Gene/Protein Disease Symptom Drug Enzyme Compound
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Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).
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PMID:A new procedure for the cryopreservation of equine embryos. 1672 58

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).
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PMID:Quick freezing of rat embryos. 1672 80

The aim of the present study was to compare the survival rates of goat morulae and blastocysts after different freezing procedures. The viability of frozen-thawed embryos was assessed both in vivo and in vitro. Two cryoprotectants, ethylene glycol and glycerol, were used and three cryoprotectant removal procedures were compared: progressive dilution in 1.0, 0.5, 0.3 and 0 M of cryoprotectant in PBS; a similar progressive dilution with cryoprotectant in PBS plus 0.25 M of sucrose; or one-step transfer in PBS containing 0.25 M of sucrose. In vitro development of frozen-thawed blastocysts was always higher than that of frozen morulae irrespective of the cryoprotectant (52 129 = 40.3% vs 23 161 = 14.3% ; P< 0.001). In vivo, however, frozen-thawed morulae developed equally as well as blastocysts after an identical freezing-thawing protocol. Development both in vivo and in vitro showed ethylene glycol to be a better cryoprotectant than glycerol for goat embryos at both developmental stages (23 vs 0%, 45 vs 35% in vitro; 34.5 vs 21%, 35 vs 23% in vivo for morulae and blastocysts, respectively).
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PMID:In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol. 1672 58

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.
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PMID:A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability. 1672 56

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.
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PMID:Sheep embryo cryopreservation by vitrification and conventional freezing. 1672 40

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.
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PMID:Cryopreservation of equine oocytes by 2-step freezing. 1672 12

The purpose of this study was to investigate the in vitro-in vivo degradation and tissue compatibility of three novel biopolymers viz. polymerized rosin (PR), glycerol ester of polymerized rosin (GPR) and pentaerythritol ester of polymerized rosin (PPR) and study their potential as implant matrix for the delivery of ciprofloxacin hydrochloride. Free films of polymers were used for in vitro degradation in PBS (pH 7.4) and in vivo in rat subcutaneous model. Sample weight loss, molecular weight decline, and morphological changes were analyzed after periodic intervals (30, 60, and 90 days) to monitor the degradation profile. Biocompatibility was evaluated by examination of the inflammatory tissue response to the implanted films on postoperative days 7, 14, 21, and 28. Furthermore, direct compression of dry blends of various polymer matrices with 20%, 30%, and 40% w/w drug loading was performed to investigate their potential for implant systems. The implants were characterized in terms of porosity and ciprofloxacin release. Biopolymer films showed slow rate of degradation, in vivo rate being faster on comparative basis. Heterogeneous bulk degradation was evident with the esterified products showing faster rates than PR. Morphologically all the films were stiff and intact with no significant difference in their appearance. The percent weight remaining in vivo was 90.70 +/- 6.2, 85.59 +/- 5.8, and 75.56 +/- 4.8 for PR, GPR, and PPR films respectively. Initial rapid drop in Mw was demonstrated with nearly 20.0% and 30.0% decline within 30 days followed by a steady decline to nearly 40.0% and 50.0% within 90 days following in vitro and in vivo degradation respectively. Biocompatibility demonstrated by acute and subacute tissue reactions showed minimal inflammatory reactions with prominent fibrous encapsulation and absence of necrosis demonstrating good tissue compatibility to the extent evaluated. All implants showed erosion and increase in porosity that affected the drug release. Increase in drug loading significantly altered the ciprofloxacin release in extended dissolution studies. PPR produced drug release >90% over a period of 90 days promising its utility in implant systems. The results demonstrated the utility of novel film forming biopolymers as implant matrix for controlled/sustained drug delivery with excellent biocompatibility characteristics.
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PMID:Novel biopolymers as implant matrix for the delivery of ciprofloxacin: biocompatibility, degradation, and in vitro antibiotic release. 1696 Aug 24

The objective of this study was to evaluate glycerol (GLY) and GLY + dimethyl sulfoxide (DMSO) to increase photonic detection of transformed Salmonella typhimurium (S. typh-lux) through porcine skin. Skin was placed on 96-well plates containing S. typh-lux, imaged (5 min) using a CCD camera, and then completely immersed in PBS, GLY, DMSO, GLY+DMSO in a dose- and time-dependent manner and re-imaged (5 min). The percent of photonic emissions detected (treated or untreated skin relative to no skin controls) was used for analysis. Treatment for 4 h with 50% GLY-PBS and 50:30:20% GLY:DMSO:PBS increased photonic detection compared to untreated skin, 100% PBS, or 30:70% DMSO:PBS. Treatment with 50% GLY in the presence of 20 and 40% DMSO (v/v with PBS) increased photonic detection compared to 50% GLY alone and in the presence of 10% DMSO: 50% GLY (v/v with PBS). Data indicate that GLY and GLY+DMSO are effective optical clearing agents on porcine skin (2-3 mm thick) when treated for 4 h to increase detection of emitted photons. Clearing agents such as GLY have the potential to minimize effects of porcine skin tissue as one of the photon transmittance barriers (i.e., skin, fat, muscle, and visceral tissues) in vivo.
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PMID:Use of glycerol as an optical clearing agent for enhancing photonic transference and detection of Salmonella typhimurium through porcine skin. 1709 76

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.
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PMID:Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract. 1741

It is of great importance to efficiently immobilize probes onto a substrate with good spot quality for fabrication of protein microarrays. Printing buffers play an essential role in the fabrication process for the microarrays. In this work, antigen (Ag)/antibody (Ab) microarrays were fabricated on 3-aminopropyltriethoxysilane (APTES) modified glass slides through glutaraldehyde (GA), a bis-aldehyde homobifunctional cross-linker. Different types of buffers such as triton X-100 and glycerol and their effects on the protein immobilization were investigated for improving the quality of microspots and the immobilization efficiency on the aldehyde-activated APTES silanized slides. In addition, the performance of the optimized printing buffer was characterized with fabricated Ag/Ab microarrays. The results indicated that the optimized printing buffer, 0.01 M PBS with additional 0.003% triton X-100 and 10% glycerol could effectively eliminate non-homogeneous morphology of the microspots and significantly improve the signal intensities. The results provide an improved approach to construct high performance Ag/Ab microarrays.
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PMID:Optimization of printing buffer for protein microarrays based on aldehyde-modified glass slides. 1748 37


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