Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (−20 °C)

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 341-351 ◽  
Author(s):  
Liliana Chacón ◽  
Martha C. Gómez ◽  
Jill A. Jenkins ◽  
Stanley P. Leibo ◽  
Gemechu Wirtu ◽  
...  
Keyword(s):  
Low Temperature ◽  
Cooling Rate ◽  
Liquid Nitrogen ◽  
First Generation ◽  
Blastocyst Stage ◽  
Live Cells ◽  
First Passage ◽  
Slow Cooling ◽  
Donor Cells ◽  
Bovine Oocytes

SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

scholarly journals Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate

2018 ◽  
Vol 70 (5) ◽  
pp. 1489-1496 ◽  
Author(s):  
J.F. Fonseca ◽  
R.I.T.P. Batista ◽  
J.M.G. Souza-Fabjan ◽  
M.E.F. Oliveira ◽  
F.Z. Brandão ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cooling Rate ◽  
Liquid Nitrogen ◽  
Developmental Stages ◽  
Slow Cooling ◽  
Air Bubble ◽  
Frozen Embryos ◽  
Glycol Solution ◽  
Fresh Embryos ◽  
Fresh Transfer

ABSTRACT The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.

39 IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 126
Author(s):  
Y. Kaedei ◽  
A. Fujiwara ◽  
F. Tanihara ◽  
Z. Namula ◽  
V. L. Vien ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Donor Cells ◽  
Chi Square Analysis ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.

Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 736-737
Author(s):  
T. Inoué ◽  
H. Koike
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Temperature ◽  
Liquid Nitrogen ◽  
Specimen Preparation ◽  
Chemical Fixation ◽  
Brown Adipose ◽  
Critical Point Drying ◽  
Temperature Scanning ◽  
Scanning Electron

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.

Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes

1989 ◽  
Vol 18 (1-3) ◽  
pp. 139-148 ◽  
Author(s):  
Y. Fukui ◽  
M. Urakawa ◽  
C. Sasaki ◽  
N. Chikamatsu ◽  
H. Ono
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Bovine Oocytes

Analysis of Micro Structure and the Resistivity of Cu / Ni Thin Coat as a Low Temperature Sensor Using Electroplating Method Assisted with Magnetic Field outside of the Ion Flow

2021 ◽  
Vol 885 ◽  
pp. 141-147
Author(s):  
Winda Noor Santi ◽  
Moh. Toifur
Keyword(s):  
Magnetic Field ◽  
Low Temperature ◽  
Liquid Nitrogen ◽  
Micro Structure ◽  
Medical Field ◽  
Cold Temperatures ◽  
Ion Flow ◽  
Resistance Temperature Detector ◽  
Temperature Detector

Preservation of materials using liquid nitrogen media has been widely used. One of them is used in the medical field, namely cryonic technology. Cryonics is a method of preservation at cold temperatures using a cryoprotectant in liquid nitrogen. To maintain the quality of the material, a sensor that can detect the temperature of liquid nitrogen is needed. Low temperature sensors with Cu and Ni based Resistance Temperature Detector with layers (RTD) have been made, but these sensors have a layer of Ni deposits that are not yet homogeneous. So quality improvement is needed by adding an external magnetic field. Based on this, the aim of this research is to synthesize a thin layer of Cu / Ni using electroplating method assisted by external magnets parallel to the ion currents

scholarly journals Influence of Bitumen Type and Asphalt Mixture Composition on Low-Temperature Strength Properties According to Various Test Methods

Materials ◽  
2018 ◽  
Vol 11 (11) ◽  
pp. 2118 ◽  
Author(s):  
Marek Pszczola ◽  
Cezary Szydlowski
Keyword(s):  
Tensile Strength ◽  
Low Temperature ◽  
Cooling Rate ◽  
Strength Properties ◽  
Test Methods ◽  
Asphalt Mixtures ◽  
Bending Test ◽  
Mixture Composition ◽  
Tension Stress ◽  
Strength Reserve

In regions with low-temperatures, action transverse cracks can appear in asphalt pavements as a result of thermal stresses that exceed the fracture strength of materials used in asphalt layers. To better understand thermal cracking phenomenon, strength properties of different asphalt mixtures were investigated. Four test methods were used to assess the influence of bitumen type and mixture composition on tensile strength properties of asphalt mixtures: tensile strength was measured using the thermal stress restrained specimen test (TSRST) and the uniaxial tension stress test (UTST), flexural strength was measured using the bending beam test (BBT), and fracture toughness was measured using the semi-circular bending test (SCB). The strength reserve behavior of tested asphalt mixtures was assessed as well. The influence of cooling rate on the strength reserve was investigated and correlations between results from different test methods were also analyzed and discussed. It was observed that the type of bitumen was a factor of crucial importance to low-temperature properties of the tested asphalt concretes. This conclusion was valid for all test methods that were used. It was also observed that the level of cooling rate influenced the strength reserve and, in consequence, resistance to low-temperature cracking. It was concluded that reasonably good correlations were observed between strength results for the UTST, BBT, and SCB test methods.

scholarly journals Correlation of Processing, Inner Structure, and Part Properties of Injection Moulded Thin-Wall Parts on Example of Polyamide 66

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Dietmar Drummer ◽  
Steve Meister
Keyword(s):  
Cooling Rate ◽  
Injection Moulding ◽  
Degree Of Crystallinity ◽  
Process Conditions ◽  
Thin Wall ◽  
Polyamide 66 ◽  
Slow Cooling ◽  
Cooling Conditions ◽  
Internal Structures ◽  
Wall Injection

In micro- and thin-wall injection moulding the process conditions affect the developed internal structures and thus the resulting part properties. This paper investigates exemplarily on polyamide 66 the interactions of different cooling conditions on the morphological and crystalline structures. The investigations reveal that a slow cooling rate of the melt results in a homogeneous morphology and a higher degree of crystallinity and also a favoured crystalline structure. Consequently, the dielectric behaviour and light transmitting part properties are affected.

scholarly journals Study of low-temperature exposure on biotissue using an elongated cryoapplicator

2021 ◽  
Vol 2103 (1) ◽  
pp. 012049
Author(s):  
A V Pushkarev ◽  
N A Andreev
Keyword(s):  
Atrial Fibrillation ◽  
Data Analysis ◽  
Low Temperature ◽  
Nitrogen Dioxide ◽  
Liquid Nitrogen ◽  
Biological Tissue ◽  
The Novel ◽  
Treatment Data ◽  
Temperature Exposure ◽  
Low Temperature Exposure

Abstract The article presents the results of a study of low-temperature exposure on animal biological tissue using the novel prototype of a liquid nitrogen cryoapplicator. The data obtained are compared with the cryoapplicator characteristics cooled by nitrogen dioxide that are currently used for the atrial fibrillation treatment. Data analysis confirmed the liquid nitrogen cryoapplicators effectiveness and made it possible to highlight their advantages.

scholarly journals 153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
B.K. Kim ◽  
H.J. Chung ◽  
B.C. Yang ◽  
D.H. Kim ◽  
J.H. Woo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Type I ◽  
Bovine Embryo ◽  
Type Ii ◽  
Bovine Embryos ◽  
Mrna And Protein Expression ◽  
Bovine Oocytes

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.

scholarly journals Cryopreservation in liquid nitrogen of Brazilian orchid seeds Miltonia flavescens Lindl.

2020 ◽  
Vol 14 ◽  
Author(s):  
Jean Carlo Baudraz de Paula ◽  
Walter Aparecido Ribeiro Júnior ◽  
Gabriel Danilo Shimizu ◽  
Gabriel Barraca Men ◽  
Ricardo Tadeu De Faria
Keyword(s):  
Low Temperature ◽  
Water Content ◽  
Liquid Nitrogen ◽  
Biological Material ◽  
Control Treatment ◽  
Preservation Methods ◽  
Lower Performance ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
Protocorm Formation

Miltonia flavescens is a species vulnerable to extinction, which justifies research on preservation methods. Cryopreservation in liquid nitrogen (LN) consists of maintaining biological material at a low-temperature (-196 °C). Thus, the aim of the experiment is to evaluate the influence of different cryoprotective solutions on cryopreservation in LN of the Brazilian orchid Miltonia flavescens seeds. The experiment was conducted in a completely randomized design (CRD), with eight treatments and six replications. The treatments were composed as follows: control; immersion in LN, no cryoprotectant adding; and immersion in LN, with the addition of cryoprotectants: sucrose 0,4 mol L-1; glycerol 2 mol L-1; protection by vitrification in solution (PVS)1; PVS2; PVS2 + 1% phloroglucinol, and PVS3. Except for the control treatment, which was kept in a freezer (10±2 °C), the others remained frozen for 15 days. After this period, the viability of the seeds was evaluated. These seeds were sown and, 30 days after germination, then the frequency of protocorm formation was verified. Before the cryopreservation, the seeds showed 75% viability and 9.5% water content. After cryopreservation, the seeds varied between 67 to 75% viability. However, treatment with glycerol 2 mol L-1 exhibited lower performance than the others (58%). The control treatment showed a higher percentage of protocorm formation (71%) followed by treatments PVS1 (63%), PVS2 (64%), and PVS2PHLO (66%). For the purpose of preserving Miltonia flavescens seeds in liquid nitrogen for a prolonged period, the treatments PVS1, PVS2, and PVS2PHLO proved to be viable and promising alternatives.

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