Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli)

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

Download Full-text

69 QUALITY OF IN VITRO PRODUCED AND CRYOPRESERVED PORCINE EMBRYOS ASSESSED BY CELL NUMBER, TUNEL-POSITIVE NUCLEI, AND CASPASE-3 ACTIVITY

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab69 ◽

2011 ◽

Vol 23 (1) ◽

pp. 140

Author(s):

M. Bryla ◽

M. Trzcinska ◽

B. Gajda

Keyword(s):

In Vitro Culture ◽

Caspase 3 ◽

Preimplantation Embryos ◽

Lower Percentage ◽

Cell Detection ◽

Large White ◽

Number Of Cells

The aim of this experiment was to investigate the quality of in vitro cultured and cryopreserved porcine expanded blastocysts from Day 5, 6, and 7 of culture. The quality of the preimplantation embryos was determined by counting the number of cells, observing a TUNEL-positive reaction (TUNEL reagent; In Situ Cell Detection kit, Roche Diagnostics, Germany) and by caspase-3 labelling (PhiPhiLuxG2D2 Kit, Calbiochem, Germany). Embryos were collected from 32 superovulated donor gilts. All were crossbreds of Polish Landrace and Large White, age 6–8 months, weighing 90–100 kg. The experiment was done on 2–4 cell embryos produced in vivo and cultured in vitro for 7 days in NCSU-23 medium until expanding blastocyst stage. The embryos of this stage were obtained on Day 5, 6, and 7 of in vitro culture and divided into two groups: control-(1) 210 nonvitrified (NV) embryos and -(2) vitrified/thawed (VT) 169 embryos. The expanded blastocysts were vitrified the open pulled straw (OPS) method (Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). The results were analyzed by Student’s t-test, and all values were significant at P ≤ 0.05. The NV group of embryos showed significant differences in the number of cells (66.5 ± 24.0 v. 54.8 ± 15.9) and in TUNEL-positive nuclei (8.8 ± 12.5 v. 16.2 ± 14.9) between Day 5 and Day 7 of culture, respectively. Analysis of VT embryos also revealed significant differences in the number of cells (65.2 ± 17.4 v. 55.5 ± 14.3) and in TUNEL-positive nuclei (25.5 ± 16.4 v. 35.8 ± 19.3) between Day 5 and Day 7 of culture, respectively. Lower percentage of NT and VT blastocysts produced on Day 5 of culture revealed caspase-3 activity (51.3 v. 64.8%) compared with embryos on Day 7 (76.8 v. 89.3%), respectively. In conclusion, blastocysts cultured in vitro for 5 days consist of a high number of nuclei, have a low incidence of TUNEL-positive nuclei, and low caspase-3 activity compared with blastocysts cultured for 6 and 7 days in all analysed groups. Our results revealed that expanding blastocysts produced on Day 5 of in vitro culture had higher ability to survive vitrification/thawing procedure. This work was supported by Grant NR 12 0036 06 from the National Centre of Research and Development, Poland.

Download Full-text

IN VITRO CULTURE OF MAMMALIAN PREIMPLANTATION EMBRYOS

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.1993.tb00114.x ◽

1993 ◽

Vol 28 (3) ◽

pp. 170-173

Author(s):

T. T. Peura

Keyword(s):

In Vitro Culture ◽

Preimplantation Embryos

Download Full-text

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media

Reproductive BioMedicine Online ◽

10.1016/j.rbmo.2017.02.001 ◽

2017 ◽

Vol 34 (5) ◽

pp. 441-454 ◽

Cited By ~ 17

Author(s):

Rebecca L. Kelley ◽

David K. Gardner

Keyword(s):

In Vitro Culture ◽

Conditioned Media ◽

Preimplantation Embryos ◽

Individual Mouse

Download Full-text

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer

International Journal of Molecular Sciences ◽

10.3390/ijms21197116 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7116

Author(s):

Ximo Garcia-Dominguez ◽

Gianfranco Diretto ◽

Sarah Frusciante ◽

José Salvador Vicente ◽

Francisco Marco-Jiménez

Keyword(s):

Embryo Transfer ◽

Unsaturated Fatty Acids ◽

Ex Vivo ◽

Krebs Cycle ◽

Cumulative Effect ◽

Developmental Trajectory ◽

Preimplantation Embryos ◽

Embryo Cryopreservation ◽

Embryo Survival

Although assisted reproduction technologies (ARTs) are recognised as safe, and most of the offspring seem apparently healthy, there is clear evidence that ARTs are associated with changes in the embryo’s developmental trajectory, which incur physiological consequences during the prenatal and postnatal stages of life. The present study aimed to address the influence of early (day-3 embryos) embryo transfer and cryopreservation on embryo survival, size, and metabolome at the preimplantation stage (day-6 embryos). To this end, fresh-transferred (FT) and vitrified-transferred (VT) embryos were compared using naturally-conceived (NC) embryos as a control reference. The results show that as in vitro manipulation was increased (NC < FT < VT), both embryo survival rate (0.91 ± 0.02, 0.78 ± 0.05 and 0.63 ± 0.05, for NC, FT, and VT groups, respectively) and embryo size (3.21 ± 0.49 mm, 2.15 ± 0.51 mm, 1.76 ± 0.46 mm of diameter for NC, FT, and VT groups, respectively) were significantly decreased. Moreover, an unbiased metabolomics analysis showed overall down-accumulation in 40 metabolites among the three experimental groups, with embryo transfer and embryo cryopreservation procedures both exerting a cumulative effect. In this regard, targeted metabolomics findings revealed a significant reduction in some metabolites involved in metabolic pathways, such as the Krebs cycle, amino acids, unsaturated fatty acids, and arachidonic acid metabolisms. Altogether, these findings highlight a synergistic effect between the embryo transfer and vitrification procedures in preimplantation embryos. However, the ex vivo manipulation during embryo transfer seemed to be the major trigger of the embryonic changes, as the deviations added by the vitrification process were relatively smaller.

Download Full-text