Enhancing developmental rate and quality of mouse single blastomeres into blastocysts using a microplatform

The development of greenhouse leaf yellowing in Easter lilies (Lilium longiflorum Thunb.) was significantly reduced by the application of growth regulator solutions containing gibberellins 4 and 7 (GA4+7) or benzyladenine (BA). Solutions containing BA alone significantly reduced leaf yellowing on plants caused by close spacing but were less effective than GA4+7. Application of BA alone, however, was not effective against root rot-induced leaf yellowing. When plants were treated with GA4+7 or BA + GA4+7 around the visible bud stage, nearly all of the leaves remained green until the end of the growing season. These growth regulators, however, increased the final height of the plants by 8–10 cm. The developmental rate and size of the flower buds, as well as the length of the pedicels were not affected by the growth regulator treatments. Thus application of these growth regulators greatly improved the quality of the leaves without compromising the quality and timing of the flowers. Chemical name used: N-(phenylmethyl)-1H-purine-6-amine (benzyladenine, BA).

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Effect of anisomycin, a protein synthesis inhibitor, on thein vitrodevelopmental potential, ploidy and embryo quality of bovine ICSI embryos

Zygote ◽

10.1017/s0967199416000034 ◽

2016 ◽

Vol 24 (5) ◽

pp. 724-732 ◽

Cited By ~ 4

Author(s):

María Elena Arias ◽

Raúl Sánchez ◽

Ricardo Felmer

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Developmental Rate ◽

Fertilization Rate ◽

Terminal Deoxynucleotidyl Transferase ◽

Protein Synthesis Inhibitor ◽

Oocyte Activation ◽

Synthesis Inhibitor

SummaryIncreasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that ofin vitrofertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%,P< 0.05) and DMAP (28.6%,P> 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.

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Quality of rabbit vitrified/thawed transgenic embryos

Zygote ◽

10.1017/s0967199411000384 ◽

2011 ◽

Vol 21 (1) ◽

pp. 53-58 ◽

Cited By ~ 1

Author(s):

P. Chrenek ◽

Z. Turanová ◽

J. Slamečka ◽

A. V. Makarevich

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Developmental Rate ◽

Control Group ◽

Cell Counts ◽

Transgenic Rabbits ◽

Significant Difference ◽

Transgenic Embryos ◽

Rabbit Embryos

SummaryThe aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous–hFVIII or exogenous–enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

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In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment

Reproduction Fertility and Development ◽

10.1071/rd09015 ◽

2009 ◽

Vol 21 (7) ◽

pp. 901 ◽

Cited By ~ 24

Author(s):

Giovanni Giuseppe Leoni ◽

Sara Succu ◽

Valentina Satta ◽

Mereu Paolo ◽

Luisa Bogliolo ◽

...

Keyword(s):

Messenger Rna ◽

Developmental Rate ◽

Cyclin B1 ◽

Developmental Competence ◽

In Vitro Production ◽

Developmental Capacity ◽

Time Required ◽

Vitro Production

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus–oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean ± s.e.m., 89.67 ± 5.74 and 26.69 ± 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90β and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

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Effect of embryo developmental stage and culture conditions on number and quality of ovine in vitro produced blastocysts

Zygote ◽

10.1017/s0967199406003728 ◽

2006 ◽

Vol 14 (3) ◽

pp. 181-187 ◽

Cited By ~ 3

Author(s):

R.M. Garcia-Garcia ◽

V. Dominguez ◽

A. Gonzalez-Bulnes ◽

A. Veiga-Lopez ◽

M.J. Cocero

Keyword(s):

Developmental Stage ◽

Developmental Stages ◽

Developmental Rate ◽

Defined Medium ◽

Culture Conditions ◽

Cell Groups ◽

Early Developmental

SummaryThis study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.

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Ovarian development of Melanoplus sanguinipes (Fab.) (Acrididae: Orthoptera) in relation to utilization of water-soluble food proteins

Canadian Journal of Zoology ◽

10.1139/z72-178 ◽

1972 ◽

Vol 50 (10) ◽

pp. 1319-1324 ◽

Cited By ~ 5

Author(s):

S. S. Krishna ◽

A. J. Thorsteinson

Keyword(s):

Amino Acids ◽

Ovarian Development ◽

Developmental Rate ◽

Disc Electrophoresis ◽

Water Soluble ◽

Melanoplus Sanguinipes ◽

Food Proteins ◽

Old Adult ◽

Layer Chromatography

Virgin, 7- to 10-day-old adult Melanoplus sanguinipes (Fab.) were fed on leaves of different plant species or on bran. Water-soluble prsoteins and amino acids in the hemolymph, ovary, or faeces were compared by disc electrophoresis, colorimetry, and thin-layer chromatography. The data were interpreted in terms of quantity and quality of the food proteins and in relation to developmental rate and survival.

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Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus

Zygote ◽

10.1017/s0967199414000161 ◽

2014 ◽

Vol 23 (4) ◽

pp. 525-536 ◽

Cited By ~ 21

Author(s):

Xue-Ming Zhao ◽

Jiang-Tao Min ◽

Wei-Hua Du ◽

Hai-Sheng Hao ◽

Yan Liu ◽

...

Keyword(s):

Formation Rate ◽

Developmental Rate ◽

Mrna Levels ◽

Developmental Potential ◽

Cumulus Oophorus ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Bovine Oocytes

SummaryThis study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10−9 M melatonin (DOs + MT), cumulus–oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

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136 Efficient in vitro embryo production system using in vivo-matured oocytes from superstimulated Japanese black cows

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab136 ◽

2019 ◽

Vol 31 (1) ◽

pp. 193

Author(s):

J. Egashira ◽

H. Tatemoto ◽

Y. Wada ◽

K. Yamanaka

Keyword(s):

Prostaglandin F2α ◽

Developmental Rate ◽

Blastocyst Stage ◽

Control Group ◽

Total Production ◽

Cleavage Pattern ◽

Cortical Granules

In this study, we examined whether in vivo matured oocytes collected by ovum pickup (OPU) from superstimulated Japanese black cows can improve the productivity and quality of in vitro-produced embryos. Cows in the stimulated group received an intravaginal progesterone-releasing device (Day 0), administration of 100μg of GnRH on Day 5, a single administration of 30 Armour units of FSH on the evening of Day 8 and prostaglandin F2α on the evening of Day 10. The progesterone device was removed on the morning of Day11, and then 100μg of GnRH was administered on the morning of Day 12 (0 h). The OPU and IVF were conducted at 25~26 and 30h, respectively. Cows in the control group received no treatment before OPU, and collected oocytes were subjected to in vitro maturation followed by IVF. The cortical granules distribution of oocytes at metaphase II stage, the cleavage pattern of embryos at the first cell cycle, the developmental rate, and the quality of blastocysts were compared between the stimulated and control groups. Oocytes with cortical granules distributing cortical cytoplasm were classified as normal distribution. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the one-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The developmental rate to blastocyst stage was measured on Day 9 of culture. The morphological quality of blastocysts was evaluated based on the IETS manual. All data were obtained from more than 3 replicates. In vitro development and cortical granules distribution data were analysed using chi-squared test. Other data were analysed using Student’s t-test. Normal cortical granules distribution rate in the stimulated group was higher than that in the control group (90.3v. 23.1%; P<0.01). Although no differences in the developmental rate to blastocyst stage (51.5v. 58.6%) was observed, the normal cleavage rate (73.4v. 51.2%) and the transferable embryo rate (98.3v. 88.0%) in the stimulated group were significantly higher (P<0.01) than those in the control group. The ratio of embryos from normal cleavage among the transferable embryos in the stimulated group was also significantly higher than in the control group (82.2v. 57.2%; P<0.01). In addition, the freezable embryo ratio (71.7v. 58.1%; P<0.072) and the total production number of embryos per head (28.0v. 15.5; P<0.106) showed a tendency to be higher in the stimulated group than in the control group. These results suggest that high quality embryos can be efficiently produced by the use of in vivo matured oocytes collected by OPU from superstimulated Japanese black cows.

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Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus–oocyte complexes exposed to heat shock during in vitro maturation

Reproduction ◽

10.1530/rep-13-0179 ◽

2013 ◽

Vol 146 (4) ◽

pp. 407-417 ◽

Cited By ~ 28

Author(s):

A Z Balboula ◽

K Yamanaka ◽

M Sakatani ◽

M Kawahara ◽

A O Hegab ◽

...

Keyword(s):

Heat Shock ◽

Cathepsin B ◽

Caspase 3 ◽

Developmental Rate ◽

Cell Number ◽

Developmental Competence ◽

Control Group ◽

Tunel Staining

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

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336 DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING SPERM CYTOSOLIC FACTOR (SCF) AT FUSION-ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab336 ◽

2007 ◽

Vol 19 (1) ◽

pp. 283

Author(s):

J. H. Shim ◽

I. S. Hwang ◽

H. J. Moon ◽

M. R. Park ◽

D. H. Kim ◽

...

Keyword(s):

Nuclear Transfer ◽

Developmental Rate ◽

Blastocyst Stage ◽

Fetal Fibroblast ◽

Statistical Analysis System ◽

Donor Cells ◽

Analysis System ◽

Cytosolic Factor

At fertilization, the sperm activates the developmental program of the oocyte by inducing an elevation in the intracellular free Ca2+ concentration ([Ca2+]i). One possible explanation is that at sperm–oocyte fusion the fertilizing spermatozoon introduces a factor into the cytoplasm of the oocyte which opens the Ca2+ release channels from the intracellular stores through a yet unidentified mechanism. This study investigated the development of porcine nuclear transfer embryos fused and activated in the presence of sperm cytosolic factor (SCF) isolated from porcine sperm. Ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h at 35–39�C, and rinsed in 0.9% NaCl. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus for use as donor cells. For parthenogenesis, matured oocytes were activated using 2 DC pulses of 1.2 kV cm-1 for 30 �s in fusion medium (0.1 mM CaCl2) supplemented with 100, 200, or 300 �g mL-1 SCF. For NT, matured oocytes were enucleated, reconstructed, and fused. Reconstructed embryos were divided into 2 groups. The embryos in one group were fused with fusion medium (1.0 mM CaCl2), and the embryos in the other group were fused with fusion medium (0.1 mM CaCl2) supplemented with 100 �g mL-1 SCF. After fusion, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5�C. A TUNEL assay was used to assess the presence of apoptotic cells (In Situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Data were subjected to a generalized linear model procedure (PROC-GLM) of the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). Oocytes activated parthenogenetically in the presence of SCF showed significantly higher developmental rate to the blastocyst stage compared to that of controls (21.3–27.6% vs. 12.5%; P < 0.05). For NT, there was no difference between treatments in developmental rate to the blastocyst stage (18.2% vs. 17.1%). However, the apoptosis rate was slightly lower in blastocysts produced in the presence of SCF than that in controls. These results indicate that the presence of SCF in fusion medium can support a higher quality of porcine nuclear transfer embryos.

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