with the BCS 1 (43.60%) group. There were no statistical differences in embryo cleavage and blastocyst rate among the BCS groups. The highest total cell number (TCN, DAPI stain) of blastocysts (P < 0.05), recorded in BCS 1 (122.27 ± 6.90) in comparison with BCS 2 (101.8 ± 3.60) or BCS 3 (105.44 ± 3.70) groups, was related to higher dead cell (DCI, TUNEL) index in this group (7.07%) when compared with BCS 2 (6.54%) or BCS 3 (6.06%), respectively. The yield of good quality oocytes during spring was lower (P < 0.05) compared with the summer season. There were significant differences (P < 0.05) in maturation and cleavage rates between autumn and summer (73.42%, 76.2% vs. 85.0%, 41.8%, respectively). The highest (P < 0.01) blastocyst rate was noted during spring and summer months. Significant difference (P < 0.05) in the TCN among spring (99.38 ± 3.90), autumn (110.1 ± 4.58) or summer (108.96 ± 3.52) was observed. The highest proportion of embryos with the best (grade I) actin cytoskeleton (phalloidin-TRITC) quality was noted during the summer months. Our results indicate that body condition affects the initial quality of oocytes, but does not affect embryo cleavage, blastocyst rate and actin quality. This finding may suggest that development in vitro can mask the influence of BCS. The season affects yield and quality of blastocysts in the way that the autumn period is more favorable for embryo development.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods.

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

An Overview of Reactive Oxygen Species Damage Occurring during In Vitro Bovine Oocyte and Embryo Development and the Efficacy of Antioxidant Use to Limit These Adverse Effects.

The in vitro production (IVP) of bovine embryos has gained popularity worldwide and in recent years and its use for producing embryos from genetically elite heifers and cows has surpassed the use of conventional superovulation-based embryo production schemes. There are, however, several issues with the IVP of embryos that remain unresolved. One limitation of special concern is the low efficiency of the IVP of embryos. Exposure to reactive oxygen species (ROS) is one reason why the production of embryos with IVP is diminished. These highly reactive molecules are generated in small amounts through normal cellular metabolism, but their abundances increase in embryo culture because of oocyte and embryo exposure to temperature fluctuations, light exposure, pH changes, atmospheric oxygen tension, suboptimal culture media formulations, and cryopreservation. When uncontrolled, ROS produce detrimental effects on the structure and function of genomic and mitochondrial DNA, alter DNA methylation, increase lipid membrane damage, and modify protein activity. Several intrinsic enzymatic pathways control ROS abundance and damage, and antioxidants react with and reduce the reactive potential of ROS. This review will focus on exploring the efficiency of supplementing several of these antioxidant molecules on oocyte maturation, sperm viability, fertilization, and embryo culture.

Upper tract imaging surveillance is not effective in diagnosing upper tract recurrence in patients followed for nonmuscle invasive bladder cancer.

PURPOSE: We evaluated the usefulness of routine upper tract imaging in patients followed for nonmuscle invasive bladder cancer. MATERIALS AND METHODS: A retrospective review of patients treated for nonmuscle invasive bladder cancer between 2000 and 2006 was conducted. Kaplan-Meier curves were calculated to determine upper tract urothelial carcinoma-free probability for stage Ta and T1 disease. Bladder cancer stage was included as a time dependent covariate. Descriptive statistics were used to report rates of imaging studies used and the efficacy in diagnosing upper tract urothelial carcinoma. RESULTS: Of 935 patients treated and followed for nonmuscle invasive bladder cancer 51 were diagnosed with upper tract urothelial carcinoma. Median followup was 5.5 years. The 5-year upper tract urothelial carcinoma-free probability among patients with Ta and T1 disease was 98% and 93%, respectively. The 10-year upper tract urothelial carcinoma-free probability among patients with Ta and T1 disease was 94% and 88%, respectively. Only 15 (29%) patients were diagnosed on routine imaging while the others were diagnosed after symptoms developed. Overall 3,074 routine imaging scans were conducted for an overall efficacy of 0.49%. CONCLUSIONS: Upper tract recurrence is a lifelong risk in patients with bladder cancer, but most cases will be missed on routine upper tract imaging. The majority of upper tract urothelial carcinoma can be diagnosed using a combination of thorough history taking, physical examination, urine cytology and sonography, indicating that routine surveillance imaging may not be the most efficient way to detect upper tract recurrence.

Reduced fluoroscopy protocol for percutaneous nephrostolithotomy: feasibility, outcomes and effects on fluoroscopy time.

PURPOSE: Radiation exposure from fluoroscopy during percutaneous nephrostolithotomy contributes to patient overall exposure, which may be significant. We compared fluoroscopy times and treatment outcomes before and after implementing a reduced fluoroscopy protocol during percutaneous nephrostolithotomy. MATERIALS AND METHODS: We retrospectively reviewed the charts of patients treated with percutaneous nephrostolithotomy at a single academic institution by a single surgeon. We compared 40 patients treated before implementation of a reduced fluoroscopy protocol to 40 post-protocol patients. The reduced protocol included visual and tactile cues, fixed lowered mAs and kVp, a laser guided C-arm and designated fluoroscopy technician, and single pulse per second fluoroscopy. Preoperative characteristics, fluoroscopy and operative time, complications and treatment success were examined using univariate and multivariate analysis. RESULTS: There was no significant difference in body mass index, stone size, success rate, operative time or complications between the groups. After protocol implementation fluoroscopy time decreased from 175.6 to 33.7 seconds (p<0.001). A longer average hospital stay was seen in the pre-protocol group (3.9 vs 3.6 days, p=0.027). Stays greater than 2 days were associated with a body mass index of greater than 30 kg/m2 on multivariate analysis. No complication in either group was attributable to fluoroscopic technique. CONCLUSIONS: Implementing a decreased fluoroscopy protocol during percutaneous nephrostolithotomy resulted in an 80.9% reduction in fluoroscopy time while maintaining success rates, operative times and complications similar to those of the conventional technique. Adopting this reduced fluoroscopy protocol safely decreased radiation exposure to patients, surgeons and operating room staff during percutaneous nephrostolithotomy.

Radiation exposure in urology: a genitourinary catalogue for diagnostic imaging.

PURPOSE: Computerized tomography use increased exponentially in the last 3 decades, and it is commonly used to evaluate many urological conditions. Ionizing radiation exposure from medical imaging is linked to the risk of malignancy. We measured the organ and calculated effective doses of different studies to determine whether the dose-length product method is an accurate estimation of radiation exposure. MATERIALS AND METHODS: An anthropomorphic male phantom validated for human organ dosimetry measurements was used to determine radiation doses. High sensitivity metal oxide semiconductor field effect transistor dosimeters were placed at 20 organ locations to measure specific organ doses. For each study the phantom was scanned 3 times using our institutional protocols. Organ doses were measured and effective doses were calculated on dosimetry. Effective doses measured by a metal oxide semiconductor field effect transistor dosimeter were compared to calculated effective doses derived from the dose-length product. RESULTS: The mean±SD effective dose on dosimetry for stone protocol, chest and abdominopelvic computerized tomography, computerized tomography urogram and renal cell carcinoma protocol computerized tomography was 3.04±0.34, 4.34±0.27, 5.19±0.64, 9.73±0.71 and 11.42±0.24 mSv, respectively. The calculated effective dose for these studies Was 3.33, 2.92, 5.84, 9.64 and 10.06 mSv, respectively (p=0.8478). CONCLUSIONS: The effective dose varies considerable for different urological computerized tomography studies. Renal stone protocol computerized tomography shows the lowest dose, and computerized tomography urogram and the renal cell carcinoma protocol accumulate the highest effective doses. The calculated effective dose derived from the dose-length product is a reasonable estimate of patient radiation exposure.

Evaluation of the Cryotech Vitrification Kit for bovine embryos.

The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production.

Ovarian Follicular Dynamics, Ovarian Follicular Growth, Oocyte Yield, In vitro Embryo Production and Repeated Oocyte Pick Up in Thai Native Heifers Undergoing Superstimulation.

The objective of this study was to compare the effectiveness of the protocols for superstimulation of follicular growth in Thai native heifers. Heifers (n = 20) were randomly divided into four groups of five heifers/group. Heifers were given a single dose by i.m. administration of 100 mg Follicle Stimulating Hormone dissolved in polyvinylpyrrolidone (FSHp) at 24 h. Ovum pick up (OPU) occurred at 72 h (F24O72 protocol; Group 1) or 96 h (F24O96 protocol; Group 2), and at 36 h and OPU at 72 h (F36O72 protocol; Group 3) or 96 h (F36O96 protocol; Group 4) after follicular ablation. The dynamics of ovarian follicular growth were monitored by twice-daily ultrasonographic examinations. Blood sample collections were performed every 12 h after initiation of treatment for assessment of FSH, E2 and P4 profiles. All heifers were subjected to eight repeated sequential sessions of OPU. The follicular deviation commenced 24±5.32 h after follicular ablation in all groups. The circulatory FSH surged quickly from 24 to 36 h (>0.8 ng/ml) after follicular ablation and circulatory estrogen levels steadily increased from 36 h until OPU in all groups. At the end of the OPU sessions, the mean number of aspirated follicles/heifer/session in F36O72 protocol (Group 3) and F36O96 protocol (Group 4) were higher than in the two other groups (p<0.05). The number of cumulus-oocyte complexes (COCs), cleaved and day 8 blastocysts rates in the F36O72 protocol (Group 3) were higher than in the other groups (p<0.05). It can be concluded that a single dose i.m. administration of 100 mg FSHp at 36 h and OPU at 72 h after follicular ablation (F36O72 protocol; Group 3) was the most effective protocol for superstimulation of follicular growth for repeated OPU and subsequent in vitro embryo production in Thai native heifers.

Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change.

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.