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PubMed2006

Long-term storage conditions for carriers with denitrifying biomass of the fluidized, methanol-fed denitrification reactor of the Montreal Biodome, and the impact on denitrifying activity and bacterial population.

The Montreal Biodome has a denitrification system to stabilize the nitrate concentration in its 3 million liter seawater aquarium. However, this microbial process has failed periodically due to various technical problems. The system can take several weeks to recover its full denitrification capacity. In order to provide the denitrification system with a backup of active biomass, different freezing conditions for the denitrifying biomass were tested. The biomass was conserved for 1 week-17 months at -20 degrees C with and without glycerol or at -80 degrees C with and without glycerol, and the denitrifying activity was tested in batch culture for 140 h periods at various intervals. Our results showed that glycerol was required for fast recovery of the microbial community's denitrifying activity. The -20 degrees C and -80 degrees C conservation temperatures with glycerol gave similar results although there was a short period of nitrite accumulation in the -20 degrees C sample. There were no substantial changes in the microbial community of any of the frozen samples after 17 months of conservation as monitored by denaturing gradient gel electrophoresis. This is the first report on the long-term conservation of a complex denitrifying population by freezing.

Biomass↗
PubMed1997

Determination of acrosin amidase activity in equine spermatozoa.

Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (microIU acrosin/10(6) sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r(2) = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 +/- 49 microU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.

Journal Article↗
PubMed2005

Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants.

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

Aldehyde Dehydrogenase↗
PubMed2001

Optimization of cytokine stability in stored amniotic fluid.

OBJECTIVE: Many studies use stored amniotic fluid samples to assay cytokines and other proteins for outcome-based research; however, there is little information on the optimal methods of storage. The objective of our study was to evaluate cytokine stability in amniotic fluid stored at different temperatures both with and without a proteolytic enzyme inhibitor. STUDY DESIGN: Patients undergoing midtrimester genetic amniocentesis for routine indications gave consent for the study. After the sample was centrifuged, the acellular portion of the sample was mixed to homogeneity and aliquoted in 0.5-mL increments and stored for 1 year at 4 degrees C, -20 degrees C, and -80 degrees C with and without the protease inhibitor aprotinin. Enzyme-linked immunoassays for angiogenin, interleukin-6, and vascular endothelial growth factor were performed simultaneously on each aliquot. RESULTS: Thirty samples were assayed for each storage condition. Results were calculated as the percentage of its own sister aliquot stored at -80 degrees C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4 degrees C and the highest at -80 degrees C (angiogenin, P =.004; interleukin-6, P <.001; vascular endothelial growth factor, P =.02). The addition of aprotinin improved stability only for angiogenin at all temperatures (all P <.05). CONCLUSIONS: Degradation of cytokines occurs when amniotic fluid samples are stored for prolonged periods at temperatures greater than -80 degrees C. The addition of a protease inhibitor helps stem the degradation of some cytokines.

Amniotic Fluid↗
PubMed2003

Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies.

BACKGROUND: Large blood-based epidemiological studies require simple, cost-effective sample collection methods. Immediate sample separation or rapid transport of chilled blood samples to a central laboratory may be impractical or prohibitively expensive. To assess the feasibility and reliability of transporting blood samples over several days at ambient temperature (e.g. by mail), we evaluated the stability of various plasma analytes in samples stored at room temperature or chilled. METHODS: Multiple vacutainers of blood, containing EDTA and aprotinin as preservative, were drawn from 12 volunteers and stored at 21 degrees C or 4 degrees C. Immediately after collection and 1, 2, 3, 4, and 7 days later, vacutainers stored at each temperature were centrifuged, and the plasma was aliquoted and stored at -80 degrees C. Subsequently, all aliquots from each individual were analysed in one analytical run for a range of chemistries. RESULTS: In whole blood stored at room temperature for up to 7 days, concentrations of albumin, apolipoproteins A1 and B (apoA1 and apoB), cholesterol, high density lipoprotein (HDL), total protein, and triglycerides changed by less than 4%, and low density lipoprotein (LDL) by less than 7%. Whilst alanine transaminase (ALT), creatine kinase (CK), creatinine, and gamma-glutamyl transferase (GGT) concentrations changed substantially at room temperature, there was less than 4% change during chilled storage up to 7 days. By contrast, aspartate transaminase (AST) concentrations increased markedly under both conditions. CONCLUSIONS: A wide range of important analytes, including lipids, change by only a few per cent in whole blood during storage at room temperature for several days. Mailed transport of whole blood samples may, therefore, be a simple and cost-effective option for large-scale epidemiological studies.

Alanine Transaminase↗
PubMed1990

Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry.

Cryo-fixation followed by freeze-substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze-substituted at -80 degrees C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well-preserved ultrastructure was observed in the outer 10-15 microns of all samples. Positive immunocytochemical localization of fixation-resistant and fixation-labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR-75-1 at both light and electron microscope levels.

Animals↗
PubMed2006

Use of reverse transcription-polymerase chain reaction to determine the stability of rabies virus genome in brains kept at room temperature.

In tropical and subtropical climates, the shipment of animal brains for rabies diagnosis may be a problem because brain specimens sometimes arrive decomposed at the diagnostic laboratory. In this situation, reverse transcription-polymerase chain reaction (RT-PCR) may serve as a potential solution because of its high sensitivity. However, little is known about the stability of rabies viral RNA in decomposed brain tissue. To determine the stability of rabies virus genomic RNA in brain samples, 72 mice were inoculated with the challenge virus strain-11 of rabies virus. After incubation period, mice were euthanized to obtain their brains. These were categorized in 2 different groups. In the first group, 36 brains were kept at room temperature (25-27 degrees C) immediately after euthanasia. In the second group, the other 36 inoculated brains were frozen at -70 degrees C and later maintained at room temperature. In both groups, RT-PCR was performed at days 0, 1, 2, 3, 4, 7, 10, 12, 16, 18, 23, and 26 by using primers previously described in the literature and a primer set specifically designed for a Mexican variant of vampire-bat rabies. Reverse-transcriptase PCR experiments were performed in 3 different inoculated brains, in which the direct fluorescent antibody (DFA) test was previously conducted to detect rabies viral antigen in the brains kept at room temperature and in the frozen brains. The DFA test resulted positive in both groups up to day 7. In brain samples stored at ambient temperature (25-27 degrees C), the intensity of the RT-PCR band started to diminish by day 12; however, rabies virus genome could be successfully amplified by RT-PCR up to 23 days. These results indicate that brain samples kept at ambient temperature (up to 27 degrees C) may reach a reference laboratory in an adequate state for rabies diagnosis by RT-PCR.

Animals↗
PubMed2001

Evaluation of membrane integrity of frozen/thawed deer spermatozoa: short communication.

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.

Acrosome↗
PubMed1998

Fertilization potential and qualitative characteristics of human spermatozoa after short-term cryostorage at 5 degrees C in two different TEST-yolk buffer preparations.

Use of the media TEST-yolk buffer (TYB) in semenology today enables the short-term incubation and cryostorage of spermatozoa and its subsequent use in the various assisted reproductive technologies (ART). Preparation of TYB media involves the addition of egg yolk (20% v/v) to a physiological solution of the zwitterion buffers TES and Tris. The TYB is usually thermoprecipitated to remove the majority of the egg yolk globules and other macromolecules from the medium. However, removal of these egg yolk constituents could possibly eliminate or reduce essential factors that could enhance the sperm viability and fertilization potential after short-term dilution and storage. Improvements in the quality of the TYB could add greater benefits to those techniques employed in the various forms of ART. The objectives of the investigation were 1) to study the sperm qualitative characteristics following short-term cryostorage at 5 degrees C in either thermoprecipitated (T-TYB) or non-thermoprecipitated (NT-TYB), and 2) to compare the fertilizing potential of spermatozoa stored for 24 hours at 5 degrees C in the two TYB preparations. In Experiment 1, semen specimens from 15 patients were collected, assessed and split into two aliquots. Sperm specimens were processed by diluting 1:1 (v/v) with the T-TYB or NT-TYB, followed by centrifugation and reconstitution of the specimen to its initial volume with the corresponding TYB medium. Sperm specimens were cryostored for 1, 2, 24, 48 and 72 hours. Samples were taken at each interval and placed in a 37 degrees C water bath and allowed to warm for 15 minutes after each cryostorage interval. Semen specimens were assessed for percentage and grade of motility. The results of this study indicated that, although the NT-TYB yielded better results than the T-TYB, overall those differences were not statistically significant. In Experiment 2, the fertilization potential of spermatozoa recovered after 24 hours of cryostorage in the two TYB preparations and further prepared via filtration, was assessed by the sperm penetration assay (SPA) using zona-free hamster oocytes. The average penetration rate (PR) and penetration index (PI) were significantly better for the NT-TYB than for the T-TYB. The PR was 54% vs. 25%, and the PI 0.78 and 0.27 for spermatozoa incubated in the NT-TYB vs. T-TYB. The range of penetration was also much lower for the T-TYB (6 to 100%) preparation when compared to the NT-TYB (22 to 100%). The highest penetrator showed 100% for both preparations. However, the lowest penetrator showed 6% for the T-TYB and 22% for the NT-TYB. The data obtained in this study suggest that both TYB preparations can be employed in short-term cryostorage (5 degrees C) of human spermatozoa and can adequately maintain the qualitative characteristics of those spermatozoa. The data also showed that the NT-TYB preparation yielded sperm samples of higher fertilization potential, thus possibly establishing the superior usefulness of the NT-TYB in an ART program.

Animals↗
PubMedDate not supplied

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.).

Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.

Journal Article↗
PubMed1993

Use of colostrum to detect antibodies against glycoprotein I of Aujeszky's disease virus.

Serum and colostrum samples taken from 499 sows from five herds of pigs endemically infected and vaccinated against Aujeszky's disease virus, were used to investigate whether colostrum could be used to detect antibodies against glycoprotein I (gI) of the virus. Using serum as the reference, the test applied to colostrum had a sensitivity of 97 per cent and a specificity of 88 per cent. When samples were taken from 50 sows from a gI seronegative vaccinated herd, one colostrum sample was gI-positive, giving a specificity for the test of 98 per cent. The mean gI antibody titres in colostrum were about six times higher than in serum. Samples of colostrum were also taken from 132 sows from eight unvaccinated herds free of Aujeszky's disease virus. All these samples were gI-negative, giving a specificity of 100 per cent. Colostrum samples can be stored for at least six weeks at -20 degrees C without compromising the test results, and the repeatability and reproducibility of the test applied to colostrum were good.

Animals↗
PubMed1993

Pulsed-field gel electrophoresis analysis of retinoic acid receptor-alpha and promyelocytic leukemia rearrangements. Detection of the t(15;17) translocation in the diagnosis of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized cytogenetically by a balanced reciprocal chromosomal translocation t(15;17) (q22;q21). This translocation involves the retinoic acid receptor-alpha (RAR-alpha) on chromosome 17 and the promyelocytic leukemia locus (PML) on chromosome 15 and results in the transcription of novel fusion messenger RNAs. In this study, pulsed-field gel electrophoresis (PFGE) was applied to the detection of the t(15;17) translocation in twenty-six clinical specimens cytologically diagnosed by French-American-British criteria as APL. This technique could readily be applied to both fresh and nonviably frozen tumor samples. In 24 of 26 samples, rearrangements of the PML and RAR-alpha, loci could be detected by Southern blotting after digestion with MluI and BssHII. Furthermore, co-migration of the rearranged fragments, detected by hybridization to probes for the PML and RAR-alpha genes, demonstrated that these loci were juxtaposed. The translocation was detected in specimens at the time of initial diagnosis, on differentiation therapy with retinoic acid and at the time of relapse. The diagnostic accuracy was compared to cytogenetics and the reverse transcriptase-polymerase chain reaction for the novel PML-RAR-alpha fusion transcript. The samples from two patients were negative by all three diagnostic methods, and both of these patients failed to respond to all-trans retinoic acid. In the other 24 APL samples, cytogenetics was positive in only 76.9% of the cases, whereas both reverse transcriptase-polymerase chain reaction and PFGE methods detected the translocation in 100% of the cases. Thus, PFGE can readily detect the t(15;17) translocation in both viable and nonviable clinical specimens and can improve the diagnostic accuracy of morphology and cytogenetics in APL. In contrast to conventional electrophoresis based on rearrangement of RAR-alpha, the ability to demonstrate directly co-migration of the PML and RAR-alpha loci enables this method to distinguish the t(15;17) translocation from variant translocations such as the t(11;15). Because PFGE can be performed on nonviable, frozen tumor samples, it could be diagnostically useful in APL when the RNA-based reverse transcriptase-polymerase chain reaction cannot be performed.

Chromosomes, Human, Pair 15↗
PubMed2003

High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years.

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSCHPC from individual CBs stored frozen for 15 yr. Average recoveries (+/- 1 SD) of defrosted nucleated cells, colony-forming unit-granulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 +/- 12, 95 +/- 16, 84 +/- 25, and 85 +/- 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2 degrees dishes was >96% and yielded 2 degrees colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34(+)CD38(-) cells isolated by FACS after thawing yielded >250-fold ex vivo expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34(+) cells and infused into sublethally irradiated nonobese diabetic (NOD)severe combined immunodeficient (SCID) mice. CD45(+) human cell engraftment with multilineage phenotypes was detected in mice after 11-13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating, ex vivo expansion and mouse NODSCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.

Animals↗
PubMed2004

Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization.

Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.

Animals↗
PubMed2004

Persistent and recurrent hyperparathyroidism.

More than 95% of patients with primary hyperparathyroidism (HPT) will be cured at initial operation by an experienced surgeon. Despite this success rate, persistent and recurrent HPT remain challenging clinical entities. The most cost effective and safest treatment for persistent and recurrent HPT is avoidance by successful first operation. The contributors to treatment failure can be categorized into factors related to the initial surgical procedure, anatomic variability, and the biology of disease. An understanding of the factors that commonly contribute to treatment failure can help prevent persistent and recurrent disease and plays an integral role in planning subsequent surgical approaches. Once a biochemical diagnosis of persistent or recurrent HPT is confirmed, a thorough evaluation of previous operative, pathology, and radiology reports is essential. Localization procedures supplement this information and help direct the reoperative approach. When complementary noninvasive studies, such as ultrasound, sestamibi, and magnetic resonance imaging are negative, equivocal, or discordant, invasive tests (eg, selective venous sampling for parathyroid hormone levels) are warranted. Intraoperative ultrasound and gamma-probe localization are of questionable value, but intraoperative parathyroid hormone assays help facilitate these challenging repeat dissections. Repeat parathyroid exploration is associated with more complications and fewer cures compared to the initial explorations and should only be undertaken by an experienced surgeon in a center that can provide expert preoperative localization, adjunctive intraoperative tools, and cryopreservation of parathyroid tissue when necessary. Although controversy exists regarding indications for reoperative treatment for persistent or recurrent HPT, parathyroidectomy remains the only curative treatment option. Surgery should be considered first-line treatment in most circumstances.

Diet↗
PubMed1998

Quantitative RNA slot-blot analysis of CCND1/cyclin D1 expression in suspected mantle cell lymphoma.

Abnormal CCND1 expression is found in the majority of mantle cell lymphomas (MCL) and in a minority of other mature B cell malignancies. Its evaluation can therefore aid diagnostic classification, in conjunction with clinical, morphological, immunophenotypic and cytogenetic analysis. We describe a rapid slot-blot hybridization technique allowing quantitative assessment of CCND1 expression relative to beta-actin, with a sensitivity cut-off of approximately 10%. This allowed clear separation (P < 0.01) of CCND1 MCL (0.89 +/- 0.4; range 0.23-1.81; n = 25) from control samples (0.02 +/- 0.04; range 0-0.09; n = 22) on limited quantities of RNA (1-3.5 microg). Of nine samples in which a potential diagnosis of MCL lymphoma was based on morphological analysis of paraffin-embedded material, without adequate immunophenotype analysis, all were CCND1 negative and subsequent immunophenotype demonstrated features compatible with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) (CD5+, CD23+, FMC7-) in all cases tested. This study demonstrates the feasibility of slot-blot CCND1 quantification and the importance of the availability of cryopreserved material.

Adult↗
PubMedDate not supplied

Investigations on the influence of bradykinin upon the motility of ram spermatozoa.

The influence of bradykinin, a component of the kallikrein-kinin system, on the motility of ram spermatozoa was examined in vitro (photometric motility evaluation, penetration test in cervical mucus of sheep, estimation of the percentage of forward-moving spermatozoa in the thermal resistance test). Whereas the motility was found to be influenced by bradykinin the acrosomal status remained undisturbed by it. With fresh as well as with frozen semen, the drug mainly increased the motility of samples with a low initial motility. With frozen semen, the penetration test revealed greater motility rises at 22 degrees C than at 38 degrees C. The importance of the results in relation to the angiotensin-converting enzyme (kininase) as well as the motility evaluation capabilities of the methods used are discussed. There was, however, no positive drug effect after adding the drug in the course of cryopreservation prior to freezing semen in straws although bradykinin solutions frozen in liquid nitrogen maintained their effectiveness.

Animals↗
PubMed1999

Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism.

Animals↗