Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter

One hundred fifty-two bitches of seven breeds were vaginally inseminated with fresh or frozen-thawed semen of 10 stud dogs of respective breeds. The semen was supplemented with prostatic fluid before insemination. In experiment 1 bitches of each breed were randomly assigned to three treatment groups, consisting of 29 females (group 1), 33 females (group 2) and 32 females (group 3). In group 1 bitches were inseminated into vagina with fresh semen using a bovine infusion pipette. In group 2 bitches were inseminated into vagina with fresh semen using the Osiris catheter. In group 3 bitches were inseminated with frozen-thawed semen with the Osiris catheter. The number of sperms in each insemination dose was adjusted to 300 x 10(6). In experiment two bitches were randomly assigned to two treatment groups, consisting of 30 females (group A) and 28 females (group B). In group A bitches were inseminated with fresh semen, whereas in group B with frozen-thawed semen. Osiris catheter was used in both groups. The total number of sperms was adjusted to provide 250 x 10(6) of progressively motile spermatozoa in each insemination dose. In experiment 1 the pregnancy rates/whelping rates were 86.2/82.8%, 81.8/81.8% and 59.4/59.4% for groups 1, 2 and 3, respectively. The differences between group 1 and 3 were statistically significant (p < 0.05). The litter sizes at birth/litter sizes at weaning were 5.8+/-2.3/5.4+/-2.0, 6.3+/-1.4/5.7+/-1.0 and 3.9+/-1.2/3.5+/-1.5 in groups 1, 2 and 3, respectively. The litter size at birth and at weaning was reduced (p < 0.05) when frozen-thawed semen was used for insemination (group 3). There were not significant (p > 0.05) differences in the litter size between groups 1 and 2. In experiment 2 pregnancy rates/whelping rates and litter sizes at birth/litter sizes at weaning were 86.7/86.7%, 60.7/57.1% (p < 0.05) and 6.1+/-1.6/5.7+/-1.7, 4.0+/-1.4/3.8+/-1.4 (p < 0.05) in groups A and B, respectively. This study shows that results of AI with a fresh semen using a bovine infusion pipette and the Osiris catheter are equivalent. The results of the use of the Osiris catheter for vaginal insemination of frozen-thawed dog semen extended with prostatic fluid after thawing are not encouraging. The pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited using the Osiris catheter.     

Autoclavable Dispenser for Disposable Pipette Tips

An autoclavable dispenser for plastic disposable pipette tips is described that allows aseptic removal of individual tips while maintaining the sterility of the remaining tips.     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

A "virtual gland" method for quantifying epithelial fluid secretion

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-microl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 microl x cm(-2) x h(-1) (n = 42), and TEP was -9.2 +/- 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6) x Jv and TEP were stimulated 64 and 59%, respectively, by 5 microM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 microM bumetanide (n = 5), 71 and 82% with 100 microM acetazolamide (n = 5), and 47 and 56% with 600 microM glibenclamide (n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3- replacement with HEPES (n = 16). The net HCO3- concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (approximately 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.     

Biochemical composition and heterogeneity of heparan sulfates isolated from AH-130 ascites hepatoma cells and fluid

The glycosaminoglycan composition of AH-130 ascites hepatoma cells and fluid were examined using enzymatic digestion, electrophoresis, and sequential partition fractionation. The cell-associated glycosaminoglycans were found to consist of 93% heparan sulfate, with the remainder consisting primarily of chondroitin sulfate. The glycosaminoglycans isolated from the ascitic fluid were found to consist of 58% heparan sulfate, 26% hyaluronic acid and 16% chondroitin sulfate. Dermatan sulfate was not detected in either cells or fluid. The heparan sulfate isolated from AH-130 cells is low-sulfate and highly heterogeneous with respect to biochemical composition. Fractions isolated by partition fractionation varied from 0.14 mol sulfate/mol uronic acid to 0.6 mol sulfate/mol uronic acid. Of the total sulfate 70–80% is N-sulfate in the former and 50% in the latter. Electrophoresis in 0.1 M HCl showed a highly heterogeneous material with mobility between that of hyaluronic acid and beef lung heparan sulfate. The heparan sulfate isolated from the fluid was similar to that isolated from the cells but was, however, somewhat more homogeneous with respect to charge.     

An inexpensive sample mold for pulsed-field electrophoresis

Specimens for pulsed-field gel electrophoresis (PFGE) are formed using a plug mold. We report a technique which uses a disposable polyethylene pipette to prepare our specimen plug. We also report a convenient technique to handle portions of the plug used for the typical PFGE manipulations.     

Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

Quantitation of surfactant protein B by HPLC in bronchoalveolar lavage fluid

A sensitive reversed phase HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of the hydrophobic surfactant protein B (SP-B) in human bronchoalveolar lavage fluid. Samples were extracted two times with CHCl(3):MeOH:HCl (2:3:0.005N) solution in a ratio of 1:2 by volume. The extract of the lower phase was separated on a C4 butyl silica gel column with an isocratic elution using a mobile phase, consisting of 97% methanol, 2.75% chloroform and 0.25% 0.1 M trifluoroacetic acid (by volume), at a flow rate of 1 ml/min. SP-B was detected by ELSD and quantified by comparison to an external standard. The duration of a run was 7 min, the quantification limit 30 ng and the limit of detection was at about 15 ng of SP-B. This method is suitable for the rapid routine quantification of SP-B in human bronchoalveolar lavage fluid samples.     

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.     

Imaging the internal and external pore structure of membranes in fluid: TappingMode scanning ion conductance microscopy.

We have constructed a combined TappingMode atomic force microscope and scanning ion conductance microscope. The design is based on a bent glass pipette that acts as both the force sensor and conductance probe. Measuring the pipette deflection allows more stable feedback than possible with previous versions of the scanning ion conductance microscope. Using this microscope, we have imaged synthetic membranes in both contact and tapping modes under fluid. Although contact mode operation is possible, we found that our microscope provided higher contrast and less apparent sample damage in the topographic and ionic conductance images in the tapping mode.     

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube, one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions. The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals in a few hours. These features enabled us to perform rapid largescale seedling genotyping required for marker-assisted selection. We have also examined the applicability of this method for other PCR-based markers: RAPDs, nuclear STS, chloroplast STS and chloroplast microsatellites.     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62·6%和86·0%。用核染料Hoechst33342对卵母细胞的去核效率进行验证,去核成功率达到73·3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62.6%和86.0%。用核染料Hoechst 33342 对卵母细胞的去核效率进行验证,去核成功率达到73.3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

Characterization of a chloride current in the larval epidermis of the beetle Tenebrio molitor

Voltage-clamp analysis of single cuticle-attached epidermal cells dissected from the newly-ecdysed mealworm revealed the presence of a large inwardly-rectifying anion (i.e. outwardly-going) current. In many cells this current formed spontaneously on breaking into the cell with the patch pipette when the bath solution was isoosmotic with the pipette solution (415 mosmol/l). The current was evoked rapidly by electrical stimulation or by bathing the cells in hyposmotic saline (335 mosmol/l). The reversal potential of the activated current shifted in agreement with the Nernst prediction for Cl(-) when the transmembrane chloride gradient was altered by partially substituting bath or patch pipette Cl(-) with gluconate(-). Substitution of Na(+) with choline(+) or K(+) with TEA and Ba(+) in the bath or pipette solutions did not alter the reversal potential. Addition of 200 &mgr;mol/l cyclic AMP or 1 mmol/l cyclic GMP to the pipette solution increased the initial current strength and reduced the time taken to reach half peak amplitude from 117 sec to 49 sec and 41 sec, respectively. Cyclic AMP also raised the threshold at which the current developed under hyperosmotic conditions by about 20 mosmol/l. Addition of the Cl(-) channel blockers diphenylamine-2-carboxylic acid (200 &mgr;mmol/l) and diisothiocyanostilbene-2,2'-disulphonic acid (250 &mgr;mol/l) to the bath solution reduced the inwardly-rectifying anion current by 50%. This current was barely detectable in cells prepared from the mid-instar integument. This non-constitutive pattern of expression suggests that cellular Cl(-) efflux (and that of other anions) may be required during moult-cycle specific processes such as moulting fluid formation and cell volume regulation. As the strength of the epidermal anion current could be raised by the exogenous application of cytosolic cyclic nucleotides, the activity of the anion channels responsible for this current may normally be regulated by yet-to-be-identified hormone(s) or neuropeptide(s) acting on this tissue.     

Determination of hydroxyurea in plasma and peritoneal fluid by high-performance liquid chromatography using electrochemical detection

A sensitive method has been developed for the determination of hydroxyurea in plasma and peritoneal fluid using reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Plasma or peritoneal fluid samples were treated with acetonitrile to precipitate proteins then injected to the HPLC. A C₁₈ analytical column was used to separate hydroxyurea from interfering substances in the biological matrix. The mobile phase, consisting of 0.2 M sodium perchlorate–methanol (95:5, v/v) adjusted to pH 5.0, was delivered isocratically at a flow-rate of 1 ml/min and hydroxyurea was detected using a glassy-carbon electrode operating at an applied potential of +800 mV. Hydroxyurea eluted with a retention time of 3 min. The cycle time for analysis is short and the assay precision is acceptable (C.V. plasma=1.4–3.9%, C.V. peritoneal fluid=2.1–9.7%). The method has been validated and is linear from 25 to 400 ng/ml in plasma and 5 to 30 ng/ml in peritoneal fluid. The method has been shown to be applicable for pharmacokinetic studies.     

Isolation of mastocytes from rat peritoneal fluid using continuous Ficoll gradients

A method for mast cells purification from rat peritoneal fluid is described. The method consists of a continuous Ficoll gradient between 20 and 22,5% (w/v) Ficoll and the cells are obtained with an 88% retrieval. Purity and viability were of 95% and 97% respectively. The cells so purified were functional against the compound 48/80 and the spontaneous secretion value was less than 8%.     

Leukocyte deformability: finite element modeling of large viscoelastic deformation.

An axisymmetric deformation of a viscoelastic sphere bounded by a prestressed elastic thin shell in response to external pressure is studied by a finite element method. The research is motivated by the need for understanding the passive behavior of human leukocytes (white blood cells) and interpreting extensive experimental data in terms of the mechanical properties. The cell at rest is modeled as a sphere consisting of a cortical prestressed shell with incompressible Maxwell fluid interior. A large-strain deformation theory is developed based on the proposed model. General non-linear, large strain constitutive relations for the cortical shell are derived by neglecting the bending stiffness. A representation of the constitutive equations in the form of an integral of strain history for the incompressible Maxwell interior is used in the formulation of numerical scheme. A finite element program is developed, in which a sliding boundary condition is imposed on all contact surfaces. The mathematical model developed is applied to evaluate experimental data of pipette tests and observations of blood flow.     

Multiple sampling for increasing the diagnostic sensitivity of nipple aspirate fluid for atypical cytology

OBJECTIVE: To determine if repeated collection of nipple aspirate fluid (NAF) can improve the diagnostic sensitivity for cytologic atypia, a marker of increased risk of breast cancer. STUDY DESIGN: Two hundred sixty-seven women without known breast disease volunteered for NAF cytology at 5 6-month intervals over 2 years. NAF samples were prepared on Millipore filters (Millipore Filter Corp., Bedford, Massachusetts, U.S.A.) and stained with a modified Papanicolaou method. Fluid availability and cellular abnormalities were evaluated for each collection attempt. Cellular findings were classified as benign, hyperplasia or atypia. RESULTS: NAF was obtained from 178 women (66.6%) at the first visit and from an additional 15, 10, 2 and 4 women at visits 2, 3, 4 and 5, respectively, for a cumulative total of 78.2% by visit 5. The number of women yielding NAF containing hyperplastic or atypical epithelial cells was determined at each visit. Hyperplastic cells were found in 34 (19.1%) at visit 1 and in an additional 20, 10, 5 and 4 women at visits 2, 3, 4 and 5, respectively. Atypical epithelial cells were present in 12 (6.7%) women at the initial visit and in an additional 11, 7, 5 and 1 women at visits 2, 3, 4 and 5, respectively, for a cumulative percent of 18.2 at visit 5. NAF could not be obtained from 58 women at any visit. CONCLUSION: These findings suggest that an optimum collection method for NAF cytology should consist of at least 3 or 4 separate fluid aspiration attempts. Reviewing repeated multiple samples instead of 1 increases the number of women who can be evaluated and the likelihood of detecting cytologic atypia.     

Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa

The effect of human follicular fluid (FF) on the incidence of spontaneous acrosome reactions (AR) in human spermatozoa was examined over a 24-25 h period using electron microscopy. Suspensions of motile spermatozoa were prepared by a swim-up method in Earle's medium, known to support in-vitro fertilization. After adjusting the concentration to 10 x 10(6) cells/ml, suspensions were diluted 1:1 with medium (control) or FF, the latter giving a final concentration of 50% FF. In addition, at 5 h and 24 h an aliquant of the control suspension was removed, diluted 1:1 with FF and incubated for 1 h; the three suspensions were examined at 6 h and 25 h. Continuous exposure to 50% FF stimulated the AR, the effect being significant (P less than 0.001) at 25 h. However, the 1-h short exposure of spermatozoa to FF did not produce an increase in AR, even after 24 h preincubation. In a separate series of experiments, the effect of continuous incubation for 24 h in increasing concentrations of FF was investigated. A significant linear dose-dependent effect on the AR was observed with all concentrations assessed (P less than 0.01 for 12.5% FF and P less than 0.001 for 25, 50, 75 and 100% FF, compared with FF-free control). Therefore, human FF can stimulate the AR, but only after a continuous exposure to FF. A short exposure to FF, even after 24 h preincubation, does not trigger an increased AR response.