scholarly journals 84 SUCCESSFUL CRYOPRESERVATION OF PORCINE EMBRYOS BY THE METAL MESH VITRIFICATION (MMV) METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 192 ◽  
Author(s):  
Y. Fujino ◽  
Y. Nakamura
Keyword(s):  
Developmental Stage ◽  
Liquid Nitrogen ◽  
Control Method ◽  
Fetal Bovine Serum ◽  
Survival Rates ◽  
Metal Mesh ◽  
Sample Container ◽  
Preservation Method ◽  
Fetal Bovine ◽  
Nitrogen Sample

The present study was designed to generate piglets from porcine embryos by the metal mesh vitrification (MMV) method. Prepuberal gilts were administered eCG and hCG and were artificially inseminated. Morulae and expanding blastocysts (diameter = approximately 200 μm) were collected at 144 h and 168 h after hCG injection, respectively. The metal mesh and the plastic plate (control) were used as sample containers for ultrarapid vitrification. The metal mesh (75 μm stainless steel mesh) was 1.5 mm wide and 10 mm long, and the 3 mm at the end of the mesh was bent at a right angle. Plastic plates, made from 0.25 mL plastic straws, were the same size and form as the metal mesh. Embryos were equilibrated with 7.5% ethylene glycol (EG) + 7.5% DMSO + 10% fetal bovine serum (FBS) in PBS for 5 min, followed by exposure to 15% EG + 15% DMSO + 0.6 M trehalose + 10% FBS in PBS for 1 min. Embryos were picked up on the metal mesh or loaded onto plastic plates with minimum volume of the solution, and then plunged into liquid nitrogen. Sample containers were placed in 1.8-mL cryotubes and stored in liquid nitrogen. Warming and dilution were performed by moving the container from liquid nitrogen into 0.5 M trehalose + 10% FBS in PBS at 37°C for 5 min. Embryos were rinsed twice in 4 mg/mL BSA + 10% FBS in NCSU37 (mNCSU37) for 5 min. Survival of the vitrified embryos was assessed after culture in mNCSU37 for 24 h (expanding blastocysts) or 48 h (morulae), and those that survived to fully expanded, hatching or hatched blastocysts were scored as viable. The vitrified warmed embryos were transferred surgically to recipient gilts. Experiment 1: The survival rates of expanding blastocysts vitrified by MMV or the control method were compared. The rate by MMV was significantly higher (84%; P < 0.01 by χ2 test) than that of the control (53%). Experiment 2: The developmental stage (morula or expanding blastocyst) suitable for vitrification was examined. Survival of expanding blastocysts was significantly higher (84%; P < 0.05) than that of morulae (55%). Experiment 3: Expanding blastocysts were vitrified by MMV, warmed, and transferred into recipients (20 embryos per recipient). Eight of 10 recipients were found to be pregnant. Seven recipients farrowed a total of 37 live and 1 stillborn piglets. The other recipient miscarried and farrowed 4 stillborn piglets. These results showed that the viability of vitrified warmed porcine embryos was affected by the material of the sample container (Experiment 1) and also by the developmental stage (Experiment 2). Since the transferred embryos showed an excellent ability to develope into piglets (Experiment 3), the MMV method developed in the present study seems to be an effective preservation method for porcine embryos.

209 VIABILITY OF PORCINE EMBRYOS VITRIFIED BY THE METAL MESH VITRIFICATION METHOD AFTER SURGICAL OR NONSURGICAL TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 221 ◽  
Author(s):  
Y. Fujino ◽  
Y. Nakamura ◽  
H. Kobayashi ◽  
S. Nakano ◽  
C. Suzuki ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Uterine Horn ◽  
Outer Diameter ◽  
Metal Mesh ◽  
Angle Bend ◽  
Fetal Bovine ◽  
Inner Diameter ◽  
Polyethylene Tubing ◽  
Practical Measure ◽  
Uterine Body

The aim of the present study was to evaluate viability of porcine embryos vitrified by the metal mesh vitrification (MMV) method after surgical or nonsurgical transfer. Prepubertal gilts were treated with eCG and hCG (= Day 0), and then inseminated artificially. Expanding blastocysts that were about 200 �m in diameter were collected on Day 7. The embryos were equilibrated in 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal bovine serum (FBS) in PBS at 37�C for 5 min, and then transferred into 15% EG + 15% DMSO + 0.6 M trehalose + 20% FBS in PBS for 1 min. Embryos in groups of 5 were transferred in a minimum volume of the vitrification solution (less than 1 �L) onto stainless steel mesh (75 �m screen size, 1.5 mm in width by 10 mm in length, with a 3-mm right-angle bend), and then plunged into liquid nitrogen. The mesh was stored in a 1.8-mL cryotube submerged in liquid nitrogen. Warming and dilution were performed by moving the mesh from liquid nitrogen into 0.5 M trehalose + 20% FBS in PBS at 37�C for 5 min. Embryos were rinsed twice in NCSU37 + 10% FBS (mNCSU37) for 5 min. After being vitrified, embryos in groups of 20 per recipient were suspended in modified NCSU37 medium and then transferred into gilts either by surgical transfer (5 gilts) or by nonsurgical transfer (6 sows). For surgical transfer, embryos suspended in 0.1 mL of medium were transferred into the uterine horn at 15 cm above the uterine body, which was about 35 cm from the external uterine orifice. For nonsurgical transfer, an intrauterine catheter made from polyethylene tubing (1.2 m long, 3.0 mm outer diameter, 0.5 mm inner diameter) was used. A spiral guide inserted through the vagina into the cervix was used to guide the catheter into one uterine horn. The catheter was moved through the cervix and along the uterine horn. Then, embryos suspended in 1 mL of medium were transferred. Pregnancy was assessed by ultrasonography at 30 days post-estrus. With surgical transfer, 4 of 5 recipients became pregnant, and 3 gilts farrowed a total of 21 (10, 8, 3) live piglets; the fourth gilt aborted one fetus on Day 34. With nonsurgical transfer, 3 of 6 sows became pregnant. The present study demonstrates that vitrified porcine embryos can develop after both surgical and nonsurgical transfer to recipients. As a practical measure, nonsurgical transfer of vitrified porcine blastocysts may be used instead of surgical transfer.

82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Beebe ◽  
S. McIlfatrick ◽  
R. Ashman ◽  
M. Nottle
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Direct Contact ◽  
Survival Rates ◽  
Genetic Material ◽  
Basic Medium ◽  
Embryo Cryopreservation ◽  
Fetal Bovine ◽  
And Storage

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5�C in an humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean � SEM; 109 � 6 and 112 � 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving embryos.

117 Kit ligand enhances growth and nuclear maturation of buffalo oocytes in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 184
Author(s):  
M. N. Islam ◽  
M. H. Alam ◽  
A. Khatun ◽  
M. A. Hashem ◽  
M. Moniruzzaman
Keyword(s):  
Serum Sodium ◽  
Bovine Serum ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Survival Rates ◽  
Sodium Pyruvate ◽  
Antral Follicles ◽  
Kit Ligand ◽  
Fetal Bovine

This study aimed to investigate the effect of Kit ligand (KL), a growth factor that regulates folliculogenesis in mammalian ovaries, on growth of buffalo oocytes in early antral follicles in vitro. Cumulus-oocyte complexes were dissected from early antral follicles (1mm) of slaughtered buffaloes and cultured in Dulbecco’s minimum essential medium supplemented with fetal bovine serum, sodium pyruvate, gentamycin, hypoxanthine, dexamethasone, cysteine, polyvinylpyrolidione, l-ascorbic acid, oestradiol-17β, and androstenedione in a 96-well culture plate at 38.5°C under an atmosphere of 5% CO2 in air for 6 days. The culture medium was supplemented with 0, 50, and 100 ng/mL KL (recombinant human SCF, Cat. No. H8416, R&amp;D Systems, Minneapolis, MN, USA). Sixty oocytes were cultured in each group with 6 replications. In vitro-grown oocytes were cultured for maturation in tissue culture medium-199 supplemented with 5% fetal bovine serum, sodium pyruvate, gentamycin, and 100 ng/mL FSH at 38.5°C for 24h under an atmosphere of 5% CO2 in air. The oocytes were then stained with aceto-orcein and examined under a differential interference contrast microscope. Data were analysed using SAS/STAT version 9.1.3 for Windows (SAS Institute Inc., Cary, NC, USA) by one-way ANOVA and means compared with Tukey’s HSD test. The mean diameter of oocytes measured at the time of seeding on the culture substrate was 100.6±0.4μm (n=180). After 6 days of culture, the diameters of oocytes increased to 110.8±0.5, 114.0±0.5, and 115.0±0.6µm in 0, 50, and 100 ng/mL KL-treated groups, respectively. The survival rates were 60.0±6, 81.2±1.2, and 92.0±4.9% in 0, 50, and 100 ng/mL KL-supplemented oocytes at Day 6. Moreover, KL pretreatment enhanced maturation of buffalo oocytes dose dependently. A small proportion of oocytes (8.4%) treated with 50 ng/mL KL reached the MII stage. This number increased to 25% when oocytes were treated with 100 ng/mL KL. These results show that KL enhances growth, viability, and meiotic progression of buffalo oocytes in vitro.

Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells

1990 ◽  
Vol 48 (3) ◽  
pp. 912-913
Author(s):  
Jaang J. Wang ◽  
Cheng C. Chen ◽  
Men F. Shaio ◽  
Chia T. Liu ◽  
Chung S. Lee ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cytopathic Effect ◽  
Fetal Bovine Serum ◽  
Virus Particles ◽  
Electron Microscopies ◽  
Embedding Technique ◽  
Fetal Bovine ◽  
Flat Embedding ◽  
20 Nm ◽  
Labeling Method

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.

Treatment of fetal bovine serum improves early development of porcine embryos by alleviating oxidative stress

2014 ◽  
Author(s):  
Seon-A Choi ◽  
Seong-Eun Mun ◽  
Pil-Soo Jeong ◽  
Hae-Jun Yang ◽  
Seung-Bin Yoon ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Early Development ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Fetal Bovine

МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

2019 ◽  
pp. 20-22
Author(s):  
T.I. KUZMINA ◽  
I.V. CHISTYAKOVA
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Egg Quality ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cultural Systems ◽  
Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

scholarly journals Effect of fetal bovine serum on erythropoietin receptor expression and viability of breast cancer cells

2020 ◽  
Vol 27 (2) ◽  
pp. 653-658
Author(s):  
Guan-Young Teo ◽  
Abdullah Rasedee ◽  
Nagi. A. AL-Haj ◽  
Chaw Yee Beh ◽  
Chee Wun How ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Bovine Serum ◽  
Breast Cancer Cells ◽  
Fetal Bovine Serum ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Fetal Bovine

scholarly journals Prevalence of Bovine Viral Diarrhea Virus Genotypes and Antibody against those Viral Genotypes in Fetal Bovine Serum

1998 ◽  
Vol 10 (2) ◽  
pp. 135-139 ◽  
Author(s):  
Steven R. Bolin ◽  
Julia F. Ridpath
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Neutralizing Antibody ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Fetal Bovine ◽  
Viral Diarrhea Virus

One thousand lots of pooled fetal bovine serum (FBS) were tested for contamination with bovine viral diarrhea virus (BVDV) and/or for contamination with neutralizing antibody against BVDV. Noncytopathic or cytopathic BVDV was isolated from 203 lots of FBS. Analysis of the viral isolates identified 115 type 1 and 65 type 2 BVDV isolates. An additional 23 virus isolates were mixtures of >2 BVDV isolates and were not classified to viral genotype. Further characterization of the type 1 viruses identified 51 subgenotype 1a and 64 subgenotype 1b BVDV isolates. Viral neutralizing antibody was detected in 113 lots of FBS. Differential viral neutralization indicated that type 1 BVDV induced the antibody detected in 48 lots of FBS and type 2 BVDV induced the antibody detected in 16 lots of FBS.

Sign in / Sign up

Export Citation Format

Share Document