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 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have hypothesized several underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (<0.3 M) than the approximately 1 M concentrations that are optimal for most mammalian cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have been able to obtain high motilities in 0.8 M glycerol provided that the exposure time is held to approximately 5 min to minimize toxicity and provided that the glycerol is added and removed stepwise to minimize osmotic shock. Since free radical damage in mouse sperm is proportional to the oxygen concentrations, we have determined the consequences of reducing the oxygen to <3% of atmospheric by maintaining the sperm in contact with an Escherichia coli membrane preparation, Oxyrase, from the moment of collection throughout the assessment of motility. Prior studies have shown that the procedure significantly reduces damage from centrifugation and osmotic shock. In the experiments reported here we obtained approximately 50% motility relative to untreated controls when suspensions containing 3.8% Oxyrase were exposed approximately 5 min to a solution of 0.8 M glycerol and 0.17 M (10%) raffinose in a supplemented PBS and then frozen at approximately 25 degrees C/min to -75 degrees C. In the absence of Oxyrase, the normalized motility dropped to 31%. The protection by Oxyrase was in part a consequence of minimizing centrifugation damage, but in part it reflected a reduction in freeze-thaw damage. Preliminary experiments indicate that the number of motile sperm after cryopreservation in Oxyrase is higher when the sperm are collected without swim-up than when they are collected by swim-up. This is in part due to the fact that more cells are collected in the absence of swim-up and in part due to a greater protective effect of Oxyrase on those cells. The minimum temperature in these initial experiments was limited to -75 degrees C to avoid the potential contribution of other injurious factors between -75 and -196 degrees C.
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PMID:The enhancement of the ability of mouse sperm to survive freezing and thawing by the use of high concentrations of glycerol and the presence of an Escherichia coli membrane preparation (Oxyrase) to lower the oxygen concentration. 1086 Jun 19

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.
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PMID:Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts. 1088 39

This study examined whether the viability, determined in vitro, of DNA-injected bovine embryos produced in vitro was affected by freezing, and if the frozen embryos developed to term following transfer to recipients. In vitro fertilized zygotes were injected with the pBL1 gene and then co-cultured with mouse embryonic fibroblasts (MEF) in CR1aa medium. Embryos were prepared for cryopreservation by exposure to a 10% (v/v) glycerol solution, loaded into 0.25 ml straws and then frozen by conventional slow freezing. Thawing was by rapid warming in water (37 degrees C) and embryos were rehydrated in PBS diluents of 6%, 3% and 0% (v/v) glycerol supplemented with 0.25 M sucrose and 0.5% (w/v) BSA. In Experiment 1, blastocysts that developed from DNA-injected embryos were individually classified into three morphological groups and three stages of development prior to freezing. DNA-injected blastocysts of excellent quality at freezing showed a higher survival rate (78.8+/-10.6%) after thawing than those of good (60. 9+/-16.4%) or fair (12.5+/-5.9%) quality (P<0.05). Post-thaw survival rate, judged in vitro, increased with more advanced stage of blastocyst development at freezing (early 48.8+/-15.9%, mid 52. 1+/-12.6% and expanded 71.2+/-1.1; P<0.05). In Experiment 2, the frozen/thawed embryos were transferred to recipients to examine in vivo viability. Following transfer of one or two embryos per recipient, pregnancy rates at 60 days of gestation were 13.6% (13/96) for frozen embryos and 26.5% (43/162) for fresh embryos (P<0. 05). Of the 12 live calves born from the frozen/thawed embryos, two males (18.3%) were transgenic. None of the live-born calves derived from fresh embryos exhibited the transgene. One of transgenic bulls did not produce transgenic sperm. Three out of 23 calves (13.0%) produced from cows inseminated with semen of the other bull were transgenic, suggesting that this animal was a germ-line mosaic. These studies indicated that the viability of in vitro produced, DNA-injected bovine blastocysts was affected by freezing and by both the quality and stage of development of the embryo prior to freezing. The generation of transgenic cattle demonstrates that it is feasible to freeze DNA-injected, in vitro produced embryos.
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PMID:Blastocyst viability and generation of transgenic cattle following freezing of in vitro produced, DNA-injected embryos. 1096 40

Transgene expression in intact animals now can be visualized by noninvasive techniques. However, the instruments and protocols developed so far have been formidable and expensive. We describe here a system for rapidly visualizing transgene expression in major organs of intact live mice that is simple, rapid, and eminently affordable. Green fluorescent protein (GFP) is expressed in the cells of brain, liver, pancreas, prostate, and bone, and its fluorescence is encoded in whole-body optical images. For low-magnification images, animals are illuminated atop a fluorescence light box and directly viewed with a thermoelectrically cooled color charge-coupled device camera. Higher-magnification images are made with the camera focused through an epi-fluorescence dissecting microscope. Both nude and normal mice were labeled by directly injecting 8 x 10(10) plaque-forming units/ml of adenoviral GFP in 20-100 microl PBS and 10% glycerol into either the brain, liver, pancreas, prostate, or bone marrow. Within 5-8 h after adenoviral GFP injection, the fluorescence of the expressed GFP in brain and liver became visible, and whole-body images were recorded at video rates. The GFP fluorescence continued to increase for at least 12 h and remained detectable in liver for up to 4 months. The system's rapidity of image acquisition makes it capable of real-time recording. It requires neither exogenous contrast agents, radioactive substrates, nor long processing times. The method requires only that the expressed gene or promoter be fused or operatively linked to GFP. A comparatively modest investment allows the study of the therapeutic and diagnostic potential of suitably tagged genes in relatively opaque organisms.
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PMID:Visualizing gene expression by whole-body fluorescence imaging. 1105 Feb 47

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.
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PMID:Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability. 1129 15

This study employed two water-soluble and nontoxic molecules, sucrose and glycerol, to enhance the permeability of PEG-PHEMA polymer gels coated onto 100 kDa molecular weight cutoff polyethersulfone (PES) microdialysis probes. Sucrose precoating of the probes prior to prepolymer coating prevented penetration of the prepolymer into the microdialysis membrane. Glycerol mixed with the prepolymer introduced porosity in the polymer coating upon curing. The sucrose and glycerol were completely removed by soaking in PBS after curing of the polymer coat on the probe tip. Polymer coated probe glucose permeability was tested by measuring glucose recovery from PBS solutions. Biocompatibility was assessed by measuring glucose recovery of polymer coated probes from heparanized whole porcine blood. Results show that the sucrose and glycerol treatments yielded polymer coated probes with glucose permeability nearly equal to bare probes when tested in PBS solution, but that this increased permeability was not observed when tested in whole blood. This suggests that the thickness of the polymer films (10-100 microm), while not a limiting factor in PBS solution, may have presented a diffusion barrier to glucose recovered from blood. Surprisingly, however, the polymer coated probes exhibited less thrombus formation that did the bare probes after blood exposure.
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PMID:Water-soluble treatments to enhance glucose permeability of protein-resistant polymer overlayers. 1146 78

The use of a simple cryopreservation method, adapted to direct transfer of thawed embryos may help to reduce the costs of embryo transfer in sheep and increase the use of this technique genetic improvement of this species. Two experiments were made to test a vitrification method that is easy to apply in field conditions. All embryos were collected at Day 7 of the estrous cycle of FSH-stimulated donor ewes and were assessed morphologically, washed in modified PBS and incubated for 5 min in 10% glycerol, for 5 min in 10% glycerol and 20% ethylene glycol and were transferred into the vitrification solution (25% glycerol and 25% ethylene glycol). All solutions were based on mPBS. Embryos were loaded in straws (1 cm central part, the remaining parts being filled with 0.8 M galactose in mPBS) and plunged into liquid N2 within 30 sec of contact with the vitrification solution. The straws were thawed (10 sec at 20 degrees C) and the embryos were either transferred directly or after 5 min of incubation in the content of the straw (followed by washing in PBS) into the uterus of a recipient ewe. In Trial 1, the pregnancy rates at term (72 vs. 72%) as well as the embryo survival rates (60 vs 50% respectively) were not different between fresh (n = 48 embryos) and vitrified (n = 50) embryos. In a second trial no difference was observed between vitrified embryos transferred after in vitro removal of the cryoprotectant (n = 86 embryos) or directly after thawing (n = 72) both in terms of lambing rate (67 vs. 75%, respectively) and embryo survival rate (lambs born/embryos transferred; 49 vs. 53%). This method of sheep embryo cryopreservation provided high pregnancy and embryo survival, even after direct transfer of the embryos.
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PMID:Successful direct transfer of vitrified sheep embryos. 1148 Jun 21

Benzo[a]pyrenebutyric acid (B[a]PBA) has been synthesized and covalently coupled to bovine serum albumin to generate monoclonal antibodies (Mab). A competitive indirect enzyme-linked immunosorbent assay (ELISA) for polycyclic aromatic hydrocarbons (PAH) has been developed with Mab B[a]P-13. It was shown by testing with 21 parent PAH and 10 compounds carrying methyl, hydroxy, or butyric acid functions that the antibody had broad specificity. Highest affinity was found for four- to six-ring PAH. Different organic co-solvents were tested. No loss in sensitivity, compared with controls in PBS, were found with methanol, dimethyl sulfoxide, and glycerol at final concentrations of 5 to 10%. Further, an observation was made that a modification (fine-tuning) of the affinity and specificity of the antibodies was possible by changing the type of the added organic co-solvent. The high susceptibility of the ELISA with regard to inorganic ions might be an indication of a more hydrophilic binding pocket e.g. involving a pi-cation interaction. Investigation of the effect of pH revealed that for pH between 6 to 9 there was no noticeable impairment. With an LOD as low as 30 pg per well for B[a]P the sensitivity of the ELISA is sufficient for analyses of solvent extracts of many environmental samples. As an example, the determination of a PAH sum parameter, given as B[a]P-equivalents, in crude aerosol extracts by both ELISA and HPLC revealed good correlation (r2=0.717) but approximately five-fold overestimation by the immunochemical method, obviously as a result of cross-reacting analytes.
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PMID:Monoclonal antibody to polycyclic aromatic hydrocarbons based on a new benzo[a]pyrene immunogen. 1176 82

Although cryopreservation of bovine embryo has made great progress in recent years, little achievement was obtained in ovine embryo freezing, especially in vitro produced embryos. However, a simple and efficient method for cryopreservation of sheep embryos will be important for application of ovine embryonic techniques such as in vitro fertilization, transgenic, cloning and etc. In this study ovine blastocysts, produced in vivo or in vitro, were cryopreserved by vitrification in EFS40 (40% ethylene glycol (EG), 18% ficoll and 0.5 M sucrose) or GFS40 (40% glycerol (GL), 18% ficoll and 0.5 Mol sucrose). In vitro produced, early blastocysts were directly plunged into liquid nitrogen (LN2) after preparation by one of the following procedures at 25 degrees C: (A) equilibration in EFS40 for 1 min; (B) equilibration in EFS40 for 2 min; (C) equilibration in EFS40 for 30 s following pretreatment in 10% EG for 5 min; (D) equilibration in EFS40 for 30 s following pretreatment in EFS20 for 2 min (E) equilibration in GFS30 for 30 s following pretreatment in 10% GL for 5 min. The survival rates observed after thawing and in vitro culture for 12 h were A 78.0% (39/50), B 50.0% (26/52), C 93.3% (70/75), D 92.0% (46/50) and E 68.0% (34/50). Survival rates were not significantly different for treatments C and D (p>0.05), but those for groups C and D were significantly higher than for A, B and E (p<0.05). After 24 h in vitro culture, hatched blastocyst rates were A 28.0% (14/50), B 21.1% (11/52), C 49.3% (37/75), D 48.0% (24/50), E 32.0% (16/50) and control 54.0% (27/50). The hatching rates for groups A, B and E were significantly lower than the control (p<0.05) in which early IVF blastocysts were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min, but for groups C and D it was similar to the control (p>0.05). The freezing procedures A, B and C were used to vitrify in vivo produced, early blastocysts recovered from superovulated ewes. The survival rates of frozen-thawed in vivo embryos were A 94.7% (72/76), B 75.0% (45/60) and C 96.4% (54/56) and for group B was significantly lower than for the other two treatment groups (p<0.05). Hatched blastocyst rates were A 46.0% (35/76), B 26.6% (16/60), C 51.8% (29/56) and the control 56.7% (34/60) in which early blastocysts from superovulation were cultured in fresh SOFaaBSA medium following treatment in PBS containing 0.3% BSA for 30 min. The hatching rate for treatment B was significantly lower than for the control (p<0.05) but did not differ between groups A, C and the control (p>0.05). Frozen-thawed embryos vitrified by procedure C were transferred into synchronous recipient ewes. Pregnancy and lambing rates were similar for embryos transferred fresh or frozen/thawed for both in vivo and in vitro produced embryos. These rates did not differ between in vivo and in vitro embryos transferred fresh (p>0.05). However, for frozen-thawed embryos, both rates were significantly lower for in vitro than for in vivo produced embryos (p<0.05).
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PMID:Vitrification of in vivo and in vitro produced ovine blastocysts. 1180 35

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.
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PMID:Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa. 1196 13


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