scholarly journals The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification

2015 ◽  
Vol 42 (3) ◽  
pp. 94 ◽  
Author(s):  
Hye Jin Kim ◽  
Ki Hwan Lee ◽  
Sung Baek Park ◽  
Young Bae Choi ◽  
Jung Bo Yang
Keyword(s):  
Cell Number ◽  
Assisted Hatching ◽  
Artificial Shrinkage

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

90 THE EFFECT OF LIPID SEGREGATION WITH OR WITHOUT ZONA PELLUCIDA LASER DRILLING ON POST-THAW EMBRYO DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 125
Author(s):  
J. H. Pryor ◽  
C. R. Looney ◽  
S. Romo ◽  
D. C. Kraemer ◽  
C. R. Long
Keyword(s):  
Zona Pellucida ◽  
Developmental Stages ◽  
Uv Light ◽  
Statistical Significance ◽  
Cell Number ◽  
Live Cells ◽  
Assisted Hatching ◽  
Embryo Viability ◽  
And Control

High levels of lipid within in vitro-produced embryos during freezing can increase intracellular damage and lower production rates (Seidel 2006 Theriogenology 65, 228). The objective of this study was to determine if lipid segregation with or without laser-assisted hatching (LAH), or zona pellucida drilling of in vitro-fertilized (IVF) embryos would enhance in vitro survivability and development 24 h post-thaw. Three replicates utilizing 1179 bovine oocytes (BOMED, Madison, WI, USA) were fertilized with frozen/thawed Tuli bull semen and cultured in G1.3/G2.3 supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 of culture, grade 1 & 2 embryos were morphologically divided into 3 developmental stages: 32-cell (n = 78), compact morula (CM, n = 223), and blastocyst (n = 56). Each group was then randomly allocated to the following treatments prior to cryopreservation in 1.5 m ethylene glycol (Vigro Freeze Plus, Bioniche, Pullman, WA, USA): no treatment (control), 7.5 µg mL–1 cytochalasin B for 20 min (CB), or CB with centrifugation (16 000g) for 20 min (CBCF). All CB treatments were extended to include embryo freezing. Embryos were loaded in sterile straws, frozen at 0.5�C min–1 from –6�C to –32�C, and then plunged into LN2. Frozen embryos were air-thawed for 7 s and then thawed in 35�C H2O for 10 s before being assessed for survivability. Immediately post-thaw, one-half of the CBCF and control groups were subjected to LAH, using a single laser pulse at 90% laser power for 600 µs using the XY Clone� system (Hamilton Thorne Biosciences, Beverly, MA, USA), creating groups CBCFLAH and LAH, respectively. All thawed embryos were cultured in G2.3 for 24 h and evaluated morphologically to determine survivability and development. Live/dead staining was performed by using Hoechst 33342 (2.5 µg mL–1) and propidium iodide (5 µg mL–1) under UV light. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student's t-test. Due primarily to low numbers in embryos in stages other than CM, no differences among treatments were detected. For CM, treatment means ranged from 89.6 to 95.0% and from 69.6 to 82.6% for survival and development, respectively, and no treatment differences were observed. Within the CM stage, CBCFLAH was not different from LAH, CBCF, and control (77.0 v. 71.9, 68.8, and 68.3%, respectively; P > 0.05), but showed a significantly greater percentage of live cells than CB (77.0 v. 65.5%; P < 0.05). CBCFLAH and LAH exhibited a significantly greater number of both total and live cells than control (total cells: 69.4, 69.3, and 53.0; live cells: 56.4, 54.7, and 39.3, respectively; P < 0.05). These data indicate that LAH post-thaw alone or in combination with CBCF improves both total cell number and embryo viability following cryopreservation. Financial support was provided by a grant from TAMU-CONACYT (USA-Mexico) and OvaGenix.

scholarly journals 85ASSISTED HATCHING IMPROVES POST-WARMING IN VITRO VIABILITY OF VITRIFIED PORCINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 164 ◽  
Author(s):  
L.F.S. Beebe ◽  
R.D.A. Cameron ◽  
A.W. Blackshaw ◽  
H. Keates ◽  
M.B. Nottle
Keyword(s):  
Ethylene Glycol ◽  
Cell Count ◽  
Zona Pellucida ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Fluorescent Microscopy ◽  
Cell Number ◽  
Assisted Hatching ◽  
Large White

The objective of this study was to improve the vitrification protocol that was used to obtain the first reported litter of pigs resulting from the transfer of vitrified zona pellucida-intact blastocysts (Beebe et al. 2000 Theriogenology 53: 249 abstr). We had previously noticed that vitrified and warmed early blastocysts rarely hatched after 48h in vitro culture, starting to degenerate after 24h. These experiments examined whether assisted hatching, initially done by removing the zona, then by zona thinning, would improve post-warming in vitro viability. Early blastocysts were surgically recovered from mature Large White x Landrace sows five days after mating. Embryos were washed 3 times in modified phosphate-buffered saline (mPBS; Quinn et al. 1982 J. Reprod. Fert. 66:161–168), and then washed twice and cultured in 40-μL droplets of NCSU23+10% fetal bovine serum under mineral oil at 39°C until ready for vitrification. Embryos were cultured for 25min with 7.5μgmL−1 cytochalasin B, placed into a 0.5-mL eppendorf tube in 30μL of the culture medium, centrifuged for 13min at 13000g and then recovered back to the culture medium. All media involved in the vitrification protocol included cytochalasin B. Centrifuged embryos were equilibrated in 2M ethylene glycol in mPBS at 25°C for 5min, washed briefly in 8M ethylene glycol+7% polyvinylpyrrolidone in mPBS for approximately 20sec, loaded into open pulled straws and plunged into liquid nitrogen. Warming and rehydration was performed by immersing the end of the straw containing the embryos into 1.2mL 1M sucrose in mPBS at 39°C, allowing the thawing media to enter the straw and recovering the embryos from the straw with a pipette, followed by 2min in 1M ethylene glycol in mPBS, and 2min in 0.5M ethylene glycol, both at 25°C. The zona-intact group (ZI) was placed in mPBS at 39°C for at least 5min, then cultured in vitro for 24h. The zona-free group (ZF) was washed briefly in mPBS, and the zona was removed with 0.5% pronase in PBS for 30sec, then washed extensively in mPBS, all at 25°C, and then washed and cultured as described for 24h. After 24h embryos that had reformed the blastocoel and expanded were considered viable. Viable embryos were fixed with 100% ethanol and the nuclei stained with 10ngmL−1 bisbenzimide, visualized and counted using fluorescent microscopy. There were 3 experiments. Removing the zona did not affect survival rates (ZI 81% n=21; ZF 91% n=23) but improved the cell count by 56% (cell number±SEM; ZI 39.1±2.8 n=17; ZF 60.8±4.3 n=20; P&lt;0.05 by ANOVA). A subsequent series of experiments found that zona-thinned embryos (0.25% pronase for 10sec) had the same survival and cell count as zona-free embryos. These experiments show that vitrified porcine blastocysts benefit from assisted hatching, whether the zona pellucida is removed or just thinned.

The ultrastructure of acinar cells in the external orbital gland of young and old male rats

1978 ◽  
Vol 36 (2) ◽  
pp. 276-277
Author(s):  
R. Carriere
Keyword(s):  
Young Adulthood ◽  
Acinar Cells ◽  
Cell Number ◽  
Male Rats ◽  
Polyploid Cell ◽  
Secretory Cells ◽  
Age Changes ◽  
Albino Rat ◽  
Golgi Membranes ◽  
Diploid Cells

The external orbital gland of the albino rat exhibits both sexual dimorphism and histological age changes. In males, many cells attain a remarkable degree of polyploidy and an increase of polyploid cell number constitutes the major age change until young adulthood. The acini of young adults have a small lumen and are composed of tall serous cells. Subsequently, many acini acquire a larger lumen with an irregular outline while numerous vacuoles accumulate throughout the secretory cells. At the same time, vesicular acini with a large lumen surrounded by pale-staining low cuboidal diploid cells begin to appear and their number increases throughout old age. The fine structure of external orbital glands from both sexes has been explored and in considering acinar cells from males, emphasis was given to the form of the Golgi membranes and to nuclear infoldings of cytoplasmic constituents.

Effects of the pseudomonad phytotoxin coronatine on tomato leaf structure and ultrastructure

1995 ◽  
Vol 53 ◽  
pp. 862-863
Author(s):  
D.A. Palmer ◽  
C.L. Bender
Keyword(s):  
Pseudomonas Syringae ◽  
Membrane Integrity ◽  
Leaf Tissue ◽  
Cell Number ◽  
Cell Ultrastructure ◽  
Response To Treatment ◽  
Prominent Symptom ◽  
Chlorophylls A And B ◽  
Cell Dimensions ◽  
Structure And Ultrastructure

Coronatine is a non-host-specific phytotoxin produced by several members of the Pseudomonas syringae group of pathovars. The toxin acts as a virulence factor in P. syringae pv. tomato, allowing the organism to multiply to a higher population density and develop larger lesions than mutant strains unable to produce the toxin. The most prominent symptom observed in leaf tissue treated with coronatine is an intense spreading chlorosis; this has been attributed to a loss of chlorophylls a and b in tobacco. Coronatine's effects on membrane integrity and cell ultrastructure have not been previously investigated. The present study describes changes in tomato leaves in response to treatment with purified coronatine, infection by a coronatine-producing strain of P. syringae pv. tomato, and infection by a cor" mutant.In contrast to H2O-treated tissue, coronatine-treated tissue showed a diffuse chlorosis extending approximately 5 mm from the inoculation site. Leaf thickness, cell number, and cell dimensions were similar for both healthy and coronatine-treated, chlorotic tissue; however, the epidermal cell walls were consistently thicker in coronatine-treated leaves (Figs, la and lb).

Apoptosis and development

2003 ◽  
Vol 39 ◽  
pp. 11-24 ◽  
Author(s):  
Justin V McCarthy
Keyword(s):  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Cell Number ◽  
Model Organisms ◽  
Biological Processes ◽  
Nematode Caenorhabditis Elegans ◽  
Apoptotic Pathway ◽  
Genetic Pathways ◽  
Evolutionarily Conserved ◽  
Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

Culture of Arterial Endothelial Cells

1975 ◽  
Vol 34 (03) ◽  
pp. 825-839 ◽  
Author(s):  
Francois M Booyse ◽  
Bonnie J Sedlak ◽  
Max E Rafelson
Keyword(s):  
Endothelial Cells ◽  
Calf Serum ◽  
Intercellular Junctions ◽  
Cell Number ◽  
Population Doubling ◽  
Homogeneous Population ◽  
Bovine Endothelial Cells ◽  
Pinocytotic Vesicles ◽  
Arterial Endothelial Cells ◽  
Doubling Times

SummaryArterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15 mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially ah (90–95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3–5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12–14 months (30–35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32–34 hours and 29–31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluent cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.

Теория гетерогенного катализа. Теория хемосорбции

2021 ◽  
Author(s):  
Василий Садовников
Keyword(s):  
Heterogeneous Catalysis ◽  
Potential Energy ◽  
Publishing House ◽  
Cell Number ◽  
Oil Refining ◽  
Lateral Interaction ◽  
Reaction Products ◽  
Crystal Face ◽  
Independent Particle ◽  
The Face

This monograph is a continuation of the monograph by V.V. Sadovnikov. Lateral interaction. Moscow 2006. Publishing house "Anta-Eco", 2006. ISBN 5-9730-0017-6. In this work, the foundations of the theory of heterogeneous catalysis and the theory of chemisorption are more easily formulated. The book consists of two parts, closely related to each other. These are the theoretical foundations of heterogeneous catalysis and chemisorption. In the theory of heterogeneous catalysis, an experiment is described in detail, which must be carried out in order to isolate the stages of a catalytic reaction, to find the stoichiometry of each of the stages. This experiment is based on the need to obtain the exact value of the specific surface area of the catalyst, the number of centers at which the reaction proceeds, and the output curves of each of the reaction products. The procedures for obtaining this data are described in detail. Equations are proposed and solved that allow calculating the kinetic parameters of the nonequilibrium stage and the thermodynamic parameters of the equilibrium stage. The description of the quantitative theory of chemisorption is based on the description of the motion of an atom along a crystal face. The axioms on which this mathematics should be based are formulated, the mathematical apparatus of the theory is written and the most detailed instructions on how to use it are presented. The first axiom: an atom, moving along the surface, is present only in places with minima of potential energy. The second axiom: the face of an atom is divided into cells, and the position of the atom on the surface of the face is set by one parameter: the cell number. The third axiom: the atom interacts with the surrounding material bodies only at the points of minimum potential energy. The fourth axiom: the solution of the equations is a map of the arrangement of atoms on the surface. The fifth axiom: quantitative equations are based on the concept of a statistically independent particle. The formation energies of these particles and their concentration are calculated by the developed program. The program based on these axioms allows you to simulate and calculate the interaction energies of atoms on any crystal face. The monograph is intended for students, post-graduate students and researchers studying work and working in petrochemistry and oil refining.

THE EFFECT OF ASSISTED HATCHING IN FROZEN-THAWED EMBRYO TRANSFER CYCLES

2016 ◽  
pp. 80-84
Author(s):  
Thi Tam An Nguyen ◽  
Minh Tam Le ◽  
Ngoc Thanh Cao
Keyword(s):  
Embryo Transfer ◽  
Zona Pellucida ◽  
Assisted Hatching ◽  
Implantation Failure ◽  
Multiple Pregnancies ◽  
Recurrent Implantation Failure ◽  
Key Words Laser ◽  
Implantation And Pregnancy Rates ◽  
Frozen Thawed Embryo Transfer ◽  
Thawed Embryo Transfer

Background: Laser assisted hatching technique based on the hypothesis to make an artificial hole on zona pellucida (ZP) that can help embryo hatching out of ZP easily. This technique has been shown to increase implantation and pregnancy rates in women of advanced age, in women with recurrent implantation failure and following the transfer of frozen–thawed embryos. This study described the outcome of frozen–thawed embryo transfers with laser assisted hatching (LAH), which is one of the safest method in nowadays. Purpose: To assess the effect of assisted hatching technique on the clinical outcomes in vitrified-warmed transfer cycles. Method: A total of 65 thawed-transfer cycles with 153 thawed-embryos undertaken within a 12-month period were analysed, Assisted hatching with laser zona thinning was performed with one-quarter of the zona pellucida circumference. The overall thawed-embryos (day 3) were kept in culture overnight. Patient were prepared the suitable endometrium and transferred embryos advantageously. Results: In which, having the rate of survival embryos were 143 occupying 94.3%, the percentage of grade 1 and 2 embryos occupied 55.9% and 29,3% respectively, and that were enrolled LAH before transfering of frozen–thawed embryos. The average transferred embryos were 2.4±0.8, The rate of implantation per transferred embryos and per transferred embryos cycles was 19.5% and 43.1% respectively. The rate of clinical pregnancies per embryo transfer cycles occupied 33.8% with percentage of early miscarriages (biochemical pregnancies and early clinical miscarriages) was 12.3%. The rate of ongoing pregnancies was 30.8% and multiple pregnancies was low just 12.3%. This result was equal or higher than other researchs in embryos transfer had or no LAH. Conclusion: LAH contributed to stable frozen–thawed embryos transfer effectiveness. Key words: Laser assisted hatching, frozen–thawed embryos transfer, zona pellucida (ZP)

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