Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85.

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pI of 4.8, and temperature and pH optima of 39 degrees C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the Vmax was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a Vmax of 10.4 U/mg. It was a basic protein with a pI of 9.4, a temperature optimum of 39 degrees C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.     

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.     

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60 h, 5.0 and 50 °C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1–M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.     

Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization

Two thermostable and alkali-stable β-1,3–1,4 glucanases (EC 3.2.1.73) EG1 and EG2 from a newly isolated Bacillus licheniformis UEB CF were purified. The molecular weights of EG1 and EG2 enzymes determined by SDS–PAGE were approximately 30 kDa and 55 kDa, respectively. The N-terminal amino acid sequences of EG1 and EG2 β-glucanases were determined to be GAAPIKKGTTKLN and DINGGGATLPQK, respectively. The optimum temperature, optimum pH, k_m and V_max of EG1 were 70 °C, 5.0, 2.1 mg/ml and 21.25 μmol/min/mg, respectively. These values for EG2 were 60 °C, 7.0, 1.82 mg/ml and 18.54 μmol/min/mg, respectively.Both endoglucanases were highly active against barley β-glucan and lichenan. However, they were inactive against CMC and laminarin. The purified β-glucanases were found to be relatively stable toward non-ionic surfactants and oxidizing agents. In addition, both enzymes showed excellent stability and compatibility with a wide range of commercial solid detergents suggesting that they are a potential candidate in detergent industries formulation.     

Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein⁻¹, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Development of a simple, portable carbon dioxide incubator for in vitro production of bovine embryos

The objective of this study was to develop a simple and portable CO2 incubator using effervescent granules (EG) and to examine the effect of negative and positive air pressure for in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocytes. In experiment 1, cumulus-oocyte complexes (COCs) were matured (22 h), fertilized (5 h) and cultured (7 days) using 0.25, 0.5 or 1.0 g of EG per 0.6 l added to maintain an optimum level of CO2 (approximately 3, 6 or 12%, respectively) for in vitro production of embryos. Control oocytes, zygotes and embryos were cultured in a standard CO2 incubator. The blastocyst production rates observed on Days 7 to 9 after insemination were 20.5+/-4.2%, 18.5+/-3.9% and 28.7+/-5.1% for the 0.25 g EG, 0.5 g EG treatments and control, respectively. These rates were significantly higher (P < 0.05) than that of the 1.0 g EG treatment (8.7+/-2.6%). The number of cells in the inner cell mass (ICM) and trophectoderm (TE) produced from blastocysts using the control procedure were 40.8+/-2.9 and 81.2+/-5.3, respectively, and were higher (P < 0.05) compared to the 0.50 g EG (34.6+/-2.9 and 66.8+/-5.7) and 1.0 g EG treatments (33.4+/-3.4 and 67.2+/-7.3). In experiment 2, COCs were placed in a small box with 0.25 g of EG so that the effects on IVM, IVF and IVC of positive or negative air pressure could be compared. The blastocyst production rate observed in the negative air pressure treatment (29.6+/-4.6%) was higher (P < 0.01) than that of the positive air pressure treatment (6.2+/-1.5%) or the normal treatment pressure (P < 0.05; 18.7+/-4.2%) but did not differ from that of the control (30.7+/-4.4%). These results indicate that this simple type of incubator with negative air pressure can be successfully used for in vitro production of bovine embryos and could be used at the field level.     

Expression and characterization of two secreted His6-tagged endo-beta-1,4-glucanases from the mollusc Ampullaria crossean in Pichia pastoris

Two endo-β-1,4-glucanase cDNAs, eg27I and eg27II , from the mollusc Ampullaria crossean were expressed in Pichia pastoris cells. The secreted His₆-tagged proteins were purified in a single chromatography step. The purified recombinant EG27I and EG27II showed enzymatic activity on carboxylmethyl cellulose sodium salt at 15.31 U/mg and 12.40 U/mg, respectively. The optimum pH levels of the recombinant EG27I and EG27II were 5.5 and 5.5–6.0, respectively, and the optimum temperatures were 50°C and 50°C–55°C, respectively. The pH stability study revealed that both EG27I and EG27II showed their highest stability at pH 8.0. Analysis of their thermostability indicated that both EG27I and EG27II were relatively stable up to 40°C. Site-directed mutagenesis of Asp43 and Asp153 of both EG27I and EG27II showed that the two Asp residues are critical for the enzymatic activity.     

Glycosylation is not needed for the intracellular transport of phytohemagglutinin in developing Phaseolus vulgaris cotyledons and for the maintenance of its biological activities

Approximately 10% of the total protein contained in Phaseolus vulgaris L. cv. Greensleeves seeds is composed of the glycoprotein lectin, phytohemagglutinin. We have investigated whether the presence of N-linked oligosaccharide side chains is a prerequisite for the correct intracellular transport of this protein and whether unglycosylated phytohemagglutinin maintains its biological activities. Excised developing cotyledons were incubated in the presence of tunicamycin to prevent glycosylation "in vivo", and the fate of the unglycosylated protein synthesized in such cotyledons determined. It was found that unglycosylated phytohemagglutinin reaches its normal site of accumulation, the protein bodies, and maintains erythro-agglutinating and mitogenic activities.     

Establishment of porcine transgenic embryonic germ cell lines expressing enhanced green fluorescent protein

The purpose of this study was to isolate porcine embryonic germ (EG) cells and establish transgenic EG cell lines. Plasmid DNA was the enhanced green fluorescent protein (EGFP) vector. Porcine EG cells in rapid proliferation (4th to 9th passage) were transfected with LipofecTamine 2000 and TransFast reagents. Porcine EG cells transfected with a complex of 1 microg of DNA and 2 microL of LipofecTamine 2000 reagent yielded four EG-EGFP cell lines, which emitted bright green fluorescence. EG-EGFP cells cultured for more than 2 weeks without passage gave rise to various differentiated phenotypes. In addition, to determine the degree to which EG cells become integrated into the inner cell mass of host embryos, 135 embryos were injected with porcine EG-EGFP cells; 110 embryos survived and developed into blastocysts (81.5%). Eighty-four chimeric embryos contained fluorescent cells after culture; 49 blastocysts contained EG-EGFP cells in the inner cell mass. Our results suggested that the chimeric rate would not be improved via using different stages of embryos for injection.     

中性内切型纤维素酶在毕赤酵母中高水平表达的研究

中性内切型纤维素酶在纺织及造纸工业具有重要的应用,为了进一步提高从我国草菇中克隆的一种新的中性内切型纤维素酶(EG1)在Pichiapastoris中的表达水平,我们进行了提高基因拷贝数及高密度发酵等多种手段实现其高水平表达的研究。在前期研究的基础上,利用对已整合eg1的重组子再转化的方法,从含有2000μgLZeocin的YPDSZ平板上筛选到高抗Zeocin转化子,在摇瓶培养条件下,该转化子表达量比原来提高了3.8倍,在pH4～8条件下均有稳定表达,接种量(OD600=5.0)表达水平最好,提高甲醇的诱导浓度对表达有显著的促进作用。用3.2L发酵罐进行了高密度发酵,甲醇诱导95.5h后达到最高值,比摇瓶培养再提高了6.4倍,因此利用高抗Zeocin转化子及高密度发酵的手段,使EG1的表达水平提高了34倍,蛋白表达量达8.80mgmL,EG1酶活达到543.36IUmL,实现了中性内切纤维素酶的高水平表达,本研究将大大促进建立我国纺织用纤维素酶大规模高效生产技术。     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Expression and characterization of full-length Ampullaria crossean endoglucanase EG65s and their two functional modules

Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K(r)) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.     

Transgenesis and nuclear transfer using porcine embryonic germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.     

脱墨用棘孢曲霉SM-L22纤维素酶系中内切酶的纯化及性质

通过Bio-Gel P-60分子筛和DEAE-与Q-sepharose离子交换层析等手段,分离纯化了棘孢曲霉SM-L22纤维素酶系中五种内切酶组分EGⅡ-1、EGⅡ-2、EGⅢ-1、EGⅢ-2和EGⅣ,并且对这五种内切酶组分的基本性质进行了研究.通过SDS-PAGE和IEF电泳测得其分子量分别为38.7,34.4,31.4,36.9和23.7kD,等电点分别为pH＜3.5,＜3.5,4.9,4.5和5.0.5个酶组分均属酸性纤维素酶,最适pH在3.5～4.0之间;最适温度分别为55℃、60℃、(60～70)℃、(60～70)℃和60℃.各酶组分有较宽的pH稳定性;温度稳定性表现为EGⅡ-1＞EGⅡ-2＞EGⅢ-1＞EGⅢ-2＞EGⅣ.EGⅡ-1和EGⅡ-2有较高的底物专一性,而EGⅢ-1、EGⅢ-2和EGⅣ对木聚糖有交叉活性.Fe2+对除EGⅣ以外的四种酶组分都有激活作用,尤其是对EGⅢ-2有强烈的激活作用.动力学分析表明各纤维素酶组分对底物亲和力的大小与酶的催化率之间并无相关性.     

Unglycosylated rat alpha 1-proteinase inhibitor has a six-fold shorter plasma half-life than the mature glycoprotein

The plasma half-lives of glycosylated and unglycosylated alpha 1-proteinase inhibitor-radioactively labeled with [35S]methionine in rat hepatocyte primary cultures - were determined in the rat. Unglycosylated alpha 1-proteinase inhibitor was synthesized by hepatocytes in the presence of tunicamycin. Media from hepatocytes containing 35S-labeled glycosylated or unglycosylated alpha 1-proteinase inhibitor were injected into the tail veins of rats. At different times after injection alpha 1-proteinase inhibitor was isolated from plasma by affinity chromatography with anti-alpha 1-proteinase inhibitor Sepharose. Radioactivity measurements revealed a plasma half-life of 170 min for glycosylated alpha 1-proteinase inhibitor and of 30 min for the unglycosylated form of the inhibitor.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

β-1,4-endoglucanases from Talaromyces amestolkiae: Production of glucooligosaccharides from different β-glucans

Abstract

This article presents the purification and characterization of two β-1,4-endoglucanases from Talaromyces amestolkiae. The cellulase activities secreted by this fungus were studied in the presence of different carbon sources, attaining the maximal levels in the presence of Avicel as carbon source. In these conditions, two glycosylated β-1,4-endoglucanases with molecular masses of 25,573?kDa (EG1) and 51,825?kDa (EG2), were purified. Both isoenzymes have acidic isoelectric points, 5.4 and 4.6, respectively. Their optimum pH and temperature, either in crudes or after purification, were in the range normally used for the simultaneous saccharification and fermentation in bioethanol production. In addition, the enzymatic hydrolysis of different β-glucans by both enzymes was studied. In the assayed conditions, both enzymes hydrolysed carboxymethylcellulose, a typical substrate for endoglucanases, although EG2 was much more efficient. However, EG1 was also able to hydrolyse lichenan and laminarin. These findings suggest the potential interest of EG2 for specific hydrolysis of cellulose, present in plant cell walls, to produce bioethanol, while the more promiscuous enzyme EG1 could be used for production of glucooligosaccharides.     

Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos

This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.     

Osmotic tolerance and freezability of isolated caprine early-staged follicles

Isolated caprine early-staged follicles were submitted to osmotic tolerance tests in the presence of sucrose, ethylene glycol (EG), or NaCl solutions and were exposed to and cryopreserved (by slow or rapid cooling) in MEM alone or MEM supplemented with sucrose, EG (1.0 or 4.0 M), or both. When follicles were exposed to 1.5 M NaCl, only 2% of the follicles were viable, whereas 87% of the follicles were viable after exposure to 4.0 M EG. Regarding exposure time, the highest percentage of viable follicles was obtained when follicles were exposed for 10 min to 1.0 M EG + 0.5 M sucrose; exposure for 60 s to 4.0 M EG + 0.5 M sucrose also maintained high percentage viability in follicles. Slow cooling in the presence of 1.0 M EG + 0.5 M sucrose (75%) or rapid cooling in the presence of 4.0 M EG + 0.5 M sucrose (71%) resulted in a significantly higher proportion of viable follicles than all other treatments (P < 0.05). A 24-h culture of frozen-thawed follicles was used to assess survival; only slow-frozen follicles showed viability rates similar to control follicles (64% vs. 69% respectively; P > 0.05). Interestingly, the percentage of viable rapid-cooled follicles (59%) was similar to that obtained after in vitro culture of conventional slow-cooled follicles but was significantly lower than that in controls. Thus, in addition to determining improved procedures for the exposure of follicles to EG and sucrose before and after freezing of caprine early-staged follicles, we report the development of rapid- and slow-cooling protocols.