Frozen–thawed ampullary cell monolayer improves bovine embryo in vitro development and quality

SummaryThe aim of this study was to evaluate the effects of different timing for frozen–thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen–thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

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Sex differences in response of the bovine embryo to colony-stimulating factor 2

Reproduction ◽

10.1530/rep-16-0336 ◽

2016 ◽

Vol 152 (6) ◽

pp. 645-654 ◽

Cited By ~ 19

Author(s):

Luiz G B Siqueira ◽

Peter J Hansen

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Cell Mass ◽

Transcript Abundance ◽

Bovine Embryo ◽

Sexually Dimorphic ◽

Dependent Manner ◽

Trophectoderm Cells ◽

Gene Assay

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

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Secretome derived from different cell lines in bovine embryo production in vitro

Reproduction Fertility and Development ◽

10.1071/rd17356 ◽

2018 ◽

Vol 30 (4) ◽

pp. 658 ◽

Cited By ~ 3

Author(s):

C. Perrini ◽

P. Esposti ◽

F. Cremonesi ◽

A. Lange Consiglio

Keyword(s):

Embryo Quality ◽

Inner Cell Mass ◽

Cell Mass ◽

Control Group ◽

Bovine Embryo ◽

Blastocyst Development ◽

Glutathione Peroxidase 1 ◽

Amniotic Cell ◽

Amniotic Cells

The present study investigated the effects of conditioned medium (CM), composed of microvesicles (MVs) and soluble factors present in the supernatant (SN), of bovine endometrial and amniotic cells on embryo quality and rate of blastocyst production. Presumptive zygotes were randomly assigned on Days 1, 3 and 5 after fertilisation to synthetic oviducal fluid with amino acids (SOFaa; control) or to SOFaa supplemented with either 20% endometrial or amniotic CM, 20% SN or 100 × 106 MVs mL−1. Embryos were evaluated on Day 7. For groups supplemented with MVs derived from either endometrial or amniotic cells on Day 1 of culture, blastocysts had developed, but at a lower rate than in the control group. Blastocysts had developed in all groups in which endometrial or amniotic cell-derived CM or MVs were added on Day 3 of culture, but the rate of blastocyst development was significantly lower in both CM groups than in the MVs groups. The addition of all secretome fractions (CM, MVs and SN) derived from either bovine endometrial or amniotic cells on Day 5 of culture resulted in blastocyst production, but only amniotic MVs resulted in a blastocyst production rate comparable to that in the control group. Supplementation of SOFaa on Day 5 resulted in a qualitatively higher number of inner cell mass cells compared with the control group only for the amniotic CM and MVs groups. At day 7, these data were confirmed by RT-qPCR evaluation of genes (Bcl-2-associated X protein (BAX) and glutathione peroxidase 1 (GPX1) involved in apoptosis and protection against reactive oxygen species. In conclusion, of the different secretome fractions tested, only amniotic MVs added to SOFaa resulted in better outcomes than in the control group.

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

10.21203/rs.3.rs-112034/v1 ◽

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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In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽

10.1242/dev.45.1.93 ◽

1978 ◽

Vol 45 (1) ◽

pp. 93-105

Author(s):

Brigid Hogan ◽

Rita Tilly

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Giant Cells ◽

Epithelial Layer ◽

Visceral Endoderm ◽

Limited Region ◽

In Vitro Development ◽

Vitro Development ◽

Embryonic Ectoderm

This paper describes the in vitro development of inner cell masses isolated immunosurgically from mouse blastocysts which had been collected on 3·5 days p.c. and then incubated for 24 h. The inner cell masses continue to grow in culture and develop through a series of stages with increasing complexity of internal organization. By day 1 all of the cultured ICMs have an outer layer of endoderm, and by day 3 some of them have two distinct kinds of inside cells; a columnar epithelial layer and a thin hemisphere of elongated cells. Later, mesodermal cells appear to delaminate from a limited region of the columnar layer, close to where it forms a junction with the thinner cells. By day 5, about 25% of the cultured ICMs have a striking resemblance to normal 7·5-day p.c. C3H embryos, with embryonic ectoderm, extra-embryonic ectoderm and chorion, embryonic and extra-embryonic mesoderm, and visceral endoderm. When mechanically disrupted and grown as attached clumps of cells in a tissue dish, these embryo-like structures give rise to trophoblast-like giant cells. These results suggest that the inner cell mass of 4·5-day p.c. blastocysts contains cells which can give rise to trophoblast derivates in culture.

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104 BIRTH OF FIRST BUFFALO (BUBALUS BUBALS) CALF FOLLOWING EMBRYO SPLITTING AND POLYMERASE CHAIN REACTION SEXING AT BLASTOCYST STAGE

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab104 ◽

2012 ◽

Vol 24 (1) ◽

pp. 164 ◽

Cited By ~ 1

Author(s):

M. Zhang ◽

H. H. Chen ◽

J. W. Tang ◽

X. W. Liang ◽

M. T. Chen ◽

...

Keyword(s):

Nested Pcr ◽

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Buffalo Calf ◽

Practical Procedure ◽

Blastocyst Quality ◽

Grade 3 ◽

Vitro Development

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).

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157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab157 ◽

2016 ◽

Vol 28 (2) ◽

pp. 208

Author(s):

N. G. Alves ◽

I. J. Ascari ◽

L. S. A. Camargo ◽

J. Jasmin ◽

C. C. R. Quintão ◽

...

Keyword(s):

Heat Shock ◽

Embryo Quality ◽

Linear Models ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Quality Data ◽

Trophoblast Cells ◽

Melatonin Concentration

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P > 0.05). The total number of cells was not affected (P > 0.05) by melatonin, but it was decreased (P < 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P > 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P < 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

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In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation

Development ◽

10.1242/dev.66.1.43 ◽

1981 ◽

Vol 66 (1) ◽

pp. 43-55

Author(s):

J. Rossant ◽

K. M. Vijh

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Giant Cells ◽

Blastocyst Stage ◽

Parietal Endoderm ◽

Trophoblast Giant Cells ◽

Vitro Development ◽

Ectoplacental Cone

Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6·5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8·5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5·5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives, It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.

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In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽

10.1242/dev.45.1.107 ◽

1978 ◽

Vol 45 (1) ◽

pp. 107-121

Author(s):

Brigid Hogan ◽

Rita Tilly

Keyword(s):

Outer Layer ◽

Normal Mouse ◽

Inner Cell Mass ◽

Cell Mass ◽

Giant Cells ◽

Visceral Endoderm ◽

Floating Structures ◽

Parietal Endoderm ◽

Vitro Development

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3·5 and 4·0 days p.c. By 24–48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm- like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

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Colony-Stimulating Factor 2 (CSF-2) Improves Development and Posttransfer Survival of Bovine Embryos Produced in Vitro

Endocrinology ◽

10.1210/en.2009-0481 ◽

2009 ◽

Vol 150 (11) ◽

pp. 5046-5054 ◽

Cited By ~ 90

Author(s):

Bárbara Loureiro ◽

Luciano Bonilla ◽

Jeremy Block ◽

Justin M. Fear ◽

Aline Q. S. Bonilla ◽

...

Keyword(s):

Embryonic Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Colony Stimulating Factor ◽

Bovine Embryos ◽

Epigenetic Effects ◽

Trophectoderm Cells ◽

Stimulating Factor

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30–35 of gestation. Moreover, treatment with CSF2 from either d 1–7 or 5–7 after insemination reduced pregnancy loss after d 30–35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

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