Polyvinyl Alcohol Medium Research Articles

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Optimization and preparation of a compound cod liver oil film former agent: an orthogonal design.

Background: Cod liver oil has anti-inflammatory properties and could help regulate recurrent aphthous stomatitis (RAS). An orthogonal experiment was used to evaluate and improve the dosage form of compound cod liver oil, which has replaced the previously used liniment preparation based on film method. Methods: An orthogonal experiment was adopted, and the appearance and film-forming time of the film coating agents were used as indicators. The optimal ratio in the preparation process for the compound cod liver oil film agent was then optimized. A method for determination of compound cod liver oil film was established using High-Performance Liquid Chromatography (HPLC). Results: The results indicate that the blank films prepared using 55mg polyvinyl alcohol (PVA) (PVA low), 45mg of PVA (PVA medium), and 10mg glycerol had the optimal performance, which was defined as PVAa. The drug-carrying film prepared from 3mL PVAa (i.e., film-forming material with the optimal proportion), 30mg dexamethasone acetate, and 30mg metronidazole had the optimal performance. The verified sample has a complete and smooth appearance, uniform thickness and color, and no evident bubbles, which meets the requirements for a film agent defined in the Chinese Pharmacopoeia, 2020 edition. HPLC was used to determine the major components: dexamethasone acetate, metronidazole, and dyclonine hydrochloride, and the optimal separation effect was obtained. The method has advantages of good specificity, good linear results, high recovery rate, and good repeatability. Conclusion: This study proposes an optimized compound cod liver oil film former agent and preparation method. The results indicate that the compound cod liver oil film former agent had good performance, reflecting the high feasibility of this research method. The detection method of compound cod liver oil film was established by HPLC. The method was feasible, and the validity and stability of the formulation and preparation technology were guaranteed. The role of the newly developed agent in patients with RAS should be investigated further.

In situ precipitation and characterization of nano zinc oxide in different polymeric media and photoluminescent properties

Zinc oxide (ZnO) nanoparticles were prepared using zinc chloride and ammonium hydroxide with proper concentration in different polymeric mediums by in situ precipitation method. The polymeric mediums used were chitosan, starch and polyvinylalcohol (PVA). The structural properties of the synthesized ZnO nanoparticles have been confirmed using the Scanning Electron Micrographs. The crystallinity and particle size of the nano particles were also determined. From the surface morphology, it is clear that the prepared ZnO is nano in nature and the shape of ZnO changes with respect to the medium of precipitation. The ZnO particles obtained from chitosan medium exhibit cuboidal shape while those obtained from starch have cylindrical shape and coral shape from PVA medium. Photoluminescence studies of the sample with three different media were also studied.

Fluorine-free superhydrophobic carbon-based coatings on the concrete

This work reports a fluorine-free superhydrophobic carbon-based emulsion coated on the concrete surface via mixing B-doped carbon particle (BCP) aggregations and natural metakaolin (MK) in the polyvinyl alcohol medium. The BCP aggregations were fabricated by two-step hydrothermal processes without using toxic chemicals such as fluorine/silicon-based materials. Structure and element analyses confirmed boron doping boosts the formation of micro-nano roughness in BCP aggregations. With MK addition, the hierarchical undulating texture was formed and endowed the carbon-based coating with excellent superhydrophobicity of WCA = 156°. When coated on the concrete surface, the resultant carbon-based materials would have a potential application in the ocean buildings to prevent from permeation and corrosion.

Effects of surface grafting of copper nanoparticles on the tensile and bonding properties of flax fibers

Abstract In the present work, functionalized copper nanoparticles (FCuNPs) were grafted onto flax fibers, and the effects on the tensile properties, bonding strength to an epoxy resin, as well as the properties of the flax fiber-epoxy model composites were investigated. The copper nanoparticles were synthesized at ambient temperature by a chemical reduction method. The reduction of solution of copper chloride salt in the polyvinylalcohol medium was done by using sodium borohydrate. Ultraviolet-visible spectroscopy, transmission electron microscopy, and X-ray diffraction studies were used to characterize the size of the synthesized particles. The synthesized copper nanoparticles were applied to saturate the unidirectional flax natural fibers, whose surfaces were previously tailored with the cationic agent triethylammonium chloride. A remarkable improvement in the tensile strength by 75% and modulus by 50% for FCuNPs grafted flax fibers was found. Thermo-mechanical properties of the flax fiber reinforced epoxy composites were studied using DMTA. Finally, the anti-microbial analysis for composites was also conducted against Aspergillus niger spores, and enhanced anti-microbial performance was observed for treated fiber-based composites.

Competent arsenicals: aqueous biphasic extractions and their application in nitrate/nitrite speciation

The present article reports on the speciation of As(V) and As(III) using aqueous biphasic extraction based on a polyethylene glycol–salt system. For the detection of these inorganic arsenicals the salts of As(V) and As(III) are converted to their respective arsenomolybdenum blues having the formulae [AsVMo4V Mo8VIO40]7− and [AsIIIMo4V Mo8VIO40]9− and measured for their absorption spectra at 820 nm. These blue species are different in their extraction behaviour with the aqueous biphasic system. Once the blue solutions are formed they could further find application in the speciation of nitrite and nitrate in aqueous solution. A high molecular weight polymer, polyvinyl alcohol (PVA) was found to have a better efficiency to differentiate the nitrite and nitrate species. Kinetic studies show a proper decay rate of the As-blues with nitrite and no sensitivity for the nitrate species in PVA medium. This has been attributed to the structural property of PVA and its changes in the presence of the heteroplyacids. The results have been explained on the basis of changes in structural topology as revealed by the scanning electron microscope images.

217 EFFECTS OF PORCINE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON PORCINE IN VITRO-FERTILIZED EMBRYOS

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in the female reproductive tract and is one of the regulatory molecules that mediate maternal effects on the growth and development of pre-implantation embryos in several species. The objective of the present study was to investigate the effects of porcine GM-CSF (pGM-CSF) on the developmental potential of porcine IVF embryos. All experiments were performed with zygotes that were produced in vitro and cultured in porcine zygote medium-3–polyvinyl alcohol-based medium. Data were analysed with PASW statistics-17 (SPSS Inc., Chicago, IL) using Duncan’s multiple range test. A total 865 zygotes in 4 replicates were used with different concentrations of pGM-CSF (0, 2, 10, 100 ng mL–1) in Experiment 1. It was demonstrated that 10 ng mL–1 of pGM-CSF could increase (15.1 ± 2.2) blastocyst development significantly (P < 0.05) compared with the control (6.1 ± 0.7). There was no effect on cleavage rate. In blastocyst formation, early and expanded blastocysts were significantly (P < 0.05) higher in the 10 ng mL–1 of pGM-CSF group compared with the control. In Experiment 2, a total 839 zygotes with at least 5 replicates in each group were used, and whether pGM-CSF would act to increase blastocyst yield before or after Day 4 development was tested. Zygotes were cultured with the following treatments: 1) zygotes cultured with fresh porcine zygote medium–polyvinyl alcohol medium from Days 0 to 7 post-insemination as a control; 2) medium supplemented with 10 ng mL–1 of pGM-CSF from Days 0 to 4 followed by no pGM-CSF from Days 4 to 7; 3) medium alone from Days 0 to 4 followed by supplementation with 10 ng mL–1 of pGM-CSF from Days 4 to 7; and 4) medium supplemented with 10 ng mL–1 of pGM-CSF from Days 0 to 7. As compared with the controls (7.8 ± 0.7), pGM-CSF influenced the percentage of blastocyst formation when pGM-CSF was added from Days 4 to 7 (14.6 ± 1.6) or Days 0 to 7 (15.2 ± 1.8), but not from Days 0 to 4 (8.7 ± 1.5). Similarly, the early blastocyst formation rates were significantly higher in the Day 4 to 7 culture period compared with the control, and expanded blastocyst formation was significantly higher in the Day 4 to 7 and Day 0 to 7 culture periods. There was no significant different in cleavage rate among these groups. In conclusion, these data suggest that supplementation of pGM-CSF in in vitro culture medium at Days 4 to 7 or Days 0 to 7 promotes the developmental potential of porcine IVF embryos. This work was supported by a grant (20070301034040) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Some studies on calcium phosphate embedded in polyvinyl alcohol matrix before and after γ-irradiation

Abstract The Ca–P ratio in polymer matrix was adjusted to 1.67 as in hydroxyapatite. The complexed polyvinyl alcohol (PVA) materials exhibit the characteristic absorption bands of phosphate at 601 and 962 cm –1 indicating the presence of (HPO 4 ) −1 anions in the structure. The X-ray diffraction (XRD) revealed that the reaction between Ca and P ions in the PVA medium has a great effect on polymeric matrix crystallization. After irradiation, XRD results for sample c showed more sharp peaks indicating that irradiation can break the principal chemical bonds and favoring additional crystallization processes and therefore the crystallinity increases. An improvement in the thermal stability of PVA doped with calcium phosphate could be seen with the increase of calcium phosphate intercalated with PVA. The surfaces of films were analyzed by scanning electron microscope (SEM) before and after γ-irradiation. The formation of hydroxyapatite is confirmed by energy dispersive X-ray spectroscopy (EDX analysis). The electric conduction data were interpreted on the basis of an intrachain one-dimensional interpolaron hopping model of Kuivalainen.

The pva operon is located on the megaplasmid of Sphingopyxis sp. strain 113P3 and is constitutively expressed, although expression is enhanced by PVA

The upstream and downstream regions of the tentative pva operon including genes encoding oxidized polyvinyl alcohol (PVA) hydrolase (oph), PVA dehydrogenase (pvaA) and cytochrome c (cytC) from Sphingopyxis sp. strain 113P3 were sequenced. The resultant 7.9 kb sequence contained orf1 in the upstream region and orf2 and orf3 in the downstream region. Reverse transcription-polymerase chain reaction (PCR) analyses revealed that the intergenic regions between orf1 and oph or between cytC and orf2 were expressed neither in PVA medium nor glucose medium, indicating that the pva operon consists of three genes. A transcription start site was determined by 5'-rapid amplification of cDNA ends to be 428 bp upstream of the start codon of the oph. The stop codon of cytC was followed by a sequence of inverted repeats that could function as a factor-independent transcription terminator. Strain 113P3 had one megaplasmid including the pva operon. Northern blot hybridization for the three genes revealed that mRNA size was approximately 3 to 4 kb and expression was elevated in PVA medium compared to glucose medium.

Cell surface structure enhancing uptake of polyvinyl alcohol (PVA) is induced by PVA in the PVA-utilizing Sphingopyxis sp. strain 113P3

Polyvinyl alcohol (PVA)-utilizing Sphingopyxis sp. 113P3 (re-identified from Sphingomonas sp. 113P3) removed almost 0.5% PVA from culture supernatants in 4 days. Faster degradation of 0.5% PVA was performed by the periplasmic fraction. The average molecular size of PVA in the culture supernatant or cell-bound PVA was gradually shifted higher, suggesting that lower molecular size molecules are degraded faster. Depolymerized products were found in neither the culture supernatant nor the cell-bound fraction; however they were recovered from the periplasmic fraction. As extracellular or cell-associated PVA oxidase activity was almost undetectable in strain 113P3, degradation of PVA must be performed by periplasmic PVA dehydrogenase after uptake into the periplasm. Following the consumption of PVA, a dent appeared on the cell surface on day 2 and increased in size and depth for 4 days and was maintained for 8 days. Ultrastructural change on the cell surface was only observed in PVA medium, but not in nutrient broth (NB), suggesting that the change is induced by PVA. Fluorescein-4-isothiocyanate-labeled PVA was bound more to cells grown in PVA than to cells grown in NB. No binding was found with PVA-grown cells treated with formaldehyde. Thus, a dent on the cell surface seems to be related to the uptake of PVA.

260 REPLACEMENT OF PVA WITH FETAL BOVINE SERUM IMPROVES FORMATION AND HATCHING OF PORCINE BLASTOCYSTS PRODUCED IN VITRO

Porcine embryos, derived from in vitro maturation and fertilization, were used to investigate the effects of timing of serum inclusion and PVA replacement in the medium for in vitro culture (IVC) on rates of blastocyst formation and hatching. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) or cleaved embryos obtained by culture in porcine zygote medium (PZM-5) containing 3 mg mL−1 polyvinyl alcohol (PVA) at 48 or 96 hpi were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 1%, 5%, or 10% fetal bovine serum (FBS) until Day 6 (Day 0 = the day of in vitro insemination). Supplementation with 1% to 10% FBS at 20 and 48 hpi reduced (P < 0.05; by ANOVA and Fisher's PLSD test) blastocyst rates on Days 5 (0% to 1%) and 6 (3% to 6%) compared with PVA supplementation (4% and 22%, respectively). However, addition of 10% FBS at 96 hpi increased (P < 0.05) blastocyst rates (30%) on Day 5 compared with PVA (11%) and 1% FBS (15%); there was no significant difference among treatments in rates of blastocyst formation on Day 6 (24% to 40%). The total number of blastomeres in Day 6 blastocysts did not differ among treatments at any timing of serum supplementation (26.5 to 48.3 cells). In Experiment 2, presumptive zygotes were cultured from 20 to 96 hpi in PVA medium, and the cleaved embryos were later transferred into PZM-5 containing PVA, or 1%, 5%, or 10% FBS for another 4 days. Hatching rates of embryos on Days 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (15% and 20%, respectively) than those in PZM-5 containing PVA (1% and 5%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (135.1 cells) than that in PVA medium (77.0 cells). In Experiment 3, at 130 hpi, blastocysts derived from IVC with PZM-5 containing PVA were transferred into PZM-5 containing PVA, 3 mg mL−1 bovine serum albumin (BSA) or 10% FBS for another 2 days. Hatching rates of blastocysts on Days 6, 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (12%, 56%, and 64%, respectively) than those in PZM-5 containing PVA (0%, 12%, and 20%, respectively) and BSA (0%, 12%, and 20%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (138.7 cells) than that in PVA (71.7 cells) and BSA medium (70.7 cells). The results indicate that the timing of serum inclusion in the culture medium markedly affects porcine embryo development in vitro and that replacement of PVA with FBS in PZM-5 at 96 hpi or later improves the subsequent development of embryos to the hatching/hatched blastocyst stage. This work was supported by MAFF, Japan, and STINT and FORMAS, Sweden.

Immobilization methods for continuous hydrogen gas production biofilm formation versus granulation

Hydrogen gas was continuously produced by treating glucose-containing synthetic wastewater with sewage digester sludge. The activity of methanogenic bacteria in the sludge was repressed by pH-control at 5.0 under anaerobic conditions. For efficient continuous hydrogen production, two immobilization methods were employed, biofilm formation on poly vinyl alcohol (PVA) and granulation of the sludge with cationic and anionic polymers. Acetic acid was the dominant fatty acid in the early operation stage in both cases. In the case of the biofilm, biogas production at 20 h hydraulic retention time was severely repressed to around 50 ml/L-reactor/h when propionic acid was sharply increased after 20 days of operation. In the case of granular sludge, propionic acid was produced only during the early operation stage and the biogas production rate was maintained at around 300 ml/L-reactor/h at 20 h of hydraulic retention time. The hydrogen gas content in the biogas was in the range of 40–60% and no methane was detected either case. Since granular sludge harboured more microorganisms and its density was higher than the biofilm on PVA medium, granulation was considered to be a better method for continuous hydrogen gas production.

Isolation and Culture Characterization of a New Polyvinyl Alcohol-Degrading Strain: Penicillium sp. WSH02-21

A fungal strain able to grow on polyvinyl alcohol (PVA) as sole carbon source was isolated from activated sludge of a textile factory. Morphological characteristics showed that this strain belonged to Penicillium sp., and, to our knowledge, this is the first report of PVA degradation by a strain of Penicillum sp. When 0.5% PVA was used as the carbon source in culture medium, it could be completely degraded after 12 days. This strain was found to produce and secrete an inducible PVA-degrading enzyme. High PVA concentration and oxygen transfer were favourable for PVA-degrading enzyme synthesis by Penicillium sp. cultured in shake-flasks. Moreover, Penicillum sp. cultured in PVA medium may spontaneously produce more catalase to decompose H2O2, a product of PVA oxidation by PVA oxidase, for protection of the cells from H2O2 damage.

Secretory Proteins from Goat Oocytes Matured in Culture

In this experiment, oocytes were collected from goat ovaries available in slaughterhouse by follicle puncture method. Morphologically culturable type of oocytes which having compact, multilayered cumulus granulosa cell complex and evenly granulated cytoplasm, was separated under a stereozoom microscope. Oocytes were washed thoroughly in maturation medium containing TCM-199, 1 µg/ml estradiol-17β, 0.5 µg/ml FSH, 100 µg/ml LH, 3 mg/ml BSA and 10% estrus goat serum. Washed oocytes were cultured into maturation medium on granulosa cell monolayer. Culture plate was then kept into CO2 incubator at 38±1°C, maximum humidity and 5% CO2 for 18 h. After maturation the oocytes were washed thoroughly with maturation medium containing polyvinyl alcohol (PVA) without serum and BSA and further cultured for 12 h for secretory proteins of oocytes. PVA medium was collected, pooled and concentrated by 5000 cut off centrisart. Secretory proteins were separated on 12.5% SDS-PAGE. A total number of 3.41 oocytes per ovary were obtained and 2.17 culturable oocytes per ovary were cultured into maturation medium. After 18 h of maturation, 4,567 oocytes (1.82 oocytes per ovary) were further cultured into serum and BSA free PVA medium for its secretory proteins. Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa were obtained on SDS-PAGE in silver staining and three proteins with approximately molecular weight of 45, 55 and 65 kDa in Coomassie brilliant blue staining. In conclusion, four secretory proteins with approximately molecular weight of 45, 55, 65 and 95 kDa was obtained from in vitro cultured oocytes of goats. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 3 : 340-345)

Effects of tissue protectants on the kinetics of lactate dehydrogenase in cells.

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. the kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (L-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation vi-v = a0A, where vi is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and 0 A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed (vi-v) against 0 A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.

Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of ≥-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of ≥-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.

Localization of high benzaldehyde dehydrogenase activity in rat upper gastrointestinal tract mucosa: a quantitative histochemical study.

An unusual aldehyde dehydrogenase (AlDH) phenotype, histochemically similar to the "tumor-associated" AlDH appearing during rat hepatocarcinogenesis, was detected in normal rat upper gastrointestinal tract tissues. This phenotype is characterized by high activities with aromatic substrates, i.e., benzaldehyde (Bz) and NADP. Frozen sections of GI tract tissue from normal rats and from liver nodules induced by a Solt-Farber protocol were evaluated for AlDH activity. A sensitive, high-resolution procedure was used in which sections are pre-incubated in nitroblue tetrazolium and then incubated at 20 degrees C in a viscous polyvinyl alcohol medium containing buffer, phenazine methosulfate, sodium azide, substrate, co-enzyme, and nitroblue tetrazolium. Incubation at a suboptimal pH of 7.0 was found to improve retention of the final reaction product and the linearity with time. Activity was quantitated by computer-assisted microscopic photometry. Intense BzDH-NADP activity was localized in the squamous epithelium of the tongue, esophagus, and fore-stomach, and in the glandular pit cells of the glandular stomach; this activity was not evident in the submucosa, muscle walls, and vessels. Little if any BzDH-NADP activity was observed in the small or large intestine, pancreas, and liver. AlDH in upper GI tissues and in liver nodules shared three characteristics: a sharp localization; a preference for Bz and NADP compared to the aliphatic substrate acetaldehyde and NAD; and a high co-enzyme-independent activity in the presence of Bz.

Isolation of Mites from House Dust1

House dust mites, Dermatophagoides farinae (Hughes) were isolated by the flotation method with the aid of an ultrasonic cleaner. Crystal violet stain facilitated the counting procedure. The mites were mounted in a polyvinyl alcohol medium. An incandescent lamp was used to separate the live mites from house dust. The mites were reared on human whiskers in a special microchamber.