137 EFFECT OF FLUNIXIN MEGLUMINE IN CO-CULTURE MEDIUM ON THE DEVELOPMENT OF IN VITRO MATURED AND FERTILIZED BOVINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
S. Goda ◽  
S. Hamano ◽  
M. Miyamura ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
Embryo Transfer ◽  
Culture Medium ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
The Other ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Cell Stage ◽  
Flunixin Meglumine

PG concentration is often increased during uterine manipulation with embryo transfer. Embryo viability is affected by the increase in the PGF2α concentration accompanying manipulation of the uterus during embryo transfer. Schrick et al. (2001 Theriogenology 55, 370 abst) observed that treatment with flunixin meglumine, an inhibitor of prostaglandin, increased pregnancy rates depending on the stage and quality of embryos transferred. On the other hand, prostaglandin was secreted by a cumulus cell monolayer in an in vitro culture of bovine oocytes. The present study aimed to assess the effects of flunixin meglumine in culture medium on the development of in vitro-matured and fertilized bovine embryos. COCs were collected from ovaries of slaughtered cows by aspiration. The COCs were matured for 20 h in TCM-199 supplemented with 5% fetal bovine serum (FBS) and antibiotics at 38.5°C under an atmosphere of 2% CO2 in air. Matured COCs were inseminated with 1.0 × 107 sperm mL−1 in BO medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 5 mM theophillin and 5 μg mL−1 heparin for 5 h. All of the inseminated oocytes were introduced into the maturation medium that had been kept with the cumulus cells in the CO2 incubator. At 48 h after insemination, all embryos over the 4-cell stage were cultured in TCM-199 plus 5% FBS supplemented with each of five concentrations of flunixin meglumine (0, 0.0025, 0.005, 0.01, and 0.025%) with a cumulus cell monolayer. Development to the blastocyst stage and quality were examined at Days 7 to 8 (Day 0 = day of insemination) using a microscope. The experiment was replicated four times. Data were analyzed by the chi-square test. The total blastocyst rates from the over-4-cell embryos were 61.2 (52/89), 53.7 (44/89), 65.6 (59/90), 57.3 (51/89), and 33.7% (31/92) for 0, 0.0025, 0.005, 0.01, and 0.025%, flunixin meglumine, respectively. The total blastocyst rate with the flunixin meglumine concentration of 0.025% was significantly lower than those with the other concentrations (P < 0.05). The proportion of grade 1 blastocysts with the flunixin meglumine concentration of 0.005% was significantly higher than that with the 0, 0.0025, and 0.025% concentrations (27.8 vs 11.2, 14.6, and 5.4%; P < 0.05). Our present results show that the addition of 0.005% flunixin meglumine to the co-culture medium is positively associated with blastocyst quality in bovine embryos.

50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
L. Ferré ◽  
C. Fresno ◽  
M. Kjelland ◽  
P. Ross
Keyword(s):  
Cytochalasin B ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Slow Freezing ◽  
Treatment Level ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Stages Of Development ◽  
Cell Layers

The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400 µL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL−1), EGF (50 ng mL−1), ovine FSH (50 ng mL−1), bLH (3 µg mL−1), cysteamine (0.1 mM), and 10% fetal bovine serum (FBS) for 22 to 24 h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90 µg mL−1), penicillamine (3 µg mL−1), hypotaurine (11 µg mL−1), and heparin (20 µg mL−1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5–6, blastocysts at Day 6–6.5, and expanded blastocysts at Day 6.5–7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45 min. The slow freezing protocol consisted of 1.5 M ethylene glycol (EG) + 20% FBS + 0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing the 0.25-mL straws to air (23°C) for 10 s and then underwater at 35°C for 1 min. The vitrification (Cryo-Top) medium was 15% (vol/vol) EG + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos from both groups were cultured in SOF-BSA + 10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0 h), re-expansion, and hatching of the zona pellucida (72 h). Three replicates were performed for each treatment level. Fisher’s l.s.d. test with Bonferroni correction was used to determine treatment differences (P < 0.05). The post-thaw survival, re-expansion, and hatching results showed that either expanded blastocysts (84.7 ± 3.2%, 74.1 ± 3.9%, and 60.9 ± 4.4%) or blastocysts (81.7 ± 3.5%, 69.6 ± 4.2%, and 55 ± 4.6%) were preferred (P < 0.05) embryo stages for cryopreservation compared with morulae (67.6 ± 4.4%, 52.5 ± 4.6%, and 33.2 ± 4.3%). Vitrification and cytochalasin-B pre-freezing exposure (61.3 ± 3.6% and 56.6 ± 3.8%) provided better (P < 0.05) hatching results compared with slow-freezing and without cytochalasin-B (37.8 ± 3.6% and 42.5 ± 3.7%).

Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 662-674 ◽  
Author(s):  
Kayla J. Perkel ◽  
Pavneesh Madan
Keyword(s):  
Culture Medium ◽  
Physiological State ◽  
Proton Nuclear Magnetic Resonance ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Isoleucine Leucine ◽  
H Nmr

SummarySpent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12–24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

91 INTERACTION OF OXYGEN TENSION AND CYSTEAMINE SUPPLEMENTATION DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 158
Author(s):  
L. M. Stauber ◽  
G. E. Seidel
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Culture Medium ◽  
Mammalian Embryos ◽  
Defined Culture

Reactive oxygen species damage early mammalian embryos, so culture of bovine in vitro-produced (IVP) embryos at 5% O2 is clearly superior to 20% O2. The thiol compound cysteamine is an antioxidant and improves in vitro blastocyst production when added to in vitro maturation (IVM) medium. The purpose of this study was to investigate supplementation of IVP embryo culture medium with cysteamine at different oxygen tensions. Bovine ovaries from feedlot heifers were collected from a local abattoir and cumulus–oocyte complexes (COC) were aspirated from 3- to 8-mm follicles. COC were matured in maturation medium with 100μM cysteamine (Reprod. Fertil. Dev. 18, 585) for 23 h in a humidified incubator at 38.5°C in 5% CO2 in air. COC at 23 h of maturation were co-incubated with sperm for 18 h. Cumulus cells were then removed and presumed zygotes were cultured in our chemically defined culture medium system (Reprod. Fertil. Dev. 18, 585) in a 2 × 2 factorial arrangement: with or without 50 μM cysteamine × atmospheres of either 5% O2, 5% CO2, 90% N2, or 5% CO2 in air. There were no treatment differences (P > 0.01) in percentages of oocytes cleaving or reaching the 8-cell stage (Table 1). However, blastocyst production rates were lower (P < 0.01) in the group cultured without cysteamine at 20% O2 compared with all the other groups. Adding cysteamine for embryo culture at 20% O2 resulted in blastocyst rates similar to those cultured at 5% O2 with or without cysteamine. Cysteamine was not beneficial at 5% O2. Table 1.Cysteamine supplementation during in vitro culture of bovine embryos

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Serum Free ◽  
Epidermal Growth ◽  
Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P &lt; 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P &gt; 0.05), whereas the combination P4 + EGF was better than P4 alone (P &lt; 0.05). Blastocyst rate was not different (P &gt; 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P &lt; 0.05) blastocyst rate than control but it was lower (P &lt; 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

178 IN VITRO CULTURE OF BOVINE EMBRYOS WITH NEUROTROPHINS

2007 ◽  
Vol 19 (1) ◽  
pp. 205
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
C. De Frutos ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
The Other ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Cell Counts

Neurotrophins (NTs) mediate human embryonic stem (hES) cell survival and may also improve methods for hES cell derivation (Pyle et al. 2006 Nature Biotech. 24, 344–350) and quality of the inner cell mass (ICM). We searched published microarray data sets for tyrosine kinase receptors (TRK) (geo data base: GSM27469, GSM27470, GSM27471). The analysis suggested that bovine embryos in vitro at unspecified stages express TRKA, for nerve growth factor (NGF); TRKC, for neurotrophin-3 (NT3); and TRKB, for both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF). NTs functionally cooperate among them and also with basic fibroblast growth factor (bFGF) (Pyle et al. 2006; Logan et al. 2006 Brain 129, 490–502). Experiments in progress include detection of TRK expression by RT-PCR at defined development stages, and analysis of embryonic development with NTs and without bFGF. In this work we cultured embryos matured and fertilized in vitro from slaughterhouse oocytes for 8 days in SOF medium with 6 g L-1 BSA and 2 ng mL-1 bFGF (negative control). Development was monitored and cells were differentially counted in the ICM and trophectoderm (TE) of expanded and hatched blastocysts. NTs were used during the whole culture at 20 ng mL-1 as single (4 experimental groups: NGF, NT3, NT4, and BDNF) or as pooled (1 group) NT compounds. Data (5 replicates; 1403 oocytes) were processed by GLM and Duncan&apos;s test, and expressed as LSM � SE (a,b: P &lt; 0.05). At Day 3, no differences were found at the 5- to 8-cell stage, but NT3 and NT4 increased the proportions of embryos at the 8- to 16-cell stage (19.1 � 2.2 and 20.5 � 2.2, respectively, vs. 12.9 � 2.2 to 13.7 � 2.2 within the other groups). On Day 6, NT4 yielded more morulae than controls, BDNF, and NGF (35.3 � 2.7 vs. 26.1 � 2.7, 27.4 � 2.7, and 27.8 � 2.7, respectively), and did not differ from the other groups. NT4 produced more total Day 7 blastocysts than NT3 and BDNF (12.5 � 2.2 vs. 8.1 � 2.2 and 9.9 � 2.2, respectively), whereas there were no differences within medium and expanded blastocysts and Day 8 blastocysts. Proportions of morulae that formed blastocysts were appreciably lower than in concomitant experiments without bFGF. Pooled NTs showed decreased values as compared to some single NTs within the ICM [13.0 � 4.0 vs. 29.1 � 4.6 (NT3) and 24.9 � 4.3 (NGF)], the TE [89.0 � 8.4 vs. 120 � 11.9 (BDNF)], total cells [102.0 � 8.5 vs. 134.0 � 9.9 (NT3), and 140.0 � 12.1 (BDNF)], and tended to differ (P = 0.08) within ICM/total cells [13.1 � 3.1 vs. 21.6 � 3.6 (controls) and 22.2 � 3.6 (NT3)]. Controls differed from BDNF (TE: 88.1 � 9.8 vs. 120.2 � 11.9; total cells: 110.8 � 10.0 vs. 140.0 � 12.1, respectively), and from NT4 for ICM/total cells (21.6 � 3.6 vs. 11.5 � 2.9, respectively). NT4 is likely to exert a role during early embryonic development. However, these blastocysts showed decreased cell counts in the ICM, probably reflected in the pooled NTs group. Targeting proliferation stimuli specifically to the ICM is difficult to get when the ICM is enclosed in the embryo, in contrast with the isolated ICM or the derived stem cells. This work was supported by Grant AGL2005-04479.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Du ◽  
R Li ◽  
Q Zhang ◽  
W Wang
Keyword(s):  
Escherichia Coli ◽  
In Vitro Culture ◽  
Clinical Outcomes ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Culture System ◽  
Microbial Contamination ◽  
Culture Medium ◽  
Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 ◽  
Author(s):  
Gabriela de Oliveira Fernandes ◽  
Marcella Pecora Milazzotto ◽  
Andrei Antonioni Guedes Fidelis ◽  
Taynan Stonoga Kawamoto ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Markers ◽  
Culture Medium ◽  
Culture Media ◽  
Ionization Mass ◽  
Metabolic Profiles ◽  
Bovine Embryos ◽  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

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