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K Hinrichs

Publications and source records attributed to K Hinrichs.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of biosensors by chemically specific optical techniques. Chemiluminescence-imaging and infrared spectroscopic mapping ellipsometry.

The standard methods currently used to read out microarrays are fluorescent and chemiluminescent imaging techniques. These methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected. A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.

Equipment Design↗
PubMed2006

Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence.

The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.

Animals↗
PubMed2006

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection.

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.

Animals↗
PubMed2006

Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation.

This study was conducted to evaluate the in vitro development of equine oocytes with compact cumuli that had been subjected to a period of meiotic suppression with roscovitine before in vitro maturation. In experiment 1, oocytes were recovered from slaughterhouse-derived ovaries and held in M199 + 10% fetal bovine serum containing 66 microM roscovitine with or without an overlay of mineral oil in 5% CO2 in air at 38.2 degrees C for 16-18 or 24 h. No oocytes treated with roscovitine in the absence of an oil overlay for 16-18 h were maturing, compared with 2-4% of oocytes in other treatments. In experiment 2, oocytes were either fixed immediately after recovery, or were cultured for 18 h in the presence or absence of roscovitine. Oocytes cultured in the absence of roscovitine had a significantly higher rate of meiotic resumption (18%) than was found in the other two treatments (0). In experiment 3, oocytes were matured immediately or after 16-18 h culture with roscovitine. Maturation rates were similar between oocytes previously treated with roscovitine (22%) and control oocytes (19%). Mature oocytes were fertilized by intracytoplasmic sperm injection and then cultured, with or without oviductal epithelial cells, for 7.5 days. There was no significant effect of roscovitine treatment on blastocyst development. Development to blastocyst of roscovitine-treated oocytes in DMEM/F-12 + co-culture (37%) was significantly higher than that of control oocytes in DMEM/F-12 without co-culture (14%). These data indicate that equine oocytes with compact cumuli can be held in roscovitine before maturation without any harmful effect on blastocyst formation.

Animals↗
PubMed2006

Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract.

We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days' gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.

Adenine↗
PubMed2005

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles.

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.

Animals↗
PubMed2005

Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence.

We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage.

Animals↗
PubMed2005

Evaluation of migration models that might be used in support of regulations for food-contact plastics.

Materials and articles intended to come into contact with food must be shown to be safe because they might interact with food during processing, storage and the transportation of foodstuffs. Framework Directive 89/109/EEC and its related specific Directives provide this safety basis for the protection of the consumer against inadmissible chemical contamination from food-contact materials. Recently, the European Commission charged an international group of experts to demonstrate that migration modelling can be regarded as a valid and reliable tool to calculate 'reasonable worst-case' migration rates from the most important food-contact plastics into the European Union official food simulants. The paper summarizes the main steps followed to build up and validate a migration estimation model that can be used, for a series of plastic food-contact materials and migrants, for regulatory purposes. Analytical solutions of the diffusion equation in conjunction with an 'upper limit' equation for the migrant diffusion coefficient, D(P), and the use of 'worst case' partitioning coefficients K(P,F) were used in the migration model. The results obtained were then validated, at a confidence level of 95%, by comparison with the available experimental evidence. The successful accomplishment of the goals of this project is reflected by the fact that in Directive 2002/72/EC, the European Commission included the mathematical modelling as an alternative tool to determine migration rates for compliance purposes.

Diffusion↗
PubMed2004

Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection.

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57-59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55-61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

Animals↗
PubMed2003

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection.

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.

Animals↗
PubMed2003

Instrumentation for FT-IR reflection spectroscopy with synchrotron radiation.

A versatile experimental set-up for infrared reflectance measurements with synchrotron radiation and its adaptation to a beamline is presented. Particular consideration is given to the collimation and the polarization of incident radiation. The performance is characterized with experimental results. Due to the high brilliance of the synchrotron radiation source, the irradiation of samples smaller than 1 mm(2) was found to be improved by more than one order of magnitude when compared to a globar.

Journal Article↗
PubMed2003

Vibrational analysis of 2-[4-(N-dodecanoylamino)phenyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole.

The infrared absorption spectra of 2-[4-( N -dodecanoylamino)phenyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole (also denoted AF51) in solution and in the solid state as well as the solid-state Raman spectrum of the powdered compound are compared with the infrared linear dichroic (LD) spectra recorded at two orthogonal polarizations. The IR-LD spectra were measured in an anisotropic solvent-the nematic liquid crystal ZLI-1695 (Merck). The solvent spectrum taken under precisely the same experimental conditions and polarized radiation azimuths was subtracted from the polarized sample spectra in order to achieve pure oriented spectra of the compound studied. These spectra were further processed using a stepwise reduction procedure that allows for assignment of vibrational modes having mutually orthogonal dipole moments.

Journal Article↗
PubMed2003

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture.

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

Amino Acids↗
PubMed2003

Fourier transform infrared synchrotron ellipsometry for studying the anisotropy of small organic samples.

An experimental setup for polarization-dependent and spectroscopic ellipsometric measurements was developed that utilizes the brilliance of synchrotron infrared radiation at the electron storage ring at BESSY II for investigations of small samples and sample areas. During commissioning of the beamline and the experimental setup, a 1 mm2 piece of a well-characterized polyimide film was studied to show the benefits of Fourier transform infrared (FT-IR) synchrotron ellipsometry. The band shapes are interpreted with respect to the anisotropic distribution of transition dipole moments within the film. In comparison to a globar source, the signal intensity has been improved by more than one order of magnitude for this example.

Anisotropy↗
PubMed2003

Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa.

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

Animals↗
PubMed2003

Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro.

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P<0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovaries of bitches in oestrus and dioestrus had higher rates of maturation to metaphase II than did oocytes from bitches at pro-oestrus or anoestrus (P<0.01). In Expt 3, no differences were observed in rates of maturation among treatment groups. Rates of maturation to metaphase II of oocytes from bitches in dioestrus were significantly higher than those from bitches in pro-oestrus (P<0.01). These results indicate that supplementation of culture medium with progesterone either during maturation or during meiotic arrest before maturation does not increase the rate of IVM of canine oocytes. However, stage of oestrous cycle is a key factor in the selection criteria for meiotically competent canine oocytes for use in in vitro experiments.

Animals↗
PubMed2003

Effects of roscovitine on maintenance of the germinal vesicle in horse oocytes, subsequent nuclear maturation, and cleavage rates after intracytoplasmic sperm injection.

This study was conducted to evaluate the effects of roscovitine on suppression of meiosis, subsequent meiotic maturation, and cleavage rates after intracytoplasmic sperm injection of horse oocytes. Oocytes were classified as having compact or expanded cumuli (Com or Exp oocytes) and were divided into three culture groups: 30 h culture in maturation medium (30 h Mat); 54 h culture in maturation medium (54 h Mat), or 24 h culture in medium containing 66 micro mol roscovitine l(-1) and then 30 h culture in maturation medium (Ros+M). After maturation, oocytes were subjected to intracytoplasmic sperm injection and cultured in G1.2 medium for 96 h. Among oocytes fixed immediately after roscovitine culture, 26 of 31 (84%) Com oocytes and 16 of 28 (57%) Exp oocytes were at the germinal vesicle stage (P<0.05). After maturation culture, there were no differences in maturation rates or morphological cleavage rates among treatments. Among Com oocytes, significantly more embryos in the Ros+M treatment than in the 54 h Mat treatment had cleaved with > or = two normal nuclei (63 versus 36%; P<0.05); whereas among Exp oocytes, significantly more embryos in the 30 h Mat treatment than in the Ros+M treatment (63 versus 42%; P<0.05) had cleaved with > or = two normal nuclei. The average number of nuclei in embryos at 96 h was significantly higher (P<0.05) in Ros+M Com oocytes (13.5) than in any other Com or Exp group. These results demonstrate that roscovitine can reversibly maintain equine oocytes in the germinal vesicle stage for up to 24 h, and that such suppression may increase the developmental potential of Com, but not Exp, oocytes.

Animals↗