124 Cell Count of Bovine In Vitro-Produced Blastocysts After In Vitro Maturation in Glass versus Plastic Vials

2018 ◽  
Vol 30 (1) ◽  
pp. 202
Author(s):  
J. O. Secher ◽  
N. Hashem ◽  
J. H. Pryor ◽  
C. R. Long ◽  
J. Docherty ◽  
...  
Keyword(s):  
Cell Count ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Differential Staining ◽  
Atmospheric Air ◽  
Cell Counts ◽  
Blastocyst Rate

Optimal bovine in vitro oocyte maturation (IVM) is a prerequisite for subsequent optimal blastocyst rates. Ovum pick-up (OPU), by which cumulus–oocyte complexes (COC) are collected in vivo, is performed outside a laboratory and often requires IVM to take place during transportation from the farm to the IVF laboratory. Hashem et al. (2017 Reprod. Fertil. Dev. 29, 179) demonstrated that blastocyst rates are affected by type of vial (glass v. plastic), number of COC per vial, and volume of medium per vial. This was achieved by maturing more than 2500 COC from slaughterhouse material under contrasting conditions, followed by standardised IVF and in vitro culture (IVC) and observation of blastocyst rates, morphology (1: poor; 2: good; 3: excellent), and kinetics (1: blastocyst; 2: expanded blastocyst ; 3: hatching/hatched blastocyst). Here we examined differential staining of a subset of expanded blastocysts (XB) from the previous study to assess the influence of vial material, medium volume, and number of COC per vial on total cell count, number and ratio of inner cell mass (ICM), and trophectoderm (TE) cells. In experiment 1 (4 groups), oocytes were matured in different vials without lids in an incubator at 5.5% CO2 in humidified atmospheric air at 38.5°C to assess plastic toxicity. In experiment 2 (6 groups) and experiment 3 (6 groups), the 2 best performing vials-polypropylene cryovials (Sigma-Aldrich, St. Louis, MO, USA) and glass vials (VWR International, Radnor, PA, USA)-containing 50% (Exp. 2) or 95% (Exp. 3) medium volume per vial and 5, 20, or 45 COC per vial were tested. In experiments 2 and 3, the vials were closed and incubated in atmospheric air at 38.5°C. All groups were evaluated for blastocyst rates, kinetics, and morphology. Because kinetics (range 2.01–2.25) and morphology (range 2.15–2.50) were similar in all groups, only XB were collected from each group. These were fixed and stained with CDX2 antibody and Hoechst (Wydooghe et al. 2011 Anal. Biochem. 416, 228-230) and their ICM and TE cells were counted. The cells were counted manually in blinded groups using an inverted fluorescence microscope and 16× magnification. Counts of total, ICM, and TE cells were compared between treatments by a two-way ANOVA analysis. A total of 240 XB from the 16 different vial groups were counted in the 3 experiments, with average total cell counts of 139 (110–211) and ICM cell counts of 44 (28–75). Even though the blastocyst rates differed between some of the groups, the cell counts within the XB did not differ statistically significantly between groups. In fact, the highest cell count was found in the glass vial group with the lowest blastocyst rate (45 COC per vial in 50% medium volume; blastocyst rate 28%, total cells 211, ICM cells 75). We have previously demonstrated that the type of vial, number of COC per vial, and the volume of medium per vial influence the subsequent blastocyst rates. It is concluded, however, that the embryos able to proceed to the blastocyst stages seem to be of the same quality in all groups, assessed by kinetics, morphology, and cell counts within XB.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

176 EFFECT OF LEUKEMIA INHIBITORY FACTOR FROM HUMAN AND MOUSE ORIGIN ON THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 204
Author(s):  
C. De Frutos ◽  
A. Rodríguez ◽  
C. Díez ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Bovine Embryos ◽  
Cell Counts

Leukemia Inhibitory Factor (LIF) is a cytokine with potential to influence embryonic quality and proliferation within the inner cell mass (ICM). However, conflicting effects of LIF have been reported with in vitro-produced (IVP) bovine embryos, in spite of LIF receptor (LIFr) and gp130 transcripts being expressed at all stages during pre-implantation development (Niemann and Wrenzycki 2000 Theriogenology 53, 21–34). As there is no commercially available bovine LIF (bLIF), researchers have used human LIF (hLIF) because of its greater sequence homology compared to murine LIF (mLIF). However, mLIF has been not compared with hLIF in culture with bovine embryos; thus this was the aim of this study. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro and presumptive zygotes cultured in modified synthetic oviduct fluid with 6 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 423 morulae (&gt;90%) and early blastocysts were cultured for 48 h with: (1) 100 ng mL-1 recombinant mLIF (Sigma-Aldrich Quimica SA, Madrid, Spain); (2) 100 ng mL-1 recombinant hLIF (Sigma); and (3) no LIF. Data (6 replicates) were processed by GLM and Duncan&apos;s test, and expressed as LSM � SE (ab: P &lt; 0.05; xy: P &lt; 0.01). Development was recorded up to the hatched blastocyst stage and cells were differentially counted in the ICM and trophectoderm (TE) following the method described by Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29). There were no differences within developmental rate on Day 7, but reduced blastocyst rates were observed on Day 8 between hLIF (42.0 � 3.9a and 27.2 � 3.3a) and controls (57.7 � 3.9b and 38.9 � 3.3b) at the medium and expanded stages, respectively, whereas mLIF had no effect (47.4 � 3.9 and 32.3 � 3.3). Contrary to development, Day 8 blastocysts showed decreased cell counts in both the ICM and the ICM/total cell proportions in the presence of mLIF (19.1 � 3.1x and 13.8 � 2.4x vs. 32.6 � 3.0y and 24.8 � 2.3y for controls, respectively), whereas hLIF had no effect (29.7 � 3.1y and 20.9 � 2.4y). No changes were seen in TE and total cell counts. The disparate effects exhibited by hLIF and mLIF during blastocyst formation may reflect the fact that these compounds are inappropriate to replace bLIF, and/or endogenous LIF probably suffices during bovine development. In fact, mouse embryonic development and blastocyst cell numbers decrease in murine embryos injected with LIF antisense nucleotides (Cheng et al. 2004 Biol. Reprod. 70, 1270–1276). Furthermore, embryonic stem (ES)-like cell derivation in bovine is possible with (Saito et al. 2003 Biochem. Biophys. Res. Com. 309, 104–113) and without (Mitalipova et al. 2001 Cloning 3, 59–67) exogenous LIF. Therefore, strategies to investigate LIF signalling in bovine embryos and stem cells should be reconsidered. This work was supported by Grant AGL2005-04479.

182 FAST STAINING METHODS FOR DIFFERENTIAL STAINING OF INNER CELL MASS AND TROPHECTODERM CELLS OF MAMMALIAN BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
S. W. Kim ◽  
J.-K. Park ◽  
J.-H. Han ◽  
C. G. Park ◽  
W.-K. Chang
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Treatment Method ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Green Color ◽  
Mixture Treatment

The present study was undertaken to develop a simple differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using the permeabilizing agent, saponin, without species-specific antibodies and complements. The nuclei of whole embryos were pre-stained to green by 5 �M SYTO 13 for 10 min. After washing, the green color of TE was turned to red by exposure to 100 �g/mL propidium iodide and 50 to 100 �g/mL saponin solution for 5 to 10 min. To confirm the exactness of staining patterns, the fluorescent nuclei of ICM and TE from mouse, pig, and bovine blastocysts were compared with 3D location by confocal microscopy. By the saponin mixture treatment method, in vitro-cultured mouse, pig, and bovine blastocysts were shown to have an ICM:TE ratio of 1:2.5, 1:4.5, and 1:3.6, with an average total cell number of 78 � 14 (n = 45), 65 � 18 (n = 49), and 150 � 20 (n = 45), respectively. Although a few TE cells were stained to a yellowish-green color, the successful protection of the green color of ICM depended on the exposure time of blastocysts to the saponin mixture. The total time lapse of the procedure did not exceed 1 h. These results indicate that saponin could be used as a practical substitute for special antibodies and complements. So this differential staining for examining the ICM:TE ratio and the total cell count of mammalian blastocysts would be a fast and reliable method.

156 IN VITRO DEVELOPMENT OF BOVINE MORULAE PRODUCED AND/OR CULTURED WITH ACTIVIN

2010 ◽  
Vol 22 (1) ◽  
pp. 236 ◽  
Author(s):  
B. Trigal ◽  
E. Gómez ◽  
C. Diez ◽  
J. N. Caamaño ◽  
I. Molina ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Morula Stage ◽  
Vitro Development ◽  
And Control

We reported that the presence of activin during in vitro culture improves embryo development without changing the cell distribution in the blastocyst (Díez et al. 2009 AETE in press). In the present work, we aimed to analyze the morula stage as a putative milestone to activin exert differential effects. Day -5 morulae were produced with IVMFC oocytes from abattoir ovaries, using SOF with amino acids, myo-inositol, and 3 g L-1 of BSA as a culture medium. Embryo culture contained 10 ng mL-1 or 0 ng mL-1 of activin from Day -3 to Day -5. Early morulae (n = 543 out of 1099 cultured oocytes) were selected and subsequently cultured with or without 10 ng mL-1 of activin up to Day -8. Embryo development was daily monitored and cells differentially counted in Day -8 expanded blastocysts. (Thouas et al. 2001 Reprod. Biomed. 2001 3, 25-29). Data were analyzed by general linear model and presented as least squares means ± SEM. Activin from Days 3 to 5 did not change Day -5 morulae rates (P > 0.8). In morulae produced without activin (Days 5 to 8 and control), a treatment with activin from Days 5 to 8 improved total blastocyst rates v. controls, both in Day -7 and Day -8 (50.9 ± 3.6 v. 32.6 ± 3.6 and 60.8 ± 2.9 v. 42.3 ± 2.9, respectively; P < 0.01). Similarly, Day -7 expansion rates with activin (Days 5 to 8) were higher than controls (14.6 ± 1.8 v. 8.6 ± 1.8; P < 0.03). However, the above effects were not the same as those observed in morulae produced with activin (Days 3 to 5 and Days 3 to 8), where blastocyst development between activin treatment and controls only significantly differed in expansion rates on Day -7 (14.9 ± 1.8 v. 5.8 ± 1.8, respectively; P < 0.03). Morulae treated with activin (Days 5 to 8) yielded Day -7, total and expanded blastocyst rates, higher than morulae produced with activin (Days 3 to 5) (50.9 ± 3.6 v. 37.4 ± 3.6 and 14.6 ± 5.8 v. 5.8 ± 1.8, respectively; P < 0.03). Expansion rates on Day -8 were numerically higher within morulae produced and/or treated with activin (Days 3 to 8, Days 5 to 8, and Days 3 to 5) (values between 26.7 ± 2.6 and 27.4 ± 2.6) than in controls without activin at any time (19.2 ± 2.6) (P > 0.05). Trophectoderm (TE) cell numbers were reduced in embryos produced and/or treated with activin (Days 3 to 8, Days 3 to 5, and Days 5 to 8) (values between 109.4 ± 7.6 and 115.3 ± 7.9) as compared with untreated controls (141.2 ± 10.1) (P < 0.05). In morulae produced without activin, total cell counts were lower with activin being present from Day -5 to Day -8 (154.0 ± 8.8 v. 128.4 ± 7.2; P < 0.05). Inner cell mass (ICM) and ICM/total cell ratio were not affected by the presence of activin (P > 0.05). Activin did not change Day -5 morulae rates, although subsequent blastocyst development was in part affected by the presence of activin before the morula stage. Interestingly, improvements in blastocyst development, including expansion rates, triggered by activin led to reduced TE and unaltered ICM cell counts, suggesting that activin inhibits TE differentiation. Support: Cajastur (B. Trigal). MCINN: M. Muñoz (RYC08-03454); D. Martín (PTA2007-0268-I); INIA (I. Molina); Project HF2007-0126.

218 DEVELOPMENT OF EVENLY AND UNEVENLY CLEAVED TWO-CELL PORCINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 188
Author(s):  
D. N. Q. Thanh ◽  
K. Kikuchi ◽  
T. Somfai ◽  
M. Ozawa ◽  
M. Nakai ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Great Influence ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Further Development

Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 � 0.8% and 91.4 � 3.6%, respectively) or between the 36E and 36U groups (98.2 � 1.1% and 88.0 � 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 � 5.7% and 61.7 � 7.8%, respectively) and total cell numbers (39.1 � 2.1 and 31.7 � 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 � 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 � 5.1%), the total cell number was not different (26.3 � 5.5 and 25.3 � 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 � 6.3%) than did the 36-h embryos (28.2 � 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 � 2.6) were significantly higher than those of the 36-h embryos (26.2 � 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 � 7.0%) and greater cell numbers (40.6 � 2.6) than those not showing a second division during this period (30– group; 46.8 � 5.0% and 19.9 � 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.

Ratio and number of inner cell mass and trophoblast cells of in vitro and in vivo produced porcine embryos

1995 ◽  
Vol 43 (1) ◽  
pp. 304 ◽  
Author(s):  
D. Rath ◽  
H. Niemann ◽  
T. Tao ◽  
M. Boerjan
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophoblast Cells

The control of trophoblastic growth in the guinea pig

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 405-418
Author(s):  
E. B. Ilgren
Keyword(s):  
Guinea Pig ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
The Other ◽  
In Vivo Analysis

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly, the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In a manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

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